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Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE
3 To whom correspondence and reprint requests should be addressed. E-mail: jzempleni2{at}unl.edu.
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ABSTRACT |
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 TOP ABSTRACT LITERATURE CITED |
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KEY WORDS: • biotin • chromatin • DNA repair • gene expression • histone
The classical role of biotin in metabolism is to serve as a covalently bound coenzyme for cytoplasmic acetyl-CoA carboxylase 
and mitochondrial acetyl-CoA carboxylase ß, 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase, and pyruvate carboxylase (1). Carboxylases play crucial roles in fatty acid homeostasis, leucine catabolism, gluconeogenesis, and other metabolic pathways. There is evidence that biotin also plays a role in cell signaling, and that biotin-dependent cell signals are mediated by biotinyl-AMP, receptor tyrosine kinases, and the transcription factors Jun/Fos, nuclear factor-
B, and Sp1/Sp3 (2). Not surprisingly, biotin affects the expression of >2000 genes in human cells (2). Pioneering studies by Dakshinamurti and Mistry (3) provided evidence that biotin-binding proteins in the cell nucleus may participate in gene regulation by biotin. Subsequently, Paparelli et al. (4) and Wolf and co-workers (5) demonstrated that biotin affects post-translational modifications of histones (DNA-binding proteins), and that biotinidase has catalytic activity to attach biotin covalently to histones. Finally, biotinylated histones were detected in human cells using streptavidin as a probe (6). This line of observations laid the groundwork for studies of the biotin-dependent chromatin remodeling processes reviewed here.
Epigenetic control of chromatin structure and function. Chromatin comprises DNA, histones H1, H2A, H2B, H3, and H4, and nonhistone proteins (7). DNA and histones form repetitive nucleoprotein units, the nucleosomal core particles. Each particle consists of 146 bp of DNA wrapped around an octamer of core histones (1 H3-H3-H4-H4 tetramer and 2 H2A-H2B dimers). The DNA located between nucleosomal core particles is associated with histone H1 (Fig. 1). This 11-nm histone fiber is then further packed into an irregular 30-nm chromatin fiber structure, which is coiled into even more complex structures to eventually assemble the chromosome.
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Histone biotinyl transferases.
Wolf and co-workers (5) proposed a reaction mechanism by which biotinidase mediates covalent biotinylation of histones. It was suggested that cleavage of biocytin (biotin-
-lysine) by biotinidase leads to the formation of a biotinyl-thioester intermediate at or near the active site of biotinidase. In a further step, the biotinyl moiety is transferred from the thioester to the -amino group of lysine residues in histones. Biocytin is generated in the breakdown of biotin-dependent carboxylases (1). Biotinidase is ubiquitous in mammalian cells (11), but its cellular localization remains somewhat controversial. Some investigators detected biotinidase in the nuclear compartment (11,12), whereas others claim that the enzyme resides primarily in extranuclear compartments (13). Notwithstanding the well-established role of biotinidase as a histone biotinyl transferase (see below), questions began to emerge concerning the existence of additional histone biotinyl transferases. These questions were fueled by observations that alterations in the abundance of biotinylated histones in response to cell proliferation were not paralleled by alterations in cellular biotinidase activity (6). Finally, Gravel and co-workers (14) succeeded in demonstrating that holocarboxylase synthetase may act as a histone biotinyl transferase.
Biotinylation sites in histones. Initial evidence for the existence of biotinylated histones H1, H2A, H2B, H3, and H4 in vivo came from studies in human lymphocytes (6). Subsequently, biotinylated histones were also detected in human lymphoma cells, small cell lung cancer cells, choriocarcinoma cells, chicken erythrocytes, and Drosophila melanogaster (1,2). All of these early studies suffered from a lack of availability of biotinylation site-specific antibodies; histone-bound biotin was detected by using avidin as a generic probe for biotin. This is a substantial limitation, given that biotinylation of distinct amino acid residues is likely associated with unique biological functions; investigations of histone methylation provide precedence for this notion (8,9). Recently, our laboratory developed a procedure in which biotinylation sites in histones were identified by incubating synthetic peptides with histone biotinyl transferases (15). Using this approach we identified 10 lysine residues in histones H2A, H3, and H4 (Fig. 2) that are targets for biotinylation (12,15,16). Some of these lysine residues are also sites for acetylation and methylation (17). Importantly, evidence was provided that biotinylation of lysine residues is affected by modifications of neighboring amino acids, a phenomenon referred to as "cross talk" (12,15,16). For example, acetylation and phosphorylation of lysine and serine residues, respectively, decrease biotinylation of adjacent lysine residues (15,16). Ongoing studies in our laboratory used MS to confirm the biotinylation sites depicted in Figure 2, to identify tentatively additional biotinylation sites, and to identify modifications (acetylation, methylation) that coexist with biotinylation in the same histone molecule (18). Antibodies were raised that are specific for biotinylation sites in histones (12,15,16); availability of these antibodies has greatly facilitated studies of biological functions of histone biotinylation.
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The enrichment of biotinylated histones in pericentromeric heterochromatin is consistent with a potential role of histone biotinylation in cell proliferation. Biotinylation of histones is greater in proliferating cells compared with quiescent cells (6). Preliminary evidence was provided that biotinylation of K8 and K12 in histone H4 shows a cell cycle–dependent pattern, and that maximal levels of biotinylation are achieved during mitotic chromosome condensation (19).
Biotinylation of histones might play a role in the cellular response to DNA double-strand breaks. Biotinylation of K12 in histone H4 decreases rapidly and transiently in response to double-strand breaks caused by etoposide and other agents in human JAr choriocarcinoma cells (20). It is currently unknown whether altered biotinylation of histones in response to DNA damage triggers repair or apoptosis.
Holocarboxylase synthetase deficiency is associated with interesting genotypes and phenotypes in human lymphoma cells and Drosophila melanogaster (21,22). We generated holocarboxylase synthetase–deficient cells by using RNAi and observed a 70% decrease in histone biotinylation. Holocarboxylase synthetase deficiency altered the expression of 
800 and 400 genes in human cells and flies, respectively. This was associated with decreased cell proliferation in human cells and with decreased temperature tolerance in flies. Currently, it is unknown whether a given genotype or phenotype is caused by decreased biotinylation of histones, decreased biotinylation of carboxylases, or both. We are seeking to overcome this limitation by investigating flies that are deficient for individual carboxylases and by conducting chromatin immunoprecipitation assays.
Biotin supply. Biotin concentrations in culture media had only a moderate effect on biotinylation of histones in JAr cells, whereas biotinylation of carboxylases correlated strongly with biotin supply (23). Similarly, other human-derived cell lines preserved histone-bound biotin at the expense of carboxylase-bound biotin when cultured in biotin-deficient media (2). The reader should note that even small changes in histone biotinylation might be physiologically meaningful. For example, quantitatively important changes in histone biotinylation may occur in confined regions of the chromatin but may escape detection if the analysis is based on Western blotting of bulk histone extracts. Hence, potential effects of biotin deficiency on chromatin structure and gene function should not be discounted until further study. In fact, one might interpret the preservation of histone biotinylation under biotin-deficient culture conditions as an indicator for the biological importance of histone biotinylation.
Histone debiotinylases. Covalent modifications of histones are typically reversible (8), but the enzymes mediating debiotinylation of histones are largely unknown. There is evidence that biotinidase may catalyze both biotinylation and debiotinylation of histones (24). Variables such as the microenvironment in chromatin, and post-translational modifications and alternate splicing of biotinidase might determine whether biotinidase acts as biotinyl histone transferase or histone debiotinylase (2).
In conclusion, our knowledge of potential biological functions of biotin was expanded dramatically by the recent demonstration of enzyme-mediated biotinylation of histones. This observation offers an exciting mechanism for epigenetic regulation of chromatin structure and gene function by biotin. Over the past 5 y, the field was advanced by the generation of a variety of analytical tools. For example, site-specific antibodies were generated on the basis of the identification of biotinylation sites in histones, and knockdown models of the enzymes mediating biotinylation of histones were generated in human cells and Drosophila. Using these tools, evidence was provided that histone biotinylation plays a role in heterochromatin structures, gene silencing, mitotic condensation of chromatin, and DNA repair. The implications of these findings for biotin nutrition are uncertain, yet exciting. For example, teratogenic and immunosuppressive effects of biotin deficiency were demonstrated by Mock's group (25) and other research teams, and one can envision that many of the known effects of biotin deficiency can be attributed to abnormal chromatin structures.
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FOOTNOTES |
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2 Manuscript received 17 March 2006.
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LITERATURE CITED |
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TOPABSTRACTLITERATURE CITED |
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1. Camporeale G, Zempleni J. Biotin. In: Bowman BA, Russell RM, editors. Present knowledge in nutrition. 9th edition. Washington, DC: International Life Sciences Institute. In press 2006.
2. Zempleni J. Uptake, localization, and noncarboxylase roles of biotin. Annu Rev Nutr. 2005;25:175–96.[Medline]
3. Dakshinamurti K, Mistry SP. Tissue and intracellular distribution of biotin-C14 OOH in rats and chicks. J Biol Chem. 1963;238:294–6.
4. Petrelli F, Coderoni S, Moretti P, Paparelli M. Effect of biotin on phosphorylation, acetylation, methylation of rat liver histones. Mol Biol Rep. 1978;4:87–92.[Medline]
5. Hymes J, Fleischhauer K, Wolf B. Biotinylation of histones by human serum biotinidase: assessment of biotinyl-transferase activity in sera from normal individuals and children with biotinidase deficiency. Biochem Mol Med. 1995;56:76–83.[Medline]
6. Stanley JS, Griffin JB, Zempleni J. Biotinylation of histones in human cells: effects of cell proliferation. Eur J Biochem. 2001;268:5424–9.[Medline]
7. Wolffe A. Chromatin. 3rd edition. San Diego, CA: Academic Press; 1998.
8. Jenuwein T, Allis CD. Translating the histone code. Science. 2001;293:1074–80.
9. Fischle W, Wang Y, Allis CD. Histone and chromatin cross-talk. Curr Opin Cell Biol. 2003;15:172–83.[Medline]
10. Dey A, Chitsaz F, Abbasi A, Misteli T, Ozato K. The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis. Proc Natl Acad Sci U S A. 2003;100:8758–63.
11. Pispa J. Animal biotinidase. Ann Med Exp Biol Fenn. 1965;43:Suppl, 5:1–39.[Medline]
12. Chew YC, Camporeale G, Kothapalli N, Sarath G, Zempleni J. Lysine residues in N- and C-terminal regions of human histone H2A are targets for biotinylation by biotinidase. J Nutr Biochem. 2006; in press.
13. Stanley CM, Hymes J, Wolf B. Identification of alternatively spliced human biotinidase mRNAs and putative localization of endogenous biotinidase. Mol Genet Metab. 2004;81:300–12.[Medline]
14. Narang MA, Dumas R, Ayer LM, Gravel RA. Reduced histone biotinylation in multiple carboxylase deficiency patients: a nuclear role for holocarboxylase synthetase. Hum Mol Genet. 2004;13:15–23.
15. Camporeale G, Shubert EE, Sarath G, Cerny R, Zempleni J. K8 and K12 are biotinylated in human histone H4. Eur J Biochem. 2004;271:2257–63.[Medline]
16. Kobza K, Camporeale G, Rueckert B, Kueh A, Griffin JB, Sarath G, Zempleni J. K4, K9, and K18 in human histone H3 are targets for biotinylation by biotinidase. FEBS J. 2005;272:4249–59.[Medline]
17. Espino PS, Drobic B, Dunn KL, Davie JR. Histone modifications as a platform for cancer therapy. J Cell Biochem. 2005;94:1088–102.[Medline]
18. Chew YC, Raza AS, Sarath G, Zempleni J. Biotinylation of K8 and K12 co-occurs with acetylation and mono-methylation in human histone H4 [abstract]. FASEB J. 2006;20:A610.
19. Kothapalli N, Zempleni J. Biotinylation of histones depends on the cell cycle in NCI-H69 small cell lung cancer cells. FASEB J. 2005;19:A55.
20. Kothapalli N, Sarath G, Zempleni J. Biotinylation of K12 in histone H4 decreases in response to DNA double strand breaks in human JAr choriocarcinoma cells. J Nutr. 2005;135:2337–42.
21. Camporeale G, Suzuki Y, Eissenberg JC, Zempleni J. Low abundance of wild-type biotinidase and holocarboxylase synthetase decreases biotinylation of histones [abstract]. FASEB J. 2005;19:A55.
22. Camporeale G, Eissenberg JC, Giordano E, Zempleni J. Lifespan and resistance to heat stress depend on histone biotinylation in Drosophila melanogaster [abstract]. FASEB J. 2006;20:A610.
23. Crisp SERH, Camporeale G, White BR, Toombs CF, Griffin JB, Said HM, Zempleni J. Biotin supply affects rates of cell proliferation, biotinylation of carboxylases and histones, and expression of the gene encoding the sodium-dependent multivitamin transporter in JAr choriocarcinoma cells. Eur J Nutr. 2004;43:23–31.[Medline]
24. Ballard TD, Wolff J, Griffin JB, Stanley JS. Calcar Sv, Zempleni J. Biotinidase catalyzes debiotinylation of histones. Eur J Nutr. 2002;41:78–84.[Medline]
25. Mock DM. Biotin. In: Rucker RB, Zempleni J, Suttie JW, McCormick DB, editors. Handbook of vitamins. 4th edition. Boca Raton, FL: Taylor and Francis. In press 2006.
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