The Journal of Nutritional Biochemistry
Volume 22, Issue 5 , Pages 470-475, May 2011

Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18☆

  • Baolong Bao

      Affiliations

    • Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE 68583, USA
    • Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, 201306 Shanghai, China
    • ,
  • Valerie Pestinger

      Affiliations

    • Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE 68583, USA
    • ,
  • Yousef I. Hassan

      Affiliations

    • Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE 68583, USA
    • ,
  • Gloria E.O. Borgstahl

      Affiliations

    • Eppley Institute for Cancer Research and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA
    • ,
  • Carol Kolar

      Affiliations

    • Eppley Institute for Cancer Research and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA
    • ,
  • Janos Zempleni

      Affiliations

    • Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE 68583, USA
    • Corresponding Author InformationCorresponding author.

Received 27 January 2010; received in revised form 29 March 2010; accepted 5 April 2010. published online 06 August 2010.

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.

Abbreviations: DAPI, 4′,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

Keywords: Biotin, Chromatin, Histone H3, Holocarboxylase synthetase

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☆ This study was supported by a contribution of the University of Nebraska Agricultural Research Division, supported in part by funds provided through the Hatch Act. Additional support was provided by NIH grants DK063945, DK077816, DK082476 and ES015206; by USDA CSREES grant 2006-35200-17138; by NSF grants MCB 0615831 and EPS 0701892; by Nebraska Research Initiative funding for the Nebraska Center for Structural Biology; and by NCI Eppley Cancer Center Support Grant P30CA036727.

PII: S0955-2863(10)00099-9

doi:10.1016/j.jnutbio.2010.04.001

The Journal of Nutritional Biochemistry
Volume 22, Issue 5 , Pages 470-475, May 2011

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