A dissertation submitted in partial satisfaction of the...

Core Promoter Recognition Complex Switching in Liver Development and Regeneration

by

Joseph Anthony D’Alessio A.B. (Bowdoin College) 1998

A dissertation submitted in partial satisfaction

of the requirements for the degree of

Doctor of Philosophy

in

Molecular and Cell Biology

in the

Graduate Division

of the

University of California, Berkeley

Committee in charge:

Professor Robert Tjian, Chair Professor Michael Levine Professor Iswar Hariharan

Professor David V. Schaffer

Fall 2009

Abstract

Core Promoter Recognition Complex Switching

in Liver Development and Regeneration

by

Joseph Anthony D’Alessio

Doctor of Philosophy in Molecular and Cell Biology

University of California, Berkeley

Professor Robert Tjian, Chair

The exquisite structural and functional complexity of the mammalian body

requires the exactingly precise spatial and temporal regulation of gene expression. The

most critically regulated step of which is the transcription of DNA to RNA by the

elaborate interplay of sequence-specific activators and repressors, chromatin modifiers,

coactivators, the basal machinery and RNA polymerase II. Until recently, developmental

stage and tissue-specific gene expression patterns were believed to be regulated primarily

by the combinatorial actions of DNA binding activators and repressors, while the core

machinery was held to be invariant. New evidence suggests that significant differences

in coactivator and core promoter recognition complex composition between cell types

and developmental time points may facilitate the global changes in gene expression

programs necessary to confer functional diversity. Because of its critical role in

vertebrate development and homeostasis, the role of coactivator and core promoter

recognition complex switching in the developing liver was examined.

This work shows that mouse liver progenitors or hepatoblasts contain significant

levels of the canonical core promoter recognition complex TFIID, including the TATA

binding protein and multiple associated factors or TAFs, and the canonical coactivator

complex Mediator. These complexes and their constitutive proteins are significantly

downregulated in adult hepatocytes. Furthermore, the promoters of several TFIID

components become enriched for repressive chromatin marks in adult hepatocytes, and an

in vitro model of liver development recapitulates the downregulation of these complexes

observed in vivo. In contrast, expression of the TBP-related factor TRF3, the TAF7

paralogue TAF7l, and TAF13 is maintained or induced upon hepatic differentiation.

Additionally, these proteins associate with high molecular weight complexes in vitro

where they are enriched at hepatocyte-specific promoters, and their depletion attenuates

hepatic gene induction. Together these observations support a model wherein liver

development and the induction of hepatic gene expression requires the down regulation

of TFIID and its replacement with one or more complexes likely containing TRF3,

TAF7l and TAF13.

For my parents

Anne and Richard D’Alessio

who have supported all my adventures

and

with appreciation for Laurie McDonough

my best friend

Table of Contents

Chapter 1: Introduction

Summary 1

TFIID, TAFs, and the Basal Transcription Machinery 3

Alternative TAF Complexes, TBP-Related Factors, 7 and Tissue-Specific TAFs

TAFs in Metazoan Development and Differentiation 11

Embryonic and Postnatal Liver Development 14

Hepatic Regeneration 18

Embryonic Stem Cells and Therapeutic Approaches 22

Figures 26

References 32

Chapter 2: TBP, TRFs, and TAFs in Liver Development and Regeneration

Summary 54

Introduction 55

Results 58

Discussion 68

Materials and Methods 71

Figures 76

References 94

Chapter 3: TRF3, TAF7l, and TAF13 in Hepatic Gene Regulation

Summary 99

Introduction 100

Results 110

Discussion 119

Materials and Methods 124

Figures 129

References 145

Chapter 1: Introduction 1

Chapter 1: Introduction

Summary

The precise spatial and temporal regulation of gene expression essential to

metazoan development and cellular homeostasis requires a complex interplay between

sequence-specific activators and repressors, coactivators, chromatin modifiers, general

transcription factors (GTFs), and RNA polymerase II (PolII). The first and most

regulated step in this process is the exacting recognition of unique gene-specific DNA

regulatory sequences by diverse activator proteins. These activators then recruit the core

promoter recognition complex TFIID consisting of the TATA box binding protein (TBP)

and 12-15 associated factors (TAFs) to the site of transcription. Subsequently,

coactivators including the Mediator complex (Med), chromatin modifiers, and the

additional GTFS TFIIA-F are recruited to the core promoter to form the preinitiation

complex (PIC). This allows for the recruitment of PolII and the start of productive RNA

transcription.

Historically, developmental stage and tissue-specific patterns of gene expression

were thought to be determined primarily by DNA regulatory sequences and their

associated activators, while the general transcription machinery including TFIID was

held to be invariant (reviewed by Thomas and Chiang 2006). However, recent studies

have shown that during skeletal muscle myogenesis a novel mechanism of regulating

global patterns of gene expression by switching of core promoter recognition complexes

is employed (reviewed by Reina and Hernandez 2007). Whether this novel mechanism


of transcriptional regulation exists in other tissues and developmental programs or is

restricted to muscle is currently unknown.

Chapter two examines patterns of TBP and TAF expression in diverse adult

mouse tissues and finds that they are significantly down regulated in the adult liver.

Further by purifying fetal liver progenitors (hepatoblasts), partially differentiated

neonatal hepatocytes, adult hepatocytes, and cycling regenerative hepatocytes, I

demonstrate that TFIID is significantly upregulated in liver progenitors and entirely

absent from the fully differentiated cell type, while at intermediate levels in slowly

dividing or not fully differentiated populations. In striking contrast, the TBP-related

factor TRF3 and TAF13 are expressed at the same levels throughout liver development

and the unique TAF paralogoue TAF7l is highly induced upon differentiation.

Importantly, these changes occur at both the transcriptional and protein levels and can be

reconstituted in an in vitro cellular model of liver differentiation.

The third chapter of this thesis confirms the presence of TRF3, TAF7l, and

TAF13 polypeptides in differentiated liver cells and provides evidence that they are

components of one or more high molecular weight complexes in vivo. Furthermore,

RNAi mediated depletion of each of these three proteins attenuates hepatic gene

induction in an in vitro model of liver differentiation, and all three proteins show varying

degrees of enrichment at the proximal promoters of hepatocyte-specific genes. Finally,

an embryonic stem cell based hepatic differentiation protocol suggests that these changes

in core promoter recognition complex architecture may extend beyond the murine model.


TFIID, TAFs, and the Basal Transcription Machinery

Proteins, the basic functional units of every living cell, are produced by the

transcription of DNA to messenger RNA, the splicing and nuclear export of this mRNA,

and the translation of mRNAs to polypeptides in a cell’s cytoplasm; a process known as

the Central Dogma (Crick 1958). The most critical and regulated step in this process is

the precise transcription of protein coding regions within a cell’s DNA by RNA

polymerase II (PolII) to form mRNA. The recruitment of PolII and initiation of

transcription at specific genes requires the exquisite coordination of hundreds of

polypeptides including the core promoter recognition complex TFIID (Figure 1) (Thomas

and Chiang 2006).

TFIID was initially identified as a biochemical activity required for in vitro

transcription by PolII (Matsui et al. 1980). Upon further purification, it was identified as

a 750kD complex containing the TATA box binding protein (TBP) and 12-15 associated

factors (TAFs) that are required for activated transcription both in vitro and in vivo

(Dynlacht et al. 1991; Pugh and Tjian 1991). It is now known that both TBP and most of

the TAFs are highly conserved in yeast (S. cerevisiae), worms (C. elegans), flies (D.

melanogaster) and humans (H. sapiens) and partially conserved in the metazoan

predecessor M. brevicollis, suggesting an ancient evolutionary origin (Burley and Roeder

1996; Albright and Tjian 2000; King et al. 2008).

The process of regulated transcription takes place by the stepwise assembly of the

preinitiation complex (PIC), followed by promoter escape, productive elongation, and

termination. PIC assembly is initiated through the precise recognition and binding of


distinct DNA sequences or enhancers within a gene’s distal or proximal regulatory

regions by a diverse set of tissue and developmental stage-specific transcriptional

activators. These activators in turn position TFIID at the core promoter through binding

of their activation domains either directly to TBP or a subset of specific TAFs (Stringer et

al. 1990; Chen et al. 1994; Thut et al. 1995). TFIID’s affinity for the core promoters is

further stabilized by its recognition of conserved motifs within the promoter itself

through both TBP and a limited number of TAFs (Smale and Kadonaga 2003). The most

widespread and important of these motifs include the TATA box (-30), the initiator (Inr,

+1), and the downstream promoter element (DPE, +30), while unique core promoter

elements continue to be discovered and their significance analyzed (Juven-Gershon et al.

2008). Currently, it is poorly understood whether distinct core promoter recognition

complexes are selectively recruited to specific genes based on their preference for certain

core promoter motifs (Muller et al. 2007).

TFIID’s association with promoter DNA is further stabilized through the

sequential recruitment of heterodimeric TFIIA, which can bind directly to TBP, TAFs

and activators, and the single subunit TFIIB, which interacts with TFIID, PolII and

promoter DNA itself (Maldonado et al. 1990). As part of this heterotrimeric complex,

TFIIB in turn recruits TFIIF, which maintains multiple contacts with, and thus brings to

the promoter, the PolII holoenzyme (Ha et al. 1993). Subsequently, TFIIF recruits

heterodimeric TFIIE, which stabilizes PolII binding and primarily functions to recruit the

large multisubunit TFIIH, which posses multiple enzymatic activities required for the

initiation of transcription (Maxon et al. 1994). Hence, TFIIH contains a helicase activity


necessary for the melting of promoter DNA, an ATPase activity necessary for formation

of the first phosphodiester bond, and a kinase domain which phosphorylates the C-

terminus of PolII to signal the transition from initiation to productive elongation (Lu et al.

1992; Holstege et al. 1996). Thus, once recruited to the core promoter by sequence-

specific activators, TFIID coordinates the stepwise assembly of all general transcription

factors required for initiation of productive PolII transcription.

Extensive structural and mapping studies have begun to provide a picture of how

TBP and the TAFs are assembled and positioned within the larger TFIID holoenzyme

(Cler et al. 2009). One of the first insights into TFIID structure came from the

identification of histone fold domains within several of the TAFs, leading to speculation

that TFIID may contain nucleosome like octomers. Biochemical studies have confirmed

that TAFs with like histone fold domains do pair to form heterodimers that make up the

basic structural subunits of the complex, including the pairs TAF4-TAF12, TAF6-TAF9,

TAF8-TAF10, and TAF11-TAF13 (Selleck et al. 2001; Leurent et al. 2002). Though the

exact stoichiometry of TAFs within the complex is unclear, biochemical studies suggest

that certain TAFs, including TAF1, TAF2, and TAF7, are present as a single copy while

the majority of TAFs are present as two or more subunits per TFIID molecule (Sanders et

al. 2002). Though X-ray crystallographic structures of intact TFIID have not yet been

solved, lower resolution electron microscopy and particle reconstruction has provided

some insight to the three dimensional structure of the complex (Andel et al. 1999; Brand

et al. 1999; Sanders et al. 2002). These structures reveal a tri-lobed horseshoe like

structure in which TBP can be localized to the central cleft, suggesting that this is the


sight of DNA binding. Further, TAF12, TAF4, TAF6, and TAF9 appear to be present in

each lobe, while TAF5 is present in the linker region joining all three lobes consistent

with a scaffolding function (Leurent et al. 2004). RNAi and overexpression studies in

insect cells suggest that TAF4, TAF5, TAF6, TAF9, and TAF12 are essential for the

formation of a stable core complex in vivo, which is then decorated by additional TAFs to

form holo-TFIID (Leurent et al. 2002; Wright et al. 2006).

Through a combination of in vitro and in vivo experiments, the specific functions

of individual TAFs have been greatly illuminated (Albright and Tjian 2000). A subset of

TAFs has been found to bind known sequence-specific activators and thus link these

proteins’ activation domains to the rest of the basal machinery. Some of these observed

interactions include binding of p53 to TAF6 and TAF9, binding of SP1 and Pygopus to

TAF4, binding of VP16 to TAF9, and binding of multiple activators to TAF7 (Goodrich

et al. 1993; Weinzierl et al. 1993a; Chiang and Roeder 1995; Thut et al. 1995; Wright and

Tjian 2009). Yet other TAFs bind specific motifs within the core promoter to help

stabilize TFIID position and PIC assembly. Hence, TAF1 and TAF2 can bind the Inr

element while TAF6 and TAF9 contact the DPE (Verrijzer et al. 1995; Burke and

Kadonaga 1997). More recently, some TAFs have been shown to interact directly with

chromatin marks such as TAF1 with acetylated histone tails and TAF3 with trimethylated

histone H3 lysine 4 residues (Jacobson et al. 2000; Vermeulen et al. 2007). Interestingly,

TAF1 possesses kinase, ubiquitin ligase, and acetylase enzymatic activities which may

act on histones or activators to further modulate transcription (Wassarman and Sauer


2001). Thus, individual TAFs serve specific functions within the context of the larger

complex, many of which are yet to be fully understood.

Alternative TAF Complexes, TBP-Related Factors, and Tissue-Specific TAFs

While TFIID is thought to be ubiquitously expressed and present at most genes,

several complexes have been discovered which contain a subset of TAFs, are free of

TBP, and likely act on a more limited number of promoters (Figure 2). The human TBP-

free TAF-containing complex (TFTC) which contains many of the TAFs as well as

additional proteins not found in TFIID, is able to bind multiple activators and facilitate in

vitro transcription, though its role in vivo is not well understood (Wieczorek et al. 1998).

The yeast Spt-Ada-GCN5 acetyltransferase complex (SAGA) contains both overlapping

and unique subunits with TFTC and appears to be important in the activation and

recruitment of free TBP to a subset of mostly stress responsive genes (Grant et al. 1998;

Huisinga and Pugh 2004). Similarly, SAGA-like (SLIK) and the human Spt3-TAF9-

GCN5L acetyltransferase complex (STAGA) have very similar composition to SAGA

and likely many of the same functions but are less well studied (Martinez et al. 1998;

Pray-Grant et al. 2002). The p300/CBP-associated factor (PCAF) complex has many

similarities to yeast SAGA and is involved in both global histone modifications and

locus-specific coactivation; additionally its disruption has been implicated in oncogenic

transformation (Ogryzko et al. 1998; Nagy and Tora 2007). Hence, while the

physiological roles of these alternative TAF containing complexes are not yet fully


understood, it is clear that TAFs have important functions outside the context of

canonical TFIID.

To date three distinct TBP-related factors (TRFs) or TBP-like proteins (TBPL)

have been discovered, from worms to humans, and their biological significance is

beginning to be unraveled (Figure 2) (Aoyagi and Wassarman 2000; Hochheimer and

Tjian 2003). TBP-related factor 1 (TRF1) was the first of these proteins to be identified

and appears to be unique to Drosophila, where it is highly expressed in the nervous

system and testes (Crowley et al. 1993). Similarly to TBP, TRF1 is found in a large

complex, binds TFIIA, TFIIB, and TATA containing DNA, and can promote PolII

transcription in vitro (Hansen et al. 1997). However, TRF1 appears to be distinct from

TBP in that its expression is restricted to a limited number of tissues, it is present at

relatively few promoters, and it also binds to a unique TC box core promoter element

(Holmes and Tjian 2000). Initially identified based on homology, TBP-related factor 2

(TRF2) is found from worms to humans and is more closely related to TBP than TRF1

(Dantonel et al. 1999; Rabenstein et al. 1999). Though it does interact with TFIIA and

TFIIB, TRF2 fails to bind a canonical TATA box and appears to bind novel promoters

not occupied by TBP or TRF1 (Hansen et al. 1997). Hence, it has been suggested that

TRF2 may also act as a repressor by disrupting PIC formation at TATA containing

promoters (Moore et al. 1999). Further, TRF2 is part of a high molecular weight

complex which lacks TAFs but contains known chromatin remodeling factors

(Hochheimer et al. 2002). Knockdown studies in worms and frogs (X. laevis) showed

that loss of TRF2 caused early embryonic arrest through the disruption of developmental


gene expression (Dantonel et al. 2000; Veenstra et al. 2000); however, TRF2 knockout

mice are viable with the only apparent phenotype being disrupted spermiogenesis (Zhang

et al. 2001).

The most recent and perhaps most interesting of these proteins to be identified is

TBP-related factor 3 (TRF3/TBPL2). Initially isolated based on the near identity of its

C-terminus to that of TBP including the DNA and GTF binding domains, TRF3 is

conserved in vertebrates from fish (Fugu) to humans but is absent in lower metazoans

(Persengiev et al. 2003). Initial reports showed that it was widely but variably expressed

in adult mouse and human tissues, migrated as part of a larger 150-200 kD complex, and

that its nuclear import was regulated during the cell cycle asynchronously from that of

TBP. Subsequent studies showed TRF3 to be highly enriched in the ovary and to a lesser

extent in the testes where it had a different pattern of nuclear import than TBP. However,

these reports conflicted as to whether TRF3 is expressed in early embryogenesis

following fertilization (Xiao et al. 2006; Yang et al. 2006; Gazdag et al. 2007; Jacobi et

al. 2007). Additionally, one of these studies found that TRF3 and TBP occupied unique

sets of promoters in mouse embryonic stem cells, while two others showed that TRF3 but

not TBP was required for the transcription of a majority of embryonic genes.

Knockdown studies in frogs and zebrafish (D. rerio) determined that TRF3 is required for

embryogenesis and may be able to partially rescue loss of TBP in the early embryo

(Bartfai et al. 2004; Jallow et al. 2004). Interestingly, regulation of the mespa gene by

TRF3 is shown to be the earliest and an essential step in the commitment of mesoderm to

hematopoietic precursors, suggesting that TRF3 plays a role in cell-type-specific


differentiation programs (Hart et al. 2007). Perhaps the best evidence for TRF3’s role in

tissue-specific differentiation comes from studies of the transition from muscle precursors

to mature muscle fibers. In this myogenic program it was found that canonical TFIID,

including both TBP and most of the TAFs, is completely degraded, while a unique

complex of TRF3 and TAF3 is retained in the adult cell type (Deato and Tjian 2007).

Furthermore, knockdown of TRF3 blocks myogenic differentiation and TRF3 but not

TBP is enriched at the promoters of key myogenic genes. Additional in vitro dissection

of this mechanism found that the TRF3/TAF3 complex cooperates with the myogenic

activator MyoD to initiate transcription of the myogenin promoter, and that this complex

is necessary and sufficient for the process (Deato et al. 2008).

In addition to TBP-related factors, a series of novel tissue-specific TAF paralogs

have been identified which further expand the diversity of core promoter recognition

complexes (Figure 2). Apparently unique to flies are the testes-specific TAFs, no hitter

(dTAF4b), cannonball (dTAF5b), ryan express (dTAF12b), and meiosis I arrest

(dTAF6b), all of which are required for spermatogenesis and normal germ cell-specific

gene expression (Hiller et al. 2001). Originally isolated from B-cells and highly

expressed in the testes and ovary, human TAF4b is the best understood tissue-specific

TAF. It associates with a slightly altered TFIID, which may or may not contain copies of

TAF4 and is required for expression of a subset of ovary-specific genes (Freiman et al.

2001; Geles et al. 2006). Furthermore, TAF4b knockout mice display defects in

folliculogenesis and spermatogenesis but no other apparent phenotypes, consistent with a

germ cell-specific pattern of expression and function (Falender et al. 2005). Similarly,


abundant expression of TAF7l coincides with reduced TAF7 expression in mouse

spermatids, and TAF7l associates with TBP and TAF1 presumably to form an altered

TFIID like complex (Pointud et al. 2003). Additionally, TAF7l knockout mice produce

abnormal sperm, have reduced fertility and overall weight, and show altered patterns of

testes-specific gene expression (Cheng et al. 2007). However, to date the full range of

phenotypes of these mice have not been characterized. The less well-studied TAF5l has

been associated with an increased risk of type I diabetes, but its pattern of expression and

association with TFIID remain to be determined (Cooper et al. 2007). Similarly, initial

characterization of TAF9l shows that it is a component of both TFIID and TBP-free

complexes, and that its loss affects the expression of a unique and overlapping set of

genes from that of TAF9 (Frontini et al. 2005). Finally, cell-type-specific alternative

splicing of TAF6 and possibly other TAFs may further expand the diversity of core

promoter recognition complexes (Weinzierl et al. 1993b).

TAFs in Metazoan Development and Differentiation

The central role TAFs and canonical TFIID in organismal development is

supported by the initial discovery of several TAFs in forward genetics screens and

confirmed by the requirement for many others as shown in depletion and knockout

experiments. Despite these findings, most TAFs, with the exception of tissue-specific

paralogs, are assumed to be ubiquitously expressed and canonical TFIID present at a

majority of promoters in nearly all tissue types. Hence, the first evidence of TFIID’s in

vivo function came from mutations in TAF4 and TAF6, which were homozygous


embryonic lethal and prevented proper complex formation in a sensitized Drosophila

suppressor screen (Karim et al. 1996; Sauer et al. 1996). Further analysis of the

heterozygous mutants showed a dosage dependent requirement for these TAFs as co-

activators of a known transcriptional activator at two important embryonic promoters

(Zhou et al. 1998; Pham et al. 1999). An independent mutation in TAF9 was found to

have dosage dependent affects on the expression of multiple genes, and these different

affects are potentially influenced by the genes differing core promoter architecture

(Soldatov et al. 1999). Finally, loss of function mutations in TAF1 were found to affect

the development of specific Drosophila tissues including the eye, ovaries, wing and

bristle by altering cell cycle progression and tissue-specific differentiation (Wassarman et

al. 2000).

Initial knockout studies in mice showed that loss of TAF8 or TAF10 destabilized

TFIID resulting in apoptosis of the blastocyst's inner cell mass and embryonic lethality,

though trophoblast viability appeared to be unaffected (Voss et al. 2000; Mohan et al.

2003). Conditional TAF knockout mice, which avoid the embryonic lethality phenotype,

are providing greater insight to tissue-specific TFIID function. As expected, a

conditional TAF4 allele failed to produce viable offspring when deleted in ES cells, but

was successfully employed to generate proliferative embryonic fibroblasts devoid of

TAF4 expression (Mengus et al. 2005). While these cell lines divided normally at routine

serum concentrations they displayed altered morphology, accelerated serum independent

growth, and disrupted expression of genes responsible for TGF signaling. Targeted

deletion of TAF4 in basal keratinocytes demonstrated that it is absolutely required for


fetal epidermis differentiation but partially dispensable for adult skin maintenance

(Fadloun et al. 2007). Furthermore, TAF4 inactivation increased the incidence of

epidermal hyperplasia, and this phenotype was traced to upregulation of multiple EGF

genes and downregulation of MAPK pathway components.

A similar approach to keratinocytes-specific deletion of TAF10 demonstrated

slightly altered requirements for this subunit relative to that of TAF4 (Indra et al. 2005).

Specifically, TAF10 is required for fetal epidermal differentiation, skin barrier formation,

and normal patterns of fetal skin gene expression, but dispensable for adult keratinocyte

homeostasis. Hence, adult epidermis lacking TAF10 showed normal gene expression

patterns, responses to UV irradiation, and proper regeneration after wounding. Recently,

the requirements of TAF10 in fetal liver development and adult homeostasis were

analyzed by targeted deletion of the same allele (Tatarakis et al. 2008). These studies

showed that TAF10 is required for the expression of most fetal hepatic genes and normal

liver organogenesis, and that its deletion disrupts normal TFIID complex assembly along

with general transcription factor promoter occupancy. Conversely, loss of TAF10 in

adult hepatocytes has no detectable adverse phenotypes, causes downregulation of only a

small subset of genes, and surprisingly derepresses genes that are normally silenced

neonatally. These observations led the authors to propose a multiclass gene model in

which some promoters require TFIID for their ongoing transcription, others are actively

repressed by TFIID occupancy, and the majority employ TFIID for the first round of fetal

transcription but continue to be activated in the adult despite its absence. New

conditional knockout models and novel approaches to in vivo TAF ablation, including


virally mediated RNA, will continue to enhance our understanding of TFIID’s function in

mammalian development and differentiation.

Embryonic and Neonatal Liver Development

The liver is unique to vertebrates and is responsible for many of the most critical

metabolic and homeostatic functions of adult organisms in addition to being required for

hematopoiesis during fetal and neonatal development. In the adult the liver is the

primary site of carbohydrate, lipid, and protein metabolism and storage. Additionally, it

is responsible for the synthesis and secretion of most serum proteins, including many

growth and coagulation factors and several digestive enzymes. Finally, the liver is a

critical site for the removal of endogenous waste products and exogenous toxins,

including most pharmaceuticals. Thus because of its central and varied role in

organismal function, there is great interest in understanding how the vertebrate liver

develops and is maintained (Zaret 2002; Zaret and Grompe 2008).

While the molecular mechanisms of liver organogenesis are not yet fully

understood, the morphogenetic steps of liver development have been well characterized.

At mouse embryonic day 6.5-7.5 or 15-16 in humans, the primitive streak forms anterior

to posterior along the ventral midline initiating gastrulation and leading to the creation of

the three germ layers (Figure 3). Of these the definitive endoderm results from

ingression of epiblast cells through anterior portions of the streak and eventually gives

rise to the organs of the embryonic gut including the liver, pancreas, lung, and thyroid; at

the same time the extra-embryonic visceral endoderm is displaced laterally (Lewis and


Tam 2006). Cell lineage experiments show that the hepatic endoderm arises soon after

gastrulation from lateral portions of the ventral foregut epithelium and a smaller group of

cells along the ventral midline epithelium, which are joined upon tube closure (Lawson et

al. 1991; Tremblay and Zaret 2005). As the hepatic endodermal cells rapidly proliferate,

they migrate into the neighboring septum transversum mesenchyme to form the liver bud,

which becomes increasingly vascularized and by embryonic day 10 develops the stromal

spaces that will eventually form the sinusoids of the adult liver. By embryonic day 9

these cells already express important early hepatic markers, including albumin (ALB), -

fetoprotein (AFP), and transthyretin (TTR) and are now considered hepatoblasts, the

bipotential progenitors that will differentiate into the major cell types of the adult liver

(Nobuyoshi Shiojiri1984; Germain et al. 1988; Gualdi et al. 1996). Hepatocytes

differentiate from hepatoblasts between embryonic day 14 and birth, and the neonatal

hepatocytes continue to proliferate and induce adult hepatic gene expression between

birth and neonatal day 21.

Hepatoblasts are small stem-like cells that are defined by their expression of both

hepatocyte and cholangiocyte markers and their ability to differentiate into both cell

types. They expand as a homogenous progenitor population in the liver bud until

embryonic day 14.5, when a subpopulation reduces its expression of hepatocyte genes

and differentiates towards the cholangiocyte or bile duct epithelial cell lineage; the

remaining hepatoblasts commit to the hepatocyte lineage between embryonic day 16 and

birth. Primary hepatoblasts can be reproducibly purified from mouse embryonic day 13.5

livers by enrichment of hepatoblast-specific markers including E-cadherin (CDH1) and


delta-like 1 (DLK), or depletion of the hematopoietic markers TER119 and CD45 (Nitou

et al. 2002; Tanimizu et al. 2003; Nava et al. 2005). Additionally human heptaoblasts

have been successfully purified and characterized by a variety of techniques, and shown

to expand and differentiate when transplanted to immunocompromised mice (Mahieu-

Caputo et al. 2004; Wauthier et al. 2008). Several groups have reported the ex vivo

culture of primary hepatoblasts and their faithful bipotential differentiation stimulated by

extracellular factors; in particular the interleukin-6 family member oncostatin M (OSM)

appears to be absolutely required for hepatocyte-specific differentiation (Kamiya et al.

1999; Kamiya et al. 2002). Further, immortalized hepatoblast cell lines that maintain

progenitor gene expression patterns and the ability to differentiate into cholangiocytes

and hepatocytes in vitro have been developed (Rogler 1997; Strick-Marchand and Weiss

2002; Tanimizu et al. 2004). Together these in vitro techniques have helped to elucidate

the signaling and transcriptional mechanisms of in vivo liver development (Strick-

Marchand et al. 2004; Tanimizu and Miyajima 2004; Ader et al. 2006). The extracellular

signaling events that lead to hepatic endoderm specification and hepatoblast commitment

have been extensively studied by tissue explant and gene knockout experiments. Hence,

hepatic fate decisions are initiated by suppression of Wnt and fibroblast growth factor 4

(FGF4) signaling in the foregut mesoderm and suppressed by active Wnt signaling in the

posterior gut mesoderm (Wells and Melton 2000). The hepatic endoderm’s anterior-

posterior position is further refined by retinoic acid signaling from the paraxial

mesoderm, and following tube closure, solidified by suppression of sonic hedgehog

(SHH) signaling in the dorsal endoderm (Apelqvist et al. 1997; Kumar et al. 2003).


Foregut hepatic differentiation is further stimulated by multiple FGFs secreted by the

cardiac mesoderm and bone morphogenetic proteins (BMPs) secreted by the septum

transversum mesenchyme (Jung et al. 1999; Rossi et al. 2001). While it is known that

specific extracellular signaling events alter intracellular mitogen-activated protein kinase

activity (MAPK) and cause tractable changes in cellular transcription factor expression

levels, the mechanism by which soluble factors transduce global transcription programs

to specify hepatoblast cell fate are not well understood (Wandzioch and Zaret 2009).

The transcriptional control of hepatic differentiation is currently attributed to a

large and dynamic network of liver-enriched classical sequence-specific activators

(Cereghini 1996; Costa et al. 2003). These proteins are categorized based on the

structural similarity of their DNA binding domains and include the onecut homeodomain

family (HNF-1), leucine zipper family (C/EBP), winged helix family (HNF-3/FOXA),

and nuclear hormone receptor family (HNF4, COUP-TFII,, LRH-1, FXR, PXR). While

these activators are widely detectable throughout multiple tissues and phases of liver

development, their onset and degree of expression correlates well with their unique

requirements in sequential phases of hepatic organogenesis (Kyrmizi et al. 2006). Hence,

the initial specification of hepatoblasts within the definitive endoderm requires HNF-3

and HNF-3 , which are expressed as early as gastrulation (Duncan et al. 1998; Lee et al.

2005). HNF-4 and GATA-6 are expressed in the developing hepatic endoderm and are

required for liver bud expansion and the hepatoblast to hepatocyte transition (Duncan et

al. 2000; Li et al. 2000; Zhao et al. 2005). HNF-1b and HNF-6 are first expressed in the

developing liver bud and are required for the lineage split of hepatoblasts to hepatocytes


and cholangiocytes (Clotman et al. 2002; Coffinier et al. 2002). Further, C/EBP , which

is first expressed at birth, is necessary for the expression of metabolic genes which will

function in the adult liver (Wang et al. 1995). The delicate spatial and temporal

regulation of liver-specific gene expression in both the developing tissue and the adult

organ is proposed to be achieved by the combinatorial activation and repression of each

unique promoter by this diverse toolbox of proteins; regulatory diversity is further

complicated by the ability of many of these factors to heterodimerize. Additionally, the

liver-enriched transcription factors have been shown to autoregulate their own and each

other’s expression throughout development and adult homeostasis (Odom et al. 2004;

Odom et al. 2006). While the circuitry of liver-enriched sequence-specific activators has

been extensively investigated, the role of core promoter recognition complexes, co-

activators, and chromatin remodelers in liver specification is remains to be understood.

Hepatic Regeneration

The liver is distinct among metazoan organs for it intrinsic regenerative ability

following acute physical, toxic, or viral injury (Fausto and Campbell 2003; Fausto et al.

2006). While much attention has been given to the potential role of resident liver stem

cells or oval cells in maintaining the chronically injured liver, the primary source of liver

regeneration in healthy animals is thought to be the limited proliferation of adult

hepatocytes (Figure 3). This unique regenerative capacity has made the liver a target of

human transplantation therapies as well as a model system for understanding adult stem

cell differentiation and organ plasticity.


Within 36-42 hours of partial hepectomy (PH), the most accepted experimental

model of hepatic injury, 95% of quiescent mouse hetapocytes synchronously transition

from G0 to G1 and enter S-phase to begin DNA replication (Grisham 1962; Mitchell and

Willenbring 2008). The hepatocytes divide once or twice and the full liver mass can be

restored within one to two weeks in rodents or approximately one to two months in

humans, although in most cases the original liver architecture is altered. Mouse models

of chronic liver injury suggest that adult hepatocytes have an even greater replicative

potential, which may allow for novel therapeutic applications. Hence, when wild type

hepatocytes are transplanted into urokinase plasminogen activaor (uPA) transgenic mice

they undergo up to 15 cell divisions almost fully replacing the necrotic host liver (Rhim

et al. 1994). Similarly, serial repopulation of the chronically injured fumarylacetoacetate

hydrolase (FAH) knockout mouse liver with wild type hepatocytes suggests that at least

80 rounds of division are possible (Overturf et al. 1996; Overturf et al. 1999).

Much like developing heptaoblasts, hepatocyte regeneration depends on a

complex network of extracellular signaling molecules and cellular transcription factors

(Fausto et al. 1995). Hence, within hours of hepatic injury nonparenchymal cell derived

tumor necrosis factor (TNF) and interleukin-6 (IL-6) stimulate the nuclear factor-kappa B

(NF B), signal transducer, and activator of transcription 3 (STAT3) pathways

respectively, inducing immediate early gene expression, promoting the G0 to G1

transition, and priming the quiescent growth factor immune hepatocytes for activation by

additional extracellular signals (Akerman et al. 1992; Webber et al. 1998; Iwai et al.

2001). Next mesenchyme derived hepatocyte growth factor (HGF) and hepatocyte


expressed transforming growth factor alpha (TGF ) activate the mitogen-activated

protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K) cascades to promote

progression past the G1-S restriction point and induce expression of additional

transcription factors (Mead and Fausto 1989; Webber et al. 1993; Borowiak et al. 2004;

Huh et al. 2004). The activation of NFkB and STAT3 is the result of post translational

modifications and occurs independent of new protein synthesis, allowing these factors to

induce immediate early gene expression within hours of hepectomy (Cressman et al.

1994; Cressman et al. 1995). The activated immediate early genes include

transmembrane receptors required for continued mitogen responsiveness, cyclins and

cyclin dependent kinases which mediate cell cycle progression, and additional

transcription factors that drive the regenerative program (Mohn et al. 1991). Among

these transcription factors are known mitogen dependent cell cycle regulators not

normally expressed in the adult liver including c-Jun, c-Fos (AP-1), and c-Myc; the

overexpression or deletion of these genes has been shown to promote or impair liver

regeneration respectively (Thompson et al. 1986; Morello et al. 1990; Behrens et al.

2002). A second class of immediate early genes includes liver-enriched transcription

factors such as C/EBP which are responsible for the reestablishment of normal

hepatocytic function and coincident with these is an increase in the expression of liver

proteins such as albumin (ALB), 1-antitrypsin (AAT) and Phosphoenolpyruvate

carboxykinase (PEPCK) (Park et al. 1990; Diehl 1998). Intriguingly, gene expression

profiles of regenerative and neonatal hepatocytes are similar suggesting, a common

transcriptional program. More recently FoxM1B was shown to be an immediate late


gene in regenerative hepatocytes which is responsible for expression of cyclins regulating

the G2-M transition and exit from the regenerative program; consistent with this

overexpression, of FoxM1B can speed up liver regeneration or restore it in older

hepatocytes that have lost their regenerative capacity (Ye et al. 1999; Wang et al. 2001).

However, as with the embryo, the precise influence of ligands and signaling cascades on

intercellular transcriptional events during hepatic regeneration is not well understood.

Chronic toxic injury, certain genetic abnormalities, and age related degeneration

may limit or eliminate the hepatocytes proliferative capacity; under these conditions rare

resident liver stem cells, or oval cells, can expand and differentiate, contributing to

hepatic regeneration (Figure 3) (Fausto and Campbell 2003). While there has been

considerable disagreement concerning the oval cell’s phenotype and capacity, a

consensus exists that the portal region of the hepatic lobule contains a bipotential

progenitor with large oval nuclei that expresses hepatic, biliary, and hematopoietic

markers (Farber 1956; Dorrell et al. 2008). Though several groups have reported the

purification of hepatic progenitors from healthy liver, most of the work on oval cells

employs a model of chronic liver injury involving dietary deficiency, extended feeding of

carcinogenic or hepatotoxic compounds, and partial hepectomy (Wilson and Leduc 1958;

Dabeva et al. 1998; Preisegger et al. 1999; Wang et al. 2003a). Expanded oval cell

populations from these injury models have been purified based on differential

centrifugation or cell surface marker enrichment and used for in vivo bipotential

differentiation studies as well as the derivation of immortalized cell lines (Wilson and

Leduc 1958; Hayner et al. 1984; Yaswen et al. 1984; Germain et al. 1985; Dabeva et al.


1998; Preisegger et al. 1999; Petersen et al. 2003; Wang et al. 2003a). Furthermore,

purified oval cells and immortalized cell lines have been successfully used to partially

repopulate chronically injured liver models, providing the greatest evidence to date for

the oval cells proliferative and regenerative capacity (Wang et al. 2003b; Song et al.

2004). While oval cell expansion is likely dependent on many of the same extracellular

signaling pathways as hepatocyte proliferation, it also appears to require additional

unique signaling events typically associated with hematopoiesis, such as the SCF/c-kit

system (Fujio et al. 1994). Intriguingly, the oval cell’s temporal pattern of liver-enriched

transcription factor expression has been shown to closely mirror that of the developing

hepatoblast, suggesting that these two populations may differentiate through shared

transcriptional mechanisms. However transcription in oval cells has not been

investigated in any greater detail and warrants further study (Nagy et al. 1994).

Importantly, oval cell populations have been identified in the chronically injured human

liver, and their bipotential and proliferative capacity are beginning to be understood

(Haruna et al. 1996; Baumann et al. 1999; Roskams et al. 2004).

Embryonic Stem Cells and Therapeutic Approaches

The liver’s central place in many aspects of organismal function, combined with a

restricted supply of human liver donors and the complications associated with whole

organ transplantation, has generated considerable interest in cell based therapeutic

approaches to liver repopulation (Grompe 2006; Nussler et al. 2006). Donor derived

mature hepatocyte transplantation has been reported for the treatment of acute liver


failure, genetic metabolic disorders, and end stage cirrhosis with some success (Mito et

al. 1992; Fox et al. 1998; Bilir et al. 2000). Hepatocyte transplantation, has considerable

advantages over whole liver transplantation including the ability to treat multiple patients

from a single donor, decreased immunogenicity, cryopreservation of donor cells, and

reduced morbidity associated with the procedure itself. However, it also suffers from

considerable drawbacks such as donor supply, low engraftment rates, limited

proliferation of transplanted cells, and the inability to restore normal liver architecture

(Fox and Chowdhury 2004). Thus, a principal aim of hepatology research is to create an

indefinite source of autologous regenerative hepatocytes from patients’ own stem or

progenitor cells. Understanding the transcriptional mechanisms of hepatic differentiation

and regeneration is critical to this effort.

Embryonic stem cells (ES) hold great promise as a source of transplantable

hepatocytes because of their indefinite proliferative capacity and ability to differentiate

into every major cell type of the body (Keller 2005; Dalgetty et al. 2009). Mouse ES

cells have been successfully differentiated into functional hepatocytes and used to

partially restore liver function in a murine model of hepatic failure (Soto-Gutierrez et al.

2006). More critically, multiple groups have reported the directed differentiation of

human ES cells into hepatocyte like cells which faithfully recapitulate the gene

expression profiles and metabolic functions of primary hepatocytes (Duan et al. 2007;

Hay et al. 2007; Hay et al. 2008b). In most cases these differentiation protocols are based

on our current understanding of key signaling events required for hepatic development

and employ the same timing and extracellular signals observed in vivo (Schuldiner et al.


2000). Furthermore, new discoveries concerning in vivo liver development have been

successfully applied to improve the efficiency and accuracy of in vitro ES cell

differentiation toward hepatocytes (Hay et al. 2008a). Recent reports demonstrate that

these human ES cell derived hepatocytes can partially restore liver function when

transplanted into an immunocompromised mouse model, further supporting their

therapeutic potential (Basma et al. 2009). Similarly, multiple groups have

transdifferntiated adult hematopoietic stem cells into functional hepatocytes by

employing our understanding of key signaling events in liver developments thereby

avoiding the ethical issues associated with ES cell based therapies (Alison et al. 2000;

Lagasse et al. 2000). Thus, our expanding knowledge of the molecular mechanisms

required for hepatic development is currently being employed to generate potentially

therapeutic hepatocytes from non-hepatic stem cells.

The recent discovery that adult somatic cells can be reprogrammed into

proliferative pluripotent stem cells termed induced pluripotent stem cells (iPS) could

contribute greatly to our understanding of hepatogenesis, and hasten the advancement of

therapeutic liver repopulation (Yamanaka 2007; Jaenisch and Young 2008). Intriguingly,

iPS cells have been successfully generated from adult hepatocytes suggesting that the

mature cell type possesses all but a few factors required for proliferation, and raising the

possibility that damaged liver cells could be dedifferentiated, modified, and transplanted

(Aoi et al. 2008). While iPS cells have not yet been differentiated into functioning

hepatocytes, they have been specifically differentiated to cardiomyocytes, adipocytes,

retinal cells, and multiple hematopoitic lineages (Schenke-Layland et al. 2008; Hirami et


al. 2009; Senju et al. 2009; Zhang et al. 2009). Furthermore, comparative studies suggest

that iPS cells can be differentiated by the same protocols and with similar efficiency as

ES cells (Taura et al. 2009). Importantly, directed in vivo differentiation of genetically

modified autologous iPS cells followed by transplantation has been used to correct mouse

models of human disease including sickle cell anemia, fanconi anaemia, and hearing loss

(Nishimura et al. 2009; Raya et al. 2009). Such experiments raise the possibility that a

patient’s own somatic cells could be used for therapeutic liver repopulation.

Understanding the transcriptional mechanisms of liver development, regeneration, and

dedifferentiation is essential to realizing this potential.


Figure 1: Model of Preinitiation Complex Assembly

Sequence-specific activators (green) bind proximal (PA) and distal (DA) enhancer

elements within a genes regulatory DNA and recruit TFIID (yellow) to the core promoter.

These activators and/or TFIID in turn recruit chromatin remodeling complexes such as

SWI/SNF (orange), coactivators including Mediator (MED, blue), and TFIIA and TFIIB

(purple). The TFIID/TFIIA/TFIIB heterotrimer sequentially recruits TFIIE, TFIIF, PolII

(red), and TFIIH (purple), allowing for promoter escape and productive transcriptional

elongation.


H

PETATA

TFIID

DE

MED

PolII

E

F

SWI/SNF

BA


Figure 2: Combinatorial Model of Core Promoter Recognition Complex Diversity

Canonical TFIID consisting of TBP (red) and 15 associated TAFs (yellow) is

believed to be present at most promoters and in most cell types. Additional regulatory

complexity comes from TBP-free TAF containing complexes such as TFTC which

contain some TAFs plus additional unique proteins (burgundy), TBP-Related Factor

complexes such as TAF3-TRF3 (green), and alternate forms of TFIID that use tissue-

specific TAFs such as TAF5b and TAF7l (blue). Novel, as yet unidentified complexes

may be present in specific tissues or developmental programs.


TAF3

-TR

F3M

yoge

nesi

s

TAF4

b-II

DFo

llicu

loge

nesi

s/Sp

erm

atog

enes

is

TFI

IA-T

RF2

TFT

C

TAF7

l-II

DSp

erm

ioge

nesi

s

Can

onic

al T

FIID


Figure 3: Hepatic Development and Regeneration

Hepatoblasts, the common bipotential liver progenitors, develop from the

definitive endoderm between embryonic day 8.5 and 13.5. Starting at embryonic day

14.5 they differentiate to cholangiocytes or bile duct epithelial cells and hepatocytes.

Hepatocytes continue to proliferate and mature from before birth until at least neonatal

day 21. Following acute liver injury, mature hepatocytes reenter the cell cycle to

regenerate liver mass and function. Rare resident liver stem cells, or oval cells, may also

expand and differentiate contributing to liver regeneration.


Hea

d

Para

xial

Mes

oder

m

Som

ites

Car

diac

Mes

oder

m

Hep

atic

End

oder

mD

orsa

l End

oder

m

Ven

tral

End

oder

m

Sept

um T

rans

vers

um

Hep

atob

last

sE

8.5-

E14

.5

Adu

lt C

hola

ngio

cyte

sO

val C

ells

Neo

nata

l Hep

atoc

ytes

Adu

lt H

epat

ocyt

es

Cyc

ling

Hep

atoc

ytesIn

jury

and

Reg

ener

atio

nFe

tal a

nd N

eona

tal D

evel

opm

ent


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Chapter 2: TBP, TRFs, and TAFs in Liver Development and Regeneration Summary

The precise spatial and temporal control of gene expression required for

organismal development and cellular differentiation is primarily determined at the level

of transcriptional regulation. Historically, diverse cellular programs of proliferation,

terminal differentiation, and specialized function have been attributed to a repertoire of

sequence-specific activators and repressors. General transcription factors including

coactivators such as Mediator and core promoter recognition factors such as TFIID were

assumed to be ubiquitous and invariant between tissues and developmental time points.

Recent work from a variety of model organisms and mammalian differentiation programs

supports an expanding role for TAF paralogs, TBP-related factors, and alternative core

promoter recognition complexes in determining tissue and developmental stage-specific

transcriptional programs. The vertebrate liver serves varied and critical functions in

metabolism, growth control, and detoxification; however, the precise transcriptional

mechanisms of hepatic development, differentiation, and regeneration remain elusive.

Herein, I examine the expression of TBP, TRFs, TAFs and other coactivators in adult

mouse tissues and purified liver cells. Furthermore, I demonstrate that during liver

development and hepatocyte commitment canonical TFIID is in toto eliminated and its

gene locus transcriptionally silenced coincident with retention of TRF3 expression.

Importantly, an in vitro model of hepatic development recapitulates these observations.


Introduction

The highly specific and exquisitely complex patterns of gene expression required

for organismal development and cellular differentiation have traditionally been attributed

to sequence-specific DNA binding activators, while the general transcription factors

including the core promoter recognition complex TFIID and the coactivator Mediator

were assumed to be invariant (Thomas and Chiang 2006). In the mammalian liver

developmental commitment and functional specialization are primarily attributed to a

large group of heterogeneous nuclear factors (HNFs) and other liver-enriched activators,

while changes in the basal machinery have remained largely uninvestigated (Schrem et

al. 2002). Recent advances in genetic, biochemical, and cell culture techniques have

allowed for a more thorough examination of core promoter recognition and coactivator

proteins in diverse cell types and differentiation programs.

The first indication that the canonical core promoter recognition complex TFIID

may not be essential at all developmental stages or for the transcription of all genes came

from multiple gene disruption studies. Hence, while depletion of TBP in the mouse or

zebrafish leads to eventual embryonic lethality it does not prevent the onset of zygotic

transcription or reduce RNA PolII activity at the earliest embryonic stages, suggesting

that an alternative mechanism of core promoter induction must take its place in this

particular setting (Muller et al. 2001; Martianov et al. 2002). Similarly, disruption of the

TBP associated factors TAF10 and TAF8 also led to eventual lethality but only after

expansion of the inner cell mass, and staining experiments suggest differing requirements

for these factors in the developing blastocyst (Voss et al. 2000; Mohan et al. 2003).


Conditional inactivation of the key TFIID subunit, TAF4 in embryonic fibroblasts

showed it to be nonessential for cellular growth and differentiation and required for the

transcription of many but not all fibroblastic genes, while depletion in developing

keratinocytes suggested that it is essential to epidermal differentiation and maintenance,

consistent with differential tissue-specific functions (Mengus et al. 2005). Intriguingly,

TAF10 is also required for embryonic keratinocyte development but unlike TAF4 is

dispensable for maintenance of the adult epidermis (Indra et al. 2005). Most recently,

TAF10 was shown to be required for embryonic liver development but dispensable for

the maintenance of adult hepatocytes and the ongoing expression of most liver-specific

genes, suggesting that an alternative mechanism of core promoter activation may replace

TFIID in the hepatogenic program (Tatarakis et al. 2008).

Additional evidence for tissue-specific differences in TFIID activity comes from

several limited observations of differences in TBP and TAF expression throughout adult

tissues. Hence, in an early study TBP and TAF12 expression were shown to vary widely

between adult mouse tissue, being highly enriched in the testes and nearly undetectable in

the heart and nervous system (Perletti et al. 1999). Furthermore, differences in TBP

abundance were inversely correlated with TRF levels suggesting that TRFs may partially

replace TBP in certain tissues. In another report, TAF4 was shown to be highly

upregulated during neuronal differentiation and required for the induction of adult

neuron-specific genes (Perletti et al. 1999). Furthermore, increased TAF8 (TBN)

expression was shown to occur during adipogenesis, and functional TAF8 shown to be

required for induction of the adipogenic gene expression program (Perletti et al. 1999).


Additionally, an investigation of the mouse TAF4 paralogue, TAF4b, demonstrated that

TAF4 is significantly donwregulated in the adult liver at least at the mRNA level

(Freiman et al. 2001). While it is important to note that many of these studies are

observational and may depend on the accuracy of specific reagents, collectively they

provide a precedent for the idea that significant differences do exist in the expression of

TFIID components between tissues and cell types.

The most recent indication of developmental stage-specific changes in the core

promoter recognition machinery occurs in the myogenic program. Hence, in both

primary cells and an in vitro model of myogenesis TBP and many TAFs are in toto

degraded and their mRNA levels significantly decreased during the transition from

myoblasts to myotubes (Deato and Tjian 2007). Importantly, levels of other general

transcription factors including TFIIA-H and RNA PolII are unchanged as are levels of

TAF3 and the TBP-related factor TRF3. Furthermore, TFIID was shown in vitro to be

dispensable for the activated transcription of the myoube-specific myogenin promoter,

suggesting that TBP and TAFs are at least in part functionally nonessential for the onset

and/or maintenance of transcription in the adult cell type (Deato et al. 2008).

Additionally, multiple components of the canonical coactivator complex mediator are

also significantly donwregulated during the myogenic transition, suggesting that like

TFIID, Med activity may vary significantly between developmental stages and tissues.

Importantly, downregulation of TFIID does not appear to be a hallmark of all

developmental transitions, as TBP and TAF levels are shown to be unaltered at both the


protein and mRNA level during the differentiation of mouse ES cells to motor neurons

(Francisco Herrera, personal communication).

Results

Varied Expression of TFIID Subunits in Diverse Mouse Tissues

The recent seminal finding that TFIID including TBP and many TAF subunits is

wholly degraded and replaced by a novel core promoter recognition complex consisting

of TRF3 and TAF3 during terminal differentiation has thus far been restricted to murine

muscle development (Deato and Tjian 2007). To better understand if this intriguing

transcriptional mechanism is unique to myogenesis or is a common feature of other

distinct differentiation programs we investigated the disposition of TBP and multiple

TAFs in diverse adult mouse tissues at both the mRNA and protein level.

To determine the relative levels of TBP and TAF protein in differentiated mouse

tissues we subjected whole tissue lystates to Western blot analysis with specific

antibodies against TBP, TRF3, TAF1, and TAF4 (Figure 1). Equal amounts of total

protein were analyzed and GAPDH was used as a loading control. Importantly, TBP and

multiple TAFs are enriched in proliferative C2C12 myoblasts and whole E13.5 embryos

which contain multiple progenitor cell types including myoblasts and hepatoblasts, but at

significantly reduced levels in differentiated C2C12 myotubes as previously reported.

Additionally, TFIID components are highly expressed in the testes and spleen which

poses significant stem and progenitor cell populations and were formerly shown to

contain high levels of TBP (Persengiev et al. 1996; Perletti et al. 1999). Intriguingly,


TBP, TAF1, and TAF4 protein appear to be all but absent from many adult tissues and at

exceedingly low levels in the whole liver; possibly at lower levels than in differentiated

myotubes. Conversely, TRF3 protein is as abundant in many adult tissues as it is in

C2C12 myoblasts or the whole embryo, and appears to be particularly abundant in the

liver sample. Hence, core promoter recognition complex protein levels vary widely

between tissues and TFIID protein levels appear to be particularly low in the

differentiated mouse liver.

To further investigate the tissue-specific expression of TFIID we determined the

relative levels of key subunit transcripts in the same tissues by reverse transcription and

quantitative real-time PCR (qPCR) (Figure 2). Because no single mRNA is expressed at

the same level in all tissues of the mammalian body, and the relative proportion of

mRNA to total RNA varies considerably between tissues, accurate normalization of cross

tissue qPCR is inherently problematic. To partially abrogate this problem, we used the

same quantity of total RNA in reverse transcription reactions and normalized against both

the 18s RNA (Figure 1A) and GAPDH transcript (Figure 2B), two of the most widely

accepted total RNA and mRNA standards respectively. As with protein levels,

significant differences are observed in TFIID mRNA levels between tissues, with adult

liver showing some of the lowest levels of both TBP and multiple TAFs. Importantly,

the whole embryo, testes and ovary, which contain more progenitors and proliferative

cells than most adult tissues have the highest transcript levels of key TFIID components.

While significant differences exist for TBP and TAF levels between the GAPDH and 18s

normalizations, the general trend of reduced TFIID expression in the liver is the same.


As with the protein analysis, levels of TRF3 transcript do not correlate with TBP or TAFs

and are at least as high in the liver and other adult tissues as the embryo or testes.

Differential Expression of TBP, TRFs and TAFs in Highly Purified Liver Cells

The embryonic and adult livers both contain multiple highly specialized cell types

with unique functions and transcriptional programs (Zaret 2002; Zaret and Grompe

2008). While hepatocytes comprise sixty to eighty percent of the adult liver mass

significant populations of cholangiocytes and Kupffer cells, and smaller populations of

several other specialized cell types are present and serve critical physiological functions.

Similarly, the bipotential hepatoblasts that eventually give rise to hepatocytes and

cholangiocytes are a minority population in the developing liver bud, where erythrocytes,

leukocytes and other hematopoietic cells are highly abundant. Consistent with their

specialized functions and gene expression patterns each of these cell types maintains a

unique transcriptional program (Cereghini 1996; Costa et al. 2003). Hence, analysis of

core promoter recognition complex subunits in whole liver tissue is likely complicated by

differences in expression between the unique cell types that comprise the tissue mass, and

may represent an average of different expression levels between the various cell types.

More generally, most differentiated tissues are composed of multiple highly specialized

cell types in addition to progenitors, such that any whole tissue expression analysis likely

misrepresents the abundance of a given gene product in a specific functional cell.

To overcome this complication we chose to focus our analysis on highly purified

embryonic and adult liver cells. Multiple protocols have been established for the


purification of hepatoblasts from the embryonic liver both by enrichment of hepatoblast-

specific markers and depletion of hematopoietic cells (Weiss and Strick-Marchand 2003;

Wauthier et al. 2008). We found that collagenase based dissociation of individually

dissected E13.5 livers followed by magnetic bead based depletion of erythrocytes

(TER119) and hematopoietic cells (CD45) consistently produced hepatoblast populations

which were ninety five percent pure based on both negative and positive FACS analysis

(Suzuki et al. 2000). Protocols for the purification of neonatal, partial hepatectomy, and

adult hepatocytes by two step in situ perfusion with collagenase and differential

centrifugation have been employed for more than 50 years (Wang et al. 2003a). The

most excepted protocol for purification of regenerative cycling hepatocyte is 2/3 partial

hepatectomy followed by in situ perfusion (Mitchell and Willenbring 2008).

To determine the expression of TBP, TRFs, and TAFs we performed quantitative

RT-PCR using equal amounts of RNA from purified hepatoblasts and adult hepatocytes

and normalized to the GAPDH transcript (Figure 3A). Intriguingly TBP, TRF2 and the

majority of TAFs are significantly downregulated at the mRNA level in hepatocytes

when compared to hepatoblasts. Importantly, his includes the donwregulation of TAF3

which was previously shown to be unaltered in the myogenic differentiation program

(Deato and Tjian 2007). Conversely, TRF3 and TAF13 expression appear to be

relatively unchanged in the adult hepatocyte while the testes-specific TAF7 paralogue,

TAF7l is significantly unregulated. Additionally, expression of a core component of a

TBP-free TAF complex, PCAF is induced in hepatocytes.


The recent finding that components of the coactivator complex Mediator are

downregulated during myogenesis led us to ask how mediator is affected in the hepatic

program (Deato et al. 2008). Hence, we analyzed the expression of several key Mediator

subunits including components of the head, middle, tail, and CDK8 domains in

hepatoblast and hepatocytes by qPCR (Figure 3B). As with TFIID each mediator mRNA

examined is significantly downregulated in hepatocytes relative to hepatoblasts. While

approximately half of the known Mediator subunits were excluded in this analysis, none

of these proteins has been yet been isolated in a complex other than canonical Mediator

and those subunits which are shown to be downregulated are positioned throughout the

known complex (Myers and Kornberg 2000; Taatjes et al. 2004).

These analyses demonstrate a clear difference in the expression of TFIID and

Mediator subunits between progenitor hepatoblasts and adult hepatocytes. This led us to

ask when in hepatic development TFIID is downregulated and how its expression might

be altered following liver injury. While hepatocytes start to develop from hepatoblasts as

early as embryonic day 14, they may not become fully differentiated until postnatal day

21. Hence, neonatal hepatocytes are smaller and less binucleate than adult hepatocytes,

do not express all adult hepatic genes, and continue to proliferate. While not fully

understood neonatal hepatocyte maturation likely involves transcriptional changes which

are distinct from the embryonic or adult programs. Following toxic or mechanical injury

adult hepatocytes reenter the cell cycle and proliferate to regenerate the liver mass.

These regenerative hepatocytes undergo significant changes in gene expression without

dedifferentiation and are thus a unique cell type from either quiescent hepatocytes or


embryonic and neonatal progenitors. While liver-enriched transcription factors and

signaling molecules are known to be upregulated during liver injury, the transcriptional

programs which govern hepatocyte proliferation are not well understood. Thus, we

compared TBP, TRF, TAF, and Mediator mRNA levels from neonatal hepatocytes and

2/3 partial hepetectomy hepatocytes to hepatoblasts and adult hepatocytes by qPCR to

further understand the role of these complexes in maturation and regeneration (Figure 4).

Based on this analysis it appears the TFIID and Med expression in neonatal and

regenerative hepatocytes mirrors that of adult hepatocytes as TBP and most TAFs are

similarly downregulated relative to hepatocytes while TRF3, TAF13, and TAF7l are

unchanged. Hence, canonical TFIID and Mediator are likely downregulated and TAF7l

upregulated at the early stages of hepatocyte commitment prior to birth and their

expression does not appear to be significantly reinitiated following liver injury and

regeneration.

While spatial and temporal patterns of gene expression as determined by analysis

of mRNA abundance often correlate with developmental and cell-type-specific function,

differences in mRNA stability, translational efficiency, and protein turnover may cause

significant fluctuations between mRNA levels and protein abundance. For this reason

relative polypeptide levels are generally assumed to more accurately reflect the

developmental stage and cell-type-specific requirements for a given protein product.

Hence, we examined TBP, TRF and critical TAF subunit protein levels by western blot of

whole fetal, adult liver, and purified liver cells (Figure 5). This analysis demonstrates

that TBP and multiple TAFs are highly enriched in purified hepatoblasts and are possibly


expressed at higher levels than in C2C12 myoblasts or whole fetal liver. Conversely,

TBP and every TAF examined is significantly downregulated in adult hepatocytes when

compared to hepatoblasts including the critical structural subunits TAF1, TAF4 and

TAF5 (Wright et al. 2006). Furthermore, neonatal and regenerative hepatocytes appear

to have more TBP and TAF protein than do purified adult hepatocytes but clearly far less

than hepatoblasts. Intriguingly, several TAFs such as TAF10 appear to be more highly

expressed in the whole adult liver than they are in purified hepatocytes suggesting liver

cells other than hepatocytes may posses higher TAF levels. Additionally, protein levels

of TRF2 appear to be relatively unchanged between hepatoblasts and hepatocytes,

suggesting that this protein may partially substitute for TBP in adult hepatocytes. In the

case of TBP and several of the TAFs the fold reduction in protein levels may be greater

than that for mRNA, but this comparison is limited by the qualitative nature of western

blotting.

To further understand changes in Mediator expression during hepatic

development we also conducted western blots of multiple Med subunits in whole liver

and purified liver cells (Figure 6). As is the case with TFIID, every Mediator subunit

examined is enriched in fetal hepatoblasts and significantly downregulated in adult

hepatocytes while at reduced but intermediate levels in neonatal and regenerative

hepatocytes. Importantly, this is true for components of the distinct head, middle, and tail

domains, and the dissociable CDK8 module (Guglielmi et al. 2004). The apparent

donwregulation of Mediator protein during hepatic development closely mirrors what

was reported for the myogenic program (Deato et al. 2008).


Transcriptional Silencing of TBP and TAF Promoters During Hepatogenesis

While qPCR and western blot analysis clearly show that TFIID levels are

significantly reduced upon hepatic development, we wished to further confirm that TBP

and TAF gene expression is specifically silenced at the promoter level. The enrichment

or removal of highly defined histone marks is increasingly understood to correlate with

the level or gene expression or silencing (Bernstein et al. 2007). Among the many

recently identified histone modifications, promoter enrichment of histone H3 lysine 4

(H3K4Me3) trimethylation has been widely shown to correlate with gene activation,

while enrichment of histone H3 lysine 9 trimethylation (H3K9Me3) is associated with

transcriptional silencing (Li et al. 2007). Hence, we crosslinked E13.5 and adult livers

and performed chromatin immunoprecipitation with specific antibodies against these two

chromatin marks followed by qPCR of precipitated material with promoter-specific

primers (Figure 7). This analysis shows that the proximal promoters of TBP, TAF3, and

TAF5 are enriched for H3K4Me3 in the fetal liver sample and that levels of this mark are

subsequently reduced in the adult liver. Similarly and more strikingly, H3K9Me3

enrichment at these promoters is low in the fetal liver and greatly increased in the adult.

Importantly, the promoter of an adult hepatocyte-specific gene, CYP7A1, shows relative

enrichment of H3K9Me3 in the fetal liver and of H3K4Me3 in the adult. Hence, TFIID

promoters appear to be specifically upregulated in the fetal liver and transcriptionally

silenced in adult cells through a mechanism which involves directed modification of

histone tails in the proximal promoters of these genes.


Expression of TBP and TAFs in a Cell Culture Model of Hepatogenesis

Defined cell culture systems have contributed greatly to our understanding of

multiple metazoan differentiation programs and provide the greatest opportunity for

functional manipulation of cellular differentiation when a suitable whole animal model is

absent. Several groups have reported systems for both the short term ex vivo culture of

primary hepatoblasts and the long term maintenance of immortalized hepatoblast derived

cell lines (Strick-Marchand and Weiss 2003; Weiss and Strick-Marchand 2003). Hence,

fetal mouse hepatoblasts purified by both negative and positive selection have been

successfully cultured for multiple divisions on extracellular matrices in the presence of

growth factors which mimic the hepatic endoderm niche (Nitou et al. 2002; Tanimizu et

al. 2003). Furthermore, these cultures have been efficiently differentiated by defined

factors into both hepatocytes and cholangiocytes that recapitulate the metabolic functions

and gene expression patterns of in vivo isolates (Kamiya et al. 1999; Kinoshita et al.

1999; Tanimizu et al. 2007). While these ex vivo models have proven useful they require

frequent isolation of fresh hepatoblasts and result in significant experimental variability.

To date three groups have established defined immortalized hepatoblast cell lines which

abrogate the complexities of hepatoblast culture while faithfully recapitulating embryonic

liver development (Rogler 1997; Strick-Marchand and Weiss 2002; Tanimizu et al.

2004). Of these we chose to employ the Hepatic Progenitors Proliferating on Laminin

(HPPL) cell line, which mimics proliferative hepatoblasts in the presence of epidermal

growth factor (EGF) and hepatocyte growth factor (HGF) but is differentiated to

functional hepatocytes following incubation with oncostatin M (OSM) and Matrigel.


We maintained and expanded the HPPL cell line for multiple generations on

laminin-coated plates in the presence of EGF and HGF with no apparent change in

morphology, proliferative capacity, or marker gene expression consistent with published

reports. When confluent cultures were incubated for five days with 20ng/ml of

recombinant oncostatin M followed by five days with growth factor reduced Matrigel

cells took on an enlarged binucleate cuboidal morphology and acquired multiple

intracellular vacuolar structures. Reverse transcription and qPCR revealed a significant

decrease in the expression of bipotential and cholangiocytes markers by five days post

induction and a greater increase by ten days (Figure 8A). Conversely, adult hepatic gene

expression was significantly and rapidly upregulated. Similarly, western blot analysis

showed a reduction of bipotential marker protein levels and an increase in proteins

associated with terminal adult hepatocytes (Figure 8B). Thus the HPPL cell model

faithfully mimics the hepatoblast to hepatocyte transition based on morphology and

marker gene expression.

To assess changes in TBP and TAF expression during HPPL differentiation we

performed reverse transcription and qPCR on RNA isolated from proliferating and

differentiated HPPL cells (Figure 9A). These analyses clearly show that TBP, many

TAFs, and at least one Mediator subunit are significantly downregulated by five days

post induction with further downregulation by ten days. Conversely, TRF3, TAF13, and

TAF7l transcript levels are maintained or increased coincident with the downregulation

of other subunits. Importantly the extent of transcript changes closely mirrors that seen

between primary hepatoblasts and hepatocytes. Western blot analysis of TBP, TRF3, and


some TAFs displays a similar pattern with significant changes in TFIID but not TRF3

occurring by day five after induction. Hence, the HPPL culture system faithfully

recapitulates the downregulation of TFIID observed the primary hepatogenic program.

Discussion

A growing body of functional data suggests that specific TAFs and potentially holo-

TFIID may be dispensable for the activation of many promoters and the maintenance of

at least some differentiated cell types. This, combined with two recent studies that

demonstrate that TBP and multiple TAFs are specifically degraded during certain

differentiation programs, cast doubt on the idea that TFIID is universally expressed and

required for all cell-type-specific transcriptional programs (Brunkhorst et al. 2005; Deato

and Tjian 2007). Because of its central role in mammalian biology, the transcriptional

mechanisms that govern liver development and function are a subject of extensive study.

At least one previous study suggested that the adult mouse liver contains

particularly low levels of the key TFIID subunit TAF4 (Freiman et al. 2001). Indeed our

current data confirms that TAF4, along with TBP and TAF1, are significantly

downregulated in the adult liver relative to the embryo and other adult tissues, suggesting

that the hepatogenic program may also require the downregulation of canonical TFIID

and potentially its replacement with an alternative core promoter recognition complex.

While methods of normalization and differences in progenitor cell populations between

tissues complicate cross tissue analysis, preliminary data suggest that the significant

downregulation of TFIID at both the protein and RNA level that is observed in muscle


and liver may not be observed in all adult tissues and thus may be specific to a subset of

differentiation programs. As new protocols for the isolation of tissue progenitors and

fully committed cells become available, it will be interesting to explore how widely the

phenomenon of TFIID downregulation and core promoter complex switching is

employed or restricted throughout the mammalian body.

Because of the considerable cell type and functional complexity of most adult

organs, changes in transcription factor expression and function are clearly best observed

in highly defined pure cell populations. Hence, hepatocytes which represent sixty to

seventy percent of the adult liver cell population may exhibit differences in transcription

factor abundance from other liver cell types which would be masked in whole organ

experiments (Daoust and Cantero 1959). Importantly, our results suggest that the

hepatogenic downregulation of TFIID at both the protein and mRNA levels is most

pronounced between hepatoblast progenitors and purified hepatocytes. Intriguingly,

whole adult livers appear to retain at least some TAF subunits relative to hepatocytes,

consistent with previous literature reports (Tatarakis et al. 2008). While the many minor

cell types of the adult and developing liver are more difficult to purify and characterize, it

will be interesting for future studies to determine if at least some of these populations

retain and require canonical TFIID in their adult form, and if this accounts for at least

part of the difference in TFIID abundance between whole liver and purified hepatocytes

(Alpini et al. 1994). Specifically, it remains to be determined if bipotential hepatoblasts

downregulate TFIID when committing to both mature hepatocytes and cholangiocytes or

if selective downregulation is itself a determinant of this lineage split. While clear


differences in TFIID expression exist between hepatocytes and hepatoblasts, these two

cell types lie at the ends of a differentiation process which requires at least two weeks and

multiple intermediate stages in the mouse, making the exact timing of TFIID

downregulation unclear (Zaret 2002). The finding that TAFs are already significantly

degraded in neonatal hepatocytes suggests that this process primarily occurs early in

differentiation and most likely prenatally, however the observation that some protein

remains in late neonatal hepatocytes may imply that at least some TFIID is required for

final maturation. Additionally, differences in the relative levels of TAFs in purified

hepatocytes and the whole liver may indicate that some subunits are retained as part of

characterized or novel TBP free TAF containing complexes including TFTC and SAGA;

the role of these complexes in liver development and function remains to be determined.

The significant decrease in TBP and TAF mRNA levels and the active silencing of these

genes promoters supports the notion that long term reduction of TFIID in adult

hepatocytes is primarily a transcriptional phenomenon. However, more extensive time

course measurements of mRNA and protein levels during the differentiation process is

needed to determine if an active mechanism of subunit proteolysis is also employed. One

recent study suggests that in some differentiation programs, targeted ubiquitination and

proteasome mediated degradation of TFIID subunits occurs independently of

transcriptional changes (Perletti et al. 2001). Thus, it is possible that both transcriptional

silencing and ubiquitin E3 ligase mediated degradation of specific TFIID polypeptides

cooperate to establish immediate and prolonged reduction of TFIID levels. Most

interestingly, it remains to be understood how known and as yet undiscovered


extracellular signaling events trigger the transcriptional silencing of TBP and TAF

promoters or the targeted degradation of these subunits in precise spatial and temporal

patterns. Recent advances in the ex vivo study of these critical signaling events may be

fruitfully combined with existing transcriptional readouts to better understand these

connections (Wandzioch and Zaret 2009).

Importantly, a cell culture model of hepatic differentiation recapitulates the

downregulation of TFIID observed in purified primary cells. This model expands the

range of functional and cell signaling experiments that can be performed to better

understand the process of core promoter complex switching in hepatogenesis.

Additionally, these observations also suggest that because core promoter changes that are

observed in vivo are conserved in vitro, novel findings may be successfully employed to

improve the efficiency and fidelity of potentially therapeutic differentiation and

transplantation protocols.

Materials and Methods

Protein Extracts and RNA

Whole tissue lysates were generated by dounce homogenization in RIPA buffer of

fresh CD-1 mouse tissues dissected in cold PBS, or purchased from Protientech Group.

Tissue-specific total RNA was purchased from Ambion and used to generate cDNA by

reverse transcription with the High Capacity cDNA Kit (ABI). Purified mouse liver cells

were homogenized in RIPA buffer or used to generate cDNA by extraction with the

RNeasy Plus Mini Kit (Qiagen) and reverse transcription with the High Capacity cDNA


Kit. Protein samples were clarified by centrifugation and total protein concentration

normalized to BSA with the BCA Protein Assay reagents (Pierce).

Quantitative PCR

Relative qPCR of total cDNA was performed on an ABI 7300 with transcript

specific FAM labeled Taqman probes and Universal Master Mix (ABI) or verified

transcript specific primer sets and Power SYBR Green Master Mix (ABI). Input was

normalized to 18s for whole tissue samples and GAPDH for all other experiments.

Samples were quantified in triplicate and represent a minimum of two biological

replicates.

Western Blotting

Equal amounts of total protein were separated by denaturing polyacryalamide gel

electrophoresis and transferred to 0.22μm nitrocellulose, blocked with 5% milk or 5%

BSA in Tris-buffered saline/0.1% Tween (TBST) and incubated overnight with

appropriate antibodies. Membranes were washed extensively with TBST and probed

with species-specific horseradish peroxidase conjugated secondary antibodies (Jackson).

The following Antibodies were used: Rabbit anti-MED1 (Bethyl), Rabbit anti-MED6

(Bethyl), Rabbit anti-MED12 (Novus), Rabbit anti-MED18 (Novus), Rabbit anti-MED23

(Bethyl), Goat anti-CDK8 (Abcam), Mouse anti-TBP (Biodesign), Rabbit anti-TRF2

(Abcam), Rabbit anti-TRF3 (Deato and Tjian 2007), Goat anti-TAF1 (Santa Cruz),

Rabbit anti-TAF3 (Deato and Tjian 2007), Mouse anti-TAF4 (BD Bioscience), Mouse

anti-TAF5 (Eurogentec), Rabbit anti-TAF6 (Abcam), Rabbit anti-TAF7 (Abnova), Goat

anti-TAF9 (Santa Cruz), Mouse anti-TAF10 (Chemicon), Rabbit anti-TAF12


(Proteintech Group), Rabbit anti-GAPDH (Cell Signaling), Goat anti-Albumin (Bethyl),

Mouse anti-AFP (R&D Systems), Mouse anti-AQP1 (BD Bioscience), Rabbit anti-

CYP7A1 (Santa Cruz).

Histone ChIP

Indivitual E13.5 livers were dissected from the embryos of timed pregnant CD-1

females and adult livers were dissected from 8-10 week old CD-1 females. Crosslinked

chromatin was prepared essentially as described (Soutoglou and Talianidis 2002; Kyrmizi

et al. 2006). Embryonic and adult liver tissue was minced in PBS and homogenized by

10-15 strokes of an A pestle in the presence of 1% formaldehyde. Crosslinking was

continued for 10 minutes and quenched by addition of glycine to 125mM. Cells were

collected at 1,000g for 10 minutes and washed twice for 20 minutes in hypotonic lysis

buffer (25mM Hepes pH7.9, 10mM KCl, 0.250M Sucrose, 2mM EDTA, 1.5mM MgCl2,

0.1% NP-40, 1mM DTT, 0.5mM Spermidine, Protease Inhibitor Cocktail (Roche)).

Nuclei were collected at 1,000g and resuspended in an equal volume of nuclear lysis

buffer (50mM Hepes pH7.9, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium

deoxycholate, 0.1% SDS, Protease Inhibitor Cocktail (Roche)). Chromatin was sheared

to 300-600 bp fragments by sonication and cleared by centrifugation at 15,000g. Equal

amounts of cleared chromatin were precipitated by overnight incubation with rabbit IgG,

rabbit anti-H3K4Me3, or rabbit anti-H3K9Me3 (Active Motif). Immunocomplexes were

incubated for two hours with pre-blocked protein A and protein G magnetic beads

(Invitrogen). Samples were washed twice with nuclear lysis buffer, twice with nuclear

lysis buffer plus 500mM NaCl, twice with Li was buffer (20mM Tris-HCl pH7.9, 1mM


EDTA, 250mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate), and once with TE.

Chromatin was eluted by addition of 50mM Tris-HCl pH 7.9, 25mM EDTA, 1% SDS,

0.2mg/ml RNase A, 0.1mg/ml Proteinase K. After incubation at 37˚C for 2 hours NaCl

was added to 500mM and samples were incubated overnight at 65˚C to reverse the

crosslinking. DNA was purified using QIAquick spin columns (Qiagen) and used in

quantitative PCR with primers surrounding the relevant proximal promoter.

Liver Cell Purification

Adult, day 15 neonatal, and partial hepatectomy hepatocytes were purified by

two-step in situ perfusion with Liberase 3 (Roche) as described (Wang et al. 2003b).

Partial hepatectomy of 8-12 week old male CD-1 mice was performed as described

(Mitchell and Willenbring 2008). Indivitual E13.5 livers were dissected from 3-5 timed

pregnant females in PBS and dissociated by incubation for 10-15 minutes at 37˚C in the

presence of 0.9mg/ml Liberase 3 (Roche) and 0.07% DNAse I (Sigma). Digestion was

quenched by the addition of FBS to 3% and EDTA to 2mM. Cells were collected at 40g

and resuspended in 1x Pharmalyse (BD Bioscience) for hypotonic lysis of erythrocytes.

Cells were resuspended in PBS/10%FBS, passed through a 70μm filter and diluted to

1x108 cells/ml. Cells were incubated for 15 minutes at 4˚C with CD45 microbeads

(Miltenyi Biotec), and in the absence of hypotonic lysis TER119 micobeads (Miltenyi

Biotec), after which labeled cells were removed by passage over an LS column attached

to a MidiMacs (Miltenyi Biotec) separator according the manufacturer’s instructions.

HPPL Cell Culture and Differentiation

Hepatic Progenitors Proliferating on Laminin (HPPL) were cultured and


differentiated essentially as described (Tanimizu et al. 2004). Proliferating cultures were

maintained in DMEM/F12 (Invitrogen), 10% heat inactivated FBS, 1x

insulin/transferrin/selenium (ITS) (Invitrogen), 10mM nicotinamide (Sigma), 0.1μm

dexamethasone (Novagen), 5mM L-glutamine (Invitrogen), 20ng/ml HGF (Peprotech),

and 20ng/ml EGF (Peprotech) on laminin-coated plates (BD Bioscience). Confluent

cultures were differentiated by incubation for five days in the same media minus HGF

and EGF and containing 20ng/ml OSM (Peprotech). For complete differentiation OSM

media was removed and cells were overlaid with fresh media containing 0.350 mg/ml

growth factor reduced Matrigel (BD Bioscience) for an additional five days as reported

(Kamiya et al. 2002).


Figure 1: TBP, TRF3, and TAF Expression in Mouse Tissues

C2C12 myoblasts and myotubes, CD-1 embryos, and whole mouse tissues were

homogenized in RIPA buffer and equal amounts of total protein were analyzed by

western blot with TBP, TRF3, TAF1, and TAF4 specific antibodies. GAPDH is used as a

protein loading control.


TBP

TRF3

TAF1

TAF4

GAPDH

Myoblast

Myotube

Embryo

Liver

Brain

Heart

Kidney

Lung

Testes

Ovary

Thymus

Spleen


Figure 2: TBP, TRF3, and TAF Expression in Mouse Tissues

Total RNA from diverse mouse tissues was reverse transcribed and used for

relative qPCR with transcript-specific Taqman probes. CT values were normalized to

18s ribosomal RNA (A) or GAPDH mRNA (B). Values for each tissue are expressed as

a percentage of the average value for all tissues combined.


Normalized to 18s

0

50

100

150

200

250

TBP TRF2 TRF3 TAF1 TAF3 TAF4 MED17

Embryo

Liver

Brain

Heart

Kidney

Lung

Testes

Ovary

Thymus

SpleenPer

cent

of A

vera

ge f

or A

ll T

issu

es

866%

A

Normalized to GAPDH

0

50

100

150

200

250

300

350

400

TBP TRF2 TRF3 TAF1 TAF3 TAF4 MED17

Embryo

Liver

Brain

Heart

Kidney

Lung

Testes

Ovary

Thymus

SpleenPer

cent

of A

vera

ge f

or A

ll T

issu

es

494%

896%

806%

B

452%


Figure 3: TBP, TRF, TAF, and Med Expression in Purified Liver Cells

Total RNA was purified from mouse E13.5 hepatoblasts and adult hepatocytes,

reverse transcribed, and used for qPCR. CT values were normalized to GAPDH and

expressed as a percentage of hepatoblast values. (A) TBP, TRFs and TAFs. (B)

Mediator subunits.


A

B

0

100

200

300

400

500

600

700

TB

PT

RF

2T

RF

3T

AF

1T

AF

2T

AF

3T

AF

4T

AF

4bT

AF

5T

AF

5LT

AF

6T

AF

6LT

AF

7T

AF

7LT

AF

8T

AF

9T

AF

9LT

AF

10T

AF

11T

AF

12T

AF

13T

AF

15G

CN

5P

CA

FB

TA

F1

Hepatoblast

Hepatocyte

0

20

40

60

80

100

120

Hepatoblast

Hepatocyte

Per

cent

of

Hep

atob

last

Per

cent

of

Hep

atob

last


Figure 4: TBP, TRF, TAF, and Med Expression in Neonatal and Regenerative

Hepatocytes.

Total RNA was purified from mouse E13.5 hepatoblasts, neonatal day 15

hepatocytes, regenerative hepatocytes following 2/3 partial hepatectomy, and adult

hepatocytes; reverse transcribed; and used for qPCR. CT values were normalized to

GAPDH and expressed as a percentage of hepatoblast values.


050100

150

200

250

300

350

Hep

atob

last

Neo

nata

l Hep

atoc

yte

Adu

lt H

epat

ocyt

e

Cyc

ling

Hep

atoc

yte

(2/3

par

tial

Hep

)

Percent of Hepatoblast


Figure 5: TBP, TRF and TAF Expression in Purified Liver Cells

Equal amounts of total protein from C2C12 cells, fetal and adult liver, and

purified liver cells were analyzed by western blotting with TBP, TRF2, and TAF-specific

antibodies.


TRF2

TBP

TAF1

TAF3

TAF4

TAF5

TAF7

TAF9

TAF10

TAF12

C2C

12

Fet

al L

iver

(E

13.5

)

Adu

lt L

iver

Hep

atob

last

Neo

nate

Hep

atoc

yte

Adu

lt H

epat

ocyt

e

Cyc

ling

Hep

atoc

yte


Figure 6: Med Expression in Purified Liver Cells

Equal amounts of total protein from C2C12 cells, fetal and adult liver, and

purified liver cells were analyzed by western blotting with antibodies against key

Mediator subunits.


MED26

MED18

MED23

MED16

MED17

MED12

MED14

MED7

MED1

MED6

CDK8

C2C

12

Fet

al L

iver

(E

13.5

)

Adu

lt L

iver

Hep

atob

last

Neo

nate

Hep

atoc

yte

Adu

lt H

epat

ocyt

e

Cyc

ling

Hep

atoc

yte


Figure 7: Transcriptional Silencing of TBP and TAF Promoters During

Hepatogenesis.

Individually dissected fetal and adult liver cells were crosslinked and used for

chromatin immunoprecipitation with histone mark-specific antibodies. Precipitated

chromatin was used in qPCR with primers surrounding the relevant proximal promoters

and values expressed as fold enrichment over an IgG control.


Fold Enrichment Fold Enrichment

E13

.5A

dult

051015202530354045

IgG

H3K

4Me3

H

3K9M

e3

TA

F3

Pro

mot

er

00.

511.

522.

533.

544.

55

IgG

H3K

4Me3

H

3K9M

e3

CY

P7A

1 P

rom

oter

05101520253035

IgG

H3K

4Me3

H

3K9M

e3

TA

F5

Pro

mot

er

0510152025

IgG

H3K

4Me3

H

3K9M

e3

TB

P P

rom

oter


Figure 8: Induction of Hepatic Gene Expression in a Cell Culture Model of

Development.

Hepatic Progenitors Proliferating on Laminin (HPPL) cells were differentiated by

incubating for five days in media containing 20ng/ml Oncostatin M and followed for five

days in media containing 0.350mg/ml Matrigel. Total RNA was reverse transcribed and

used in qPCR to analyze bipotential and hepatocyte marker gene expression (A). Values

are expressed as a percentage of undifferentiated control cells on a log scale. Whole cell

lysates were used in western blots with antibodies specific for -fetoprotein (AFP),

albumin (ALB), aquporin (AQP1), and CYP7A1 (B).


Day 0 5 10

AFP

ALB

AQP1

CYP7A1

A

B

1

10

100

1000

10000

100000

Day 0

Day 5

Day 10

Per

cent

Day

0 (

Log

Sca

le)


Figure 9: TBP, TRF, and TAF Expression in Differentiated HPPL Cells.

HPPLs were differentiated for five or ten days and total RNA was used for

quantitative PCR of TBP, TRF3, and TAF expression levels (A). CT values were

normalized to GAPDH and expressed as a percentage of an undifferentiated control.

Whole cell lysates were made and equal amounts of total protein analyzed by western

blot with antibodies specific to TBP, TRF3, TAF3, and TAF4 (A). GAPDH is used as a

protein loading control.


TBP

TAF4

TAF3

TRF3

GAPDH

Day 0 5 10

1

10

100

1000

10000

Day 0

Day 5

Day 10

A

B

Per

cent

Day

0


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Summary

The near infinite diversity of cell types and biological functions intrinsic to the

mammalian body plan is primarily determined at the level of transcriptional regulation.

In the mammalian liver, a diverse set of tissue-enriched and extensively studied

sequence-specific activators are known to regulate embryonic and adult gene expression

programs. While the spatial and temporal patterns of liver-enriched transcription factor

expression have been determined, and their requirements in hepatic gene expression

demonstrated, the exact mechanisms by which multiple factors collaborate to tune global

patterns of expression are not well understood. In contrast, the molecular mechanisms of

gene activation by canonical core promoter recognition and coactivator complexes such

as TFIID and Mediator are well established in cell culture and in vitro systems, but have

not yet extended to a tissue-specific context. Now that tissue-specific TAF paralogs,

TBP-related factors, and alternative core promoter recognition complexes have been

identified, it is critical to determine how these assemblies alter gene expression patterns

to specify tissue and cell-type-specific functions in collaboration with known activators.

To date, liver-specific core promoter recognition and coactivator complex subunits have

not been specifically characterized. If canonical TFIID is indeed absent from the adult

liver, than the isolation of such liver-enriched subunits and an understanding of their

coordination with existing liver-enriched transcription factors would greatly elucidate our

picture of liver development, disease, and regeneration.


Introduction

While TBP and the canonical TAFs which comprise TFIID were historically

assumed to be invariant between tissues, cell types and developmental time points, tissue-

specific TAF paralogs have since their discovery been known to exhibit highly restricted

expression patterns and transcriptional functions. Though a variety of TAF paralogs

were previously identified by bioinformatic means, the first concrete evidence of a tissue-

specific TFIID subunit came with the discovery of a substoichiometric TAF4 paralog in

cultured B cells (Dikstein et al. 1996). Subsequently, TAF4b was found to be highly

enriched in mouse testes and ovary and to be an intrinsic component of an altered ovary-

specific TFIID (Freiman et al. 2001). TAF4b knockout females, while viable, are

infertile as a result of deficient folliculogenesis, and knockout males show age-dependent

infertility resulting from a defect in spermatogenic maintenance (Falender et al. 2005).

Consistently, gene expression analysis revealed a requirement for TAF4b in regulating a

subset of ovarian granulosa cell-specific genes and genes required for spermatogonial

stem cell maintenance (Geles et al. 2006). Furthermore, TAF4b containing TFIID does

not necessarily require unique tissue-specific activators, but rather induces the expression

of and cooperates with ubiquitous general activators such as c-Jun to regulate cell-type-

specific gene expression (Geles et al. 2006). More detailed developmental analysis

confirmed that TAF4b is the primary transcriptional integrator of extracellular signals

that specify the developmental and proliferative program of granulosa cells (Voronina et

al. 2007). While not explicitly required in B cells, TAF4b cooperates with known

regulators of B cell-specific transcription such as OCA-B and NF B, and may serve a


redundant function to TAF4 in this context (Yamit-Hezi and Dikstein 1998; Wolstein et

al. 2000; Freiman et al. 2002). Promoter-specific differences in both basal and activated

transcription by canonical and TAF4b containing TFIID were demonstrated in vitro, and

these differences shown to correlate with TAF4b target genes identified in vivo (Liu et al.

2008). Intriguingly, a TAF4b homologue is enriched in Xenopus ovaries but absent from

lower metazoans, suggesting that unlike canonical TAFs, tissue-specific paralogs may

have evolved to direct species-specific transcriptional programs (Xiao et al. 2006)

The importance of tissue-specific TAFs was further validated by the identification

of five testis-restricted (tTAF) subunits in Drosophila, no hitter (paralog of TAF4),

cannonball (paralog of TAF5), meiosis I arrest (paralog of TAF6), spermatocyte arrest

(paralog of TAF8), and ryan express (paralogue of TAF12) (Lin et al. 1996). Disrupting

each of these proteins alters expression of testis-specific genes, and together they form a

stable complex, which is required for meiotic cell cycle progression and normal

spermatid differentiation (Hiller et al. 2001; Hiller et al. 2004). Intriguingly, the tTAF

complex mediates spermatid gene activation at least in part by displacing the repressive

polycomb complex and promoting its sequestration to the nucleolus (Chen et al. 2005);

hence, a derepressive tTAF strategy may represent a novel cell-type-specific mechanism

of TAF dependent coactivation.

While tTAFs are restricted to Drosophila, a testis-linked paralogue of TAF7

appears unique to vertebrates. High level expression of mouse TAF7l in late

spermatocytes and haploid spermatids is coincident with a reduction in TAF7 expression,

and as spermatogenesis proceeds TAF7l, is relocalized to the nucleus where it associates


with TBP and other TAFs to form an alternative TFIID- like complex (Pointud et al.

2003). Viable TAF7l knockout mice display altered patterns of spermatocyte gene

expression and defects in spermiogenesis which lead to reduced fertility, consistent with

a testes function (Cheng et al. 2007). Additional TAF paralogs, including TAF9b, while

shown to associate with TFIID and promote alternative gene expression programs, are

just beginning to be characterized, and the full repertoire of tissue-specific TAFs remains

to be discovered (Frontini et al. 2005).

While noncanonical TAF paralogs were originally identified in a subset of tissues,

TBP-related factors or TRFs were originally assumed to be widely expressed but are

now known to exhibit tissue-specific expression patterns and regulatory functions

(Hochheimer and Tjian 2003). Hence TRF1, the first identified TBP-related factor to be

discovered, is restricted to Drosophila, where it is enriched in the nervous system and

gonads and thus seems to have evolved to suite a specific purpose in these insect tissues

(Hansen et al. 1997). Polytene chromosome staining and genome-wide promoter

occupancy experiments suggest that TRF1 regulates a highly restricted set of genes

relative to TBP, and that these loci are primarily associated with RNA PolIII transcription

(Isogai et al. 2007b). However, TRF1 has been isolated in association with BRF and a

larger complex containing novel proteins and been shown to direct both RNA PolII and

PolII transcription in vitro and in vivo, much like TBP (Holmes and Tjian 2000; Takada

et al. 2000). Hence, TRF1 represents an evolutionarily restricted alternative core

promoter recognition complex which directs promoter-selective transcription by multiple

polymerases in a subset of tissues.


TBP-related factor 2 (TRF2, also called TLF, TLP, or TRP), though more widely

distributed evolutionarily from C. elegans to humans, also exhibits tissue-specific

expression patterns and possibly species-specific transcriptional specialization (Dantonel

et al. 1999; Rabenstein et al. 1999). While TRF2 binds TFIIA and TFIIB and displays

high conservation of the TBP DNA binding domain, it does not bind TATA box

containing DNA and fails to complement TFIID in basal or activated in vitro

transcription assays (Moore et al. 1999; Teichmann et al. 1999). Biochemically, TRF2

associates with the DRE binding factor (DREF) and components of the NURF chromatin

remodeling complex, and has been shown to selectively regulate the transcription of

DRE-containing promoters of cell cycle and proliferation genes in coordination with

these proteins (Hochheimer et al. 2002). Intriguingly, several genes have been shown to

contain tandem transcriptional start sites whose promoters are specifically and

differentially regulated by TRF2 and TFIID. Disruption experiments in C. elegans,

Drosophila, and Xenopus demonstrate a requirement for TRF2 in early embryonic

development, but also confirm that TRF2 regulates a subset of developmental genes

responsible for these phenotypes, while being dispensable for the expression of others

(Dantonel et al. 2000; Kaltenbach et al. 2000; Veenstra et al. 2000; Kopytova et al. 2006).

Conversely, mouse TRF2 is highly enriched in the testis, and while knockout animals

display spermiogenesis defects and altered patterns of testes-specific gene expression,

they show no discernable developmental defects, consistent with an evolutionarily

divergent function in mammals (Martianov et al. 2001; Zhang et al. 2001). Recent

genome-wide localization of Drosophila TRF2 showed that it occupies over a thousand


promoters, most of which are not bound by TBP and are enriched for DREs but lack a

canonical TATA box (Isogai et al. 2007a). Intriguingly, the Drosophila histone H1

promoter is specifically regulated by TRF2 while the reminder of the histone gene cluster

is TFIID dependent, suggesting that differences in core promoter recognition complex

activity may alter histone ratios. Hence, TRF2 exhibits evolutionarily diverse roles, and

clearly extends the diversity of core promoter recognition complexes in its regulation of

both tissue-specific and promoter-selective gene expression patterns. Additionally, the

existence of separate TRF2 and TFIID-dependent promoters within related gene clusters,

or in some cases single genes, may allow a limited set of promoters and core promoter

recognition complexes to produce dramatically varied expression patterns in response to

signaling events or between tissues. This alternative regulation likely results from

TRF2’s association with novel transcription factors through its divergent N and C-

terminal protein interaction domains and its recruitment to novel core promoter elements

not occupied by TBP.

The most recently identified and perhaps most intriguing member of the TBP-

related factor family is the vertebrate-specific TRF3 (also called TBP2). Initially

identified based on homology, mammalian TRF3 shows near identity to TBP in its C-

terminus including, the TATA box, TFIIA, and TFIIB binding domains, and considerable

divergence from all known TBPs in its N-terminus (Persengiev et al. 2003). Preliminary

characterization showed TRF3 to be widely expressed in adult mammalian tissues,

though at varying levels relative to TBP, and to be primarily contained in a larger 150-

200kD complex. Many studies of TRF3 function have focused on oogenesis and early


embryonic development. Hence, murine oocyte TRF3 expression has been shown to

increase rapidly during oocyte growth from primordial follicle formation until

preovulation at the same time that TBP expression is effectively eliminated (Yang et al.

2006; Gazdag et al. 2007). However, these observational reports reach conflicting

conclusions on the role of TRF3 in murine embryogenesis, as one shows a steady

increase in both TRF3 and TBP expression following fertilization, while the other

concludes that TRF3 remains silent as TBP expression is induced post fertilization; this

conflict in immunofluorescence data will likely be resolved by more extensive studies

and additional approaches.

The role of TRF3 in early development has been most extensively investigated in

Xenopus and zebrafish. Hence, Xenopus and zebrafish TRF3 bind TFIIA, TFIIB, and

TATA containing DNA, and Xenopus TRF3 support TATA-dependent transcription in

egg extracts (Bartfai et al. 2004; Jallow et al. 2004). While highly expressed in the

Xenopus oocyte, TRF3 is expressed at lower levels in the developing embryo and is

recruited to a subset of genes not occupied by TBP in a time-dependent manner.

Importantly, TRF3 depletion causes defects in Xenopus gastrulation and blastopore

closure and alters expression of many oocyte and embryo-specific genes, a subset of

which also require TBP. Intriguingly, Xenopus TRF3 overexpression can partially rescue

TBP knockdown phenotypes and gene expression patterns, consistent with a partial

redundancy. Further genome wide analysis found that Xenopus TBP primarily regulates

the synthesis of maternally deposited transcripts while a majority of embryonically

transcribed genes require TRF2 or TRF3 (Jacobi et al. 2007). Additionally, TBP


dependent Xenopus genes are widely conserved from yeast to humans while TRF3-

specific genes are generally restricted to vertebrates. Xenopus knockdown and ChIP

experiments demonstrate that many promoters while exclusively dependent on either

TRF3 or TFIID are occupied by both, suggesting that differences in complex-specific

transcription may occur at the level of activation rather than recruitment. Expression of

zebrafish TRF3 increases steadily from fertilization to gastrulation, after which it declines

rapidly but remains detectable in all adult tissues and enriched in the ovary. As with

Xenopus, knockdown inhibits normal gastrulation and results in mesodermal patterning

defects. Intriguingly, both species show an enrichment of TRF3 in the ventral embryo

consistent with a role in dorsoventral patterning. Additional knockdown studies in later

stage zebrafish embryos demonstrate a specific requirement for TRF3 in the initiation of

hematopoiesis, and partially define a lineage-specific transcriptional cascade in which

TRF3 is the master regulator of this particular differentiating program (Verdorfer et al.

2007). Thus, multiple studies in lower vertebrates reveal a requirement for TRF3 in early

development and elucidate both redundant and overlapping functions with TBP in

embryonic transcription. However, it is intriguing to note that the striking embryonic

phenotypes of TRF2 depletion in Xenopus and zebrafish are not conserved in the

knockout mice and that the important embryonic functions of TRF3 in lower vertebrates

likewise may not be preserved in mammals.

A study investigating the differentiation of mouse C2C12 cells found that while

TBP, along with several TAFs, are degraded upon differentiation, at least some TRF3 is

retained in myotubes, some of which is found in a complex with TAF3 (Deato and Tjian


2007). Depletion of TRF3 and TAF3 by RNAi suggested that this putative core promoter

recognition complex may be required for myogenesis. Consistent with a requisite role in

differentiation, ChIP analysis found TRF3/TAF3 occupying a key muscle-specific gene

promoter. Further biochemical analysis of the complex suggested that TRF3/TAF3 is

sufficient for modest activation of the myogenin promoter in vitro by the myogenic

transcription factors MyoD and E47 (Deato and Tjian 2008). Protein-protein interaction

studies, along with additional in vitro transcription assays, revealed that MyoD directly

targets TAF3, and that this interaction is necessary for in vitro activity. It will be

interesting to determine how widely the TRF3/TAF3 complex may be used in other

tissues.

The Mediator coactivator complex was also recently found to display

considerable subunit diversity, which ultimately may be shown to correlate with tissue

and cell-type-specific transcriptional programs. Hence, biochemical fractionation of

HeLa nuclear extracts yielded an altered CRSP/Med2 complex lacking two key canonical

Mediator subunits previously shown to be critical for nuclear hormone receptor (NHR)

dependent transcriptional activation (Taatjes and Tjian 2004). This complex, while

unable to support NHR dependent transcription, supports transcription by activators

which target other Mediator subunits and therefore may be responsible for limiting

coactivation to a subset of extracellular signals or promoters. Similarly, the ARC-L/Med

complex which contains most canonical Mediator subunits in addition to a dissociable

CDK8/cyclin C/Med230/Med240 submodule may fine tune Mediator activity in response

to environmental or developmental cues (Taatjes et al. 2002). Importantly, reversible


association of the ARC-L/Med submodule transitions C/EBPb dependent Mediator

activity from a stimulatory to a repressive state (Mo et al. 2004). While ARC-L/Med has

generally been associated with transcriptional repression it appears to stimulate a limited

subset of promoters, and thus the ratio of ARC-L/Med to Mediator may lead to the

activation of some genes and subsequent repression of others (Akoulitchev et al. 2000;

Zhou et al. 2002). Furthermore, the finding that the ARC-L/Med submodule enhances

Mediator’s association with a ubiquitin ligase and may enhance its proteasome-dependent

degradation suggests that submodule abundance may regulate coactivation globally by

influencing Mediator turnover (Brower et al. 2002) Thus, combinatorial control of

subunit composition could allow for cell-type and promoter-specific transcription

dependent on distinct classes of activators. However, it remains to be seen whether these

complexes are utilized in a cell-type-specific way.

Additional precedent for the idea of tissue and developmental stage-specific

changes in transcription factor complexes that were previously held to be invariant comes

from the recent observation that Swi/Snf chromatin remodeling complexes also vary

greatly between tissues. Hence, the finding that specific subunits of the mammalian

PBAF complex are enriched in cardiac progenitors and required for normal heart

maturation provided the earliest support for the idea of cell-type-specific chromatin

remodelers (Wang et al. 2004; Yan et al. 2005). Conversely, knockout studies of unique

subunits from the related BAF-A and BAF-B complexes demonstrated overlapping

deficiencies in ES cell pluripotency and lineage-specific differentiation, while disruption

of PBAF showed no specific ES cell phenotype, consistent with distinct developmental


requirements for each complex (Gao et al. 2008; Huang et al. 2008; Yan et al. 2008).

The first evidence of a requisite developmental stage-specific switch in subunit

composition came with the discovery that homologous BAF45 and BAF53 subunits must

be exchanged during the transition from neuronal progenitors to postmitotic neurons

(Lessard et al. 2007; Wu et al. 2007). Moreover, this exchange is now known to depend

on microRNA-mediated repression of the progenitor-specific subunits, adding an even

greater level of regulatory complexity to the neuronal transition (Yoo et al. 2009). Most

recently, a novel esBAF complex was isolated from embryonic stem cells which has a

unique subunit composition, is required for pluripotency and self-renewal, and is

downregulated upon differentiation (Ho et al. 2009b). Further genome-wide localization

experiments demonstrate the existence of a cooperative transcriptional network between

esBAF and known ES cell-specific transcription factors which defines self-renewal and

pluripotency (Ho et al. 2009a). The extensive evidence that TAF1 can bind and modify

histones, combined with the more recent finding that TAF3 shows a specific affinity for

active chromatin marks, further suggests that diverse cell or developmental stage-specific

chromatin remodelers and core promoter recognition complexes may cooperate with

specific chromatin states to direct highly specialized transcriptional programs (Mizzen et

al. 1996; Jacobson et al. 2000; Vermeulen et al. 2007).


Results

Hepatic Expression of TRF3, TAF7l, and TAF13

Because our previous qPCR analysis suggested that TRF3, TAF7l, and TAF13

expression may not be as dramatically downregulated as TBP and other TAFs in adult

hepatocytes or may in fact be induced upon differentiation, we chose to further explore

the expression and function of these specific subunits in hepatic transcription (Chapter 2,

Figure 3). Hence, we performed western blots of purified hepatoblasts and hepatocytes

with multiple antibodies specific for these three subunits (Figure 1). At least two

separate anti-TRF3 antibodies recognize strong expression of TRF3 expression in both

developing hepatoblasts and committed hepatocytes (Figure 1A). Within the quantitative

limits of western blot analysis, TRF3 expression in liver cells does not appear to differ

greatly from that in the whole ovary, a tissue in which TRF3 was previously shown to be

highly enriched (Gazdag et al. 2009). While qPCR data suggest that TRF3 expression

may be slightly greater in hepatocytes than hepatoblasts, quantitative western blotting or

other techniques need to be employed to determine if this is true at the protein level.

Similarly, western blots with two different TAF7l antibodies suggest that this

paralog is expressed at similar levels in hepatocytes and developing hepatoblasts and at

comparable levels to the testes, the tissue from which TAF7l was first isolated and shown

to have a specific function (Figure 1B). Again, more quantitative proteomic methods will

need to be employed to determine if the significant upregulation of TAF7l transcript

observed in adult hepatocytes leads to an increase in protein levels. Initial western blot

comparison of TAF13 expression in developing hepatoblasts and mature hepatocytes also


suggests that this subunit may be retained or induced in the adult liver, in contrast to the

majority of canonical TFIID components (Figure 1C). While further quantitation of

TAF13 polypeptide is needed, initial data suggest that this subunit warrants further

investigation.

RNAi Mediated Depletion of TRF3, TAF7l, and TAF13 Attenuates Hepatic Gene

Induction in HPPLs

While spatial and temporal patterns of transcription factor expression often

correlate with tissue-specific function, this is not always the case, and some factors may

be inactive or dispensable even where they are highly abundant. To explore the

possibility that TRF3, TAF7l, and TAF13 may play a role in adult hepatic gene

transcription, we depleted these proteins by lentiviral RNAi in the HPPL cell line.

pLKO.1 vectors which are part of the Broad Institute RNAi consortium and express short

hairpin RNAs targeting these three transcripts under the U6 promoter, along with a

puromycin resistance gene, were used to generate replication-incompetent lentiviral

particles by transient transfection of HEK293T cells (Figure 2A). Proliferating HPPLs

were infected for 24 hours with viral supernatants and then selected for four days in fresh

growth medium containing puromycin to eliminate uninfected cells. Cells were then

harvested to determine the extent of gene knockdown by qPCR or differentiated in the

presence of Oncostatin M. By this approach we consistently achieved knockdown of

TRF3, TAF7l, and TAF13 with sixty to eighty percent efficiency (Figure 2B).

Importantly, infection with a control lentivirus targeting GFP had no discernible affect on


the levels of these three transcripts, and reduction of any one transcript did not influence

the expression of another, suggesting that off target effects and lentivirally induced

cytotoxicity are minimal in this system.

Because the most significant changes in both TFIID downregulation and hepatic

gene induction in the HPPL cell model were observed after five days of differentiation,

we reasoned that this represented the most critical period for transcription factor activity

in this particular system (Chapter 1, Figure 8 and 9). Following four days of selection,

confluent lentivirally transduced HPPLs were differentiated for five days in the presence

of Oncostatin M and RNA harvested for qPCR analysis. This data shows that even

moderate depletion of TRF3, TAF7l, and TAF13 has a significant impact on the

induction of multiple adult hepatic genes in the HPPL model, as judged by transcript

abundance (Figure 3). Qualitatively, differentiated TRF3, TAF7l, and TAF13 shRNA

expressing HPPLs contained fewer large cuboidal, binucleate, and vacuolated cells than

the GFP control, suggesting that these cultures failed to fully differentiate. Though it

cannot be concluded from this data that TRF3, TAF7l, or TAF13 specifically regulate the

hepatic genes in question, this approach suggests that these factors have some influence

on the HPPLs’ competence to differentiate towards a mature hepatic cell and that in their

absence, hepatic maturation is at least partially compromised.


TRF3, TAF7l, and TAF13 are Enriched at Hepatic Promoters in the Adult Liver

and Differentiated HPPLs.

The physical recruitment of sequence-specific activators to their target enhancers

and of the general machinery to a gene’s proximal promoters is a requisite step in

transcriptional activation (Thomas and Chiang 2006). Hence, the ability to localize

proteins and complexes at the regulatory elements of a given gene supports a role for

such factors in that gene’s transcriptional regulation. Chromatin immunoprecipitation is

a powerful technique for investigating the relative occupancy of defined DNA domains

by specific proteins and has been extensively employed to elucidate the functions of

single transcription factors and the complexities of large regulatory networks (Massie and

Mills 2008; Park 2009). Specifically, this approach has been invaluable in unraveling the

role of liver-enriched activators and general transcription factors in liver development

and maintenance (Kyrmizi et al. 2006; Tatarakis et al. 2008).

To investigate the role of TRF3, TAF7l, and TAF13 in hepatic transcription we

isolated formaldehyde crosslinked chromatin from individually dissected E13.5 and adult

mouse livers, and subjected it to immunoprecipitation with antibodies against these three

factors. Precipitated material was analyzed by qPCR with primers surrounding the

promoters of adult hepatic genes (Figure 4). Intriguingly, TRF3, TAF7l, and TAF13

show overlapping and distinct patterns of promoter occupancy on multiple hepatocyte-

specific genes. Most notably all three factors are significantly and specifically enriched

on the promoters of 1-antitrypsin (AAT) and tryptophan-2,3-dioxygenase (TDO2), two

genes which are completely silent in the embryo and are highly induced late postnatally.


Importantly, these promoters show no enrichment of the factors at day E13.5, a time

when they are known to be transcriptionally silenced. Intriguingly, a second class of

promoters, including apolipoprotein C3 (APOCIII) and possibly apolipoprotein B

(APOB) and albumin (ALB), are occupied by TRF3, TAF7l, and possibly TAF13 in both

the embryo and adult. In contrast to AAT and TDO2, these promoters are expressed in

the embryo and continue to be expressed at high levels postnatally. Recruitment of

TRF3, TAF7l, and TAF13 is less pronounced and varies between factors on the other

promoters examined; however the extent of factor recruitment is significant and specific

and its timing is consistent with the known developmental upregulation for these

promoters.

The primary drawback of chromatin immunoprecipitation is its dependence on the

specificity and efficiency of available antibodies for the transcription factor in question.

Because of this, overexpression of exogenous epitope-tagged proteins followed by

precipitation with well characterized antibodies against those tags has proven valuable to

corroborate data obtained with antibodies against the endogenous factor. To this end, we

generated recombinant TRF3, TAF7l, and TAF13 constructs with N-terminal FLAG tags

in the doxycycline-inducible pTRIPZ vector; these vectors were used to produce

lentiviral particles which were used to transduce proliferative HPPLs. Two days post

infection confluent cells were differentiated for five days by the addition of oncostatin M,

and pTRIPZ expression was induced by the addition of doxycycline to 2μg/ml on the last

two days of differentiation. Cells were crosslinked and purified chromatin was

immunoprecipitated with IgG or anti-FLAG polyclonal antibodies. Precipitated material


was analyzed by qPCR and results expressed as fold enrichment over the IgG control

(Figure 5).

While the promoter enrichment of FLAG-tagged TRF3, TAF7l, and TAF13 in

differentiated HPPLs does not exactly mirror that seen in adult liver, this data clearly

demonstrates that all three factors are specifically recruited to adult hepatic genes (Figure

5). The efficiency of FLAG-tagged protein immunoprecipitation may be impacted by the

presentation of the N-terminal FLAG epitope on these proteins in a native environment;

likewise the extent of promoter occupancy may be impacted by the ability of these

exogenous proteins to associate with other factors required for their recruitment or the

presence of these factors in not fully differentiated HPPL. In particular, the reduced

recruitment of TAF7l likely results from the relatively low expression and cytotoxicity

that we observe with this construct and that may inhibit differentiation or adequate

promoter binding. However, the data show that all three factors are enriched at multiple

hepatic promoters by an approach that is independent of antibody specificity.

Furthermore, the relative fold enrichment is similar to that seen in whole liver and

previously reported for TAFs, as well as general transcription factors on hepatic genes in

whole liver and liver cell line samples (Tatarakis et al. 2008).

TRF3 Associates with One or More High Molecular Weight Complexes in the Adult

Liver.

The finding that TRF3, TAF7l, and TAF13 are expressed in the adult liver, are

enriched at overlapping hepatic promoters, and may be required for hepatic gene


expression raised the possibility that they interact to form a coordinated multisubunit

regulatory complex. Indeed, one of the defining characteristics of known core promoter

recognition and coactivator complexes including TFIID and Mediator is their existence as

stable high molecular weight assemblies under native conditions (Albright and Tjian

2000; Myers and Kornberg 2000). Additionally, TRF3 has previously been shown to

associate with a 150kD complex in HeLa cells and to form a 150kD complex with TAF3

in myotubes (Persengiev et al. 2003; Deato and Tjian 2007).

To explore this possibility, we prepared nuclear extracts from adult mouse livers

and subjected them to size exclusion chromatography on a Superdex-200 column

previously calibrated with known sizing standards. Western blot analysis of those

fractions corresponding to molecular weights from 440 to 13.7kD reveal that TRF3

migrates at approximately 150kD as previously reported in the HeLa and myotube

systems. However, TAF7l and TAF13 do not appear, under these conditions, to

significantly associate with slower migrating TRF3, as they both migrate at or near their

calculated monomeric molecular weights of 42 and 18kD respectively. Our finding that

TAF3 is downregulated transcriptionally during hepatic development and seemingly

absent from adult hepatocytes at the protein level led us to believe that the TRF3/TAF3

complex previously identified in myotubes would be absent in the hepatic system

(Chapter 2, Figure 3 and 5). However, the observation that TRF3 remains associated

with a 150kD complex in liver nuclear extracts suggests that either trace amounts of

TAF3 remain, or that TRF3 is associated with other as yet to be identified subunits.

While the slower migration of TRF3 clearly does not depend on its association with the


majority of TAF7l or TAF13 under these conditions, this does not rule out the possibility

that these three proteins interact more transiently in vivo or that minor amounts of TAF7l

and TAF13, which avoid detection in this setting, are associated with higher molecular

weight assemblies. It is important to note that neither TAF7l nor TAF13 is observed in

the void fraction that would contain canonical TFIID. Though many general

transcription factor assemblies survive biochemical fractionation under the conditions

employed, activator-coactivator interactions are typically weaker, more transient, and

refractory to efficient biochemical copurification. Clearly, further investigation is needed

to determine the full range of protein-protein interactions by TAF7l and TAF13.

Core Promoter Recognition Complex Changes in Human ES Cell Derived

Hepatocytes.

Canonical TFIID structure and function has been a subject of almost equally

intensive study in both the mouse and human systems, and its role in transcription has

been shown to be highly conserved between these and lower vertebrates. In contrast,

investigation of TBP-related factors and TAF paralogs, including TRF3 and TAF7l, has

been largely restricted to the murine, Xenopus, and zebrafish models. Furthermore,

significant differences in the mechanisms and processes of liver development and

regeneration have been identified between mice and humans (Wauthier et al. 2008).

Hence, novel mechanisms of hepatic regulation uncovered by murine experimentation

must be recapitulated and confirmed in the human system for their therapeutic potential

to be realized. The directed in vitro differentiation of human embryonic stem cells to


functioning hepatocytes with the potential for therapeutic transplantation has been

reported by several groups and is a subject of intensive ongoing investigation (Duan et al.

2007; Hay et al. 2007; Hay et al. 2008a). Thus, we wished to determine if the changes

we observe in core promoter recognition complex expression during murine liver

development are conserved in an ES cell-based model of human hepatic commitment.

Human H9 ES cells were differentiated using a three step, sixteen day protocol

which mimics in vivo signaling events and has a reported efficiency of 90% (Hay et al.

2008a). In the first stage, proliferating ESCs are committed to the definitive endoderm

by incubation for five days with Activin A and Wnt3A, in the second stage they are

differentiated to hepatoblast like cells in defined growth factor free media, and in the

final stage matured to hepatocyte-like cells by the addition of hepatocyte growth factor

and oncostatin M (Figure 7A). Analysis of known marker gene expression in these four

cell populations by qPCR mirrors which is observed in vivo and is consistent with

published reports of directed hepatocyte differentiation, suggesting that the procedure

does faithfully model the hepatogenic process (Hay et al. 2008a; Hay et al. 2008b).

Intriguingly, qPCR analysis of TBP, three critical TAF subunits, and MED17 in these

four cell populations shows that TFIID and Mediator levels are held constant or induced

during the first two stages and reduced dramatically during hepatogenic maturation

(Figure 7B). Thus, as is observed in the purified mouse liver cell samples and HPPL

protocol, this model suggests that human hepatogenesis requires significant changes in

TFIID and Mediator expression or composition.


Discussion

Intensive and ongoing study of liver-enriched sequence-specific activators has

contributed greatly to our understanding of the transcriptional changes that govern

hepatic development and regeneration (Schrem et al. 2002; Costa et al. 2003). However,

the molecular mechanisms by which unique combinations of activators alter global

patterns of gene expression in response to developmental inputs or hepatic injury remains

to be elucidated. Core promoter recognition and coactivator complexes are a critical link

between activators, chromatin modifiers, core promoter DNA, and the general PolII

machinery. Despite this, the composition and function of these complexes in liver

development, regeneration, and homeostasis has not been thoroughly explored.

Our observation that canonical TFIID, including TBP and multiple TAFs, is

significantly downregulated during hepatic development raised the possibility that

alternative core promoter recognition proteins exist in the adult liver and at least partially

replace the critical functions of TFIID (Chapter 2). This hypothesis was informed by

several recent studies that demonstrate a role for TBP-related factors and TAF paralogs is

defined differentiation programs or cell types. Specifically, muscle cell development and

embryonic hematopoiesis were independently shown to require a novel TRF3/TAF3 core

promoter recognition complex which at least partially replaces TFIID in the regulation of

developmental stage-specific genes (Deato and Tjian 2007; Hart et al. 2009).

Additionally, TRF3 was found to be a critical regulator of oocyte-specific genes during

primary folliculogenesis, while TAF7l was shown to be essential for spermiogenesis,

potentially as part of a novel TBP containing complex (Cheng et al. 2007; Gazdag et al.


2009). Our qPCR analysis of known TRF and TAF transcripts in hepatoblasts and

hepatocytes suggests that, unlike TBP and the majority of TAFs, TRF3, TAF7l, and

TAF13, expression is maintained or induced in committed liver cells and that the role of

these factors in hepatic transcription therefore warrants further investigation.

While more accurate quantitation of TRF3, TAF7l, and TAF13 polypeptide is

required, initial data confirms expression of these three proteins in adult hepatocytes and

suggests that they are therefore unique from the majority of TFIID subunits which are

significantly downregulated. A more thorough understanding of liver organogenesis

should include expression studies of TFIID and these three factors in the other major cell

types, including Cholangiocytes and Kupffer cells, to determine if retention of these

factors is specific to the hepatocyte lineage or a hallmark of multiple liver cells. Initial

RNAi experiments demonstrate that depletion of TRF3, TAF7l, and TAF13 alters

patterns of hepatic gene induction, suggesting that their presence is in fact functionally

relevant. More detailed HPPL studies will be needed to determine if RNAi of these

factors alters cell proliferation or competence prior to hepatocyte commitment,

suggestive of a role in hepatoblast transcription, or if the observed phenotype reflects a

specific requirement for these factors solely during the process of hepatic gene induction.

While gene expression is an important indirect readout of hepatic development,

differentiated HPPLs have been shown to exhibit hepatocyte like metabolic functions

such that it will be interesting to determine if RNAi impacts more functional readouts,

including albumin secretion and cytochrome P450 metabolism (Kamiya et al. 2002).

Additionally rescue experiments are needed to confirm the specificity of shRNA


targeting. Mouse knockout and knockdown experiments have helped to confirm the in

vivo relevance of both liver-enriched transcription factors and TFIID components in other

tissues (Lee et al. 2005; Mengus et al. 2005; Tatarakis et al. 2008) Whole animal

knockdown studies are currently underway and should help to corroborate and expand the

findings of TRF3, TAF7l, and TAF13 RNAi experiments conducted in the HPPL system.

Morphological and functional phenotypes resulting from gene ablation

experiments often reflect secondary or tertiary effects and thus fail to prove that a given

promoter is the direct target of the transcription factor in question. The chromatin

immunoprecipitation data presented demonstrates that at least some adult hepatic genes

are specifically occupied by TRF3, TAF7l, and TAF13 and suggests that the observed

RNAi phenotype is not exclusively the product of secondary effects. In light of these

results, ChIP and Re-ChIP analysis should be extended to determine more accurately the

timing and potential co-occupancy of additional adult and embryonic promoters in

multiple liver cell types. A major goal of hepatology research has been to uncover the

overlapping and autoregulatory actions of all known liver-enriched transcription factors

throughout development, and to use this data in defining an all inclusive regulatory

network (Odom et al. 2004; Kyrmizi et al. 2006). If novel core promoter recognition

complexes prove to be global regulators of hepatic transcription, then chromatin

immunoprecipitation combined with recent deep sequencing techniques has the potential

to identify all promoters regulated by these factors and refine or expand considerably our

understanding of the hepatogenic transcription factor network (Park 2009). Reconstituted

in vitro transcription using defined factors was recently employed to confirm the


regulation of a key myogenic promoter by the TRF3/TAF3 complex in coordination with

a known myogenic activator, and was previously shown to be a valuable technique in the

mechanistic study of a tissue-specific noncanonical TAF4 paralogs (Deato et al. 2008;

Liu et al. 2008). Hence, if liver-specific core promoter recognition proteins and their

target promoters can be sufficiently defined, then in vitro assays with known liver-

enriched transactivators may prove valuable in further uncovering the mechanisms of

hepatocyte-specific transcriptional activation.

While our data confirm the association of TRF3 with a high molecular weight

complex in adult liver as previously reported for other cell types, initial experiments

suggest that it does not physically associate with either TAF7l or TAF13 under the

conditions employed (Hart et al. 2009). Clearly, further investigation is needed to

determine if TAF7l and TAF13 possess unique or overlapping binding partners in

committed hepatocytes and if such proteins are required for their promoter recruitment or

coactivator function. One outstanding question is whether either of these factors

associates with canonical TFIID in the fetal or adult liver and if this association is

strengthened or eliminated during hepatogenic differentiation. While specific TAFs have

been shown to bind known transcriptional activators and thus facilitate recruitment of

holo-TFIID to the core promoter, no specific interaction has yet been reported between

canonical TFIID subunits and liver-specific activators (Albright and Tjian 2000). It will

be interesting for future studies to determine if known liver-specific activators exhibit

preferential binding to TFIID or novel hepatocyte enriched core promoter recognition


proteins and if these interactions are consistent with previously identified target genes

and patterns of expression for the activator in question.

Ongoing efforts to develop therapeutically relevant transplantable hepatocytes

from human embryonic stem cells have benefitted greatly from basic advances in our

understanding of liver development (Hay et al. 2007; Hay et al. 2008a). Our initial

finding that hepatogenic in vitro differentiation of human ESC involves significant

downregulation of TFIID as first observed in the mouse system, suggests that core

promoter recognition complex changes are not unique to the murine system and may

prove useful in the refinement of ESC differentiation protocols. In light of these

observations and the recent generation of iPS cells from mature hepatocytes, it will be

interesting to determine the function of TFIID and potentially novel core promoter

recognition factors, not only in hepatocyte commitment but in the process of iPS

induction, pluripotency, and self-renewal (Aoi et al. 2008).

It is now clear that TFIID, Mediator, and potentially other general transcription

factor complexes, which were once thought to be a hallmark of all cell types and gene

expression programs, are dispensable in at least some developmental strategies and

tissue-specific cell types. The role of TBP-related factors, TAF paralogs, and

noncanonical core promoter recognition or coactivator complexes have become

increasingly important as new techniques have allowed us to study rarer and more

defined populations of cells. The recent finding that myogenesis and early hematopoiesis

employ a stripped down TRF3/TAF3 complex in place of canonical TFIID, and that germ

cell development relies on multiple TFIID independent factors including TRF2, TRF3


and TAF7l raises the possibility that additional differentiation programs will require these

and as yet unidentified proteins which supplant TFIID at critical developmental junctions.

Our current data suggest that the commitment of hepatogenic precursors to mature

hepatocytes likewise involves the downregulation of canonical TFIID subunits and their

possible replacement with TRF3, TAF7l, or TAF13 and potentially other unknown core

promoter recognition elements.

Materials and Methods

Antibodies

Fresh TRF3, TAF7l, and TAF13 antibodies were generated by multiple strategies.

Rabbits, Guinea pigs, and chickens were injected with an N-terminal TRF3 peptide

coupled to Keyhole Limpet Hemocyanin (Pierce) or a recombinant TRF3-Pseudomonas

exotoxin fusion protein expressed from pVCH6 and purified under denaturing conditions.

Rabbit and chicken anti-TAF7l antibodies were raised against a Pseudomonas exotoxin

fusion-TAF7l fusion protein purified under denaturing conditions. Mouse anti-TAF13

antibodies were raised against full length recombinant Drosophila TAF13 purified under

denaturing conditions. Rabbit and mouse polyclonal antibodies were purified on Protein

A sepharose (Amersham) and chicken antibodies were purified using the IgY Purification

Kit (Pierce). Mouse monoclonal anti-TBP/TRF3 antibody 58C9 was used for western

blotting at a dilution of 1:1,000 (Sigma, (Hoey et al. 1993).


Plasmids

shRNA constructs in the pLKO.1 plasmid are part of the Broad Institute RNAi

library and were purchased from Open Biosystems. The lentiviral packaging plasmids

pSPAX2 and pMD2.G were purchased from Addgene. The TRF3, TAF7l, and TAF13

open reading frames were PCR amplified to contain N-Terminal FLAG tags and C-

Terminal HA, V5, and MYC tags respectively. PCR products were cloned into the AgeI

and MluI sites of the Doxycycline inducible pTRIPZ vector (Open Biosystems). All

constructs were sequenced and purified using Endotoxin Free Maxi or Giga kits

(Qiagen).

Size-Exclusion Chromatography

Nuclear extracts were prepared from 8-10 week old CD-1 males as reported

(Conaway et al. 1996) and fractionated on a 24ml Superdex-200 column (GE Healthcare)

using an AKTAexplorer. Fractions were analyzed by western blot and calibrated using

the Gel Filtration Calibration Kit according the manufacturer’s instructions (GE

Healthcare).

Lentiviral RNAi

Recombinant lentivirus was generated by transient cotransfection of 293T cells

with pLKO.1 or pTRIPZ (Openbiosystems) and pSPAX2/pMD2.G (Addgene) using

Lipofectamine 2000 (Invitrogen). Viral supernatants were used to infect proliferating

HPPL cells in the presence of 8ug/ml hexadimethrine bromide and 24 hours post

infection cells were selected with 1.5ug/ml Puromycin (Sigma). RNA from


lentivirus infected cells was isolated using RNeasy kits (Qiagen) and used for RT-

qPCR.

Chromatin Immunoprecipitation

Fetal and adult liver chromatin was prepared essentially as reported (Kyrmizi et

al. 2006). Individually dissected adult and E13.5 livers were minced with razor blades in

PBS and homogenized in 1% formaldyde by 10-15 strokes of an A pestle. Crosslinking

was continued for 10 minutes at room temperature and quenched by addition of glycine

to 125mM. Cells were washed twice in PBS and twice for 20 minutes in cell lysis buffer

(25mM Hepes pH7.9, 10mM KCl, 0.250M Sucrose, 2mM EDTA, 1.5mM MgCl2, 0.1%

NP-40, 1mM DTT, 0.5mM Spermidine, Protease Inhibitor Cocktail (Roche) followed by

passage through a 100μm filter. Nuclei were collected by centrifugation resuspended in

two pellet volumes of nuclear lysis buffer (50mM Hepes pH7.9, 140mM NaCl, 1mM

EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, Protease Inhibitor

Cocktail (Roche)), and chromatin was sonicated for 10 minutes (30 second on cycles) on

high with a Bioruptor (Diagenode). Chromatin was clearly by centrifugation at 12,000g

for 10 minutes and equal A260 units were precipitated overnight with protein A purified

rabbit anti-TRF3, TAF7l, and TAF13 antibodies or Rabbit IgG (Jackson

Immunoresearch). Protein A and Protein G beads were blocked overnight with 5mg/ml

BSA and 5mg/ml herring sperm DNA (Invitrogen), and added to the chromatin

immuncomplexes for 1 hour. Beads were washed three times with IP buffer, once with

IP buffer plus 500mM NaCl, twice with LiCl wash buffer (20mM Tris-HCl pH7.9, 1mM

EDTA, 250mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate), and once with TE.


Complexes were eluted by incubation for 30 minutes at 37˚C in elution buffer plus

10ug/ml RNase A (Qiagen) followed by two hours at 65˚C in the presence of 10ug/ml

Proteinase K (Invitrogen). Crosslinking was reversed overnight at 65 and DNA purified

using Qiagen spin columns before qPCR with promoter-specific primers. Chromatin

from pTRIPZ infected cell lines was prepared by addition of formaldehyde to the cell

culture media to a final concentration of 1% for 10 minutes at room temperature followed

the addition of glycine to 125mM. Chromatin was prepared as above and precipitated

with rabbit IgG or rabbit anti-FLAG polyclonal antibody (Sigma).

H9 hESC Differentiation

Human H9 hESC cells were maintained on irradiated mouse embryonic

fibroblasts (MEFs) following WICell protocols and differentiated as reported (Hay et al.

2007; Hay et al. 2008a). Cells were passaged to Matrigel (BD Bioscience) coated plates

in 10% MEF conditioned media and at 70% confluence induced to differentiate by the

addition of priming media containing RPMI1640 (Invitrogen), 1xB27 (Invitrogen),

50ng/ml Wnt3a (R&D Systems), and 100ng/ml Activin A (Peprotech). After five days

cells were split 1:2 onto fresh Matrigel and cultured in differentiation media containing

knockout-DMEM (Invitrogen), 20% Serum Replacement (Invitrogen), 1mM glutamine,

1% nonessential amino acids (Invitrogen), 0.1mM -mercaptoethanol, and 1% DMSO.

Finally, cells were matured in L15 medium (Invitrogen) supplemented with 8.3% FBS,

8.3% tryptose phosphate broth (Sigma), 10μM hydrocortisone 21-hemisuccinate (Sigma),

1μM insulin, 2mM glutamine, 10ng/ml hepatocyte growth factor (Peprotech), and


20ng/ml Oncostatin M. After seven days RNA was harvested for reverse transcription

and qPCR with validated gene-specific primers.


Figure 1: Expression of TRF3, TAF7l, and TAF13 in purified liver cells.

Recombinant TRF3, whole ovary lysate, adult liver lysate, hepatoblasts, and

hepatocytes were subjected to western blotting with mouse monoclonal antibody 58C9

and rabbit anti-TRF3 antibodies (A). Recombinant TAF7l, testes lysate, fetal and adult

liver lysate, hepatoblasts and hepatocytes were subjected to western blotting with two

different polyclonal anti-TAF7l antibodies (B). Hepatoblast and hepatocyte lysates were

subjected to western blotting with mouse anti-TAF13 antibody (C).


rTR

F3

Ova

ry

Adu

lt L

iver

Hep

tobl

ast

Hep

atoc

yte

Mouse Anti-TRF3(58C9)

Rabbit Anti-TRF3(22598)

A

rTA

F7l

Tes

tes

Fet

al L

iver

(E

13.5

)

Adu

lt L

iver

Hep

tobl

ast

Hep

atoc

yte

Rabbit Anti-TAF7l(HYRB1)

Rabbit Anti-TAF7l(23266)

B

Hep

tobl

ast

Hep

atoc

yte

Mouse Anti-TAF13(31)

C


Figure 2: shRNA Mediated Depletion of TRF3, TAF7l, and TAF13 in HPPLs.

The pLKO.1 vector, which contains lentiviral 5’ and 3’ leader and other

regulatory sequences, expresses short hairpin RNAs from the human U6 promoter and

puromycin N-acetyl-transferase from the PKG promoter (A). Proliferating HPPLs were

infected for 24 hours with pLKO.1 viral supernatants expressing the indicated shRNA,

selected for four days with 1.5μg/ml Puromycin and knockdown assessed by qPCR (B).


5’ LTR 3’ LTRRRE cPPT PGK-PuroU6 shRNA

A

B

0

20

40

60

80

100

120

TRF3 TAF7l TAF13

shGFP

shTRF3

shTAF7l

shTAF13

Per

cent

of

shG

FP

Con

trol


Figure 3: Expression of Adult Hepatic Genes in Differentiated and pLKO.1

Tranduced HPPLs.

Following four days of selection, five days post infection, HPPLs were

differentiated for five days in the presence of 20ng/ml Oncostatin M and the induction of

hepatic genes was analyzed by qPCR.


020406080100

120

Cyp

2F2

Cyp

7B1

Fas

nIG

F1

Mup

3Sl

co1a

1F

oxA

2H

NF

1aH

NF

4aG

6PC

TA

TT

DO

2

shG

FP

shT

RF

3

shT

AF

7l

shT

AF

13

Percent of shGFP Control


Figure 4: Chromatin Immunoprecipitation in Fetal and Adult Liver

E13.5 and adult livers were individually dissected, crosslinked, and chromatin

was immunoprecipitated with IgG or TRF3, TAF7l, and TAF13 antibodies. Crosslinking

was reversed and precipitated DNA was analyzed by qPCR. Signals were normalized to

input and expressed as fold enrichment over the IgG control.


0

1

2

3

4

5

6

7

8

E13.5-IgG

Adult-IgG

E13.5-TRF3

Adult-TRF3

AF

old

Enr

ichm

ent

02468

101214161820

E13.5-IgG

Adult-IgG

E13.5-TAF7l

Adult-TAF7l

B

Fol

d E

nric

hmen

t

0

10

20

30

40

50

60

E13.5-IgG

Adult-IgG

E13.5-TAF7l

Adult-TAF7l

C

Fol

d E

nric

hmen

t


Figure 5: Chromatin Immunoprecipitation in Differentiated HPPLs.

Proliferating HPPLs were infected for 24 hours with pTRIPZ based lentivirus

expressing FLAG tagged TRF3, TAF7l or TAF13 and differentiated for five days in the

presence of 20 ng/ml Oncostatin M. Cells were crosslinked and chromatin was subjected

to immunoprecipitation with IgG or anti-FLAG polyclonal antibody. Crosslinking was

reversed and precipitated DNA analyzed by qPCR. Signals were normalized to input and

expressed as fold enrichment of the IgG control.


0510152025

IgG

FL

AG

-TR

F3

FL

AG

-TA

F7l

FL

AG

-TA

F13

Fold Enrichment


Figure 6: Size Exclusion Chromatography of Adult Liver Nuclear Extract.

Adult mouse liver nuclear extract was fractionated on a Superdex-200 column and

fractions analyzed by western blot with TRF3, TAF7l, and TAF13 antibodies. Relative

molecular weights were calibrated with the standards in HMW and LMW Gel Filtration

Calibration Kit.


0 1 2 3 4 5 6 7 8 9 10 11 12

Inpu

t (N

E)

Voi

d44

0 kD

158

kD

43 k

D

13.7

kD

TRF3

TAF7l

TAF13


Figure 7: Hepatic Differentiation of Human H9 ES Cells.

Human H9 ES cells were differentiated to mature hepatocyte-like cells by a three

stage protocol that mimics in vivo signaling events (A). Analysis of known marker genes

for ES cells, definitive endoderm, hepatoblasts, and mature hepatocytes by qPCR shows

that this protocol faithfully mimics human hepatogenesis (B).


110100

1000

1000

0

1000

00

1000

000

1000

0000

Oct

4

Sox1

7C

XC

R4

Met

Krt

19A

qp1

Pro

x1A

fpA

lbG

6PC

HN

F3b

AA

TT

DO

2C

PS1

hESC

(H9)

Stag

e 1

Stag

e 2

Stag

e 3

Percent of hESC(H9) (Log Scale)

hESC

(H9)

Stag

e 1

Stag

e 2

Stag

e 3

A B


Figure 8: Expression of TBP, TRFs, and TAFs in H9 Differentiation

Expression of TBP, TAF1, TAF3, TAF5, and MED17 as judged by qPCR,

remains stable during definitive endoderm and hepatoblast commitment, but decreases

significantly during hepatocyte maturation in the H9 system.


0

50

100

150

200

250

TBP TAF1 TAF3 TAF5 MED17

hESC(H9)

Stage 1

Stage 2

Stage 3Per

cent

of

hESC

(H9)


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