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      Automethylation of G9a and its implication in wider substrate specificity and HP1 binding

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          Abstract

          Methylation of lysine residues on histones participates in transcriptional gene regulation. Lysine 9 methylation of histone H3 is a transcriptional repression signal, mediated by a family of SET domain containing AdoMet-dependent enzymes. G9a methyltransferase is a euchromatic histone H3 lysine 9 methyltransferase. Here, G9a is shown to methylate other cellular proteins, apart from histone H3, including automethylation of K239 residue. Automethylation of G9a did not impair or activate the enzymatic activity in vitro. The automethylation motif of G9a flanking target K239 (ARKT) has similarity with histone H3 lysine 9 regions (ARKS), and is identical to amino acids residues in EuHMT (ARKT) and mAM (ARKT). Under steady-state kinetic assay conditions, full-length G9a methylates peptides representing ARKS/T motif of H3, G9a, mAM and EuHMT efficiently. Automethylation of G9a at ARKT motif creates a binding site for HP1 class of protein and mutation of lysine in the motif impairs this binding. In COS-7 cells GFP fusion of the wild-type G9a co-localized with HP1α and HP1γ isoforms whereas the G9a mutant with K239A displayed poor co-localization. Thus, apart from transcriptional repression and regulatory roles of lysine methylation, the non-histone protein methylation may create binding sites for cellular protein–protein interactions.

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          TANDEM: matching proteins with tandem mass spectra.

          Tandem mass spectra obtained from fragmenting peptide ions contain some peptide sequence specific information, but often there is not enough information to sequence the original peptide completely. Several proprietary software applications have been developed to attempt to match the spectra with a list of protein sequences that may contain the sequence of the peptide. The application TANDEM was written to provide the proteomics research community with a set of components that can be used to test new methods and algorithms for performing this type of sequence-to-data matching. The source code and binaries for this software are available at http://www.proteome.ca/opensource.html, for Windows, Linux and Macintosh OSX. The source code is made available under the Artistic License, from the authors.
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            Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome.

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              Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin.

              Histone H3 lysine 9 (H3-K9) methylation and DNA methylation are characteristic hallmarks of mammalian heterochromatin. H3-K9 methylation was recently shown to be a prerequisite for DNA methylation in Neurospora crassa and Arabidopsis thaliana. Currently, it is unknown whether a similar dependence exists in mammalian organisms. Here, we demonstrate a physical and functional link between the Suv39h-HP1 histone methylation system and DNA methyltransferase 3b (Dnmt3b) in mammals. Whereas in wild-type cells Dnmt3b interacts with HP1 alpha and is concentrated at heterochromatic foci, it fails to localize to these regions in Suv39h double null (dn) mouse embryonic stem (ES) cells. Consistently, the Suv39h dn ES cells display an altered DNA methylation profile at pericentric satellite repeats, but not at other repeat sequences. In contrast, H3-K9 trimethylation at pericentric heterochromatin is not impaired in Dnmt1 single- or Dnmt3a/Dnmt3b double-deficient ES cells. We also show that pericentric heterochromatin is not transcriptionally inert and can give rise to transcripts spanning the major satellite repeats. These data demonstrate an evolutionarily conserved pathway between histone H3-K9 methylation and DNA methylation in mammals. While the Suv39h HMTases are required to direct H3-K9 trimethylation and Dnmt3b-dependent DNA methylation at pericentric repeats, DNA methylation at centromeric repeats occurs independent of Suv39h function. Thus, our data also indicate a more complex interrelatedness between histone and DNA methylation systems in mammals. Both methylation systems are likely to be important in reinforcing the stability of heterochromatic subdomains and thereby in protecting genome integrity.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                December 2007
                25 October 2007
                25 October 2007
                : 35
                : 21
                : 7313-7323
                Affiliations
                1New England Biolabs, 240 County Road, Ipswich, MA 01938-2723 and 2Department of Biological Chemistry, Gene Regulation Program, Jonsson Cancer Center, 10833 LeConte Ave., UCLA School of Medicine, Los Angeles, CA 90095-1737, USA
                Author notes
                * To whom correspondence should be addressed. +1 978 380-7227+1 978 921-1350 pradhan@ 123456neb.com

                The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

                Article
                10.1093/nar/gkm726
                2175347
                17962312
                b7d739aa-54fb-42d0-932a-4f65763f4f65
                © 2007 The Author(s)

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-com

                Categories
                Nucleic Acid Enzymes

                Genetics
                Genetics

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