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Control of Gene Expression

Shaw-Jenq Tsai

Department of Physiology

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Thinking about Gene Regulation

Humans begin life from a single cell; all the genetic information needed to create an adult is in our genome.

Embryonic cells undergo differentiation to produce specific cell types such as muscle, nerve, and blood cells.

Different cell types are the consequence of differential gene expression.

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A typical differentiated mammalian cell makes about 100,000 proteins from approximately 35,000 genes.

Most of these are housekeeping proteins needed to maintain all cell types.

Certain proteins can only be detected in specific cell types.

How is gene expression regulated?

Regulation of gene expression is very complex

Presently – we have a superficial understanding

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Synthesis of a protein involves discrete steps

Several levels at which control mechanisms work

Transcriptional control

RNA processing control

Translation control

Protein activity control

Control of Gene Expression

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Differential gene transcription – the major mechanism of selective protein synthesisGoverned by a large number of proteins known as transcription factorsTwo functional classes of transcription factors

General transcription factorsSpecific transcription factors

Transcriptional level control

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A single gene controlled by many regulatory sites – bind different regulatory proteins

A single regulatory protein may become attached to numerous sites on the genome

Cells respond to environmental stimuli by synthesizing different transcription factors

Bind to different sites on DNA

Specific transcription factors

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PEPCK

A key enzyme of gluconeogenesis (conversion of pyruvate to glucose)

Synthesized in liver in response to low glucose

Synthesis drops sharply after a meal

Level of synthesis of PEPCK controlled by different transcription factors

e.g. receptors for hormones involved in regulating carbohydrate metabolism

Specific Transcription Factors

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Closest upstream sequenceTATA box – major element of the gene’s promoter

Region from TATA box to start of transcription site is the core promoter

Site of assembly of preinitiation complex – RNA polymerase II and general transcription factors

Two other promoter sequencesCAAT boxGC box

Promoter Structure

Core

promoter

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PEPCKTATA box determines the site of initiation of transcription

CAAT and GC boxes regulate the frequency of transcription

All located within 100 to 150 base pairs upstream of the transcription start site – proximal promoter elements

Control of PEPCK Gene Expression

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Hormones which affect transcription of PEPCK include insulin, thyroid hormone, glucagon and glucocorticoids

All affect transcription factors which bind DNA

DNA sites bound by transcription factors are termed – response elements

Glucocorticoids stimulate PEPCK expression by binding to a specific DNA sequence termed – a glucocorticoid response element (GRE)

Activation of Transcription

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Same GRE is located upstream from different genes on different chromosomes

Thus – a single stimulus – elevated glucocorticoid concentrations – simultaneously activates a range of genes needed in a comprehensive response to stress

Activation of Transcription

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Activation of TranscriptionExpression of genes also regulated by more distant DNA elements termed enhancers

Can be experimentally moved without affecting their ability to enhance gene expression

May be 1000s or 10000s base pairs upstream or downstream from the gene

How??Brought into close proximity to the gene as DNA can form loops

Promoters and enhancers cordoned off from other genes by sequences called insulators

Enhancers

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Control of Gene ExpressionActivation of Transcription

Transcription factor

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Activation of TranscriptionEnhancers

Control of Gene Expression

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Action of an Insulator

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Figure 12.34

Two hypotheses for the mechanism of insulator activity.

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A transcription factor bound to an enhancer may act via the following mechanisms:

Recruit general transcription factors and DNA polymerase II to the core promoter

Stabilize the transcription machinery located in the core promoter

Via an intermediary termed a coactivatorCoactivators are large complexes with 15 to 20 subunits

Do not directly bind DNA

Interact with a range of transcription factors

Action of Transcription Factor

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Contain different domains which mediate the different functions – at least two domains

DNA-binding domain

Activation domain

Commonly form dimers

ExampleGlucocorticoid receptor

Binds DNA at the glucocorticoid response element (GRE)

Ligand-binding domain / DNA-binding domain / Activation domain

Structure of Transcription Factors

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GRE

A palindrome

Two-fold nature is important

Pairs of GR polypeptides bind to DNA forming dimers

Transcription Factors Binding Element

5’-AGAACAnnnTGTTCT-3’

3’-TCTTGTnnnACAAGA-5’

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Transcription factors belong to each of several classes based upon specific types of binding domains or motifsMany contain an -helix which is inserted into the DNA major groove

Recognizes the particular nucleotide sequence lining the grooveBinding between aa and DNA (including DNA backbone) via:

Van der Waals (hydrophobic) forcesIonic bondsAnd hydrogen bonds

Transcription Factor Motifs

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Transcription Factor MotifsCommon transcription factor motifs

Zinc finger

Helix-loop-helix

Leucine zipper

HMG box

Shared featureStructurally stable framework

Specific DNA recognizing sequences are correctly positioned

Control of Gene Expression

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Types of DNA binding proteins

DNA and RNA polymeraserepair enzymes

structural proteinstranscription factors

Types of DNA binding proteins

DNA and RNA polymeraserepair enzymes

structural proteinstranscription factors

DNA binding motifs

zinc fingersleucine zippershelix-turn-helixhelix-loop-helix

DNA binding motifs

zinc fingersleucine zippershelix-turn-helixhelix-loop-helix

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Zn ion coordinated to two cysteines and two histidines

Each contains multiple zinc finger domains

Zinc finger

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Two a helices separated by a loopOften preceded by a stretch of basic aa which interact with a specific nucleotide stringAlways occur as dimers

homodimersheterodimers

Helix-loop-helix (HLH)

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Leucines every seven aa along an -helix

All leucines face the same direction

Two -helices can zip together forming a coiled coil

Basic aa on opposite side of coils

Leucine zipper motif

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Cells also possess negative regulatory elements

Mechanisms:Binding to promoter elements

Blocking assembly of the preinitiation complex

Inhibiting binding or functioning of transcriptional activators

Modifying DNA and its interaction with nucleosomes

Some transcription factors activate some genes and repress others

Repression of Transcription

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Binding to promoter elementsBlocking assembly of the preinitiation complex

Mechanisms of Transcription Repression

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Inhibiting binding or functioning of transcriptional activators

Mechanisms of Transcription Repression

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DNA MethylationMethyl groups may be attached to cytosine (C5 position)

Methyltransferases

Methyl groups provide a tag In mammals always part of a symmetrical sequenceConcentrated in CG-rich domains

Often in promoter regions

Methylation of promoter DNA highly correlated with gene repression

Repression of Transcription

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Maintains a gene in inactive state rather than initiating gene repression – Example:

Inactivation of genes of one X chromosome in female mammals occurs prior to a wave of methylation

Shifts throughout life in DNA-methylation levels

Early Zygote – most methylation tags removed

Implantation – a new wave of methylation occurs

Important example – Genomic Imprinting

DNA Methylation

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Genomic ImprintingCertain genes are active or inactive during early development

Depending on whether they are paternal or maternal genes

e. g.– IGF-2 is only active in the gene from the male parent

The gene is imprinted according to parental origin

Mammalian genome has > 100 imprinted genes in clusters

Imprinted due to selective methylation of one of the alleles

DNA Methylation

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Genomic ImprintingIn the early embryo the waves of demethylation and new methylation do not affect the methylation of imprinted genes

Thus the same alleles are affected in the zygote through to the adult stage in the individual

DNA Methylation

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DNA is not naked – but wrapped around histone complexes to form nucleosomes

How are transcription factors and RNA polymerases able to interact with DNA tightly associated with histones?

Apparently nucleosome structure does inhibit initiation of transcription

Initiation of transcription requires assembly of large complexes and nucleosomes block assembly at the core promoter

Chromatin structure and transcription

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Genes which are actively transcribed are bound by histones which are acetylatedEach of the histones has a flexible N-terminal tail

Extends outside the core particle and the DNA helixAcetyl groups are added to lysine residues by enzymes

Histone acetyl transferases (HATs)

Acetylation has two functionsNeutralize the positive charge on the lysine residuesDestabilize interactions between histone tails and structural proteins

Role of Acetylation

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Some coactivators have HAT activityLinks histone acetylation, chromatin structure and gene activation

HAT activity of coactivator acetylates core histones bound to promoter DNA causing

release of nucleosome core particles or loosening of histone-DNA interactionSubsequent binding of transcription factors and RNA polymeraseOnce transcription is initiated – RNA polymerase is able to transcribe DNA packaged into nucleosomes

Acetylation is dynamic – enzymes also remove acetyl groups

Role of Acetylation

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Removal of acetyl groupsHistone deacetylases (HDACs)

HDACs associated with transcriptional repression

HDACs are subunits of larger complexes – corepressors

HDACs guided to regions of DNA by methylation patterns

Role of Deacetylation

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Role of DeacetylationExample:

Inactive X chromosome of femaleLargely deacetylated histones

Active X chromosome has a normal level of histone acetylation

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Control of Acetylation / Deacetylation

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Control of Acetylation / Deacetylation

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Recall that the formation of multigene families is a mechanism that generates protein diversity

Protein diversity also generated via alternate splicing

Regulates gene expression at the level of RNA processing

A mechanism by which a single gene can encode two or more related proteins

Most genes (and their primary transcripts) contain multiple introns and exons

Often – more than one pathway for processing of primary transcript

Processing-Level Control

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Transcripts from approx 35% of human genes may be subjected to alternate splicing

Simplest case – a specific segment either spliced out or retained – Example:

Fibronectin:

Synthesized by fibroblasts – two additional peptides compared to that synthesized by liver

Extra peptides encoded by pre-mRNA retained in fibroblast

Processing-Level Control

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Wide variety of mechanisms – affecting mRNA previously transported from the nucleus

Subjects include:Localization of mRNA in the cell

mRNA translation

Half-life of mRNA

Mediated via interactions between mRNA and cytosolic proteins

Translational-Level Control

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mRNA noncoding segments – untranslated regions (UTRs)

5’ – UTR – from methylguanosine cap to AUG initiation codon

3’ – UTR – from termination codon to end of poly(A) tail

UTRs contain nucleotide sequences which mediate translational-level control

Translational-Level Control

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Cytoplasmic localization of mRNAs – Example ferritin

Translation regulated by iron regulatory protein (IRP)

Activity of IRP dependent on cellular iron concentrationAt low iron concentration – IRP binds the 5’ UTRBound IRP interferes physically with the binding of a ribosome to the 5’ end of the mRNAAt high iron concentration the IRP changes conformation and looses affinity for the 5’ UTR

Translational-Level Control

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Half-life of mRNA is variable – 10 minutes to 24 hours

Specific mRNAs are recognized in the cytoplasm and treated differentially

mRNAs lacking the poly(A) tail are rapidly degraded

Poly(A) tail is not naked mRNA but bound by the poly(A) binding protein (PABP)

Each PABP bound to about 30 adenosine residues

Control of mRNA stability

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PABP protects poly(A) tail from general nuclease activity

But – increases its sensitivity to poly(A) ribonuclease

mRNA in cytoplasm is gradually reduced in length by poly(A) ribonuclease

When the tail is reduced to approx 30 residuesmRNA is rapidly degraded

Degradation occurs from the 5’ endSuggests two ends held in close proximity

Control of mRNA stability