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Eskeland, Ragnhild (2007): Histone H3 lysine 9 methylation: A signature for chromatin function. Dissertation, LMU München: Faculty of Biology
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Abstract

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.