Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18 |
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Authors: | Baolong Bao Valerie PestingerYousef I. Hassan Gloria E.O. BorgstahlCarol Kolar Janos Zempleni |
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Affiliation: | a Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE 68583, USAb Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, 201306 Shanghai, Chinac Eppley Institute for Cancer Research and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA |
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Abstract: | Holocarboxylase synthetase (HCS) mediates the binding of biotin to lysine (K) residues in histones H2A, H3 and H4; HCS knockdown disturbs gene regulation and decreases stress resistance and lifespan in eukaryotes. We tested the hypothesis that HCS interacts physically with histone H3 for subsequent biotinylation. Co-immunoprecipitation experiments were conducted and provided evidence that HCS co-localizes with histone H3 in human cells; physical interactions between HCS and H3 were confirmed using limited proteolysis assays. Yeast two-hybrid (Y2H) studies revealed that the N-terminal and C-terminal domains in HCS participate in H3 binding. Recombinant human HCS was produced and exhibited biological activity, as evidenced by biotinylation of its known substrate, recombinant p67. Recombinant histone H3.2 and synthetic H3-based peptides were also good targets for biotinylation by recombinant HCS (rHCS) in vitro, based on tracing histone-bound biotin with [3H]biotin, streptavidin and anti-biotin antibody. Biotinylation site-specific antibodies were generated and revealed that both K9 and K18 in H3 were biotinylated by HCS. Collectively, these studies provide conclusive evidence that HCS interacts directly with histone H3, causing biotinylation of K9 and K18. We speculate that the targeting of HCS to distinct regions in human chromatin is mediated by DNA sequence, biotin, RNA, epigenetic marks or chromatin proteins. |
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Keywords: | DAPI, 4&prime ,6-diamidino-2-phenylindole Gal4 AD, Gal4 activation domain Gal4 BD, Gal4 binding domain GFP, green fluorescent protein HCS, holocarboxylase synthetase H3K4bio, histone H3, biotinylated at lysine-4 H3K9bio, biotinylated at lysine-9 H3K18bio, biotinylated at lysine-18 H3K4me3, histone H3, trimethylated at lysine-4 H3K9me2, histone H3, dimethylated at lysine-9 H3K9me3, histone H3, trimethylated at lysine-9 H4K12bio, histone H4, biotinylated at lysine-12 K, lysine rHCS, recombinant human holocarboxylase synthetase Y2H assay, yeast two-hybrid assay |
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