Transcript RNA Polymerase II mediated modifications - CS

RNA Polymerase II mediated
modifications
What RNA Pol II does to histones
and why?
Asaf Peer
Seminar in Computational Biology, 2007
histones
• Histones pack the
DNA
• exposed to
posttranslational
modifications
• some modifications
are abundant than
other
Histones
• The existence of a “histone code” is
controversial
• modifications marks heterochromatin,
especially Methylation of K9, K27 of H3
• marks active genes with acetylation in the
promoter region
• Phosphorylation of ser10 H3 correlates
with mitosis
Transcription Initiation
Structure and mechanism of the RNA polymerase II transcription machinery
Steven Hahn. Nature Structural & Molecular Biology 11, 394 - 403 (2004)
Transcription Elongation
http://www.smbs.buffalo.edu/bch/images/figures/ponticelli_fig.jpg
Modifications in Elongation
• Control Chromatin remodeling
• Recruitment of elongation and termination
factors
• Prevent initiation from within the gene
Chromatin remodeling
•
•
Evidence:
- Chromatin lost in highly transcribed
genes
- accumulation of H3.3 in highly
transcribed genes
Finding:
- histones are evicted and replaced
during elongation
Jerry L. Workman Genes Dev. 2006; 20: 2009-2017
Chromatin remodeling
Jerry L. Workman Genes Dev. 2006; 20: 2009-2017
Chromatin remodeling
H3/H4
H2A/H2B
RNA Pol II
Ac
Ac
Ac
spt6
Ac
spt16
spt16
spt16
spt16
spt6
Ac
spt6
spt6
spt16
Ac
spt16
spt16
spt6
Ac
spt16
Histone Acetylation
• Acetylation may facilitate the disassembly
of nucleosomes
• It may also facilitate their reassembly
• Acetylation is done using Gcn5 and Elp3
• In hypoacetylated genes expression level
is reduced
• Mutations in spt6/spt16 with mutations in
gcn5/elp3 has an alleviating effect
Svejstrup Q. Jesper Biochimica et Biophysica Acta 1677 (2004) 64-73
Elongator
Pi
Pi
Pi Gcn5
Pi
Elp3
RNA Pol II
Ac
Elongator
H3K36 Methylation
• Lys 36 of Histone H3
is hypermethylated
• apparently, set2 has
to do with the
methylation
Bhargavi Rao
et. al. Mol Cell Biol. 2005 25(21): 9447–9459.
Nevan J. Krogan et. al. Mol Cell Biol. 2003 23:4207-4218
Set2 and Ser2 Phosphorylation
• Deleting CTK1 prevents set2 recruitment
• CTK1 replaces ser5 Phosphorylation with ser2
Phosphorylation
• Methylation is higher at the 3’ region of genes
Svejstrup Q. Jesper Biochimica et Biophysica Acta 1677 (2004) 64-73
The role of H3K36 Methylation
Conflict
Histone deAcetylation
• Rpd3- histone deacetylase
• found in the complexes Rpd3(L) and
Rpd3(S)
• The complexes share the proteins Rpd3,
Sin3 and Ume1
• What are the differences between the two
complexes?
Histone H3 methylation by Set2
directs deacetylation of coding
regions by Rpd3S to suppress
spurious intragenic transcription.
Michael J. Carrozza, Bing Li, Laurence Florens, Tamaki
Suganuma, Selene K. Swanson, Kenneth K. Lee, WeiJong Shia, Scott Anderson, John Yates, Michael P.
Washburn and Jerry L. Workman
Cell. 2005 123(4):581-92.
Histone deAcetylation
Jason D. Lieb and Neil D. Clarke cell 2005 123:1187-1190
deacetylation
wt dep1Δ eaf3Δ rco1Δ set2Δ sin3Δ eaf3chdΔ
wt dep1Δ eaf3Δ rco1Δ set2Δ sin3Δ eaf3chdΔ
wt dep1Δ eaf3Δ rco1Δ set2Δ sin3Δ eaf3chdΔ
Michael J. Carrozza et. al. cell 2005 123:581-592
H3K36 mutant
WT
Michael J. Carrozza et. al. cell 2005 123:581-592
H3K36A
set2Δ
acetylation at promoter
wt
dep1Δ
eaf3Δ
rco1Δ
wt
Michael J. Carrozza et. al. cell 2005 123:581-592
set2Δ
dep1Δ
sin3Δ
eaf3Δ
rco1Δ
set2Δ
sin3Δ
Preventing initiation from cryptic
TATA box
Spt6 mutant
deacetylation mutants
Michael J. Carrozza et. al. cell 2005 123:581-592
Craig D. Kaplan, Lisa Laprade and Fred Winston science 2002 301:1096-1099
Preventing initiation from cryptic
TATA box
promoter
5’
ORF
3’
Histone Acetylation
Initiation
Elongation
Histone disassembly
Histone deacetylation
Histone reassembly
Histone 3 K36 Methylation
H3K4 Methylation and H2B
Ubiquitylation
Histone H2B Ubiquitylation Is
Associated with Elongating RNA
Polymerase II
Tiaojiang Xiao, Cheng-Fu Kao, Nevan J. Krogan, Zu-Wen
Sun, Jack F. Greenblatt, Mary Ann Osley, and Brian D.
Strahl
Mol.Cell biol. 2005 25(2):637-651
H2B Ubiquitylation
• H2B goes ubiquitylation in transcribed
genes at lysine 123
• It has to do with H3 methylation on lysine
4 and 79
• In the promoter region H2B goes
ubiquitylation which is rapidly removed by
ubp8
H2B Ubiquitylation
Tiaojiang Xiao et.al. Mol Cell Biol. 2005 25(2): 637–651
Pi
Leo1
Pi
Pi
Pi
Paf1
Ctr9
RNA Pol II
CDC73
Rtf1
Rad6
Ub
Rad6 directly interacts with
Elongating RNA Pol II
Ser5 Phosphorylated
CTD (5’)
Ser2 Phosphorylated
CTD (3’)
Unphosphorylated CTD
Rad6 interacts only with
phosphorylated CTD of RNA Pol II
and throughout the elongation process
Tiaojiang Xiao et.al. Mol Cell Biol. 2005 25(2): 637–651
The effect of Ubiquitylation
Bre1 recruits Rad6 to the promoter
Tiaojiang Xiao et.al. Mol Cell Biol. 2005 25(2): 637–651
Ubiquitylation permits Methylation
Tiaojiang Xiao et.al. Mol Cell Biol. 2005 25(2): 637–651
Elongation- Gal1 paradigm
H3K4 Methylation
• Lysine 4 of Histone 3 is being Methylated
by set1
• set1 interacts with Ser5 phosphorylated
CTD RNA Pol II
• Therefore, H3K4 is hypermethylated at the
5’ regions of active genes
• As we will see, some factor binds di and tri
Methylated H3K4 residues
Methylation of Histone H3 K4
Mediates Association of the
Isw1p ATPase with Chromatin
Helena Santos-Rosa, Robert Schneider, Bradley E.
Bernstein, Nickoletta Karabetsou, Antonin Morillon,
Christoph Weise, Stuart L. Schreiber, Jane Mellor
and Tony Kouzarides
Molecular Cell 2005,12:1325-1332
Isw1 binds Methylated H3K4
Nevertheless, Isw1 can bind chromatin in alternative ways!
Helena Santos-Rosa et. al. Mol Cell 2003 12:1325-1332
Set1 and Isw1- the MET16
paradigm
Helena Santos-Rosa et. al. Mol Cell 2003 12:1325-1332
What goes wrong?
Poly(A) mRNA
level
Helena Santos-Rosa et. al. Mol Cell 2003 12:1325-1332
What goes wrong?
RNAPII in the mutant is more abundant in the 3’ region
maybe the termination process gets stuck?
Helena Santos-Rosa et. al. Mol Cell 2003 12:1325-1332
What goes wrong?
ChIP of the termination
factor Rna15p
Helena Santos-Rosa et. al. Mol Cell 2003 12:1325-1332
H3K4 methylation- summary
MeMe Me
Isw1
?
Set1 methylates H3K4
?
?ORF
?
?
Isw1 binds methylated H3K4
Recruitment of ployadenylation
factors and termination factors
For ending- a mystery
H3K9Me- What are you saying?
Histone H3 Lysine 9 Methylation
and HP1γ Are Associated with
Transcription Elongation through
Mammalian Chromatin
Christopher R. Vakoc, Sean A. Mandat, Benjamin A.
Olenchock and Gerd A. Blobel
Molecular Cell 2005,19(3):381-391
H3K9 Methylation
• H3K9 Methylation is known to mark
heterochromatin
• HP1 protein binds di and tri-methyl H3K9
and takes a part in gene silencing
• In Mammals, 3 types of HP1 are found:
HP1α HP1β and HP1
H3K9 Methylation
• G1E cells were used
• These cells are arrested in their
differentiation due to lack in GATA-1
transcription factor
• GATA-1 fused to Estrogen receptor was
expressed
• Estrogen controls the differentiation and
makes it fast and synchronized
H3K9 Methylation
Vakoc et. al. Mol Cell 2005 19:381-391
H3K9 Methylation
Vakoc et. al. Mol Cell 2005 19:381-391
HP1 binding
TriMethyl H3K9
Vakoc et. al. Mol Cell 2005 19:381-391
HP1
IgG (control)
Conclusions
• Acetylation is important for Chromatin
disassembly and reassembly
• Methylation of H3K36 is larger in the 3’ region of
genes and important for deAcetylation
• Ubiquitylation of H2B is throughout the gene and
permits H3K4 and H3K79 Methylation
• H3K4 involves with positioning of Ist1 and has to
do with proper poly-Adenylation and termination
• H3K9 Methylation is observed in active genes
thought its reason is not yet clear
Have a nice Post-snow day!