View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by UNL | Libraries University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln U.S. Department of Agriculture: Agricultural Publications from USDA-ARS / UNL Faculty Research Service, Lincoln, Nebraska 8-3-2005 K4, K9 and K18 in human histone H3 are targets for biotinylation by biotinidase Kenya A. Kobza University of Nebraska-Lincoln Gabriela Camporeale University of Nebraska-Lincoln Brian Rueckert University of Nebraska-Lincoln Alice Kueh University of Nebraska-Lincoln Jacob B. Griffin University of Nebraska-Lincoln See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub Part of the Agricultural Science Commons Kobza, Kenya A.; Camporeale, Gabriela; Rueckert, Brian; Kueh, Alice; Griffin, Jacob B.; Sarath, Gautam; and Zempleni, Janos, "K4, K9 and K18 in human histone H3 are targets for biotinylation by biotinidase" (2005). Publications from USDA-ARS / UNL Faculty. 36. https://digitalcommons.unl.edu/usdaarsfacpub/36 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Publications from USDA-ARS / UNL Faculty by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Kenya A. Kobza, Gabriela Camporeale, Brian Rueckert, Alice Kueh, Jacob B. Griffin, Gautam Sarath, and Janos Zempleni This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/ usdaarsfacpub/36 K4, K9 and K18 in human histone H3 are targets for biotinylation by biotinidase Keyna Kobza1, Gabriela Camporeale1, Brian Rueckert1, Alice Kueh1, Jacob B. Griffin1, Gautam Sarath2 and Janos Zempleni3 1 Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA 2 USDA-ARS and Department of Entomology, University of Nebraska at Lincoln, Lincoln, NE, USA 3 Departments of Biochemistry, and Animal Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA Keywords Histones are modified post-translationally, e.g. by methylation of lysine biotin; biotinidase; histone H3; lysine and arginine residues, and by phosphorylation of serine residues. These modifications regulate processes such as gene expression, DNA repair, and Correspondence mitosis and meiosis. Recently, evidence has been provided that histones are J. Zempleni, Department of Nutrition and Health Sciences, University of Nebraska at also modified by covalent binding of the vitamin biotin. The aims of this Lincoln, 316 Ruth Leverton Hall, Lincoln, study were to identify biotinylation sites in histone H3, and to investigate NE 68583-0806, USA the crosstalk among histone biotinylation, methylation and phosphoryla- Fax: +1 402 472 1587 tion. Synthetic peptides based on the sequence of human histone H3 were Tel: +1 402 472 3270 used as substrates for enzymatic biotinylation by biotinidase; biotin in pep- E-mail: [email protected] tides was probed using streptavidin peroxidase. These studies provided evidence that K4, K9 and K18 in histone H3 are good targets for biotiny- Note K. Kobza and G. Camporeale contributed lation; K14 and K23 are relatively poor targets. Antibodies were generated equally to this work to histone H3, biotinylated either at K4, K9 or K18. These antibodies localized to nuclei in human placental cells in immunocytochemistry and Note immunoblotting experiments, suggesting that lysines in histone H3 are biot- Presented in part at Experimental Biology inylated in vivo. Dimethylation of R2, R8 and R17 increased biotinylation 2004, Washington DC [Sarath G, Kobza K, of K4, K9 and K18, respectively, by biotinidase; phosphorylation of S10 Rueckert B, Camporeale G, Zempleni J & abolished biotinylation of K9. These observations are consistent with cross- Haas E (2004) Biotinylation of human histone H3 and interactions with biotinidase. talk between biotinylation of histones and other known modifications of FASEB J 18, A103] histones. We speculate that this crosstalk provides a link to known roles for biotin in gene expression and cell proliferation. (Received 15 June 2005, accepted 1 July 2005) doi:10.1111/j.1742-4658.2005.04839.x Histones are small proteins (11–22 kDa) that mediate nucleosomal core particles. Nucleosomes are stabilized the folding of DNA into chromatin. The following five by electrostatic interactions between negatively charged major classes of histones have been identified in euk- phosphate groups in DNA and positively charged aryotic cells: H1, H2A, H2B, H3 and H4 [1]. DNA is e-amino groups (lysine residues) and guanidino groups wrapped around octamers of core histones, each con- (arginine residues) in histones. sisting of one H3–H3–H4–H4 tetramer and two H2A– Histones consist of a globular C-terminal domain H2B dimers, to form the nucleosomal core parti- and a flexible N-terminal tail [1]. The amino terminus cle. Histone H1 associates with the DNA connecting of histones protrudes from the nucleosomal surface; Abbreviation DAPI, 4¢,6-diamidino-2-phenylindole. FEBS Journal 272 (2005) 4249–4259 ª 2005 FEBS No claim to original US government works 4249 Biotinylation sites in human histone H3 K. Kobza et al. lysines, arginines, serines, and glutamates in the amino generated. Such antibodies are invaluable tools to: (a) terminus are targets for acetylation, methylation, phos- study the cross-talk among modifications of histones, phorylation, ubiquitination, poly(ADP-ribosylation) e.g. biotinylation and acetylation of lysine residues [7]; and sumoylation [1–5]. These modifications play (b) investigate cellular distribution patterns of biotinyl- important roles in chromatin structure, regulating pro- ated histones by using immunocytochemistry; and (c) cesses such as transcriptional activation or silencing of investigate roles for biotinylation of histones in the genes, DNA repair, and mitotic and meiotic condensa- regulation of transcriptional activity of genes by using tion of chromatin. chromatin immunoprecipitation assays. Recently, a novel covalent modification of histones Recently we have developed a peptide-based proce- has been identified in human cells, namely the bio- dure to identify biotinylation sites in histones [7]. In tinylation of lysine residues [6,7]. Two enzymes can this study we applied this procedure to identify bio- independently catalyze biotinylation of histones: tinylation sites in human histone H3. As a secondary biotinidase [8] and holocarboxylase synthetase [9]. Bio- goal we investigated interactions among histone bio- tinidase belongs to the nitrilase superfamily of enzymes tinylation, methylation and phosphorylation. Histone [10]; biotinylation of histones by biotinidase depends H3 was chosen as a model because of its pivotal role on the hydrolytic cleavage of biocytin (biotinyl-e- in regulating gene expression [20–22]. lysine), coupled to the transfer of the biotinyl residue to free amino groups in histones [11]. In contrast, bio- Results tinylation of histones by holocarboxylase synthetase depends on ATP and biotin [9]. Preliminary studies Biotinylation sites in histone H3 suggest that biotinylation of histones might play a role in processes such as gene silencing [12], cell prolifer- The N-terminal tail of histone H3 was efficiently bio- ation [6,9], and DNA repair or apoptosis [12,13]. tinylated by biotinidase. The binding of biotin was These observations could have important implications substantially greater in peptide N1)25 than in peptide for human health, based on the following lines of rea- N15)39, if equal amounts of both peptides were incuba- soning. First, preliminary evidence has been provided ted with biotinidase and biocytin for 45 min (Fig. 1; that biotinylation of K12 in histone H4 decreases rap- idly in response to double-stranded DNA breaks caused by the cancer drug etoposide [13]. This observa- tion is consistent with the hypothesis that alterations in the biotinylation pattern of histones might be an early signaling event in response to DNA damage. Second, mutations of the genes encoding biotinidase [14–16] and holocarboxylase synthetase [17] have been documented; some of these mutations are fairly com- mon [18,19]. Fibroblasts from individuals with mutated holocarboxylase synthetase are deficient in histone biotinylation [9]. Likewise, in vitro studies provided evidence that mutated biotinidase is not capable of catalyzing biotinylation of histones [8]. Future studies might unravel abnormal patterns of gene silencing [12], cell proliferation [6,9], and DNA repair or apoptosis [12,13] in individuals carrying mutations of genes cod- ing for biotinidase and holocarboxylase synthetase. Although all five major classes of histones appear to be biotinylated in human cells [6], only two biotinyla- Fig. 1. Biotinylation targets amino acids in the N-terminal tail of tion sites have been identified so far: K8 and K12 in human histone H3. Synthetic peptides based on the N- and C-ter- histone H4 [7]. This gap in our understanding of his- minal region of histone H3 were biotinylated enzymatically, and bio- tone biotinylation has created a significant obstacle for tin was probed using gel electrophoresis and streptavidin peroxidase. N ) , peptide spanning amino acids 1–25 in histone investigating roles of biotinylated histones in cell bio- 1 25 H3 (lanes 1a and b); N15)39, peptide spanning amino acids 15–39 in logy,