Transcript Next lectures: Differential Gene expression

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
Before we begin…..
• The material on pages109-116….not new?
• Websites:
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5.1
5.3
5.4
5.5
Transcription through nucleosomes
Promoter structure (TBPs/TAFs)
Families of transcription factors
Histone acetylation/chromatin remodeling
A few words about enhancers
• Heavily studied since the early 1980’s
• 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
Example (Fig 5.6 from Gilbert)
Gilbert’s 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
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
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
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
General domain structure of
many transcrption factors
Activation
DNA binding dimerization/interaction
(example only)
Different regions of transcription factor proteins are responsible
for discrete functions involved in its regulatory activity
Major families of transcription factors
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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
Conserved structures are often DNA
binding domains (From Wolffe, Chromatin, 3rd ed.)
Some transcription factors contain motifs from chromatin proteins
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. 12:58-62
Figure 1 from Pruss and Wolffe
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
Structure of the winged helix domain of linker
histones and HNF3 (From Wolffe, Chromatin, 3rd Ed.)
HNF3 and the serum albumin enhancer
Lab of K.S. Zaret@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
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
Example of IFN-beta enhancer
(From Wolffe, Chromatin, 3rd Ed.)
Illustrates model of an assembled enhancer interacting with pol II
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
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
Role of HAT/HDAC in transcriptional
regulation in chromatin (From Wolffe, Chromatin, 3rd Ed.)
Year 2001 model of the IFN-b
enhanceosome (From Agalioti, et. al. (2000) Cell 103:667-678
Chromatin “remodeling machines”
• SWI/SNF (yeast, mammals)
• NURF and CHRAC (Drosophila)
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All are multi-subunit complexes
Their activity is ATP dependent (energy)
Cause nucleosome disruption in vitro
Little evidence of targeting specificity
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)
From Francastel, et. al. (1999) Cell 99:259-269
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)
Summary: Enhancers
• Enhancers and their associated proteins
(transcription factors) are important
determinants of gene expression patterns
• They affect transcription by many mechanisms
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Direct interaction with RNA polymerase
Regulation of nucleosomal positioning
Recruitment of histone acetylase/deacetylase
Prevention of gene localization to centromeric
heterochromatin