Week 8 gene regulation in eukaryotes

cis-acting elements: promoters and enhancers Promoters – usually directly adjacent to the gene • Include transcription initiation site • Often have TATA box: TATAATAAT • Allow basal leve

8TH WEEK, BIO-1053

GENE REGULATION IN EUKARYOTES

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Overview of eukaryotic gene regulation

Eukaryotes use complex sets of interactions

• Regulated interactions of large networks of genes

• Each gene has multiple points of regulation

Themes of gene regulation in eukaryotes:

• Environmental adaptation in unicellular eukaryotes

• Maintenance of homeostasis in multicellular

eukaryotes

• Genes are turned on or off in the right place and time

• Differentiation and precise positioning of tissues and organs during embryonic development

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Control of transcription initiation

Three types of RNA polymerases in eukaryotes

• RNA pol I – transcribes rRNA genes

• RNA pol II – transcribes all protein-coding genes

(mRNAs) and micro-RNAs

• RNA pol III – transcribes tRNA genes and some small regulatory RNAs

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The three polymerases in eukaryotes

These enzymes are eluted at different salt concentration during ion exchange

chromatography

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Difference in their sensitivity to α -amanitin Pol I is very insensitive, Pol II is very sensitive, Pol III is intermediate sensitivity

Comparison of three-dimensional structures of bacterial and eukaryotic RNA polymerases

cis-acting elements: promoters and

enhancers

Promoters – usually directly adjacent to the gene

• Include transcription initiation site

• Often have TATA box: TATAA(T)AA(T)

• Allow basal level of transcription

Enhancers – can be far away from gene

• Augment or repress the basal level of transcription

trans-acting factors interact with cis-acting

elements to control transcription initiation

Use of reporter genes to identify

promoters and enhancers in eukaryotes

Use of reporter genes to identify

trans-acting factors in transcriptional regulation

Localization of activator domains using recombinant

DNA constructs

lexA: the bacterial repressor, contains only DNA-binding domain

Yeast promoter

Basal transcription factors

Basal transcription factors assist the binding of RNA pol II

to promoters

Key components of basal factor complex:

• TATA box-binding protein (TBP)

• Bind to TATA box

• First of several proteins to assemble at promoter

• TBP-associated factors (TAFs)

• Bind to TBP assembled at TATA box

RNA pol II associates with basal complex and initiates

basal level of transcription

Basal factors bind to promoters of all

Activators are transcription factors that

bind to enhancers

Increase levels of transcription by interacting directly or

indirectly with basal factors at the promoter

• Stimulate recruitment of basal factors and RNA pol II

Binding of activators to enhancers increases

transcriptional levels

Steroid hormones are co-activators

Polypeptides and other molecules that play a role in

transcriptional activation without binding directly to DNA are called coactivators

Domains within activators

Activator proteins have two functional domains

Sequence-specific DNA binding domain

• Binds to enhancer

Transcription-activator domain

• Interacts with other transcriptional regulatory proteinsSome activators have a third domain

Responds to environmental signals

• Example - steroid hormone receptors

DNA-binding domains of activator proteins

Interacts with major groove

of DNA

Specific amino acids have

high-affinity binding to

specific nucleotide sequence

The three

best-characterized motifs:

•Helix-loop-helix (HLH)

•Helix-turn-helix (HTH)

•Zinc finger

Many activators must form dimers to function

Repressors diminish transcriptional activity

Repressor proteins suppress transcription initiation through different mechanisms:

Some repressors have no effect on basal transcription but suppress the action of activators

- Compete with activator for the same enhancer

- Block access of activator to an enhancer

Some repressors eliminate virtually all basal transcription from a promoter

- Block RNA pol II access to promoter

- Bind to sequences close to promoter or distant from

promoter

Repressor proteins that act through competition with an activator protein

Repressor binds to the same enhancer sequence as the activator

• Has no effect on the basal transcription level

Repressor proteins that act through quenching

an activator protein

• Type I: Repressor blocks the DNA-binding domain

• Type II: Repressor blocks the activation domain

The same transcription factor can play

different roles in different cells

Example: α 2 repressor in yeast

• In α haploids, α 2 binds to enhancers of “a”-specific genes but not to enhancers

of haploid-specific genes

• In α /a diploids, α 2 dimerizes with a1 factor and is altered so that it can bind to enhancers of

haploid-specific genes

The Myc-Max mechanism can activate or

repress transcription

Myc − identified as an oncogene

• Regulates transcription of genes involved in cell

proliferation

• Doesn't have DNA binding activity on its own

Max − identified through its binding to Myc

• Max/Max homodimers and Myc/Max heterodimers bind

to the same enhancers

• Max/Max represses transcription

• Myc/Max activates transcription

Comparative structures of Myc and Max

Max/Max acts as a repressor when Myc is

not expressed

Max is constitutively expressed but Myc expression is tightly regulated

When Myc is present, Myc/Max heterodimers form and activate transcription

• Myc - Max affinity is higher than Max - Max affinity

Gene activation occurs when a cell

makes both Max and Myc

The yeast GAL system is another complex regulatory mechanism

Enhanceosome: a multimeric complex of proteins and other small molecules associated with an enhancer element

The multimeric complex of protein can include activators,

coactivators, repressors and corepressors

Chromatin structure and epigenetic effects

Chromatin structure can affect transcription

• Nucleosomes can sequester promoters and make

them inaccessible to RNA polymerase and transcription factors

• Histone modification and DNA methylation

• Chromatin remodeling and hypercondensation

Epigenetic changes – changes in chromatin structure that are inherited from one generation to the next

• DNA sequence is not altered

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Chromatin reduces transcription

Chromatin remodeling can expose the

promoter

Nucleosomes can be repositioned of removed by chromatin remodeling complexes

• After remodeling, DNA at promoters and enhancers

becomes more accessible to transcription factors

• Can be assayed using DNase digestion (DNase

hypersensitive sites)

The SWl-SNF remodeling complex

Hypercondensation of chromatin results

Determining methylation state of DNA using

two restriction enzymes and Southern blotting

HpaII and MspI

have the same recognition

sequence (CCGG), but different

sensitivity to DNA

methylation

Gene silencing by the SIR complex in yeast

Gene for α2 repressor is at the MAT (mating type) locus

HML and HMR are copies of MAT locus but are silenced by SIR complex

SIR mutations cause

Effects of chromatin structure on transcription:

Histone modification and DNA methylation

N-terminal tails of histones H3 and H4 can be modified

• Methylation, acetylation, phosphorylation, and

ubiquitination

• Can affect nucleosome interaction with other

nucleosomes and with regulatory proteins

• Can affect higher-order chromatin structure

DNA methylation occurs at C5 of cytosine in a CpG

dinucleotide

• Associated with gene silencing

Histone Modification

• The histones can be modified

by acetylation or methylation.

• This can modify the impact of

the histones on transcriptional

activators, and enzymes that

deacetylate histones repress

transcription

• DNA methylation is also

associated with repression

Genomic imprinting results from

transcriptional silencing

Genomic imprinting – expression of a gene depends on whether it was inherited from the mother or father

• Occurs with some genes of mammals

• Epigenetic effect (no change in DNA sequence)

Paternally imprinted gene is transcriptionally silenced if it

Paternally imprinted gene is transcriptionally silenced if it was transmitted from the father

• Maternal allele is expressed

Maternally imprinted gene is transcriptionally silenced if it was transmitted from the mother

• Paternal allele is expressed

In mice, deletion of Igf2 causes a mutant

phenotype only when transmitted by father

Maternal imprinting of Igf2 gene

•Maternally-inherited Igf2 allele is silenced

•Igf2 deletion heterozygotes have normal phenotype if the

mutant allele was inherited from the mother

Insulators limit the chromatin region over

which an enhancer can operate

Methylation of the H19 promoter and the insulator occurs in

spermatogenesis, thus the H19 promoter is not available to the enhancer and

is not expressed -> lgf2 is expressed in male mice

Methylation of complementary strands

of DNA in genomic imprinting

Epigenetic state can

Regulation

after

transcription

RNA splicing helps regulate gene expression

Sex lethal (Sxl) gene encodes a protein required for

female-specific development

In early embryos, Sxl is transcribed only in females

Later in development, Sxl gene is transcribed in both sexes

Sxl protein regulates alternative splicing of its own mRNA

RNA splicing helps regulate gene expression

RNA editing alters the sequences of pre-mRNAs

The sequence of a pre-mRNA can be changed by a

process, called RNA editing

RNA editing occurs in the mitochondrial of protozoans and plants, also in chloroplasts In higher eukaryotes, RNA

editing is rarer

RNA editing of apo-B pre-mRNA

apoB gene encodes two alternative forms of the serum protein

apolipoprotein B: apoB-100 ( ≈ 5000 kDa) expressed in hepatocytes and apoB-48 ( ≈ 240 kDa) expressed in intestinal epithelial cells

N-terminal domain (green): associates with lipids, C-terminal domain (orange) binds to LDL receptors on cell membranes

Control of cytoplasmic polyadenylation and

translation initiation

CPE: cytoplasmic polyadenylation element

CPEB: CPE-binding protein

PABPI: poly (A)-binding protein

CPSF: cleavage and polyadenylation specific factor

PAP: poly (A) polymerase

Immature oocytes Mature oocytes

Pathways for degradation of eukaryotic mRNAs

An ion-sensitive binding protein

Some small RNAs are responsible for

RNA interference (RNAi)

Specialized RNAs that prevent expression of specific

genes through complementary base pairing

• Small (21 – 30 nt) RNAs

• Micro-RNAs (miRNAs) and small interfering RNAs

(siRNAs)

• First miRNAs (lin-4 and let-7) identified in C elegans

• Nobel prize to A Fire and C Mello in 2006

Posttranscriptional mechanisms for gene regulation

• mRNA stability and translation

• May also affect chromatin structure

Primary transcripts containing miRNA

Most miRNAs are transcribed by RNA polymerase II from noncoding DNA regions that generate short dsRNA

hairpins

miRNA processing

Drosha excises stem-loop from primary miRNA (pri-miRNA)

to generate pre-miRNA of ~ 70 nt

Dicer processes pre-miRNA to a mature duplex miRNA

One strand is incorporated into miRNA-induced silencing complex (RISC)

Two ways that miRNAs can down-regulate

expression of target genes

When complementarity is

perfect:

• Target mRNA is degraded

When complementarity is imperfect:

•Translation of mRNA target is repressed

RNAi provides

potential treatment for previously

incurable diseases

miR-26a Triggers Cell-Cycle Arrest

Adeno-associated viruses (AAV) exist as episomal DNA in the nucleus of cells A primary miR-26a transcript expressed by the viral vector is processed by

Drosha/DGCR8 and is exported to cytoplasm

where the pre-miRNA is further processed by

Dicer/TRBP The guide strand is selected for entry into the RNA-induced silencing complex (RISC)

Use of RNAi in anti-cancer gene therapy

into the RNA-induced silencing complex (RISC) The miR-26a guide strands pairs with sequences in the 3′ untranslated regions (UTRs) of target

transcripts encoding the cyclins D2 and E2,

thereby reducing their expression This results in the arrest of cells in G1 and inhibits proliferation

of hepatocarcinoma cells.

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Posttranslational modifications of

proteins

Ubiquitination – covalent attachment of ubiquitin to other proteins targets those proteins for degradation by the proteosome

Cascades of phosphorylation and dephosphorylation

• Transmission of signals across the cell membrane to the nucleus

• Sensitization – tissues exposed to hormones for long periods of time lose ability to respond to the hormone

• Example: binding of epinephrine to β-adrenergic receptors on surface of heart muscle cells

Phosphorylation and desensitization of

Phosphorylation of receptor has no effect on its

binding to epinephrine,

but blocks its downstream functions