tfiid and saga roles in transcription machinery
DESCRIPTION
TFIID and SAGA roles in transcription machinery. Inna Weiner Reading group in Computational Molecular Biology 16/11/06. Core promoter. Proximal promoter. Enhancer/silencer. Global structure of RNA polymerase II promoters. TSS. The core promoter. (Butler and Kadonaga, 2002). - PowerPoint PPT PresentationTRANSCRIPT
TFIID and SAGA rolesin transcription machinery
Inna WeinerReading group in Computational Molecular Biology
16/11/06
Enhancer/silencer
Global structure of RNA polymerase II promoters
Proximal promoter
Core promoter
TSS
The core promoter
(Butler and Kadonaga, 2002)
A9.30
How to recruit RNA Polymerase?
Assembly of general transcription factors is required to recruit RNA Polymerase to promoters
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.
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
TBP
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
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.
Pol II starts mRNA synthesis
Very few factors accompany the elongating transcript
Summary
Localization to promoters
Melting the DNA, transcription initiation and elongation
Is the general transcription machinery really general?
(Holstege et al., 1998)
Outline
• Introduction to general transcription machinery
• TFIID and SAGA – unique or redundant function?
• TFIID and SAGA roles in transcription regulation
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?
Percentage of genome dependent on subunits of SAGA and TFIID
Whole genome analysis of shared TAFIIs mutations
Shared vs Specific TAFIIs Influence
~70% of the genome depends on one or more of the shared
TAFs
~30% of the genome is dependent on TFIID specific sub-units
~12% of the genome is dependent on SAGA specific sub-units
How do TFIID and SAGA interact?
TFIID and SAGA have compensatory functions
w.tw.t..
Genetic Interactions
∆∆YY ∆∆XX
∆∆X YX Y
∆∆X Y=∆X*∆YX Y=∆X*∆Y∆∆X YX Y
∆∆X Y>∆X *∆YX Y>∆X *∆Y
∆∆X YX Y
∆∆X Y<∆X*∆YX Y<∆X*∆Y
Aggravating Aggravating
interactioninteraction
Alleviating Alleviating
interactioninteraction
No No interactioninteraction
YYXX
XX
YY
YYXX
Slide by Ariel
How do TFIID and SAGA interact?
TFIID and SAGA have compensatory functions
Conclusions
• There are distinct requirements for specific sub-units of TFIID and SAGA in global expression
• The functions of TAFII145 and GCN5 are redundant
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
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
GCN5 mutants
rbp1-1: gene expression without pol II activity
• gcn5Δ strain displayed a general decrease in expression
• Over 60% of the genes decreased expression by >4 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
SPT3 mutants and TAF1 mutants• spt3Δ 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 >4 std’s
• 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
GCN5/TAF1 double mutants
GCN5 makes a positive modest contribution to the expression of most of the genes
HAT activity of GCN5 plays a redundant or minor role
• TAF1/GCN5 double mutants shift the population like rbp1-1
• TAF1 and GCN5Δ interaction is expected
• GCN5hat sensitivity suggests that HAT activity of GCN5 is important when TAF1 is absent
GCN5 and TAF1 are associated to the same HAT activity
SPT3 mutants and TAF1 mutants
spt3 plays a small or redundant role
• 97% of genes in the TAF1/SPT3Δ mutant decreased by >4 std’s: nearly complete shutdown of transcription
taf1 makes a positive contribution to genes expression, but its action is not absolute
both TAF1 and SPT3 contribute to the expression of all measurable genes
SAGA and TFIID are the only redundant complexes in transcriptional general activity
Conclusions
1. TFIID and SAGA each contribute to the expression of nearly all genes
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
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%
Stress-Induced Genes Tend to be SAGA Dominated
• Enrichment in stress-dependent conditions– Genes that are commonly
upregulated biased (p_value < 10-30) to SAGA-dominated
– Genes that are commonly downregulated biased (p_value < 10-10) to TFIID-dominated
• What advantage might SAGA provide in environmental stress response that TFIID does not?
Conclusions1. TFIID and SAGA each contribute to the
expression of nearly all genes
TFIIDSAGA90%10%
Stress-RepressedStress-Induced
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
Conclusions1. TFIID and SAGA each contribute to the
expression of nearly all genes
TFIIDSAGA90%10%
Stress-RepressedStress-InducedLow
stressH4 high acetylation
pattern
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
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
SAGA-dominated genes are Coordinately Regulated
Stress-induced gened
Summary1. TFIID and SAGA each contribute to the
expression of nearly all genes
TFIID-dominated genes
SAGA-dominated genes
90%10%
Stress-RepressedStress-InducedLow
stressH4 high acetylation
patternTAF-dependentCoordinately and
tightly regulated
Two distinct mechanisms