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Mammalian Tethered Catalysis: a novel in vivo approach to identify and characterize histone post‐translational modification binding proteins
Author(s) -
Tuzon Creighton T,
Spektor Tanya M,
Rice Judd C
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.460.4
Subject(s) - histone , histone methylation , histone h4 , in vivo , nucleosome , chemistry , methylation , histone methyltransferase , microbiology and biotechnology , histone h3 , acetylation , histone h2a , biology , biochemistry , computational biology , genetics , dna methylation , dna , gene expression , gene
Post‐translational modifications (PTMs) of nucleosomes are critical for genome architecture and function. Remarkably, more than 70 different sites for histone PTMs have been reported; however, the identification of histone PTM binding proteins (PTMBPs) and the mechanistic understanding of how various PTMs translate in to biological outputs is less understood. We developed mammalian tethered catalysis (MTeC) as an in vivo approach to identify novel histone PTMBPs. To validate this approach, we first used MTeC to identify H3K9 PTMBPs in an unbiased manner. Surprisingly, we isolated G9a as an H3K9me1 binding protein in vivo . Given this selectivity, we then tested the binding specificity of methyl‐specific histone PTMBPs to two different histones – H3 and H4. To our surprise, only one of the three histone PTMBPs assayed, HP1β, exhibited specificity for H3K9 methylation status in vivo . Interestingly, both tandem tudor‐domain proteins, JMJD2A and 53BP1, did not demonstrate binding specificity for H4K20 methylation status. Instead, we discovered that JMJD2A and 53BP1 interacts with all three methylated states of H4K20 in vivo , unlike previously reported in vitro assays. Thus, the utility of MTeC extends beyond the impartial identification of histone PTMBPs and allows an objective evaluation of the binding specificity of histone PTMBPs in vivo .