Differential Gene Expression

IBS 8102 – Cell, Molecular, and Developmental Biology

January 22, 2008

Differential Gene Expression

Chromatin structure

Gene anatomy

Gene sequences

Control of gene transcription

enhancers

transcription factors

RNA processing

methylation

imprinting

Epigenetic processes (modification of structure of DNA rather than sequence)

epigenetics

Chromatin Structure

~140 bp

~60 bp

chromatin

Histone lysines are protonated = net positive charge

DNA PO 4 deprotonated = net negative charge

electrostatic attraction ensures tight binding ­ DNA inaccessible to polymerases, etc

Nucleosome

DNA bound to an octameric complex composed of two each of histones H2A, H2B, H3, and H4. From Luger et al, 1997. Nature 389:251­260.

Tails

Core

Histone H1 – associates nucleosomes into higher­order folded complex

Chromatin Acetylation

3. Histone methylation (CH 3 ) – transcriptional repression

Transcriptional Regulation:

1. “Packing” prevents access

­ acetylated histone (histone acetyltransferase; HAT) destabilizes = transcription allowed

­ deacetylated histones (histone deacetylases; HDAC) stabilizes = transcription repressed

2. Acetylation ( ) state of histones controls DNA binding CH 3 C O

­ e.g. acetylation of K14 and K9 lysines on tail of histone H3 correlated with transcriptional competence

­ methylation of K9 lysine on H3 associated with heterochromatin

Multiple histone regulatory modifications may occur at once; ­ work together to change the behavior of the chromatin ­ dynamic ­ systematic ­ reproducible

= Histone Code

­remains tightly condensed throughout most of the cell cycle

­ replicates later

John H. Frenster

Chromatin Configuration

Heterochromatin Euchromatin ­ “active” chromatin

Anatomy of the Gene

5’ 3’

core promoter region

transcription initiation site

+1 TATAT

upstream downstream

transcribed region

Transcription produces heterogenous nuclear RNA (hnRNA)

promoter exon 1 intron 1 exon 2 intron 2 termination region

gene

hnRNA Processing

splicing

exon 1 exon 2 7­methyl­Gppp AAAAAAA(n)

= mature mRNA, ready to be transported to the cytoplasm and translation

7­methyl­Gppp

cap 3’ cleavage, polyadenylation

AAAAAAA(n) 7­methyl­Gppp

cleavage signal

poly(A)polymerase

cleavage factor

AAUAA primary transcript

transcription, 5’ capping

Human β­globin Sequence

Production of β­globin

Promoters and Enhancers RNA polymerase II binds to the promoter region at the TATA box

­ various proteins bind to regulatory sequences upstream and downstream of transcription initiation site

­ promoter region binds Basal Transcription Factors

­ basal transcription factors facilitate Pol II binding and activity

promoter region

5’ 3’

­ however, Pol II cannot initiate transcription alone

Eukaryotic Transcription Initiation Complex

Basal (General) Transcription Factors: ­ small nuclear proteins ­ bind to promoter region ­ constitutive, ubiquitous ­ bind sequentially ­ binding of individual TFs mediated by small proteins ­ TBP­associated factors (TAFs) ­ mediator complex (~25 proteins)

(AKA Preinitiation Complex)

Eukaryotic Transcription Initiation Complex 1. TFIID complex binds to the TATA box through its TATA Binding Protein (TBP) subunit

2. TFIID is stabilized by TFIIA

3. TFIIB and TFIIH join the complex on the TATA box; TFIIE and TFIIF associate with RNA polymerase II

Eukaryotic Transcription Initiation Complex

5. The CTD is phosphorylated by TFIIH and is released by TFIID; RNA polymerase II can now transcribe mRNA

4. RNA polymerase II is positioned by TFIIB, and its carboxy­terminal domain (CTD) is bound by TFIID

Stabilization of Transcriptional Initiation Complex by TAFs

TBP – TATA binding protein

TAF(s) – TBP­associated factors

SP1, NTF­1 = Transcription factors

TAF Histone Acetyltransferase Activity

TBP – TATA binding protein

TAF(s) – TBP­associated factors

Enhancers

Function ­ bind specific regulatory proteins (i.e. specific Transcription Factors) ­ TFs generally interact with mediator complex proteins – recruit RNA polII

Enhancers: DNA sequences that regulate gene expression by affecting the transcription initiation complex on the promoter.

Location ­ highly variable with respect to the transcribed portion of the gene. ­ upstream (5’), downstream (3’), or within transcribed region ­ close proximity or as many as 10 6 bp away

Enhancers are cis­acting regulatory elements

cis – (same or same side); elements that reside on the same DNA strand; e.g. DNA sequences

trans – (other side); elements that originate from another DNA strand, e.g. regulatory proteins

Enhancers

Enhancers differ from promoters:

3) can work in reverse orientation

2) can work at a distance

1) need a promoter to work

Enhancer Generalizations 1. Most gene transcription requires enhancers.

2. Enhancers are the major determinants of differential transcription in cell types and through developmental stages.

3. There can be multiple signals (e.g. multiple enhancer sites) for a given gene, and each enhancer can be bound by more than one transcription factor (not at the same time).

4. Transcription is regulated by the interaction of transcription factors bound to enhancers and the transcription initiation complex assembled at the promoter.

5. Enhancers are combinatorial. Various DNA sequences regulate temporal and spatial gene expression; these can be mixed and matched.

6. Enhancers are modular. A gene can have several enhancer elements, each of which may turn it on in different sets of cells.

7. Enhancers generally activate transcription by remodeling chromatin to expose the promoter, or by facilitating the binding of RNA polymerase to the promoter by stabilizing TAFs.

8. Enhancers can also inhibit transcription (aka Silencers).

Transcription Factors

Proteins that bind to enhancer or promoter regions ­ activate or repress transcription

Most bind to specific DNA sequences (e.g. enhancers)

Transcription factors are grouped together in families, based on structural similarities ­ families share common framework in DNA binding sites ­ slight differences in binding sites cause differences in recognition

Arabidopsis ­ currently 41 TF families listed eukaryotes > 75

estrogen receptor zinc finger

TF/DNA Interactions

helix­loop­helix

leucine zipper

homeodomain

TF/DNA Interactions

T­box Transcription Factor

NFkB/Rel Transcription Factor

Transcription Factor Domains

Three major domains:

1. DNA­binding ­ recognizes particular DNA sequence

transcription factor ­ engrailed

Transcription Factor Domains

Three major domains:

1. DNA­binding ­ recognizes particular DNA sequence

2. Trans­activation – activates or represses transcription ­ often involved with transcription coregulator proteins involved in binding RNA polymerase II; e.g. TFIIB, TFIIE ­ often involved with enzymes that modify histones

Transcription Factor Domains

3. protein­protein interaction domain ­ promotes dimerization ­ allows it to be modulated by TAFs or other transcription factors

TBP – TATA binding protein

TAF(s) – TBP­associated factors

Three major domains:

1. DNA­binding ­ recognizes particular DNA sequence

2. Trans­activation – activates or represses transcription ­ often involved with transcription coregulator proteins involved in binding RNA polymerase II; e.g. TFIIB, TFIIE ­ often involved with enzymes that modify histones

NOTE – alternate categories combine 2 and 3; also add “signal sensing domain” for some TFs

Transcription Factor Domains Transcription factor MITF (microphthalmia) ­ active in ear and pigment­forming cells of eye and skin; osteoclasts ­ pigment­cell­specific tyrosinases expressed ­ MITF mutation = deafness, multicolored irises, white forelock

Structural Family ­ basic helix­loop­helix leucine zipper

Functional protein is a homodimer ­ dimerization common among TFs

The transactivating domain is located near the amino terminal ­ when bound to a promoter or enhancer, the protein is able to bind TAF p300/CBP ­ TAF p300/CBP is a histone acetyltransferase

Functional Classification of TFs I. Constitutively active – e.g. SP1, NF1 CCAAT

II.B.3.a. Resident nuclear factors – reside in the nucleus regardless of activation state; e.g. CRDEB, AP­1, Mef2

II.B.3.b. Latent cytoplasmic factors – inactive form reside in the cytoplasm, but when activated are translocated into the nucleus; e.g. STAT, R0SMAD, NF­kB, Notch, TUBBY, NFAT

II.B3. Cell membrane receptor­dependent – second messenger signaling cascades resulting in the phosphorylation of the transcription factor

II. Conditionally active – require activation

II.A. Developmental (cell specific) ­ expression tightly controlled, but once expressed requires no additional activation; e.g. GATA, HNF, PIT­1, MyoD, MNyf5, Hox, Winged Helix

II.B1. Extracellular ligand­dependent – nuclear receptors

II.B2. Intracellular ligand­dependent ­ activated by small intracellular molecules; e.g. SREBP, p53, orphan nuclear receptors

II.B. Signal dependent ­ requires external signal for activation

A. H. Brivanlou et al., Science 295, 813 ­818 (2002)

A. H. Brivanlou et al., Science 295, 813 ­818 (2002)

Functional Classification of TFs

Enhancer Modules Enhancers are modular: e.g. Pax6 gene expressed in the eye, pancreas,

nervous system. ­ also expressed in different genes within these tissues

Within these modules, transcription factors work in a combinatorial fashion.

Transcription factors operate in cascades: one stimulates the production of several others.

Combinatorial Enhancers

combinatorial transcription factors

NOTE – enhancers can act as

repressors/silencers

Kaltoff, 2001

Transcription Factor Competition

Kaltoff, 2001

Epigenetic Mechanisms

Epigenetics

epi – above, etc.

control of gene expression not involving sequence

DNA methylation

imprinting

siRNA

alternative RNA splicing

histone methylation

translational control

“Development is an epigenetic, not a genetic, process.”

Mechanisms

Histone Modification

Acetylation/deacetylation – acetylation destabilizes nucleosome ­ targets: lysines on histone “tails”

­ methylated histones recruit DNA methylation enzymes ­ further stabilizes nucleosome

Methylation – stabilized nucleosome (transcription repressed) ­ hypermethylation of H3 lysine 9 induces ectopic heterochromatin

­ demethylated by specific demethylating enzymes

Ubiquitination ­ vertebrates, yeast; core histones H2A, H2B, H3 ­ destabilizes nucleosome

Phosphorylation ­ histone serine phosphorylation correlated with gene activation

Others?

DNA Methylation

­ found in all vertebrates; not Drosophila, nematodes, inverts

­ degree of methylation is proportional to degree of transcription

DNA methylation stabilizes nucleosomes; prevents transcription

­ in adults tissues cytosine methylation occurs only in CpG sequences ­ 1­5% of CpG in adult somatic cells are methylated

­ in embryonic tissues and stem cells non­CpGs nucleotides (CpA, CpT) are also methylated

­ tumor suppressor genes silenced by methylation in many cancers

Embryonic DNA Methylation

­ methylation correlates inversely with expression

Methylation patterns are established and maintained throughout cell division by DNA (cytosine­5)­methyltransferases

Methylation patterns differ in stage­ and tissue­specific manner

­ humans: DNMT3a, DNMT3b act as de novo methyltransferases DNMT1 maintains pattern during DNA replication

­ methylation pattern changes during development

Genomic Imprinting Special case of DNA methylation:

­ alleles from maternal and paternal genome are differentially methylated

­ methylation patterns can be distinguished based on resulting phenotypes

Approximately 80 imprinted genes in humans; many involved with early developmental processes

No mammalian parthenogenesis because of imprinting; in mice:

­ in insects, imprinting silences paternal genome ­ produces males (functionally haploid)

Imprinting seen in mammals, insects, flowering plants

­ androgenetic (two male pronuclei) embryos showed poor embryonic development, but normal placental development

­ gynogenetic (two female pronuclei) embryos showed normal embryonic development, but poor placental development

Genomic Imprinting Imprinted alleles from maternal and paternal genomes are differentially methylated. ­ both methylation patterns are necessary for proper development

Chromosome 15 differentially methylated in males and females

Deletions in either parental chromosome 15 result in predominance of reciprocal pattern

Female deletion = Angelman syndrome ­ intellectual and developmental delay ­ speech impediment ­ replicated by mutation of Ube3a gene (ubiquitin pathway)

Male deletion = Prader Willi syndrome ­ extreme or insatiable appetite ­ small stature ­ learning disabilities ­ imprinted genes: necdin & SNRPN ­ small nuclear ribonucleoprotein polypeptide N

Angelman and Prader Willi syndromes:

Dosage Compensation

Mammals ­ Inactivation of a single X chromosome in mammalian XX cells

Barr bodies

Mammals, Drosophila, nematodes – XX = female, XY = male; diploid XXs have double the X gene products as diploid XYs

Drosophila – transcription rate of male X is doubled

C. elegans – both Xs partially repressed ( = hermaphrodite)

XX cell early embryo – both active later embryo – only one active

X Chromosome Inactivation Lyon hypothesis:

1. Both X chromosomes active in very early female development.

2. One X is inactivated in each cell

3. Inactivation is random

4. The process is irreversible. All progeny cells will retain the same inactivation pattern

calico cat: ­ heterozygous for coat color ­ genes contained on X chromosome X chromosome inactivation

early late

Differential RNA Processing Nuclear RNA selection ­ more genes transcribed than processed into mRNA ­ unprocessed genes degraded in nucleus

Alternate splicing ~75% of all human genes may be alternatively spliced (splice isoforms; splice variants)

= one gene – one protein family ­ nRNA splicing mediated through spliceosomes

Spliceosomes – constructed for each new hnRNA ­ small nuclear RNAs (snRNA) ­ splicing factor proteins

Introns contain recognition sequences ­ GU at 5’ end splice site, AG at 3’ end splice site (typical) ­ also variable length polypyrimidine­polypyrimidine tract (PPT) ­ recruit spliceosome factors to site

Cell­specific splicing factors differ in ability to recognize intron sequences ­ exon in one cells type might be recognized as an intron in another = alternative splicing

α­Tropomyosin Alternative Splicing

striated muscle

striated muscle’

myoblast

smooth muscle

neuroblast/muscle

hepatoma

brain

Translational Control

Rhoads, R. E. et al. Mechanism and regulation of translation in C. elegans (January 28, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.63.1, http://www.wormbook.org.

Differential mRNA longevity ­ stability dependent on length of poly(A) tail ­ poly(A) length dependent on 3’­UTR

Selective inhibition of message translation (e.g. stored maternal messages) ­ messages lacking 5’ cap or 3’ poly(A) tail are not translated (may be degraded)

­ mRNAs circularize ­ mediated by proteins bound to 5’ cap and poly(A) tail ­ no 5’ cap = no protein binding

­ proximity of 5’ and 3’ ends important for ribosomal recognition, initiation

poly(A) binding protein

eukaryotic initiation factors

ribosomal 40 S subunit

mRNA

5’ methylated guanosine cap ­ stored mRNAs often lack methylated cap

­ at fertilization, methyltransferase completes cap

MicroRNAs

NOTE ­ Drosha/Dicer/RISC mechanism also used to process siRNA

stem­loop 70 nt

miRNAs complementary to part of one or many mRNAs ­ animals ­ 3’ UTR; plants ­ coding region) ­ inhibits translation or promotes degradation