TFIID and SAGA roles in transcription machinery

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TFIID and SAGA rolesin transcription machinery
Inna WeinerReading group in Computational
Molecular Biology16/11/06
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Global structure of RNA polymerase II promoters
TSS
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The core promoter
(Butler and Kadonaga, 2002)
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How to recruit RNA Polymerase?
Assembly of general transcription factors is
required to recruit RNA Polymerase to promoters
A9.30
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General transcription factors

• Factors that permit efficient selective
  initiation by Pol II in vitro
• Promoters recognition
• DNA melting
• Isolated by biochemical fractionation
  experiments.
• TFIIA, TFIIB, TFIID, TFIIE,TFIIF and TFIIH
  complexes were isolated and are sufficient to
  permit in vitro specific transcription.

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TBP

• TATA box - Binding Protein
• TBP is a single polypeptide that sits astride
  the TATA box as a molecular ''saddle,'' inducing
  a sharp bend in the DNA
• Regarded as a universal transcription factor,
  essential for initiation by RNA Pol I, II, and III

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TAFs (TBP Associated Factors)

• The role of the TAFs in the TFIID complex
• - Specific interactions with activators
• - Binding to the promoter Inr and DPE elements
• - Modifying chromatin

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Pre Initiation Complex (PIC) formation
Transition from PIC formation into active
transcription 1. Initiation one of TFIIH
subunits has an helicase activity and is able to
melt the double helices DNA. 2. Elongation this
step is triggered by the phosphorilation of the
CTD repeats of the polymerase by one of the TFIIH
subunit that has a kinase activity.
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Pol II starts mRNA synthesis
Very few factors accompany the elongating
transcript
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Summary
Localization to promoters
Melting the DNA, transcription initiation and
elongation
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Is the general transcription machinery really
general?
(Holstege et al., 1998)
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Outline

• Introduction to general transcription machinery
• TFIID and SAGA unique or redundant function?
• TFIID and SAGA roles in transcription regulation

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TFIID and SAGA

• Multi-unit complexes
• Perform two actions essential for Pol II
  initiation
• Contain a subunit with histone acetyltransferase
  activity
• Possess TBP binding activity
• Question

Are the functions of TFIID and SAGA in vivo
unique or overlapping?
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Percentage of genome dependent on subunits of
SAGA and TFIID
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Whole genome analysis of shared TAFIIs mutations
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Shared vs Specific TAFIIs Influence
30 of the genome is dependent on TFIID specific
sub-units 12 of the genome is dependent on SAGA
specific sub-units
70 of the genome depends on one or more of the
shared TAFs
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How do TFIID and SAGA interact?
TFIID and SAGA have compensatory functions
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Genetic Interactions
Aggravating interaction
Alleviating interaction
No interaction
Slide by Ariel
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How do TFIID and SAGA interact?
TFIID and SAGA have compensatory functions
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Conclusions

• There are distinct requirements for specific
  sub-units of TFIID and SAGA in global expression
• The functions of TAFII145 and GCN5 are redundant

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Improvements

• Statistical test instead of 2-fold
• Check which genes respond to each element
  deletion?
• Define environmental changes

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Research Objective

• TFIID and SAGA
• Share a common set of TAFs
• Regulate chromatin
• Deliver TBP to promoters
• What is their distinct function and relationship
  in genome-wide regulatory network?
• Tested organism S.cerevisiae

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Experimental Setup

• Create single and double-mutants
• SAGA-specific mutant GCN5, SPT3
• TFIID-specific mutant TAF1 (TAFII145)
• Compare gene expression of single and double
  mutants by performing high-throughput analysis

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GCN5 mutants

• gcn5? strain displayed a general decrease in
  expression
• Over 60 of the genes decreased expression by gt4
  standard devations
• Gcn5hat did not change expression significantly

? GCN5 makes a positive modest contribution to
the expression of most of the genes ? HAT
activity of GCN5 plays a redundant or minor role

• rbp1-1 gene expression without pol II activity

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SPT3 mutants and TAF1 mutants

• spt3? and spt3E240K strains do not differ
  substantially from wild type

? spt3 plays a small or redundant role

• Taf1ts2 strain causes leftward shift of the
  distribution
• 84 of the genome decreased depression by gt4
  stds
• But population shift is not as severe as for
  rbp1-1

? taf1 makes a positive contribution to genes
expression, but its action is not absolute
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GCN5/TAF1 double mutants

• TAF1/GCN5 double mutants shift the population
  like rbp1-1
• TAF1 and GCN5? interaction is expected
• GCN5hat sensitivity suggests that HAT activity of
  GCN5 is important when TAF1 is absent

? GCN5 makes a positive modest contribution to
the expression of most of the genes ? HAT
activity of GCN5 plays a redundant or minor role
? GCN5 and TAF1 are associated to the same HAT
activity
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SPT3 mutants and TAF1 mutants
? spt3 plays a small or redundant role

• 97 of genes in the TAF1/SPT3? mutant decreased
  by gt4 stds nearly complete shutdown of
  transcription

? both TAF1 and SPT3 contribute to the expression
of all measurable genes ? SAGA and TFIID are the
only redundant complexes in transcriptional
general activity
? taf1 makes a positive contribution to genes
expression, but its action is not absolute
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Conclusions

1. TFIID and SAGA each contribute to the expression
   of nearly all genes

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TFIID or SAGA dominated genes

• TFIID-dominated genes 90 that showed greater
  dependency on TAF1 than SPT3
• SAGA-dominated genes 10 that are SPT3-dependent
• TAF1 appears to be inactive in SAGA-dominated
  genes

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Conclusions

1. TFIID and SAGA each contribute to the expression
   of nearly all genes
2. TFIID dominates at 90 of all genes, and SAGA
   Dominates at 10

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Stress-Induced Genes Tend to be SAGA Dominated

• Enrichment in stress-dependent conditions
• Genes that are commonly upregulated biased
  (p_value lt 10-30) to SAGA-dominated
• Genes that are commonly downregulated biased
  (p_value lt 10-10) to TFIID-dominated
• What advantage might SAGA provide in
  environmental stress response that TFIID does not?

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Conclusions

• TFIID and SAGA each contribute to the expression
  of nearly all genes

SAGA TFIID
10 90
Stress-Induced Stress-Repressed


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Histone Acetylation

• Acetylation of H3 and H4 is associated with
  transcriptional activation
• H4 under-acetylated regions were biased to
  SAGA-dominated genes whereas H4 overacetylated
  regions biased to TFIID-dominated genes
• Hda1 and Rpd3 appear to assist in keeping low
  acetylation at SAGA-dominated genes and high
  acetylation at TFIID-dominated genes

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Conclusions

• TFIID and SAGA each contribute to the expression
  of nearly all genes

SAGA TFIID
10 90
Stress-Induced Stress-Repressed
H4 high acetylation pattern Low stress

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SAGA-dominated genes are largely TAF-independent

• TAFs (TBP Associated Factors) are subunits of
  TFIID but a subset are also present in SAGA
• Genes that are positively regulated by TAFs were
  biased toward the TFIID-dominated class
• TAF-independent promoters are
  likely to be
  SAGA-dominated

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SAGA-dominated genes are highly regulated

• How do other transcription factors function?
• SRB10 phosphorilates a number of stress response
  regulators. Genes that are most inhibited by
  SRB10 are SAGA-dominated
• Genes regulated by stress activators Msn2 and
  Msn4 are also SAGA-dominated

?There is a coordinated stress response pathway
that is up-regulated by gene-specific activators
like Msn2/4 and down-regulated by Srb-10 -
regulated phosphorylation and histone
de-acetylation
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SAGA-dominated genes are Coordinately Regulated
Stress-induced gened
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Summary

• TFIID and SAGA each contribute to the expression
  of nearly all genes

SAGA-dominated genes TFIID-dominated genes
10 90
Stress-Induced Stress-Repressed
H4 high acetylation pattern Low stress
Coordinately and tightly regulated TAF-dependent
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Two distinct mechanisms