Epigenetic Regulation of Chromatin Structure and Gene Function by Biotin^,

Abstract

Covalent modifications of histones are a crucial component of epigenetic events that regulate chromatin structures and gene function. Evidence has been provided that distinct lysine residues in histones are modified by covalent attachment of the vitamin biotin, catalyzed by biotinidase and holocarboxylase synthetase. Biotinylation of histones appears to be conserved across species. The following biotinylation sites have been identified by using both mass spectrometry and enzymatic biotinylation of synthetic peptides: K9, K13, K125, K127, and K129 in histone H2A, K4, K9, and K18 in histone H3, and K8 and K12 in histone H4. Evidence has been provided that biotinylated histone H4 is enriched in pericentrometric heterochromatin, and that biotinylation of histone H4 participates in gene silencing, mitotic condensation of chromatin, and the cellular response to DNA damage. Biotinylation of histones is a reversible process and depends on exogenous biotin supply, but the identities of histone debiotinylases remain uncertain. We propose that some effects of biotin deficiency can be attributed to abnormal chromatin structures.

The classical role of biotin in metabolism is to serve as a covalently bound coenzyme for cytoplasmic acetyl-CoA carboxylase α and mitochondrial acetyl-CoA carboxylase β, 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. Evidence has been provided 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, NF-κB, and Sp1/Sp3 (2). Not surprisingly, biotin affects the expression of >2,000 genes in human cells (2). Pioneering studies by Dakshinamurti and co-workers provided evidence that biotin-binding proteins in the cell nucleus may participate in gene regulation by biotin (3). Subsequently, Paparelli, Wolf, and co-workers demonstrated that biotin affects posttranslational modifications of histones (DNA-binding proteins), and that biotinidase has catalytic activity to attach biotin covalently to histones (4, 5). 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 non-histone proteins (7). DNA and histones form repetitive nucleoprotein units, the nucleosomal core particles. Each particle consists of 146 basepairs of DNA wrapped around an octamer of core histones (one H3-H3-H4-H4 tetramer and two 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.

Fig. 1.

Chromatin structure.

The amino terminal tails of histones protrude from the nucleosomal surface; covalent modifications of these tails affect the structure of chromatin and form the basis for the epigenetic regulation of chromatin structure and gene function (8, 9). Epigenetics is defined as the study of heritable changes of gene function that occur without a change in the nucleotide sequence of DNA. For example, amino acid residues in histone tails are modified by covalent acetylation, methylation, phosphorylation, and ubiquitination to regulate gene transcription, mitotic condensation of chromatin, and DNA repair (7-9). These modifications are deciphered by proteins containing motifs that target them to chromatin. For example, some transcription factors contain bromodomains with affinity of acetylated histones, increasing gene expression (10). Modifications of distinct amino acid residues in histones have unique functions. For example, trimethylation of lysine (K)⁴ 4 in histone H3 is associated with transcriptional activation of surrounding DNA, whereas di-methylation of K9 is associated with transcriptional silencing (8, 9). Covalent modifications of histones can be reversed by a large variety of enzymatic processes (8).

Histone Biotinyl Transferases

Wolf and co-workers proposed a reaction mechanism by which biotinidase mediates covalent biotinylation of histones. It was suggested that cleavage of biocytin (biotin-ε-lysine) by biotinidase leads to the formation of a biotinyl-thioester intermediate at or near the active site of biotinidase (5). In a next step, the biotinyl moiety is transferred from the thioester to the ε-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 extra-nuclear compartments (13). Notwithstanding the well-established role of biotinidase as a histone biotinyl transferase (see below), questions began to emerge as to whether additional histone biotinyl transferases exist. 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 succeeded in demonstrating that holocarboxylase synthetase may act as a histone biotinyl transferase (14).

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 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 significant 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 has developed a procedure in which biotinylation sites in histones are identified by incubating synthetic peptides with histone biotinyl transferases (15). Using this approach we have identified ten 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, decreases biotinylation of adjacent lysine residues (15, 16). Ongoing studies in our laboratory have used mass spectrometry to confirm biotinylation sites depicted in Figure 2; to tentatively identify additional biotinylation sites; and to identify modifications (acetylation, methylation) that co-exist with biotinylation in the same histone molecule (18). Antibodies have been 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.

Fig. 2.

Modification sites in histones H2A, H3, and H4 (1, 17). Abbeviations: Ac, acetate; B, biotin; M, methyl; P, phosphate; U, ubiquitin.

Biological Functions of Histone Biotinylation

K8- and K12-biotinylated species of histone H4 appear to be involved in the formation of heterochromatin structures and in gene silencing (Camporeale and Zempleni, submitted). This notion is based on chromatin immunoprecipitation studies in human lymphoid cells, using biotinylation site-specific antibodies. Specifically, if chromatin was immunoprecipitated with antibodies to K8- and K12-biotinylated histone H4, sequences from pericentromeric heterochromatin were enriched by up to 100% compared with various control antibodies. Moreover, chromatin from silent gene loci (e.g., interleukin-2) was enriched by up to 50% if precipitated with antibodies to K8- and K12-biotinylated species of histone H4. Importantly, stimulation of interleukin-2 expression by using mitogens was associated with a rapid depletion of biotinylated histones at the interleukin-2 locus in lymphoid cells. This suggests that gene silencing by histone biotinylation can be rapidly reversed by removal of the biotin tag. Currently, it is uncertain as to whether gene activation is mediated by enzymatic debiotinylation of histones or by removal of biotinylated histones from the gene locus.

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 has been 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 about 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 impact on biotinylation of histones in JAr cells, whereas biotinylation of carboxylases correlated strongly with biotin supply (23). Likewise, other human-derived cell lines preserved histone-bound biotin at the expense of carboxylase-bound biotin if 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 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. Evidence has been provided that biotinidase may catalyze both biotinylation and debiotinylation of histones (24). Variables such as the microenvironment in chromatin, and posttranslational modifications and alternate splicing of biotinidase might determine whether biotinidase acts as biotinyl histone transferase or histone debiotinylase (2).

Conclusion

Our knowledge of potential biological functions of biotin has been 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 five years the field has been advanced by the generation of a variety of analytical tools. For example, site-specific antibodies have been generated on the basis of the identification of biotinylation sites in histones, and knockdown models of the enzymes mediating biotinylation of histones have been generated in human cells and Drosophila. Using these tools, evidence has been 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 have been demonstrated by Mock's group and other research teams (25), and one can envision that many of the known effects of biotin deficiency can be attributed to abnormal chromatin structures.