Transcriptional Gene Regulation in Eukaryotes

Sreeremya.s

Overview

Gene expression Transcription Regulation of eukaryotic transcription Influence of chromatin structure Oncogenes Techniques

Orphanides, Cell 2002

Control of gene expression at any stage:

Activation of gene structure

Initiation of transcription

What is a gene?

“The entire nucleic acid sequence that is necessary for the synthesis of a functional polypeptide or RNA molecule”

Overview

Gene expression Transcription Regulation of eukaryotic transcription Influence of chromatin structure Oncogenes Techniques

Transcription

Initiation, elongation, terminationCatalyzed by RNA polymerase

“Transcription bubble”: DNA transiently separated into single strands

One strand is used as a templateUnwinding point & rewinding pointRate 40 nucleotides/second at 37 for bacteria

RNA polymeraseMany subunits: catalytic site, CTD with

(YSPTSPS)n

pol I, pol II, pol III

Bacteria

Molecular details of gene expression control in bacteria: lac operon E. coli (Jacob & Monod 1960s)

Glutamine synthase

Eukaryotes

Basal transcription apparatus (general factors & RNA polymerase)

Proximal cis-regulatory moduleDistal cis-regulatory modulesModules = discrete DNA elements that

contain specific sequence motifs with which DNA binding proteins interact and transmit molecular signals to genes

Promoter

Enhancer

BTA

General factors: TFIIxMechanics of initiating RNA

synthesis at all promotersDetermines location of

transcription startpointComplex with RNA

polymeraseTATA

~ 25bp upstream 8bp consensus of A•T pairs Tends to be surrounded by

G•C rich regions TBP, 11 TAFs : TFIID (~800kD)

TATA-less promoters: Inr Py2CAPy5 (-3 to +5)

Promoter-prediction: TATA-box, C-G enrichment

50%

TATA Inr

Inr

TATA

6 4

132

23

3

12 19

Eponine

CpG islands

Promoter Inspector

Promoter-proximal region

Efficiency and specificity of transcription depend on binding of transcription factors

Promoter recognition

Function = to be recognized by proteins; so differs from exon, …

Any essential nucleotide sequence should be conservedSome variation is permittedWhen is it sufficiently conserved?Idealized sequence with base most often present:

consensus sequence by aligning all known examples

Only conservation of very short sequences; 60 bp associated with RNA pol lack conservation

Variety of elements can contribute, none is essential for all promoters (mix & match principle)

CAAT box ~ -80bp GGCCAATCTincreases promoter strengthBound by CTF/NF1 family, CP1 & CP2, C/EBP, ACF

GC box GGGCGGSP1

Octamer (8bp) ATTTGCATBound by Oct1 (ubiquitous): activates histon H2BBound by Oct2 (lymphoid cells): Ig kappa light

chaincontext is important

Modular nature of the promoter:Equivalent regions can be exchangedMain purpose = to bring the factors they bind

into the vicinity of the initiation complexProtein-protein interactions determine the

efficiency of the initiation reactionSequence elements influence the

frequency of initiationRepression of transcription:

Generally by influencing chromatin structureBy repressors, e.g. Dr1/DRAP1 binds to TBP

and CAAT displacement protein (CDP)

Modules

Enhancers, silencers5’ region, distal

Modules50 bp to 1.5 kbp in size4-8 TFs (often multiple sites); higher density of

regulatory elements than in the promoterMany elements are common elements in

promoters, e.g. AP1 and the octamerCan stimulate any promoter placed in its

vicinityCan function anywhere (cfr -globin: 200 fold

in vivo) ; Position relative to promoter can vary substantially; can function in either orientation

Binding sites for activators that control transcription of the mouse transthyretin (TTR) promoter in hepatocytes. HNF = hepatocyte nuclear factor. [See R. Costa et al., 1989, Mol. Cell Biol. 9:1415; K. Xanthopoulus et al., 1989,Proc. Nat’l. Acad. Sci. USA 86:4117.]

Example: muscle specific modules

Creatine kinase, myosin light chain, skeletal actin, myosin heavy chain

Model for the control of the human -globin gene. Some of the gene regulatory proteins shown, such as CP1, are found in many types of cells, while others, such as GATA-1, are present in only a few types of cells including red blood cells and therefore are thought to contribute to the cell-type specificity of -globin gene expression. (Adapted from B. Emerson, In Gene Expression: General and Cell-Type-Specific [M. Karin, ed.], pp. 116-161. Boston: Birkhauser, 1993.)

Current view: same sort of interaction with basal

apparatus as the proximal promoter moduleIncrease the concentration of transcription

factors in the vicinity of the promoter

Intervening DNA: extruded as a large “loop”

Generality: not yet clear (what proportion of promoters require an enhancer?)

Four activators enriched in hepatocytes plus the ubiquitous AP1 factor bind to sites in the hepatocytespecific enhancer and promoter-proximal region of the TTR gene.

The activation domains of the bound activators interact extensively with co-activators, TAF subunits of TFIID, Srb/Mediator proteins, and general transcription factors, resulting in looping of the DNA and formation of a stable activated initiation complex.

Cooperative assembly

Limited knowledge

Experimentally verified binding sitesExperimentally verified “composite elements”

or CE’sGR site + AP-1 in proliferin promoterSynergistic: result in non-additively high levelAntagonistic: overlapping sites, masking an

activation domain,…Direct or through coactivator

Few modules characterized that have multiple elements, some in developmental biology

Side-track: Transcription factors

5% of our proteinsActivities controlled in regulatory

pathwaysIndependent domains responsible for

activities:Recognition of specific target sequencesBinding to other components

of the transcription apparatusE.g. yeast GAL4

Protein-DNA interactionsProteins with high affinity for a specific

sequence also possess a low affinity for any (random) DNA sequence

E.g. Lac repressor E. coli: Free:bound = 10-4

High-affinity site competes with the large number of low-affinity sites; repressor binds 107 times better to operator DNA (bound 96% of time for 10 molecules/cell)

How the different base pairs in DNA can be recognized from their edges without the need to open the double helix.

The binding of a gene regulatory protein to the major groove of DNA.

Typically, a protein-DNA interface consists of 10 to 20 such contacts, involving different amino acids, each contributing to the binding energy of the protein-DNA interaction.

Zinc finger motifCommon motif in DNA binding, e.g. SP1 has 3

(A) The structure of a fragment of a mouse gene regulatory protein bound to a specific DNA site. This protein recognizes DNA using three zinc fingers of the Cys-Cys-His-His type arranged as direct repeats. (B) The three fingers have similar amino acid sequences and contact the DNA in similar ways. In both (A) and (B) the zinc atom in each finger is represented by a small sphere. (Adapted from N. Pavletich and C. Pabo, Science252:810-817, 1991. © 1991 the AAAS.)

All of the proteins bind DNA as dimers in which the two copies of the recognition helix (red cylinder) are separated by exactly one turn of the DNA helix (3.4 nm). The second helix of the helix-turn-helix motif is colored blue. The lambda repressor and cro proteins control bacteriophage lambda gene expression, and the tryptophan repressor and the catabolite activator protein (CAP) control the expression of sets of E. coli genes.

Helix-Turn-Helix

HomeodomainsRelated to helix-turn-helix bacterial repressorsHomeobox = 60 AA residuesE.g. en, eve, Hox, Oct-1, Oct-2 (Oct also have

Pou domain next to homeodomain)

The homeodomain is folded into three alfa helices, which are packed tightly together by hydrophobic interactions (A). The part containing helix 2 and 3 closely resembles the helix-turn-helix motif, with the recognition helix (red) making important contacts with the major groove (B). The Asn of helix 3, for example, contacts an adenine. Nucleotide pairs are also contacted in the minor groove by a flexible arm attached to helix 1. The homeodomain shown here is from a yeast gene regulatory protein, but it is nearly identical to two homeodomains from Drosophila, which interact with DNA in a similar fashion. (Adapted from C. Wolberger et al., Cell 67:517-528, 1991. © Cell Press.)

Helix-loop-helix (HLH)DNA binding (helix) & dimerizationClass A: ubiquitouslyh expressed proteins,

e.g. E12/E47Class B: tissue-specific expression, e.g.

MyoD, myogenin, Myf-5Myc proteins (separate class)

Leucine zippers fig 21.15Dimerization motifE.g. Jun+Fos = AP1Gcn4 ->

Steroid receptors Independent domains: DNA binding,

hormone binding, and dimerization

Cortisol - glucocorticoid receptor (GR).

Retinoic acid - retinoic acid A receptor (RAR).

Thyroxine - thyroid hormone receptor (TR).

Figure 1 Genome-wide comparison of transcriptional activator families in eukaryotes. The relative sizes of transcriptional activator families among Homo sapiens, D. melanogaster, C. elegans and S. cerevisiae are indicated, derived from an analysis of eukaryotic proteomes using the INTERPRO database, which incorporates Pfam, PRINTS and Prosite. The transcription factors families shown are the largest of their category out of the 1,502 human protein families listed by the IPI.

Transcription factories

cfr. replication factories Active RNA polymerases are concentrated in discrete

'factories' where they work together on many different templates

Complexes for transcription and RNA processing are likely to be immobile structures within the gel-like nucleoplasm (Burns et al, 2001; Kimura et al, 1999)

Transcriptional interference: phenomenon where transcription of one gene prevents transcription of an adjacent gene. Discovery: Cells were transfected with a retroviral vector encoding resistance to neomycin and azaguanine, and clones harboring a single copy of the vector selected. Expression of the 3' gene was suppressed when selection required expression of the 5' gene, and vice versa. In addition, hardly any cells grew in both neomycin and azaguanine

Cook, 1999 (Science)• Enhancers •dynamic equilibrium•enhancing the probability of the key transcription cycle interactions•Element 5’ or 3’ doesn’t matter!

Recap: evolution of understanding of eukaryotic transcription

Lemon and Tjian, Genes Dev. 14: 2551-2569 (2000)

Termination

Bacteria

Eukaryotes (?); by RNA processing

Overview Gene expression Eukaryotic transcription

Regulation of eukaryotic transcription

Influence of chromatin structure Oncogenes Techniques

Activate/inactivate a TF

Transport through nuclear pores from cytoplasm to nucleus (e.g. masking NLS, nuclear localization signal, can regulate this transport)

Link to Ubiquitin protease systemRapid turnover of promoter bound TF: resets signaling

pathway: cell can continuously monitor its environment

Tissue-specific synthesisDevelopment, e.g. homeodomain proteins

ModificationPhosphorylation, acetylation, methylationE.g., AP1 (= Jun+Fos) active form by

phosphorylationE.g., p53 acetylated (modulates interactions with

coactivator and repressor proteins

Ligand bindingE.g. Steroid receptorsInfluence: localization or DNA-binding ability

CleavageInhibitor release

E.g. NF-B + I- B (release in B lymphocytes)

Change of partner (active partner displaces inactive partner)

Examples: GATA-1 CAP NtrC Adenovirus E1A NF-KB/

+ CBP/p300 glucocorticoid receptor

Pathways…

1

2

Level 1 = active/inactive factorLevel 2 = cooperation of multiple factors

within a module (all present and active, and all repressors inactive or absent)

Level 3 = multiple autonomous modules per geneEach module can independently activate the geneEach has a specific function (e.g. activation in

certain cell type or at particular stage in dvl)different circuits of regulation, e.g.

metallothionein gene (MT): heavy metals and steroids, fig 21.1

Gene can respond to multiple signaling pathways Facilitates fine-tuning of transcript levels

Combinatorial and context dependent regulation of transcription one factor can induce transcription of one gene

while repressing that of another

Experiment demonstrating the modular construction of the eve gene regulatory region. (A) A 480-nucleotide-pair piece of the eve regulatory region was removed and inserted upstream of a test promoter that directs the synthesis of the enzyme -galactosidase (the product of the E. coli lacZ gene). (B) When this artificial construct was reintroduced into the genome of Drosophila embryos, the embryos expressed -galactosidase (detectable by histo-chemical staining) precisely in the position of the second of the seven eve stripes (C). (Metamerization)

-

+

rho

• Dorsal (Dl)• Twist (HLH)• a HLH• Snail (-)

Principles for specification

1. cis-regulatory transformation of input patterns into spatial domains of differential gene expression

2. Always assemblages of diverse target sites because multiple inputs are required

3. Output=novel with respect to any one of the incident inputs + more precise in space and time => “information processing”

4. Every specific type of interaction that can be detected in vitro is fundamentally significant (it is unlikely that highly specific site clusters, which are of improbable random occurrence would have no function)

5. Negative & positive inputs

(Davidson, 2001)

Cis-regulatory logic deviceendo16 of Strongylocentrotus (zee-egel)Secreted embryonic gut protein“hardwired biological computational

device”

Overview Gene expression Initiation of transcription Regulation of transcription

Influence of chromatin structure

Oncogenes Techniques

ChromatinEukaryotic genomes

are packaged with chromatin proteins

Heterochromatin (highly condensed, untranscribed)

Euchromatin (more accessible, transcribed)

Each cell: unique pattern of heterochromatin and euchromatin

Nucleosomes

Workman and KingstonAnn. Rev. Biochem. 67: 545 (1998)

•146 bp• H2A, H2B, H3, H4

Chicken and egg scenario

TF binding requires chromatin decompaction by certain factors but the latter also need to interact with DNA

Solution: probably some TFs can bind to their recognition sequences even when they are packaged (e.g. glucocorticoid receptor: only contacts DNA on one side NF1 surrounds double helix)

Narlikar, 2002 (Cell)

1. ATP-dependent remodeling

Modify chromatin structure

2. Histone-modifying complexes Phosphorylation, methylation, acetylation Histone acetyltransferase (HAT), histone

deacetylase (HDAC) How do they impact the structure of the

template and the ability of the transcription machinery to function?

lowered positive charge on acetylated N termini, lowered stability of interaction with DNA

Disrupting internucleosomal interactions Recruiting additional TFs A lot of combinatorial possibilities: histon code?

Jenuwein T, Allis CD. Science 293:1074-80 (2001).

Model of the protein interactions and functions of the Myc/Max/Mad transcription network.

Myc-Max and Mad-Max (along with Mnt-Max and Mga-Max) complexes bind to DNA to E-boxes. Binding can be affected by the context, sequence, cooperativity, and location of the E-boxes. Myc-Max heterodimers activate transcription by recruiting HAT's via TRRAP. This leads to the acetylation of histone tails and the opening of local chromatin structure. Additionally, Myc-Max appears to repress transcription through Inr elements via an undefined mechanism. As a result of these activities at target genes, Myc affects proliferation, cell cycle, growth, immortalization, and apoptosis. When deregulated, Myc cooperates with other oncogenes to cause a variety of cancers. Mad-Max and Mnt-Max heterodimers repress transcription by recruiting HDAC's via mSin3A. This leads to the deacetylation of histone tails and the closing of local chromatin structure. As a result of target gene repression, Mad causes an increased cell doubling time, growth arrest, and the maintenance of differentiation.

Grandori C, Cowley SM, James LP, Eisenman RN.Annu Rev Cell Dev Biol. 2000;16:653-99

Cytosine methylation

mCG often in inactive vertebrate genesAfter replication of methylated DNA, methyl

groups are added to daughter strands

CpG islandsimprinting

Imprinting

Imprinted genes are genes whose expression is determined by the parent that contributed them.

Imprinted genes violate the usual rule of inheritance that both alleles in a heterozygote are equally expressed.

Examples of the usual rule: If a child inherits the gene for blood group A from either parent and the

gene for group B from the other parent, the child's blood group will be AB.

If a child inherits the gene encoding hemoglobin A from either parent and the gene encoding hemoglobin S from the other parent, the child's red blood cells will contain roughly equal amounts of the two types of hemoglobin.

But there are a few exceptions to this rule. A small number of genes in mammals (~50 of them at the most recent count) have been found to be imprinted. Because most imprinted genes are repressed, either the maternal (inherited from the mother) allele is expressed exclusively

because the paternal (inherited from the father) allele is imprinted or vice-versa.

Link between DNA methylation and histone deacetylation

Chromatin compartmentalization

Heterochromatin: gene poor, inaccessible, perifery, transcr. inactive

Euchromatin: gene rich, transc. active

Consistent correlation between gene silencing (e.g. in B en T lymphocytes) and presence in heterochromatin regionsLCR, enhancers, insulators: act by maintaining

endogenous loci in a chromatin compartment that is either transcr. permissive or nonpermissive?

Position variegation

Position effects can be observed for the Drosophila white gene. Wild-type flies with a normal white gene have red eyes. If the white gene is inactivated by mutation, the eyes become white (hence the name of the gene). In flies with a chromosomal inversion that moves the white gene near a heterochromatic region, the eyes are mottled, with red and white patches. The white patches represent cells where the white gene is silenced and red patches represent cells that express the white gene. (After L.L. Sandell and V.A. Zakian, Trends Cell Biol. 2:10-14, 1992.)

Overview Gene expression Initiation of transcription Regulation of transcription Alteration of chromatin structure during

transcription

Oncogenes Techniques

The development and metastasis of human colorectal cancer and its genetic basis.

A mutation in the APC tumor-suppressor gene in a single epithelial cell causes the cell to divide, although surrounding cells do not, forming a mass of localized benign tumor cells called a polyp. Subsequent mutations leading to expression of a constitutively active Ras protein and loss of two tumor-suppressor genes, DCC and p53, generates a malignant cell carrying all four mutations; this cell continues to divide and the progeny invade the basal lamina that surrounds the tissue. Some tumor cells spread into blood vessels that will distribute them to other sites in the body. Additional mutations cause exit of the tumor cells from the blood vessels and growth at distant sites; a patient with such a tumor is said to have cancer. [Adapted from B. Vogelstein and K. Kinzler, 1993, Trends Genet. 9:101.]

Overview Gene expression Initiation of transcription Regulation of transcription Alteration of chromatin structure during

transcription Oncogenes

Techniques

Techniques

Mutational analysisEMSA electorphoretic mobility shift assay

Microarrays: level of mRNAChIP: Chromatin ImmunoPrecipitationYeast2HybridSequence analysis, phylogenetic footprinting

Free DNA probe

*

*Protein-DNA complex

Sources

B Lewin, Genes VIILodish et al. Molecular Cell BiologyEH Davidson: Genomic Regulatory SystemsAlberts et al. Essential Cell Biology EM Blackwood & JT Kadonaga: Going the

distance: a current view of enhancer actionCell, February 22, 2002: 108 (4) "Reviews on

Gene Expression"