Fundamental Aspects of Transcription and Chromatin Dynamics

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Fundamental Aspects of Transcriptionand
Chromatin Dynamics

• Jim Kadonaga
• Section of Molecular Biology
• University of California, San Diego

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Human Genome Website
http//www.ornl.gov/TechResources/Human_Genome/
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landscape
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Old Transcriptional Control Region
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Grand Scheme for Transcription
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There are four nuclear RNA polymerases in
eukaryotes
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Three perspectives on transcriptional regulation
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Cis-acting transcriptional control elements
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Trans-acting factors (grand scheme)
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Three perspectives on transcriptional regulation
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Basal transcription process
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Sequence-specific DNA-binding factors
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Three perspectives on transcriptional regulation
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Genetic component of regulation
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Epigenetic components
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Grand Scheme for Transcription
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Topics
Basal transcription by RNA polymerase II
Sequence-specific DNA-binding factors How
might enhancers work? Chromatin structure
Introduction Covalent modification of
histones Chromatin remodeling
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Diagram of DNA Regulatory Elements
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Core promoter April 2006
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Basal (General) Transcription Factors for RNA
Polymerase II
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Role of Core Promoters in Enhancer Specificity
(diagram)
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Runoff Transcription Assay
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Runoff transcription assay (G-less cassette)
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Primer extension assay
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Mapping of Start Sites of MTE-containing Promoter
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Basal Transcription Topics 1
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Basal Transcription - Factors and Concepts
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Design of SCP1
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Topics
Basal transcription by RNA polymerase II
Sequence-specific DNA-binding factors How
might enhancers work? Chromatin structure
Introduction Covalent modification of
histones Chromatin remodeling
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Diagram of DNA Regulatory Elements
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Grand Scheme for Transcription
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Sequence-specific DNA-binding Transcription
Factors Are the Apex at the Interface of Genetic
Regulatory Information and the Inverted Cone of
Other Transcription Factors
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Sequence-specific Transcription Factors Are
Modular
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Chromatin Is an Integral Component of
Transcription
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Sequence-specific Factors Typically Bind in
Clusters
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Nuclear receptors
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Sequence-specific factors appear to work by
recruitment of coregulators.
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How do sequence-specific factors work?
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Dnase I footprinting assay
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eTFIID Footprints - wt vs. mutant DPE
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Gel mobility shift assay
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Sequence-specific DNA Affinity Chromatography
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Chromatin Immunoprecipitation (ChIP) Analysis
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Sequence-specific DNA-binding transcription
factors (RNA pol II)
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Grand Scheme for Transcription
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Topics
Basal transcription by RNA polymerase II
Sequence-specific DNA-binding factors How
might enhancers work? Chromatin structure
Introduction Covalent modification of
histones Chromatin remodeling
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How do enhancers work?
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Enhancer-Promoter specificity vs.
Insulator/Boundary Function
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What happens when you see an increase in
transcription in a population of cells?
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Facilitated tracking model for enhancer function
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Topics
Basal transcription by RNA polymerase II
Sequence-specific DNA-binding factors How
might enhancers work? Chromatin structure
Introduction Covalent modification of
histones Chromatin remodeling
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Variety of Phenomena Involve Chromatin
A Variety of Biological Phenomena Involve
Chromatin
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Antirepression Model for Gene Activation
(or, unrepressed state Naked DNA in vitro)
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Packaging of DNA into Chromatin
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Fun facts about chromatin
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More facts about chromatin
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Schematic Diagram of Chromatin
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Crystal Structure of Nucleosome Core
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The 30 nm diameter chromatin filament
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Models for the 30 nm filament
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Solenoid Model for Structure of the 30 nm
Chromatin Fiber
Thoma, F., Koller, Th., and Klug, A. (1979) J.
Cell. Biol. 83, 403-427.
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Translational vs. rotational positioning
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Mnase Digestion and Indirect End-labelling
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Translational vs. rotational positioning
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Remodeling of a Mononucleosome
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Histone H1
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Histone variants - overview
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Histone variants - examples
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HMG proteins - overview
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HMGB (HMG1/2) proteins
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HMGN (HMG14/17) proteins
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Binding of HMG17 to mononucleosomes
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HMGA (HMG-I/-Y) proteins
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Heterochromatin
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Grand Scheme for Transcription
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Topics
Basal transcription by RNA polymerase II
Sequence-specific DNA-binding factors How
might enhancers work? Chromatin structure
Introduction Covalent modification of
histones Chromatin remodeling
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Core histone modifications
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Models for Transcriptional Regulation by Protein
Acetylation
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Model 1 Acetylation induces a conformational
change in the core histones.
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Model 2 Acetylation is a transducing signal
(histone code)
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Model 3 Acetylation affects the activity of a
nonhistone protein.
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Covalent modifications of histones
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Covalent Modifications of Histones (continued)
Different histone modifications are involved in
different nuclear processes. Acetylation and
methylation correlate with transcription, whereas
phosphorylation correlates with the maintenance
of genome integrity. Histone modifications
modulate the binding affinity for histone
(chromatin) binding modules. For example, the
chromodomain of HP1 binds to K9 methylated H3.
The bromodomain of TAF1 binds to acetylated
histone H4.
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Grand Scheme for Transcription
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Topics
Basal transcription by RNA polymerase II
Sequence-specific DNA-binding factors How
might enhancers work? Chromatin structure
Introduction Covalent modification of
histones Chromatin remodeling
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Chromatin remodeling factors - intro
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Helicases and Related ATPases (SNF2-like family)
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Some SNF2 subfamily proteins are in chromatin
remodeling complexes.
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Helicases and Related ATPases (SNF2-like family)
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Shared and unique activities for Snf2 and ISWI
complexes
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A DNA-translocating Model for Chromatin
Remodeling Factors
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Diagram of Restriction Enzyme Accessibility Assay
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Restriction enzyme accessibility assay
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Nucleosome sliding assay
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Schematic of MNase Digestion Assay
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Mnase Digestion and Indirect End-labelling
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Nucleosome positioning by the R3 Lac repressor
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Remodeling of a Mononucleosome
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Chromatin remodeling factors - intro
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Grand Scheme for Transcription
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Current topics in eukaryotic transcriptional
regulation
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