Transcriptional Gene Regulation in Eukaryotes

Transcriptional Gene

Regulation in Eukaryotes

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, termination

 Catalyzed by RNA polymerase

– “Transcription bubble”: DNA transiently separated into single strands

– One strand is used as a template

– Unwinding point & rewinding point

– Rate  40 nucleotides/second at 37 for bacteria

 RNA polymerase

– Many subunits: catalytic site, CTD with (YSPTSPS)

– pol I, pol II , pol III

Initiation

Bacteria



 Molecular details of

gene expression control

in bacteria: lac operon

E. coli (Jacob & Monod

1960s)

Bacterial enhancer

Glutamine synthase

Eukaryotes

 Basal transcription apparatus (general factors & RNA polymerase)

 Proximal cis-regulatory module

 Distal cis-regulatory modules

 Modules = 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: TFIIx

– Mechanics of initiating RNA synthesis at all promoters

– Determines location of transcription startpoint

– Complex with RNA polymerase – TATA

• ~ 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 Py

CAPy

(-3 to +5)

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

– 50%

TATA Inr

Inr TATA

6 4

132

3

12 19

Eponine 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 conserved

– Some variation is permitted

– When 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 GGCCAATCT

– increases promoter strength

– Bound by CTF/NF1 family, CP1 & CP2, C/EBP, ACF

 GC box GGGCGG

– SP1

 Octamer (8bp) ATTTGCAT

– Bound by Oct1 (ubiquitous): activates histon H2B

– Bound by Oct2 (lymphoid cells): Ig kappa light chain

– context is important

 Modular nature of the promoter:

– Equivalent regions can be exchanged

– Main purpose = to bring the factors they bind into the vicinity of the initiation complex

– Protein-protein interactions determine the efficiency of the initiation reaction

 Sequence elements influence the frequency of initiation

 Repression of transcription:

– Generally by influencing chromatin structure

– By repressors, e.g. Dr1/DRAP1 binds to TBP and

CAAT displacement protein (CDP)

Modules

 Enhancers, silencers

 5’ region, distal

Modules

 50 bp to 1.5 kbp in size

 4-8 TFs (often multiple sites); higher density of regulatory elements than in the promoter

 Many elements are common elements in promoters, e.g.

AP1 and the octamer

 Can stimulate any promoter placed in its vicinity

 Can 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: TTR

Example: muscle specific modules

Example: -globin

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.)

How?

 Current view:

– same sort of interaction with basal apparatus as the proximal promoter module

– Increase 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 sites

 Experimentally verified “composite elements” or CE’s

– GR site + AP-1 in proliferin promoter

– Synergistic: result in non-additively high level

– Antagonistic: 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 proteins

 Activities controlled in regulatory pathways

 Independent domains responsible for activities:

– Recognition of specific target sequences – Binding to other components

of the transcription apparatus

– E.g. yeast GAL4

 Protein-DNA interactions

– Proteins 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

– High-affinity site competes with the large number of low-affinity sites; repressor binds 10

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 motif

– Common 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

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

 Helix-Turn-Helix

 Homeodomains

– Related to helix-turn-helix bacterial repressors – Homeobox = 60 AA residues

– E.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

 Helix-loop-helix (HLH)

– DNA binding (helix) & dimerization

– Class A: ubiquitouslyh expressed proteins, e.g.

E12/E47

– Class B: tissue-specific expression, e.g. MyoD, myogenin, Myf-5

– Myc proteins (separate class)

 Leucine zippers fig 21.15

– Dimerization motif

– E.g. Jun+Fos = AP1

– Gcn4 ->

 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

Alternative model

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,

Cook, 1999 (Science)

• Enhancers

•dynamic equilibrium

Recap: evolution of understanding of

eukaryotic transcription

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 system

– Rapid turnover of promoter bound TF: resets signaling pathway: cell can continuously monitor its environment

 Tissue-specific synthesis

– Development, e.g. homeodomain proteins

 Modification

– Phosphorylation, acetylation, methylation

– E.g., AP1 (= Jun+Fos)  active form by phosphorylation – E.g., p53 acetylated (modulates interactions with coactivator

and repressor proteins

 Ligand binding

– E.g. Steroid receptors

– Influence: localization or DNA-binding ability

 Cleavage

 Inhibitor 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 factor

 Level 2 = cooperation of multiple factors within a module (all present and active, and all repressors inactive or absent)

 Level 3 = multiple autonomous modules per gene

– Each module can independently activate the gene

– Each 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

Example: eve

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

-

+

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

 Cis-regulatory logic device

 endo16 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

Chromatin

 Eukaryotic genomes are packaged with chromatin proteins

 Heterochromatin (highly condensed, untranscribed)

 Euchromatin (more accessible, transcribed)

 Each cell: unique pattern of heterochromatin and

euchromatin

Nucleosomes

•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)

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?

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.

Grandori C, Cowley SM, James LP, Eisenman RN.

Annu Rev Cell Dev Biol. 2000;16:653- 99

Cytosine methylation

 mCG often in inactive vertebrate genes

 After replication of methylated DNA,

methyl groups are added to daughter strands

 CpG islands

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 regions

– LCR, 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 analysis

 EMSA electorphoretic mobility shift assay

 Microarrays: level of mRNA

 ChIP: Chromatin ImmunoPrecipitation

 Yeast2Hybrid

 Sequence analysis, phylogenetic

footprinting

Free DNA probe

*

*

Protein-DNA complex

EMSA

Sources

 B Lewin, Genes VII

 Lodish et al. Molecular Cell Biology

 EH Davidson: Genomic Regulatory Systems

 Alberts et al. Essential Cell Biology

 EM Blackwood & JT Kadonaga: Going the distance: a current view of enhancer action

 Cell, February 22, 2002: 108 (4) "Reviews