Subhashinee S.K. Wijeratne

Biotin and biotinidase deficiency

Biotin is a water-soluble vitamin that serves as an essential coenzyme for five carboxylases in mammals. Biotin-dependent carboxylases catalyze the fixation of bicarbonate in organic acids and play crucial roles in the metabolism of fatty acids, amino acids and glucose. Carboxylase activities decrease substantially in response to biotin deficiency. Biotin is also covalently attached to histones; biotinylated histones are enriched in repeat regions in the human genome and appear to play a role in transcriptional repression of genes and genome stability. Biotin deficiency may be caused by insufficient dietary uptake of biotin, drug–vitamin interactions and, perhaps, by increased biotin catabolism during pregnancy and in smokers. Biotin deficiency can also be precipitated by decreased activities of the following proteins that play critical roles in biotin homeostasis: the vitamin transporters sodium-dependent multivitamin transporter and monocarboxylate transporter 1, which mediate biotin transport in the intestine, liver and peripheral tissues, and renal reabsorption; holocarboxylase synthetase, which mediates the binding of biotin to carboxylases and histones; and biotinidase, which plays a central role in the intestinal absorption of biotin, the transport of biotin in plasma and the regulation of histone biotinylation. Symptoms of biotin deficiency include seizures, hypotonia, ataxia, dermatitis, hair loss, mental retardation, ketolactic acidosis, organic aciduria and also fetal malformations. This review focuses on the deficiencies of both biotin and biotinidase, and the medical management of such cases.

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.

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.

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.

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.

Epigenetic regulation of chromatin structure and gene function by biotin: are biotin requirements being met?

Histones H2A, H3, and H4 are modified by covalent binding of the vitamin biotin to distinct lysine residues. Binding of biotin to histones is mediated by holocarboxylase synthetase (HCS) and perhaps biotinidase. Biotinylation of lysine-12 in histone H4 (K12BioH4) plays roles in gene repression, stability of repeat regions and transposable elements, and regulation of biotin transporter expression in eukaryotes. Decreased biotinylation of histones in biotin-deficient and HCS-deficient human cells and Drosophila melanogaster impairs stress resistance, life span, and biotin homeostasis. Chromatin is comprised of DNA and DNA-binding proteins, i.e., histones and non-histone proteins. Histones play a predominant role in the folding of DNA into chromatin.1 Five major classes of histones have been identified in mammals: H1, H2A, H2B, H3, and H4. Histones consist of a globular domain and a more flexible amino terminus (histone “tail”). DNA and histones form repetitive nucleoprotein units, the nucleosomes.1 Each nucleosome (“nucleosomal core particle”) consists of 146 base pairs of DNA wrapped around an octamer of core histones (one H3-H3-H4-H4 tetramer and two H2A-H2B dimers). The amino terminal tail of histones protrudes from the nucleosomal surface; covalent modifications of this tail affect the structure of chromatin and form the basis for gene regulation.1–3 Amino acid residues in histone …

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.

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.

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.

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.