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...

The Polycomb group (PcG) proteins are epigenetic suppressors of gene expression that function through modification of histones to change chromatin structure and modulate gene expression and cell behavior. Recent studies show that PcG proteins are expressed in epidermis, that their levels change during differentiation and in disease states, and that PcG expression is regulated by agents that influence cell proliferation and survival. The results indicate that PcG proteins regulate keratinocyte cell-cycle progression, apoptosis, senescence, and differentiation. These proteins are expressed in progenitor cells, in the basal layer, and in suprabasal keratinocytes, and the level, timing, and distribution of expression suggest that the PcG proteins have a central role in maintaining the balance between cell survival and death in multiple epidermal compartments. Additional studies indicate an important role in skin cancer progression.

Background: Sulforaphane is an important cancer preventative in skin cancer. Results: SFN induces p21Cip1 via a p53-dependent mechanism that involves p53 stabilization. Conclusion: p21Cip1 expression is differentially regulated in normal keratinocytes versus cancer cells. Significance: SFN treatment will slow proliferation of normal epidermal keratinocytes but will cause resident cancer cell apoptosis.

The polycomb group (PcG) genes encode a family of proteins that methylate and ubiquitinate histones to close chromatin and suppress gene expression. PcG proteins are present at elevated levels in cancer cells, and this is associated with reduced tumor suppressor protein level and enhanced cell survival. Agents that reduce PcG protein level are regarded as potentially cancer-preventative agents. Sulforaphane (SFN) is a biologically important isothiocyanate found in cruciferous vegetables that is an important candidate chemopreventive agent. However, the impact of SFN on the level and function of PcG proteins in skin cancer cells has not been assessed. We show that SFN treatment causes a concentration-dependent reduction in PcG protein (Bmi-1, Ezh2) expression in SCC-13 skin cancer cells and also reduces trimethylation of lysine 27 of histone H3. This is associated with accumulation of cells in G 2 /M phase; reduced levels of cyclin B1, cyclin A, cyclin dependent kinases 1 and 2; and increased p21Cip1 expression. Sulforaphane treatment also increases cleavage of procaspase 3, 8, and 9 and enhances PARP cleavage and apoptosis. Similar results are observed in other skin-derived cell immortalized and transformed cell lines. Forced expression of the Bmi-1 polycomb protein in SCC-13 cells reverses these effects. The SFN-dependent loss of Bmi-1 and Ezh2 is due to proteasomeassociated degradation. These results suggest that dietary isothiocyanates may suppress cancer progression by reducing PcG protein level via a proteasome-dependent mechanism, thereby inhibiting PcG-dependent pro-survival epigenetic events.

Secretory proteins such as apolipoprotein B-100 (apoB) undergo oxidative folding (formation of disulfide bonds) in the endoplasmic reticulum (ER) before secretion. Oxidative folding depends on flavoproteins in eukaryotes. Here, human liver (HepG2) cells were used to model effects of riboflavin concentrations in culture media on folding and secretion of apoB. Cells were cultured in media containing 3.1, 12.6, and 300 nmol/L of riboflavin, representing moderately deficient, physiological, and pharmacological plasma concentrations in humans, respectively. When cells were cultured in riboflavin-deficient medium, secretion of apoB decreased by >80% compared with controls cultured in physiological medium. The nuclear translocation of the transcription factor ATF-6 increased by >180% in riboflavin-deficient cells compared with physiological controls; this is consistent with ER stress. Nuclear translocation of ATF-6 was associated with activation of the unfolded protein response. Expression of stress-response genes coding for ubiquitin-activating enzyme 1, growth arrest and DNA damage inducible gene, and glucose regulated protein of 78 kDa was greater in riboflavin-deficient cells compared with other treatment groups. Finally, phosphorylation of the eukaryotic initiation factor (eukaryotic initiation factor 2alpha) increased in riboflavin-deficient cells, consistent with decreased translational activity. We conclude 1) that riboflavin deficiency causes ER stress and activation of unfolded protein response in HepG2 cells, and 2) that riboflavin deficiency decreases protein secretion in HepG2 cells. Decreased secretion of apoB in riboflavin-deficient cells might interfere with lipid homeostasis in vivo.