Download PolII Initiation TFIID and SAGA

TFIID and SAGA roles
in transcription machinery
Inna Weiner
Reading group in Computational Molecular Biology
16/11/06
Global structure of RNA
polymerase II promoters
TSS
Core promoter
Proximal promoter
Enhancer/silencer
The core promoter
(Butler and Kadonaga, 2002)
How to recruit RNA Polymerase?
Assembly of general transcription factors is
required to recruit RNA Polymerase to promoters
A9.30
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.
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
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
Genetic Interactions
∆Y
∆X
w.t.
X
X
Y
∆X Y<∆X*∆Y
Aggravating
interaction
Slide by Ariel
Y
∆X Y
∆X Y
∆X Y=∆X*∆Y
No
interaction
∆X Y
X Y>∆XY *∆Y
∆X
Alleviating
interaction
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 highthroughput analysis
GCN5 mutants
• 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
rbp1-1: gene expression without pol II activity
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
• TAF1/GCN5 double mutants
shift the population like rbp1-1
GCN5and
makes
a positive
• TAF1
GCN5Δ
interaction is
expected
modest
contribution to the
•expression
GCN5hat sensitivity
of most of suggests
the genes
that HAT activity of GCN5 is
when
TAF1 is
absent
 important
HAT activity
of GCN5
plays
a
redundant or minor role
 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
 both TAF1 and SPT3 contribute to the expression of all
measurable
genes
 taf1 makes
a positive
contribution
genesare
expression,
 SAGA andtoTFIID
the only redundant complexes in
but
its action isgeneral
not absolute
transcriptional
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 SAGAdominated 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?
Conclusions
1. TFIID and SAGA each contribute to the
expression of nearly all genes
TFIID
SAGA
90%
10%
Stress-Repressed
Stress-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 TFIIDdominated genes
• Hda1 and Rpd3 appear to assist in
keeping low acetylation at SAGAdominated genes and high acetylation at
TFIID-dominated genes
Conclusions
1. TFIID and SAGA each contribute to the
expression of nearly all genes
TFIID
SAGA
90%
10%
Stress-Repressed
Stress-Induced
Low H4 high acetylation
stress
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 TFIIDdominated 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
Summary
1. TFIID and SAGA each contribute to the
expression of nearly all genes
TFIID-dominated
genes
90%
SAGA-dominated
genes
10%
Stress-Repressed
Stress-Induced
Low H4 high acetylation
stress
pattern
TAF-dependent
Coordinately and
tightly regulated
Two distinct mechanisms