Suv39h1 and Suv39H1 possess histone methyltransferase (HMTase) activity towards histone H3 lysine 9 (H3K9) through their conserved SET domains. Point mutations in the catalytic motif of the SET domain abolish this HMTase activity. Suv39h1 specifically mono-, di- and trimethylates H3K9 in vitro but not other histone sites or proteins. Prior modifications of H3K9 like acetylation or serine 10 phosphorylation inhibit Suv39h1 HMTase activity at this site. Suv39h1 plays a role in chromatin structure and gene silencing through this site-specific H3K9 methylation in native

1. Regulation of chromatin structure by site
specific histone H3 methyltransferases
Stephen Rea*, Frank Eisenhaber*, Do nal O'Carroll*, Brian D. Strahl², Zu-Wen Sun², Manfred
Schmid*, Susanne Opravil*, Karl Mechtler*, Chris P. Ponting³, C. David Allis² & Thomas Jenuwein
Presented by- Budha Ratna Rav
MP17001
NATURE |VOL 406 | 10 AUGUST 2000 |www.nature.com © 2000 Macmillan Magazines
* Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, Dr. Bohrgasse 7, A-1030 Vienna, Austria
² Department of Biochemistry and Molecular Genetics, University of Virginia Health Science Center, Charlottesville, Virginia 22908, USA
³ MRC, Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, OX1 3QX, UK

2. Contents
• Introduction of Chromatin structure.
• Position effect variegation (PEV).
• Genes function as nucleosome modification.
• SET domain Alignment.
• SUV39H1 and Suv39h1 posses HMTase activity.
• HMTase activity is an intrinsic property of Suv39h1.
• Catalytic motif of SET domain.
• Introduction of point mutation in conserved motif.
• Specific methylation of H3 by Suv39h1 HMTase.
• Relation between Lysine9 methylation and serine10 phosphorylation.
• Role of site specific histone H3 methyltransferase(Suv39h) in native chromatin.
• Conclusion

3. Introduction of Chromatin structure.
• Chromatin= DNA + Histone
proteins.
• Covalent modification of histone
tails regulate transcriptional ‘ON’
or ‘OFF’ states.
• Post- translational modifications
of histone N-termini (H4 & H3)
are well documented for
acetylation, methylation,
phosphorylation.

4. Position effect variegation (PEV) Modification
• PEV is a Heterochromatin induced
gene inactivation.
• Discovered by H.J Muller in
Drosophila.
• e.g.- The inversion on the X
chromosome of w+ gene is get
silenced in some cells due to
Heterochromatin induced the w+
gene inactivation.

5. Heterochromatic associated proteins
• Humans (SUV39H1) and mice (Suv39h1) encode
heterochromatic proteins.
• SUV39H1 have two evolutionary conserved domain of
chromatin regulator that is Chromo & SET domains.
• Chromo domain: Made up of 60 A.A residues that directs
heterochromatic or euchromatic localization and at that
time SET role unknown.
Structure of SUV39H1

6. SUV39H1 (SET domain) alignment
• SET domain of SUV39H1 were
performed multiple sequence alignment
(MSA).
Sequence and 2° structure of protein is
similar to six plant methyltransferases.
These plant sequences shows Histone
lysine-N-methyltransferase activity.

7. SUV39H1 HMTase activity
HeLa cell(type of tumor cells) lines which expressing the triple myc-
tagged full length SUV39H1 (residues 3-412).
Isolated SUV39H1 proteins by immunoprecipitation with anti myc-
beads.
Subjected to in-vitro methylation assay by using SAM probed with
fluorescent dye and free histones in the reaction mixture.
Performed the SDS-PAGE and visualizes by fluorography.
NOTE-When SUV39H1(residues 3-118) no HMTase activity were
observed.
RESULT:SUV39H1 posses SET-domain dependent HMTase activity.

8. HMTase activity is SET-dependent only
Performed the invitro-HMTase reaction again with
recombinants protein Suv39h1 that were purified as
GST-fusion proteins from E.coli.
Performed the SDS-PAGE and visualize by
fluorography.
Result:SUV39H1-HMTase activity is SET domain
dependent.

9. Catalytic motif of SET domain
• SUV1(82-412) similar to human is
catalytically active.
• Introduces the short internal deletions
into the conserved region of SET domain
and also generated mutant that lack the
SET associated cysteine rich regions.
• Observation: None of the mutant proteins
HMTase activity.
• Result: Cysteine rich region is important
for HMTase activity.

10. Introduction of point mutation in conserved motif
• Highly conserved motif of SET domain is
320HffNHSC326 motif (where f are hydrophobic
residues) of GST±SUV1(82±412).
• Point mutation of histidine 324 to leucine (H324L or
lysine (H324K), and also changed cysteine 326 to
alanine (C326A) or replaced histidine 320 by arginine
(H320R).
• In vitro HMTase assays showed that all the point
mutations, except H320R, inhibit enzymatic activity.
• In H320R mutation resulted in an hyperactive enzyme
with activity increased 20-fold or more.
• Result: 320HffNHSC326 motif of SET domain is
important for HMTase activity.

11. Specific methylation of H3 by Suv39h1 HMTase
• Performed invitro methylation
assay with individual histones.
• RESULT- No signal were
detected with H2A, H2B, H4
but signal were observed by
H3.
• Note- Weak signal also
detected with H1.

12. Site-specific methylation of Suv39H1 HMTase in H3
• Performed Invitro methylation assay:
Sr.N Reaction mixture with
peptide
Result
1 H3 unmodified amino acid
residues from (1-20) N-
terminal
Methylated
2 H3 modified (mutated K9 to
L9) amino acid residues from
(1-20) N-terminal
No methylation
3 Insulin peptides No methylation
4 Macro H2A No methylation
5 CENP-A No methylation
RESULT-Suv39H1 HMTase activity at K9 position of the H3.

13. Relation between Lysine9 methylation and serine10 phosphorylation
• Performed the in-vitro HMTase assay with GST-SUV1(82-412)
and H3(N-terminal peptides) with unmodified, phosphorylated
at S-10, di-methylated at K9 or Acetylated at K9.
• RESULT- SUV39H1- dependent methylation of unmodified
peptide as 100%.
• K9 di-methylated shows compromised enzymatic tri-
methylated activity.
• K9 acetylation peptide was not substrate.
• Similarly, pre-existing acetylation at K9 prevented HMTase
activity.
• Unexpectedly Ser-10 phosphorylation inhibits Suv39H1
dependent methylation of adjacent K-9.

14. Modification of H3 in native chromatin
• Murine Suv39h protein is encoded by two loci by Suv39h1 & Suv39h2.
• Suv39h gene disrupted in mice germ line.
Single Suv39h mutant mice are viable but double mutant mice are growth
retarded animal.
This shows that Suv39h gene is important for normal growth.

15. Conclusion
• Lys-9 methylation in H3 might recruited a binding site of
heterochromatin associated specific protein that suppress the gene
activity.
• SU(VAR) group proteins play role in induction and assembly of higher
order chromatin.

16. Important references:
Strahl, B. D. & Allis, C. D. The language of covalent histone modi®cations. Nature 403,
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Turner, B. M. Histone acetylation as an epigenetic determinant of long-term
transcriptionalcompetence. Cell. Mol. Life Sci. 54, 21±31 (1998).
Karpen, G.& Allshire, R. C. The case for epigenetic effects on centromere identity and
function. TrendsGenet. 13, 489±496 (1997)..
Strahl, B. D., Ohba, R., Cook, R. G. & Allis, C. D. Methylation of histone H3 at lysine 4 is
highly conserved and correlates with transcriptionally active nuclei in Tetrahymena. Proc.
Natl Acad. Sci. USA96, 14967±14972 (1999).
Jenuwein, T., Laible, G., Dorn, R. & Reuter, G. SET-domain proteins modulate
chromatin domains ineu- and heterochromatin. Cell. Mol. Life Sci. 54, 80±93
(1998).
Ball, L. J. et al. Structure of the chromatin binding (chromo) domain from
mouse modi®er protein 1.EMBO J. 16, 2473±2481 (1997).