No abstract

Transposable elements such as long terminal repeats (LTR) constitute about 45% of the human genome; transposition events impair genome stability. Fifty-four promoter-active retrotransposons have been identified in humans. Epigenetic mechanisms are important for transcriptional repression of retrotransposons, preventing transposition events and abnormal regulation of genes. Here, we demonstrate that the covalent binding of the vitamin biotin to lysine-12 in histone H4 (H4K12bio) and lysine-9 in histone H2A (H2AK9bio), mediated by holocarboxylase synthetase (HCS), is an epigenetic mechanism to repress retrotransposon transcription in human and mouse cell lines and in primary cells from a human supplementation study. Abundance of H4K12bio and H2AK9bio at intact retrotransposons and a solitary LTR depended on biotin supply and HCS activity, and was inversely linked with the abundance of LTR transcripts. Knockdown of HCS in Drosophila enhances retrotransposition in the germline. Importantly, we demonstrated that depletion of H4K12bio and H2AK9bio in biotin-deficient cells correlates with increased production of viral particles, transposition events, and ultimately decreases chromosomal stability. Collectively, this study reveals a novel diet-dependent epigenetic mechanism that could affect cancer risk.

In eukaryotic cell nuclei, DNA associates with the core histones H2A, H2B, H3, and H4 to form nuclosomal core particles. DNA binding to histones is regulated by posttranslational modifications of N-terminal tails, e.g., acetylation and methylation of histones. These modifications play important roles in the epigenetic control of chromatin structure. Recently, evidence has been provided that biotinidase and holocarboxylase synthetase catalyze the covalent binding of biotin to histones. Primary aim of this study was to identify biotinylation sites in histone H2A and its variant H2AX. Secondary aims were to determine whether acetylation and methylation of histone H2A affect subsequent biotinylation, and to determine whether biotinidase and holocarboxylase synthetase localize to the nucleus in human cells. Biotinylation sites were identified using synthetic peptides as substrates for biotinidase. These studies provided evidence that K9 and K13 in the N-terminus of human histone H2A and H2AX are targets for biotinylation, and that K125, K127, and K129 in the C-terminus of histone H2A are targets for biotinylation. Biotinylation of lysine residues was decreased by acetylation of adjacent lysines, but was increased by dimethylation of adjacent arginines. The existence of biotinylated histone H2A in vivo was confirmed by using modificationspecific antibodies. Antibodies to biotinidase and holocarboxylase synthetase localized primarily to the nuclear compartment, consistent with a role for these enzymes in regulating chromatin structure. Collectively, these studies have identified five novel biotinylation sites in human histones; histone H2A is unique among histones in that biotinylation sites include amino acid residues from the Cterminus.

Polycomb group (PcG) protein-dependent histone methylation and ubiquitination drives chromatin compaction leading to reduced tumor suppressor expression and increased cancer cell survival. Green tea polyphenols and S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors are important candidate chemopreventive agents. Previous studies indicate that (-)-epigallocatechin-3-gallate (EGCG), a potent green tea polyphenol, suppresses PcG protein level and skin cancer cell survival. Inhibition of AdoHcy hydrolase with 3-deazaneplanocin A (DZNep) inhibits methyltransferases by reducing methyl group availability. In the present study, we examine the impact of EGCG and DZNep cotreatment on skin cancer cell function. EGCG and DZNep, independently and in combination, reduce the level of PcG proteins including Ezh2, eed, Suz12, Mel18 and Bmi-1. This is associated with reduced H3K27me3 and H2AK119ub formation, histone modifications associated with closed chromatin. Histone deacetylase 1 level is also reduced and acetylated H3 formation is increased. These changes are associated with increased tumor suppressor expression and reduced cell survival and are partially reversed by vector-mediated maintenance of Bmi-1 level. The reduction in PcG protein level is associated with increased ubiquitination and is reversed by proteasome inhibitors, suggesting proteasome-associated degradation.

PKC␦ increases keratinocyte differentiation and suppresses keratinocyte proliferation and survival. However, the mechanism of proliferation suppression is not well understood. The present studies show that PKC␦ overexpression increases p21Cip1 mRNA and protein level and promoter activity and that treatment with dominant-negative PKC␦, PKC␦-siRNA, or rottlerin inhibits promoter activation. Analysis of the p21Cip1 promoter upstream regulatory region reveals three DNA segments that mediate PKC␦-dependent promoter activation. The PKC␦ response element most proximal to the transcription start site encodes six GC-rich DNA elements. Mutation of these sites results in a loss of PKC␦-dependent promoter activation. Gel mobility supershift and chromatin immunoprecipitation reveal that these DNA elements bind the Kruppel-like transcription factor KLF4. PKC␦ increases KLF4 mRNA and protein level and KLF4 binding to the GC-rich elements in the p21 Cip1 proximal promoter. In addition, KLF4-siRNA inhibits PKC␦-dependent p21 Cip1 promoter activity. PKC␦ increases KLF4 expression leading to enhanced KLF4 interaction with the GC-rich elements in the p21 Cip1 promoter to activate transcription.PKC isoforms include three subfamilies of kinases that play a central role in the regulation of cell growth and differentiation (1). Classical PKCs (␣, ␤, and ␥) are calcium-, phospholipid-, and diacylglycerol-dependent; novel PKCs (nPKC ␦, ⑀, , and ) 2 are activated by diacylglycerol and phospholipids, but they do not respond directly to calcium; and atypical PKCs ( and ) are calcium-and diacylglycerol-independent but undergo allosteric activation (2, 3). Epidermal keratinocytes express PKC␣, ␤II, ␦, ⑀, , and (4 -10). These kinases have been studied in cultured keratinocytes and in animal models (11)(12)(13)(14)(15)(16)(17)(18)(19). A number of laboratories have shown that nPKC isoforms stimulate keratinocyte differentiation (15, 20 -24, 26). Consistent with this role, studies from our group show that the novel PKC (nPKC) isoforms stimulate keratinocyte differentiation by activating MAPK signaling, which results in increased nuclear levels of AP1, CCAAT enhancer-binding protein, and Sp1 transcription factors and binding of these factors to target genes to increase transcription (27)(28)(29). Involucrin is a classical marker of differentiation, and our studies show that PKC␦ is a potent activator of involucrin expression (21, 27, 30 -32).PKC isoforms have also been implicated in the regulation of keratinocyte proliferation (13,20,23,(33)(34)(35). This role is particularly important, because keratinocyte differentiation is associated with cessation of proliferation, and it would make mechanistic sense to have a common kinase activate both processes. A limited number of studies have examined the mechanism of nPKC regulation of keratinocyte proliferation. For example, the nPKC isoform PKC⑀ binds to and activates Fyn, a Src kinase, and this is associated with reduced keratinocyte proliferation (36). PKC⑀ forms a complex with cyclin E-cdk2-p21Cip1...