Histone H3 lysine 9 methylation: A signature for chromatin function

In most eukaryotes the histone methyltransferases SU(VAR)3-9, G9a
and their orthologues play major roles in transcriptional
regulation. Histone H3 lysine 9 methylation is associated to
transcriptional silencing in vivo. SU(VAR)3-9 is the main H3K9
HMTase in Drosophila heterochromatin whereas G9a was found to be an
euchromatic H3K9 methyltransferase in mammalian cells. In this work
SU(VAR)3-9 and a new HMTase homologous to G9a were characterized in
vitro. A detailed analysis of the reaction products shows that
recombinant SU(VAR)3-9 adds three methylgroups to full-length H3
and only two methylgroups to an H3-tail peptide. The transfer of
two methylgroups to an unmethylated H3-tail peptide is achieved in
a nonprocessive manner. The full-length enzyme elutes with an
apparent molecular weight of 160 kDa from a gel filtration column,
which indicates the formation of a dimer. The N-terminus was shown
to be required for this dimerisation and to retrieve full activity
in vitro. We show that the interaction occurs by domain swapping of
two motifs within the N-terminus. The fact that the N-terminus is
responsible for a concentration dependent dimerisation of
SU(VAR)3-9 may indicate a role for this domain in the
dosage-dependent effect on position effect variegation. Drosophila
G9a adds three methyl groups to unmethylated H3 in vitro as has
been described for mouse G9a. In vitro, a N-terminal truncation of
dG9a adds three methylgroups toward H3K9 and K27, with a preference
for K9. Surprisingly, dG9a also methylates H4 with specificity for
K8, K12 or K16. In vivo, dG9a is present in complexes with a
molecular mass of 440-670 kDa and we show that it specifically
interacts with the histone deacetylase Rpd3. HP1a is predominantly
associated with centromeric heterochromatin in Drosophila.
Supporting the histone code hypothesis, the chromo domain of HP1
recognises and binds H3K9 methylated peptides. Here we show the
mechanism for binding to H3K9Me chromatin by recombinant Drosophila
HP1a. HP1a requires a bimodal interaction of the chromo domain with
H3K9Me and a simultaneous interaction of the chromo shadow domain
with auxiliary factors (SU(VAR)3-9 and ACF) for stable association
with H3K9Me chromatin. The two HP1 paralogs HP1a and HP1c bind to
distinct chromatin structures and we identify distinct interaction
partners for these two proteins.