208 Euk Regulation of Gene Expression 2012

7/31/2019 208 Euk Regulation of Gene Expression 2012

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Regulation of Gene Expression in

Eukaryotes


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Eukaryotic aspects that affect gene expression

Eukaryotic chromosomes have DNA wound around histone

proteinscondensation/decondensation is a level of regulation

Eukaryotes have a nucleus

transport of mRNA into cytoplasm can be regulated

Eukaryotes process their RNA

alternative splicing is a level of regulation

Eukaryotic mRNA has a longer half-lifeallows for more translational control of regulation

Eukaryotes are multicellular

differential gene expression allows for specialization

of cell types


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Levels of Gene Expression Regulation in Eukaryotes


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Regulation of Transcription

in Eukaryotes

Activator Regulatory Transcription Factorsbind

to Enhancers

Repressor Regulatory Transcription Factors bindto Negative Regulatory Elements

Chromatin Remodelers


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Basal vs. Regulatory Transcription Factors in Eukaryotes

Basal Transcription

Factors

Recruit and Stabilize

RNA Polymerase

Bind to the basal promoter

region (-30 to -90 bp)

( TATA box, CAAT box,

or GC box)

ARE NOT directlyresponsive to signals

DO NOT regulate

transcription

RegulatoryTranscription Factors

Can Affect Basal

Transcription Factor Binding

Bind to Enhancers and

Negative Regulatory

Elements (which can be far

from the basal promoter

region)

ARE responsive to signals

DO Regulate transcription

Both are types of proteins


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Properties of Enhancers or Negative Regulatory Elements

DNA sequences

Can act at long distances (kilobases) in either orientation May be upstream, downstream, or within genes regulated

Recruit activator or repressor Regulatory Transcription Factors

Can act on heterologous (unrelated) promoters

Enhancers are bound by activator regulatory transcription factors

Negative Regulatory Elements are bound by

repressor regulatory transcription factors


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Design an experiment to ectopically express Gene Nin muscle cells.

(Gene N is usually only expressed in neurons)


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Jang, C.-C. et al. Development 2003;130:2939-2951

Ectopic eye formation due to ectopic eyeless expression

Dpp

Enhancer

Heterologous

promoter Eyeless gene


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Mechanism of Activator Regulatory Transcription Factors


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Basal vs. Regulatory Transcription Factors in Eukaryotes

Basal Transcription

Factors

Recruit and Stabilize

RNA Polymerase

Bind to the basal promoterregion (-30 to -90 bp)

( TATA box, CAAT box,

or GC box)

ARE NOT directlyresponsive to signals

DO NOT regulate

transcription

RegulatoryTranscription Factors

Can Affect Basal

Transcription Factor Binding

Bind to Enhancers and

Negative Regulatory

Elements (which can be far

from the basal promoter

region)

ARE responsive to signals

DO Regulate transcription

Both are types of proteins


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Figure 11.25: Transcriptional activation by recruitment


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Mechanism of Activator Regulatory Transcription Factors


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Regulatory Transcription Factors

Contain Domains and MotifsDomains

Regions of the protein that have a particular

function-DNA binding, protein-proteininteraction, interaction with basal transcriptionfactors

MotifsConserved three-dimensional structural patternsthat allow for a specific function-in this case DNA

binding


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Activator Transcription Factor

DomainsTrue Activators have:

1) A DNA Binding Domain that binds to a

specific Enhancer DNA sequence2) A Transactivating Domain that

positively interacts with basal

transcription factors or chromatinremodeling complexes


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TransactivationDomain

Transactivation domain binds to basal transcription factors


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DNA Binding Motifs

Helix-turn-Helix

Zinc finger

Leucine zipper

basic Helix-Loop-Helix


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Homeobox Helix-turn-Helix Activators


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Zinc Finger


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Leucine Zipper


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Basic Helix-Loop-Helix

Dimerizes

Protein-protein interaction

domain in helix 1

Basic region binds DNA


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Regulation of Transcription

in EukaryotesRegulatory Transcription Factors binding

to Enhancers or Negative RegulatoryElements

Chromatin Remodelers:Anti-repressor Activators

Repressors


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Chromatin Remodeling


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Chromatin Remodeler Systems

SWI/SNF, NURF complex

ATP hydrolysis dependent

Histone acetyltransferases (HATs)


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Models for SWI/SNF

Mediated chromatin remodeling


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Chromatin Remodeling


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Chromatin Remodeler Systems

SWI/SNF, NURF complex

ATP hydrolysis dependent

Histone acetyltransferases (HATs)

Ch ti R d li b


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Chromatin Remodeling by

Histone Acetylases and Histone Deacetylases


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Post-transcriptional regulation

RNAiAlternative splicing

selective mRNA export to cytoplasm

mRNA stability

Post-translational modifications

Proteolysis

Phosphorylation

MethylationAcetylation


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RNA interference (RNAi)

Small RNA molecules specifically block

translation of genes Two types of RNAs

siRNA

miRNA


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RNAi

siRNA

small/short interfering RNAs or silencing

RNAs

Double stranded (ds)RNAs 20-25 bases

Endogenous siRNAs identified/occur in plants

Synthetic siRNAs now made to knockout gene

products

miRNA microRNAs

22ish nts- single stranded RNA with stem-loop


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Figure 11.35: Mechanisms of gene silencing by the

siRNA and miRNA pathways


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When are genes regulated?

In response to environmental factors

(nutrients, heat, toxic compounds etc.)

During development as cells become

specialized

In response to inter-cellular signals involved in

MANY cellular processes


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Transcription

Selective nuclear RNA processing

Selective mRNA translation

Post-translational protein modification

Levels of

Gene Expression Regulation


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Techniques to study transcription

Individual Genes

Levels of expression

Northern Blots

Reporter AssaysReal-Time RT-PCR

Regulatory Transcription Factors binidng DNA

Gel shifts and ChIP assays

Genome-wide

Microarrays

Expression profiling


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Northern Blots

R t G T l


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Reporter Genes as Tools

to Study Transcriptional Regulation

Luciferase (tissue culture)

lacZ: b-galactosidase (animals- in vivo) Green Fluorescent Protein (animals-in vivo)

GUS-b-glucoronidase (plants- in vivo)


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The Genetic Elements Regulating Tissue-Specific

Transcription Can Be Identified by Fusing Reporter

Genes to Suspected Enhancer Regions of the GenesExpressed in Particular Cell Types


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Real Time RT PCR

Reverse transcriptase (RT)

mRNA DNA

Real Time PCR


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Techniques to study transcription

Individual Genes

Levels of expression

Northern Blots

Reporter AssaysReal-Time RT-PCR

Regulatory Transcription Factors binidng DNA

Gel shifts and ChIP assays

Genome-wide

Microarrays

Expression profiling


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Gel Shifts


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Microarrays


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Techniques to study protein levels

Western Blots

Immunohisto(cyto)chemistry

Documents

208 Euk Regulation of Gene Expression 2012