Nielsen, S.J. et al. Rb targets histone H3 methylation and HP1 to promoters. Nature 412, 561-565

Article (PDF Available)inNature 412(6846):561-5 · September 2001with64 Reads
DOI: 10.1038/35087620 · Source: PubMed
Abstract
In eukaryotic cells the histone methylase SUV39H1 and the methyl-lysine binding protein HP1 functionally interact to repress transcription at heterochromatic sites. Lysine 9 of histone H3 is methylated by SUV39H1 (ref. 2), creating a binding site for the chromo domain of HP1 (refs 3, 4). Here we show that SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma (Rb) protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter, and in fibroblasts that are disrupted for SUV39, the activity of the cyclin E and cyclin A2 genes are specifically elevated. Chromatin immunoprecipitations show that Rb is necessary to direct methylation of histone H3, and is necessary for binding of HP1 to the cyclin E promoter. These results indicate that the SUV39H1-HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other co-repressor proteins.
Rb targets histone 3 methylation
and HP1 to promotors
S.J. Nielsen, R. Schneider, U. Bauer, A. J. Bannister, A. Morrison, D.
O‘Carrol, R. Firesteink, M. Clearyk, T. Jenuwein R.E. Herrera, T. Kouzarides
2001, letters to nature
MBE
18. September 2007
BONIFERT, Tobias
YEDIKAPU, Mine
Overview
Introduction
Experiments
Conclusions
Comment
2
Comment
Introduction
Retinoblastoma-(Rb)-Protein was
discovered in context with
retinoblastoma disease
3
Introduction
Rb regulates cellcycle by
binding and repression of
transcription-regulating
proteins
In presence of mitogen
activity the Rb
-
protein stops
4
activity the Rb
-
protein stops
its inhibiting function which
leads to cell proliferation
cancer
Some of the Rb-associated
proteins necessary for
repression are situated on
histones.
Introduction
Deacetylated histones have a negative effect on the
transcription rate
Rb recruits this deacetylase activity to repress
5
Does the Rb-protein influence
histone-methylase activity?
transcription
Histone-methylase activity also leads to a diminished
transcription rate
Pull down
Prey protein = histone methylase
+ Rb-GST
GST fusion proteins
Immobilized on glutathione-
sepharose columne
H3-derived peptides bound to
Sulfonic beads
GST
GST
Rb
Incubation
Chromatograpy
6
Rb captures prey protein while
Incubation
Isolation of Tagged particles by
Chromatography
SDS-Page gelelectrophoresis
separation of captured proteins
GST
GST-Rb fusion can purify histone
methylase activity
samples purified with GST and
GST-Rb are added to
H3, H4 and GAR
For visualization of methylase
activity at H3, H4 or GAR,
radioactive marked CH3 is added
7
radioactive marked CH3 is added
--> autoradiography
Rb associated methylase activity
is not a GAR dependent arginine
methylase --> it must be a lysine
methylase.
H3
DNA
Nucleosome
Met
Methylase
H3
8
Where at H3 happens the methylation?
Rb
Methylation site of H3
Edman degradation
of radioactively methylated histone H3
9
methylation at lysine 9 of histone H3
H3
DNA
Nucleosome
Met
Methylase
H3
Lys 9
10
Which is the methylase associated with Rb?
Rb
SUV39H1 - The Rb associated methylase
Hints
possesses methylase activity
conserved in its SET domain
has specifity for lysine 9 of
Histone H3
11
Results
GST-Rb fusion can bind
haemagglutinin(HA)-tagged
SUV39H1
Methods
transfection of HEK-293 cells
with HA-SUV39H1 or empty
expression vector (Mock)
extracts incubated with GST or
GST-Rb --> Pull-down
Bound SUV39H1 western
blotted using anti-HA antibody
H3
DNA
Nucleosome
Met
SUV39H1
H3
Haemagglutinin (HA)
Rb
GST
Methylase
12
Rb
Acts SUV39H1 as a co-repressor with Rb?
Lys 9
SUV39H1 - An Rb co-repressor
Methods
HeLa cells transfected with
reporter, containing the cyclin E
promotor driving luciferase
together with combinations of
expression vectors for E2F1, Rb,
SUV39H1 and SUV39H1∆SET
Results
13
Results
cyclin E promoter is
stimulated by E2F
SUV39H1 is a co-repressor
Rb represses E2F activity slightly
SUV39H1 + Rb leads to further
repression of E2F activity
SUV39H1 with deleted SET-motif
(SUV∆SET) does not lead to
repression
Does a lack of the co-repressor SUV39H1 lead to
elevated amount of mRNA?
14
elevated amount of mRNA?
Repressive functions of SUV39H1
Methods
Double-knockout of genes for SUV39H1
and related SUV39H2 in mice fibroblasts
RT-PCR
15
Rb repressor needs SUV39H1 to repress
cyclin E and cyclin A genes
In double knockout cells cyclin E and A
mRNA amounts are elevated
Genes Cdc25, HPRT, GAPDH are not
regulated by Rb and show no differences
compared to wild-type
Is there another factor in the Rb-SUV39H1
interaction involved?
16
interaction involved?
The role of HP1
Hints
SUV39H1 is known to be in a complex with the HP1 protein
HP1 function downstream of SUV39H1
HP1 recognizes methylated Lys9 on histone H3
17
Methods
Co-immunoprecipitation
Westernblott
Co-immunoprecipitation
Rb/HP1-
Complex (?)
solution
Anti-Rb
Anti-HP1
Western-blotting
+ Anti Rb
+ Anti HP1
Precipitation
18
Precipitation of
HP1-X +?
Precipitation of
Rb-X + ?
HP1-X
Rb-X
Rb and HP1 form a complex
?
HP1 associates with Rb
Results
co-immunoprecipitation proves that HP1
and Rb form a complex
HP1 contains an LXCXE motif
competes for the methylase binding to Rb
19
competes for the methylase binding to Rb
but does not effect its own binding to the
methylase
Recognizing Met and Rb
In a further step, the
scientists proved by
using recombinant
Rb as well as
recombinant HP1,
H
DNA
Nucleosome
Met
HP1
20
recombinant HP1,
that HP1 can
recognize the
methylated Lys9 of
H3 and Rb at the
same time.
H
3
Rb
H3
SUV39H1
Lys 9
Are Rb regulated Promoters associated with
21
Are Rb regulated Promoters associated with
HP1?
DNA
Nucleosome
Chromatin-Immunoprecipitation
22
H
3
H3
Antibody to recognize
Lys 9 methylated H3
Cyclin Epr
HP1
in the presence of
Rb, methylase
activity and HP1 are
targeted to the cyclin
E promoter.
E2F
Conclusion
Rb binds to transcription factors which drive specific genes.
When Rb deacetylates lysine 9 of histone H3, the methylase SUV39H1
is able to methylate this amino acid.
The Rb/SUV39H1
-
complex is able to increase Rb
-
induced repression
23
The Rb/SUV39H1
-
complex is able to increase Rb
-
induced repression
activity.
After the Rb/SUV39H1-induced Methylation of Lysine 9, the Protein
HP1 can Bind to the complex.
Although the role of HP1 is not sufficiently clear, the experiments
showed that HP1, linked to the Rb/SUV39H1 -complex is involved in
Gene-repression.
Comment
Introduction is missing.
Unsufficient explanation of used methods
24
No significance tests
references
B. Alberts et al. (2002): Molecular biology of the cell, 4. edition, Garland
Science, New York
M.A. Becky Golat et al. (2004): In Vitro His
-
Tag Pull
-
Down Assay MagZ
25
M.A. Becky Golat et al. (2004): In Vitro His
-
Tag Pull
-
Down Assay MagZ
Particles. Promega Notes, 88
http://www.eyecancer.com/Doctor/Gallery.aspx?nID=4&Category=Retinal
+Tumors
http://www.ncbi.nlm.nih.gov/SCIENCE96/chr.cgi?13
Thank you for listening.
    • "Since Suv39H1 is one of the proteins that show dynamic interaction with heterochromatin regions and some inactive promoters of silenced genes (25,41–43), we next assessed whether phosphorylation of Suv39H1 affects its binding capacity to heterochromatin regions using a ChIP assay. Under asynchronous growth conditions, phospho-mimic Suv39H1 (Suv39H1-S391E) had decreased binding affinity to heterochromatin regions relative to wild-type or phospho-defective Suv39H1 (S391A) proteins in both transiently and stably expressing cells (Figure 3A and B, Supplementary Figure S7), suggesting that phospho-mimic Suv39H1 protein preferentially dissociates from heterochromatin regions. "
    [Show abstract] [Hide abstract] ABSTRACT: Although several studies have suggested that the functions of heterochromatin regulators may be regulated by post-translational modifications during cell cycle progression, regulation of the histone methyltransferase Suv39H1 is not fully understood. Here, we demonstrate a direct link between Suv39H1 phosphorylation and cell cycle progression. We show that CDK2 phosphorylates Suv39H1 at Ser391 and these phosphorylation levels oscillate during the cell cycle, peaking at S phase and maintained during S-G2-M phase. The CDK2-mediated phosphorylation of Suv39H1 at Ser391 results in preferential dissociation from chromatin. Furthermore, phosphorylation-mediated dissociation of Suv39H1 from chromatin causes an enhanced occupancy of JMJD2A histone demethylase on heterochromatin and alterations in inactive histone marks. Overexpression of phospho-mimic Suv39H1 induces early replication of heterochromatin, suggesting the importance of Suv39H1 phosphorylation in the replication of heterochromatin. Moreover, overexpression of phospho-defective Suv39H1 caused altered replication timing of heterochromatin and increases sensitivity to replication stress. Collectively, our data suggest that phosphorylation-mediated modulation of Suv39H1-chromatin association may be an initial step in heterochromatin replication.
    Full-text · Article · Apr 2014
    • "However, they can do it both directly and indirectly, and in a very broad scale, recruiting co-repressors/activators to specific transcription factors. Recent studies propose Rb proteins to bind co-repressors: histones deacetylases (HDAC1, HDAC2) (56–58), histone de-methylases (RBP2) (59), DNA methyl transferases (DNMT1) (60), helicases (Brg1, Brm) (61, 62), histone methyl transferases (Suv39h1, RIZ, suv4-20h1/h2) (63–65), and histone binding proteins, like HP1, regulating chromatin structure and transcription (63, 66). In this context, the interaction with E2F directs pocket proteins to DNA domains leads to transcriptional repression. "
    [Show abstract] [Hide abstract] ABSTRACT: The Rb1 gene was the first bona fide tumor suppressor identified and cloned more than 25 years ago. Since then, a plethora of studies have revealed the functions of pRb and the existence of a sophisticated and strictly regulated pathway that modulates such functional roles. An emerging paradox affecting Rb1 in cancer connects the relatively low number of mutations affecting Rb1 gene in specific human tumors, compared with the widely functional inactivation of pRb in most, if not in all, human cancers. The existence of a retinoblastoma family of proteins pRb, p107, and p130 and their potential unique and overlapping functions as master regulators of cell cycle progression and transcriptional modulation by similar processes, may provide potential clues to explain such conundrum. Here, we will review the development of different genetically engineered mouse models, in particular those affecting stratified epithelia, and how they have offered new avenues to understand the roles of the Rb family members and their targets in the context of tumor development and progression.
    Full-text · Article · Dec 2013
    • "Global gene expression analysis comparing rag2-hKRASG12D/rag2-hSUV39H1 with rag2-hKRASG12D/rag2-mCherry 7dpf larvae revealed that cyclin B1 is downregulated in the SUV39H1-overexpressing tumors, which was confirmed by qPCR. SUV39H1 is already known to silence cyclins E and A [30]. Silencing of cyclin B1 may explain how SUV39H1 promotes senescence and growth arrest. "
    [Show abstract] [Hide abstract] ABSTRACT: Epigenetics, or the reversible and heritable marks of gene regulation not including DNA sequence, encompasses chromatin modifications on both the DNA and histones and is as important as the DNA sequence itself. Chromatin-modifying factors are playing an increasingly important role in tumorigenesis, particularly among pediatric rhabdomyosarcomas (RMS), revealing potential novel therapeutic targets. We performed an overexpression screen of chromatin-modifying factors in a KRAS(G12D)-driven zebrafish model for RMS. Here, we describe the identification of a histone H3 lysine 9 histone methyltransferase, SUV39H1, as a suppressor of embryonal RMS formation in zebrafish. This suppression is specific to the histone methyltransferase activity of SUV39H1, as point mutations in the SET domain lacked the effect. SUV39H1-overexpressing and control tumors have a similar proliferation rate, muscle differentiation state, and tumor growth rate. Strikingly, SUV39H1-overexpressing fish initiate fewer tumors, which results in the observed suppressive phenotype. We demonstrate that the delayed tumor onset occurs between 5 and 7 days post fertilization. Gene expression profiling at these stages revealed that in the context of KRAS(G12D) overexpression, SUV39H1 may suppress cell cycle progression. Our studies provide evidence for the role of SUV39H1 as a tumor suppressor.
    Full-text · Article · May 2013
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