208 Euk Regulation of Gene Expression 2012
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Transcript of 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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Levels of Gene Expression Regulation in Eukaryotes
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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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Levels of Gene Expression Regulation in Eukaryotes
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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