Next lectures: Differential Gene expression

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Next lectures Differential Gene expression

• Chapter 5 and websites on syllabus
• Epigenetic control mechanisms
• Histone modification
• DNA methylation
• Nucleosome disruption machines
• Promoters and enhancers
• Old and new models of enhancer function
• Novel transcriptional control sequences

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Before we begin..

• The material on pages109-116.not new?
• Websites
• 5.1 Transcription through nucleosomes
• 5.3 Promoter structure (TBPs/TAFs)
• 5.4 Families of transcription factors
• 5.5 Histone acetylation/chromatin remodeling

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A few words about enhancers

• Heavily studied since the early 1980s
• Most involve minimal enhancers/promoters or
  minimal enhancer/heterologous promoter
  combinations
• Most involve studies on plasmid DNA transiently
  transfected into cells in culture
• Most of our models of enhancer activity derive
  from these kinds of experiments

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Example (Fig 5.6 from Gilbert)
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Gilberts 7 generalizations concerning enhancer
function

• Most genes require enhancers for activity
• Enhancers are the major determinant of
  differential transcription in time and space
• Enhancers can work far from the promoter so
  multiple signals can be integrated to determine
  if a gene will be transcribed. Genes can have
  several enhancers and each enhancer can bind
  multiple proteins

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The 7 generalizations (continued)

• Interaction between proteins bound to the
  enhancer sites and the transcription initiation
  complex assembled at the promoter is thought to
  regulate transcription
• Enhancers are modular. Particular combinations
  of factors (rather than any one factor)
  determines enhancer function

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The 7 generalizations (continued)

• A gene can have several enhancer elements, each
  turning it on in a different set of cells
• Enhancers can be used to inhibit transcription.
  In some cases factors that activate the
  transcription of one gene represses other genes.
  (Silencers negative enhancer

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My generalization of enhancers

• Sequences with enhancer activity bind an enormous
  array of sequence-specific DNA binding proteins
  called transcription factors
• Transcription factors fall into families with
  shared structural and functional properties
• Enhancers affect transcription efficiency and can
  do so over great distances of DNA via the binding
  of transcription factors

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General domain structure of many transcrption
factors
Activation DNA binding dimerization/interacti
on (example only)
Different regions of transcription factor
proteins are responsible for discrete functions
involved in its regulatory activity
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Major families of transcription factors

• Homeodomain (helix-turn-helix) (Pax, Hox)
• Basic helix-loop-helix (E proteins, MyoD)
• Winged helix proteins (HNF-3, Ets)
• Basic leucine zipper (fos/jun, C/EBP)
• Zinc finger proteins (SP1, CTCF, EKLF)
• Nuclear hormone receptors (RAR, RXR, ER, GR, PR)
  bind steroid hormones

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Conserved structures are often DNA binding
domains (From Wolffe, Chromatin, 3rd ed.)
Some transcription factors contain motifs from
chromatin proteins
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Motifs shared by transcription factors and
chromatin proteins

• The histone fold
• Histone H3 and TAF(II)-40
• Histone H4 and TAF(II)-60
• Histone H2B and CBF (CCAAT binding factor)
• Wolffe and Pruss (1996) Deviant nucleosomes the
  functional specialization of chromatin. Trends
  Genet. 1258-62

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Figure 1 from Pruss and Wolffe
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Motifs shared by transcription factors and
chromatin proteins (continued)

• HMG-box
• Shared by HMG1 and numerous factors including
  LEF-1, TCF, UBF, HMG-I/Y
• These tend to be DNA-bending proteins that
  facilitate enhanceosome assembly
• Winged helix domain
• Shared by HNF-3 and Linker histones (H1,H5)
• Role in nucleosome spacing/positioning

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Structure of the winged helix domain of linker
histones and HNF3 (From Wolffe, Chromatin, 3rd
Ed.)
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HNF3 and the serum albumin enhancer Lab of K.S.
Zaret_at_Brown University

• Nucleosomes are randomly positioned on albumin
  enhancer DNA without HNF3
• HNF3 precisely positions the nucleosome such that
  it lies under it right at the HNF3 binding site
  of the enhancer. Adjacent nucleosomes are also
  positioned as a result
• HNF3 has a domain that interacts with linker
  histone binding sites of the nucleosome core

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A closer look

• Interaction between proteins bound to the
  enhancer sites and the transcription initiation
  complex assembled at the promoter is thought to
  regulate transcription
• Enhancers are modular. Particular combinations
  of factors (rather than any one factor)
  determines enhancer function

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Example of IFN-beta enhancer(From Wolffe,
Chromatin, 3rd Ed.)
Illustrates model of an assembled enhancer
interacting with pol II
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New models of enhancer function

• Regulation of nucleosomal positioning
• Recruitment of histone acetylase/deacetylase to
  disrupt nucleosome structure
• Prevention of gene localization to centromeric
  heterochromatin

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Reversible histone acetylation

• Histones H3 and H4 are acetylated on lysine
• Histone acetyltransferases (HAT)
• p300/CBP
• PCAF/GCN5
• TAF(II)-250
• Histone deacetylases (HDAC)
• RPD-3
• Interacts with Sin3 and NcoR co-repressors
• Former interacts with Mad/Max family, latter
  interacts with steroid receptor family members

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Role of HAT/HDAC in transcriptional regulation in
chromatin (From Wolffe, Chromatin, 3rd Ed.)
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Year 2001 model of the IFN-b enhanceosome (From
Agalioti, et. al. (2000) Cell 103667-678
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Chromatin remodeling machines

• SWI/SNF (yeast, mammals)
• NURF and CHRAC (Drosophila)
• All are multi-subunit complexes
• Their activity is ATP dependent (energy)
• Cause nucleosome disruption in vitro
• Little evidence of targeting specificity

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Groudine and Martin

• Found that an active enhancer increased the
  probability of establishing transgene expression,
  not necessarily the rate of transcription
• Searched for a structural correlate of this
  activity
• Not DNA methylation
• Not chromatin accessibility
• Yes, proximity to centromeric DNA
  (heterochromatin)

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From Francastel, et. al. (1999) Cell 99259-269
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Why care about centromeres?

• Silent genes are found associated with
  centromeric heterochromatin
• Ikaros family of transcription factors (Zn
  finger) play a role in centromeric localization
  of inactive genes.
• Work from the labs of A.G. Fisher (London) and
  S.T. Smale (UCLA)

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Summary Enhancers

• Enhancers and their associated proteins
  (transcription factors) are important
  determinants of gene expression patterns
• They affect transcription by many mechanisms
• Direct interaction with RNA polymerase
• Regulation of nucleosomal positioning
• Recruitment of histone acetylase/deacetylase
• Prevention of gene localization to centromeric
  heterochromatin