Biotinylation of lysine 16 in histone H4 contributes toward nucleosome condensation.

Holocarboxylase synthetase (HLCS) is part of a multiprotein gene repression complex and catalyzes the covalent binding of biotin to lysines (K) in histones H3 and H4, thereby creating rare gene repression marks such as K16-biotinylated histone H4 (H4K16bio). We tested the hypothesis that H4K16bio contributes toward nucleosome condensation and gene repression by HLCS. We used recombinant histone H4 in which K16 was mutated to a cysteine (H4K16C) for subsequent chemical biotinylation of the sulfhydryl group to create H4K16Cbio. Nucleosomes were assembled by using H4K16Cbio and the 'Widom 601' nucleosomal DNA position sequence; biotin-free histone H4 and H4K16C were used as controls. Nucleosomal compaction was analyzed using atomic force microscopy (AFM). The length of DNA per nucleosome was ∼30% greater in H4K16Cbio-containing histone octamers (61.14±10.92nm) compared with native H4 (46.89±12.6nm) and H4K16C (47.26±10.32nm), suggesting biotin-dependent chromatin condensation (P<0.001). Likewise, the number of DNA turns around histone core octamers was ∼17.2% greater in in H4K16Cbio-containing octamers (1.78±0.16) compared with native H4 (1.52±0.21) and H4K16C (1.52±0.17), judged by the rotation angle (P<0.001; N=150). We conclude that biotinylation of K16 in histone H4 contributes toward chromatin condensation.

Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity.

Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.

Biotinylation is a natural, albeit rare, modification of human histones.

Previous studies suggest that histones H3 and H4 are posttranslationally modified by binding of the vitamin biotin, catalyzed by holocarboxylase synthetase (HCS). Albeit a rare epigenetic mark, biotinylated histones were repeatedly shown to be enriched in repeat regions and repressed loci, participating in the maintenance of genome stability and gene regulation. Recently, a team of investigators failed to detect biotinylated histones and proposed that biotinylation is not a natural modification of histones, but rather an assay artifact. Here, we describe the results of experiments, including the comparison of various analytical protocols, antibodies, cell lines, classes of histones, and radiotracers. These studies provide unambiguous evidence that biotinylation is a natural, albeit rare, histone modification. Less than 0.001% of human histones H3 and H4 are biotinylated, raising concerns that the abundance might too low to elicit biological effects in vivo. We integrated information from this study, previous studies, and ongoing research efforts to present a new working model in which biological effects are caused by a role of HCS in multiprotein complexes in chromatin. In this model, docking of HCS in chromatin causes the occasional binding of biotin to histones as a tracer for HCS binding sites.

Biotin regulates the expression of holocarboxylase synthetase in the miR-539 pathway in HEK-293 cells.

Holocarboxylase synthetase (HCS) catalyzes the covalent binding of biotin to carboxylases and histones. In mammals, the expression of HCS depends on biotin, but the mechanism of regulation is unknown. Here we tested the hypothesis that microRNA (miR) plays a role in the regulation of the HCS gene. Human embryonic kidney cells were used as the primary model, but cell lines from other tissues and primary human cells were also tested. In silico searches revealed an evolutionary conserved binding site for miR-539 in the 3 prime -untranslated region (3 prime -UTR) of HCS mRNA. Transgenic cells and reporter gene constructs were used to demonstrate that miR-539 decreases the expression of HCS at the level of transcription rather than translation; these findings were corroborated in nontransgenic cells. When miR-539 was overexpressed in transgenic cells, the abundance of both HCS and biotinylated histones decreased. The abundance of miR-539 was tissue dependent: fibroblasts gt kidney cells gt intestinal cells gt lymphoid cells. Dose-response studies revealed that the abundance of miR-539 was significantly higher at physiological concentrations of biotin than both biotin-deficient and biotin-supplemented media in all cell lines tested. In kidney cells, the expression of HCS was lower in cells in physiological medium than in deficient and supplemented medium. In contrast, in fibroblasts, lymphoid cells, and intestinal cells, there was no apparent link between miR-539 abundance and HCS expression, suggesting that factors other than miR-539 also contribute to the regulation of HCS expression in some tissues. Collectively, the results of this study suggest that miR-539 is among the factors sensing biotin and regulating HCS.

Repression of transposable elements by histone biotinylation.

Transposable elements constitute >40% of the human genome; transposition of these elements increases genome instability and cancer risk. Epigenetic mechanisms are important for transcriptional repression of retrotransposons, thereby preventing transposition events. Binding of biotin to histones, mediated by holocarboxylase synthetase (HCS), is a novel histone mark that plays a role in gene regulation. Here, we review recent findings that biotinylation of lysine-12 in histone H4 (H4K12bio) is an epigenetic mechanism to repress long terminal repeat (LTR) retrotransposons in human and mouse cell lines, primary cells from human adults, and in Drosophila melanogaster. Further, evidence is summarized that supports a causal relationship between the repression of LTR in H4K12bio-depleted cells and increased production of viral particles, increased frequency of retrotransposition events, and increased frequency of chromosomal abnormalities in mammals and Drosophila. Although HCS interacts physically with histones H3 and H4, the mechanism responsible for targeting HCS to retrotransposons to mediate histone biotinylation is uncertain. We hypothesize that HCS binds specifically to genomic regions rich in methylated cytosines and catalyzes increased biotinylation of histone H4 at lysine-12. Further, we hypothesize that this biotinylation promotes the subsequent dimethylation of lysine-9 in histone H3, resulting in an overall synergistic effect of 3 diet-dependent covalent modifications of histones in the repression of LTR.

Biotin.

Biotin is a water-soluble vitamin and serves as a coenzyme for five carboxylases in humans. Biotin is also covalently attached to distinct lysine residues in histones, affecting chromatin structure and mediating gene regulation. This review describes mammalian biotin metabolism, biotin analysis, markers of biotin status, and biological functions of biotin. Proteins such as holocarboxylase synthetase, biotinidase, and the biotin transporters SMVT and MCT1 play crucial roles in biotin homeostasis, and these roles are reviewed here. Possible effects of inadequate biotin intake, drug interactions, and inborn errors of metabolism are discussed, including putative effects on birth defects.

Improved extraction of peanut residues from a wheat flour matrix for immunochemical detection.

The efficacy of different buffers in extracting peanut from a solid model food incurred with peanut and subjected to processing was evaluated using two commercial ELISA kits: Veratox® for peanut allergen and peanut ELISA from Morinaga. Average percentage recoveries of peanut from unprocessed samples using the kit supplied buffers were 46 ±â€¯5 and 28 ±â€¯2 with the Veratox and Morinaga kits, respectively. However, Na2CO3, pH 9.6 and PBS containing 1 M GuHCl recovered 65% ±â€¯4% and 77% ±â€¯10% of peanut, respectively from unprocessed samples with the Veratox kit. These two buffers also performed better than the Veratox buffer with fried, high pressure processed, and baked samples. PBS containing SDS and ß-ME, performed significantly better than the Morinaga buffer in recovering peanut from unprocessed, boiled and fried samples. Thus, the use of alternative extraction buffers provides better recovery of peanut residues from a processed solid food matrix.

A cell death assay for assessing the mitochondrial targeting of proteins.

The mitochondrial proteome comprises 1000 to 1500 proteins, in addition to proteins for which the mitochondrial localization is uncertain. About 800 diseases have been linked with mutations in mitochondrial proteins. We devised a cell survival assay for assessing the mitochondrial localization in a high-throughput format. This protocol allows us to assess the mitochondrial localization of proteins and their mutants, and to identify drugs and nutrients that modulate the mitochondrial targeting of proteins. The assay works equally well for proteins directed to the outer mitochondrial membrane, inner mitochondrial membrane mitochondrial and mitochondrial matrix, as demonstrated by assessing the mitochondrial targeting of the following proteins: carnitine palmitoyl transferase 1 (consensus sequence and R123C mutant), acetyl-CoA carboxylase 2, uncoupling protein 1 and holocarboxylase synthetase. Our screen may be useful for linking the mitochondrial proteome with rare diseases and for devising drug- and nutrition-based strategies for altering the mitochondrial targeting of proteins.

Potential of rosemary (Rosemarinus officinalis L.) diterpenes in preventing lipid hydroperoxide-mediated oxidative stress in Caco-2 cells.

The effects of 24 h supplementation of Caco-2 cells with carnosic acid and carnosol, and their activities against 5 microM oleic acid hydroperoxide (OAHPx)-mediated oxidative stress, were investigated. At 24 h of incubation, under nonstressed and stressed conditions, both compounds at 25, 50, and 100 microM supplement concentrations reduced catalase activity, whereas changes in glutathione peroxidase and superoxide dismutase activities varied depending upon the concentrations. Relative to control cultures, carnosic acid and carnosol reduced membrane damage by 40-50% when stressed by OAHPx. Carnosic acid and carnosol inhibited lipid peroxidation by 88-100% and 38-89%, respectively, under oxidative stress conditions. Both compounds significantly lowered DNA damage induced by OAHPx. Results of this study suggest that antioxidant activities of carnosic acid and carnosol could be partly due to their ability to increase or maintain glutathione peroxidase and superoxide dismutase activities.

Soy isoflavones protect the intestine from lipid hydroperoxide mediated oxidative damage.

The effects of 24 h supplementation of human colon carcinoma cells (Caco-2) with isoflavones, genistein, and daidzein and their activities against oleic acid hydroperoxide mediated oxidative stress were investigated. Genistein, at 25, 50, and 100 microM, and daidzein, at 25 and 50 microM, did not induce cell injury to Caco-2 cells. Both compounds reduced cell injury and DNA damage mediated by 5 microM oleic acid hydroperoxides in Caco-2 cells. The effects of genistein and daidzein on antioxidant enzymes were dependent upon the compound and its concentration.

Lipid hydroperoxide induced oxidative stress damage and antioxidant enzyme response in Caco-2 human colon cells.

It has been demonstrated that reactive oxygen species, free radicals, and oxidative products, such as lipid hydroperoxides, participate in tissue injuries and in the onset and progression of degenerative diseases in humans. Studies were conducted using Caco-2 colon carcinoma cells to evaluate cellular damage caused by exposing cells for 30 min to oleic acid hydroperoxides (OAHPx) at concentrations varying from 0 to 25 microM. Cell membrane damage and DNA damage were significantly high even at the lowest concentration of 2.5 microM OAHPx compared to the control. Cell lipid peroxidation, indicated by conjugated diene concentration, increased exponentially with increasing OAHPx concentration. Antioxidant mechanisms in Caco-2 cells were evaluated by measuring catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities. Cellular catalase and GPx activities were not significantly different from each other at 0 to 25 microM OAHPx concentrations. SOD activity decreased with increasing OAHPx concentration. These results show that existing enzymatic antioxidant mechanisms are not sufficient for complete detoxification of 5-25 microM lipid hydroperoxides.

Antioxidant polyphenols in almond and its coproducts.

Antioxidant efficacy of defatted almond whole seed, brown skin, and green shell cover extracts was evaluated by monitoring inhibition of human low-density lipoprotein (LDL) oxidation, inhibition of DNA scission, and metal ion chelation activities. The total phenolic contents of ethanolic extracts of brown skin and green shell cover of almond were 10 and 9 times higher than that of the whole seed, respectively. Brown skin extract at 50 ppm effectively inhibited copper-induced oxidation of human LDL cholesterol compared to whole seed and green shell cover extracts, which reached the same level of efficacy at 200 ppm. Green shell cover extract at 50 ppm level completely arrested peroxyl radical-induced DNA scission, whereas 100 ppm of brown skin and whole seed extracts was required for similar efficiencies. All three almond extracts exhibited excellent metal ion chelation efficacies. High-performance liquid chromatographic (HPLC) analysis revealed the presence of quercetin, isorhamnetin, quercitrin, kaempferol 3-O-rutinoside, isorhamnetin 3-O-glucoside, and morin as the major flavonoids in all extracts.

Hydrogen peroxide induced oxidative stress damage and antioxidant enzyme response in Caco-2 human colon cells.

Studies were conducted to evaluate the cell damage caused by exposing human colon carcinoma cells, Caco-2, to hydrogen peroxide at concentrations varying from 0 to 250 microM for 30 min. Evaluation of cell viability, as measured by trypan blue dye exclusion test, showed that the loss of viability was < 5% at concentrations up to 250 microM hydrogen peroxide. Cell membrane damage and DNA damage as measured by the leakage of lactate dehydrogenase and the comet assay, respectively, were significantly high at concentrations >100 microM hydrogen peroxide compared to those of the control. Antioxidant mechanisms in Caco-2 cells were evaluated by measuring catalase, superoxide dismutase, and glutathione peroxidase activities. Catalase activities remained constant in cells treated with 50-250 microM hydrogen peroxide. Superoxide dismutase activity decreased, whereas glutathione peroxidase activity increased in cells treated with H(2)O(2) concentrations of >50 microM. This study showed that with increasing hydrogen peroxide concentration, cell membrane leakage and DNA damage increased, whereas the three antioxidant enzymes responded differently, as shown by mathematical models.

Holocarboxylase synthetase synergizes with methyl CpG binding protein 2 and DNA methyltransferase 1 in the transcriptional repression of long-terminal repeats.

Holocarboxylase synthetase (HLCS) is a chromatin protein that facilitates the creation of histone H3 lysine 9-methylation (H3K9me) gene repression marks through physical interactions with the histone methyltransferase EHMT-1. HLCS knockdown causes a depletion of H3K9me marks in mammalian cell cultures and severe phenotypes such as short lifespan and low stress resistance in Drosophila melanogaster. HLCS displays a punctuate distribution pattern in chromatin despite lacking a strong DNA-binding domain. Previous studies suggest that the binding of HLCS to chromatin depends on DNA methylation. We tested the hypothesis that HLCS interacts physically with the DNA methyltransferase DNMT1 and the methyl CpG binding protein MeCP2 to facilitate the binding of HLCS to chromatin, and that these interactions contribute toward the repression of long-terminal repeats (LTRs) by H3K9me marks. Co-immunoprecipitation and limited proteolysis assays provided evidence suggesting that HLCS interacts physically with both DNMT1 and MeCP2. The abundance of H3K9me marks was 207% greater in the LTR15 locus in HLCS overexpression human embryonic kidney HEK293 cells compared with controls. This gain in H3K9me was inversely linked with a 87% decrease in mRNA coding for LTRs. Effects of HLCS abundance on LTR expression were abolished when DNA methylation marks were erased by treating cells with 5-azacytidine. We conclude that interactions between DNA methylation and HLCS are crucial for mediating gene repression by H3K9me, thereby providing evidence for epigenetic synergies between the protein biotin ligase HLCS and dietary methyl donors.

A 96-well plate assay for high-throughput analysis of holocarboxylase synthetase activity.

BACKGROUND: Holocarboxylase synthetase (HCS) catalyzes the covalent binding of biotin to both carboxylases and histones. Biotinylated carboxylases and biotinylated histones play crucial roles in the metabolism of fatty acids, amino acids, and glucose, and in gene regulation and genome stability, respectively. HCS null mammals are not viable whereas HCS deficiency is linked to developmental delays in humans and phenotypes such as short life span and low stress resistance in Drosophila. METHODS: HCS-dependent biotinylation of the polypeptide p67 was detected and quantified in a 96-well plate format using IRDye-streptavidin and infrared spectroscopy. RESULTS: Biotinylation of p67 by recombinant HCS (rHCS) and HCS from human cell extracts depended on time, temperature, and substrate concentration, all consistent with enzyme catalysis rather than non-enzymatic biotinylation. The Michaelis-Menten constant of rHCS for p67 was 4.1±1.5 µmol/l. The minimal concentration of rHCS that can be detected by this assay is less than 1.08 nmol/l. Jurkat cells contained 0.14±0.02 U of HCS activity [µmol of biotinylated p67 formed/(nmol/l HCSh)] in 400 µg of total protein. CONCLUSIONS: We developed a 96-well plate assay for high-throughput analysis of HCS activity in biological samples and studies of synthetic and naturally occurring HCS inhibitors.

Novel histone biotinylation marks are enriched in repeat regions and participate in repression of transcriptionally competent genes.

Covalent histone modifications play crucial roles in chromatin structure and genome stability. We previously reported biotinylation of lysine (K) residues in histones H2A, H3 and H4 by holocarboxylase synthetase and demonstrated that K12-biotinylated histone H4 (H4K12bio) is enriched in repeat regions and participates in gene repression. The biological functions of biotinylation marks other than H4K12bio are poorly understood. Here, novel biotinylation site-specific antibodies against H3K9bio, H3K18bio and H4K8bio were used in chromatin immunoprecipitation studies to obtain first insights into possible biological functions of these marks. Chromatin immunoprecipitation assays were conducted in human primary fibroblasts and Jurkat lymphoblastoma cells, and revealed that H3K9bio, H3K18bio and H4K8bio are enriched in repeat regions such as pericentromeric alpha satellite repeats and long-terminal repeats while being depleted in transcriptionally active promoters in euchromatin. Transcriptional stimulation of the repressed interleukin-2 promoter triggered a rapid depletion of histone biotinylation marks at this locus in Jurkat cells, which was paralleled by an increase in interleukin-2 mRNA. Importantly, the enrichment of H3K9bio, H3K18bio and H4K8bio at genomic loci depended on the concentration of biotin in culture media at nutritionally relevant levels, suggesting a novel mechanism of gene regulation by biotin.

K12-biotinylated histone H4 is enriched in telomeric repeats from human lung IMR-90 fibroblasts.

Covalent modifications of histones play a role in regulating telomere attrition and cellular senescence. Biotinylation of lysine (K) residues in histones, mediated by holocarboxylase synthetase (HCS), is a novel diet-dependent mechanism to regulate chromatin structure and gene expression. We have previously shown that biotinylation of K12 in histone H4 (H4K12bio) is a marker for heterochromatin and is enriched in pericentromeric alpha satellite repeats. Here, we hypothesized that H4K12bio is also enriched in telomeres. We used human IMR-90 lung fibroblasts and immortalized IMR-90 cells overexpressing human telomerase (hTERT) in order to examine histone biotinylation in young and senescent cells. Our studies suggest that one out of three histone H4 molecules in telomeres is biotinylated at K12 in hTERT cells. The abundance of H4K12bio in telomeres decreased by 42% during telomere attrition in senescent IMR-90 cells; overexpression of telomerase prevented the loss of H4K12bio. Possible confounders such as decreased expression of HCS and biotin transporters were formally excluded in this study. Collectively, these data suggest that H4K12bio is enriched in telomeric repeats and represents a novel epigenetic mark for cell senescence.