K12-biotinylated histone H4 marks heterochromatin in human lymphoblastoma cells.

Covalent modifications of histones play crucial roles in chromatin structure and genomic stability. Recently, we reported a novel modification of histones: biotinylation of lysine residues. Here we provide evidence that K12-biotinylated histone H4 (K12Bio H4) maps specifically to both heterochromatin (alpha satellite repeats in pericentromeric regions) and transcriptionally repressed chromatin (gamma-G globin and interleukin-2) in human lymphoblastoma cells. The abundance of K12Bio H4 in these regions was similar to that of K9-dimethylated histone H3, a known marker for heterochromatin. Likewise, K8-biotinylated histone H4 (K8Bio H4) mapped to heterochromatin, but the relative enrichment was smaller compared with K12Bio H4. Stimulation of interleukin-2 transcriptional activity with phorbol-12-myristate-13-acetate and phytohemagglutinin caused a rapid depletion of K12Bio H4 in the gene promoter. These data are consistent with a novel role for biotin in chromatin structure and transcriptional activity of genes.

Biotin supplementation decreases the expression of the SERCA3 gene (ATP2A3) in Jurkat cells, thus, triggering unfolded protein response.

Protein folding in the endoplasmic reticulum (ER) depends on Ca(2+); uptake of Ca(2+) into the ER is mediated by sarco/endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3). The 5'-flanking region of the SERCA3 gene (ATP2A3) contains numerous binding sites for the transcription factors Sp1 and Sp3. Biotin affects the nuclear abundance of Sp1 and Sp3, which may act as transcriptional activators or repressors. Here we determined whether biotin affects the expression of the SERCA3 gene and, thus, protein folding in human lymphoid cells. Jurkat cells were cultured in media containing 0.025 nmol/L biotin (denoted "deficient") or 10 nmol/L biotin ("supplemented"). The transcriptional activity of the full-length human SERCA3 promoter was 50% lower in biotin-supplemented cells compared to biotin-deficient cells. Biotin-dependent repressors bind to elements located 731-1312 bp upstream from the transcription start site in the SERCA3 gene. The following suggest that low expression of SERCA3 in biotin-supplemented cells impaired folding of secretory proteins in the ER, triggering unfolded protein response: (i) sequestration of Ca(2+) in the ER decreased by 14-24% in response to biotin supplementation; (ii) secretion of interleukin-2 into the extracellular space decreased by 75% in response to biotin supplementation; (iii) the nuclear abundance of stress-induced transcription factors increased in response to biotin supplementation; and (iv) the abundance of stress-related proteins such ubiquitin activating enzyme 1, growth arrest and DNA damage 153 gene, X-box binding protein 1 and phosphorylated eukaryotic translation initiation factor 2alpha increased in response to biotin supplementation. Collectively, this study suggests that supplements containing pharmacological doses of biotin may cause cell stress by impairing protein folding in the ER.

The expression of genes encoding ribosomal subunits and eukaryotic translation initiation factor 5A depends on biotin and bisnorbiotin in HepG2 cells.

Biotin affects gene expression at both the transcriptional and the posttranscriptional level; biotin metabolites might have biotin-like activities with regard to gene expression. Here, human hepatocarcinoma (HepG2) cells were used (i) to identify clusters of biotin-dependent genes, (ii) to determine whether the naturally occurring metabolite bisnorbiotin affects gene expression and (iii) to determine whether biotin and bisnorbiotin affect the expression of genes coding for ribosomal subunits and translation initiation factors. HepG2 cells were cultured in media containing deficient (0.025 nmol/L), physiological (0.25 nmol/L, control) and pharmacological (10 nmol/L) concentrations of biotin; a fourth treatment group consisted of cells cultured in biotin-deficient medium (0.025 nmol/L) supplemented with bisnorbiotin (0.225 nmol/L). Gene expression was quantified by using DNA microarrays and reverse transcriptase polymerase chain reaction. The expression of 1803 genes depended on biotin concentrations in culture media; the expression of 618 genes depended on bisnorbiotin. Biotin deficiency was associated with increased expression of a gene cluster encoding ribosomal subunits and eukaryotic translation initiation factor 5A; this effect was reversed by supplementation with biotin and bisnorbiotin. Additional prominent clusters of (bisnor)biotin-dependent genes included DNA-, RNA-, and nucleotide-binding proteins, consistent with a role for biotin in cell signaling and gene expression. Collectively, these data suggest that bisnorbiotin has biotin-like activities regarding gene expression, and that clusters of (bisnor)biotin-dependent genes include genes that play roles in translational activity.

HepG2 cells develop signs of riboflavin deficiency within 4 days of culture in riboflavin-deficient medium.

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential coenzymes in redox reactions. For example, FAD is a coenzyme for both glutathione reductase and enzymes that mediate the oxidative folding of secretory proteins. Here we investigated short-term effects of moderately riboflavin-deficient culture medium on flavin-related responses in HepG2 hepatocarcinoma cells. Cells were cultured in riboflavin-deficient (3.1 nmol/l) medium for up to 6 days; controls were cultured in riboflavin-sufficient (532 nmol/l) medium. The activity of glutathione reductase decreased by 98% within 4 days of riboflavin-deficient culture. Transport rates of riboflavin increased in response to riboflavin depletion, whereas expression of enzymes mediating flavocoenzyme synthesis (flavokinase and FAD synthetase) decreased in response to depletion. The oxidative folding and synthesis of plasminogen and apolipoprotein B-100 was impaired within 4 days of culture in riboflavin-deficient medium; this is consistent with impaired processing of secretory proteins in riboflavin-deficient cells. Riboflavin depletion was associated with increased DNA-binding activities of transcription factors with affinity for endoplasmic reticulum stress elements and nuclear factor kappaB (NF-kappaB) consensus elements, suggesting cell stress. Moreover, the abundance of the stress-induced protein GADD153 was greater in riboflavin-deficient cells compared with controls. Riboflavin deficiency was associated with decreased rates of cell proliferation caused by arrest in G1 phase of the cell cycle. These studies are consistent with the hypothesis that HepG2 cells have a great demand for riboflavin and that cell stress develops rapidly if riboflavin supply is marginally low.

High-throughput immunoblotting identifies biotin-dependent signaling proteins in HepG2 hepatocarcinoma cells.

Biotin affects the abundance of mRNA coding for approximately 10% of genes expressed in human-derived hepatocarcinoma (HepG2) cells. Here, we determined whether effects of biotin on gene expression are associated with changes in the abundance of distinct proteins in cell signaling and structure. HepG2 cells were cultured in media containing the following concentrations of biotin: 0.025 nmol/L (denoted "deficient"), 0.25 nmol/L ("physiological" = control), and 10 nmol/L ("pharmacological") for 10 d before harvesting. The abundance of 1009 proteins from whole-cell extracts was quantified by using high-throughput immunoblots. The abundance of 44 proteins changed by at least 25% in biotin-deficient and biotin-supplemented cells compared with physiological controls. One third of these proteins participate in cell signaling. Specifically, proteins associated with receptor tyrosine kinase-mediated signaling were identified as targets of biotin; the abundance of these proteins was greater in biotin-deficient cells than in controls. This was associated with increased DNA-binding activities of the transcription factors Fos and Jun, and increased expression of a reporter gene driven by activator protein (AP)1-binding elements in biotin-deficient cells compared with physiological controls. The abundance of selected signaling proteins was not paralleled by the abundance of mRNA, suggesting that biotin affects expression of these genes at a post-transcriptional step. Additional clusters of biotin-responsive proteins were identified that play roles in cytoskeleton homeostasis, nuclear structure and transport, and neuroscience. This study is consistent with the existence of clusters of biotin-responsive proteins in distinct biological processes, including signaling by Fos/Jun; the latter might mediate the proinflammatory and antiapoptotic effects of biotin deficiency.

Roles for nutrients in epigenetic events.

The field of epigenetics is the study of modifications of DNA and DNA-binding proteins that alter the structure of chromatin without altering the nucleotide sequence of DNA; some of these modifications may be associated with heritable changes in gene function. Nutrients play essential roles in the following epigenetic events. First, folate participates in the generation of S-adenosylmethionine, which acts as a methyl donor in the methylation of cytosines in DNA; methylation of cytosines is associated with gene silencing. Second, covalent attachment of biotin to histones (DNA-binding proteins) plays a role in gene silencing and in the cellular response to DNA damage. Third, tryptophan and niacin are converted to nicotinamide adenine dinucleotide, which is a substrate for poly(ADP-ribosylation) of histones and other DNA-binding proteins; poly(ADP-ribosylation) of these proteins participates in DNA repair and apoptosis. Here we present a novel procedure to map nutrient-dependent epigenetic marks in the entire genomes of any given species: the combined use of chromatin immunoprecipitation assays and DNA microarrays. This procedure is also an excellent tool to map the enzymes that mediate modifications of DNA and DNA-binding proteins in chromatin. Given the tremendous opportunities offered by the combined use of chromatin immunoprecipitation assays and DNA microarrays, the nutrition community can expect seeing a surge of information related to roles for nutrients in epigenetic events.

Biotin deficiency stimulates survival pathways in human lymphoma cells exposed to antineoplastic drugs.

Cells may respond to nutrient deficiency or death signals with nuclear translocation of the transcription factor nuclear factor kappaB (NF-kappaB), which activates transcription of anti-apoptotic genes. Here we tested the hypothesis that biotin deficiency stimulates NF-kappaB-dependent survival pathways in human lymphoma cells, enhancing resistance to antineoplastic agents. Lymphoma (Jurkat) cells were cultured in biotin-deficient (0.025 nmol/L) and biotin-supplemented (10 nmol/L) media. If cells were treated with antineoplastic agents (taxol, doxorubicin or vinblastine), nuclear translocation of two NF-kappaB proteins (p50 and p65) was >25% greater in biotin-deficient compared with biotin-supplemented cells. The transcriptional activities of the following NF-kappaB-dependent reporter genes were 16-59% greater in biotin-deficient compared with biotin-supplemented cells treated with various antineoplastic agents: (1) reporter expression driven by a TATA box and five NF-kappaB repeats and (2) reporter expression driven by the regulatory region of the anti-apoptotic Bfl-1 gene. Collectively, these findings are consistent with activation of survival pathways in biotin-deficient lymphoma cells. Finally, cells were treated with antineoplastic agents for 48 h and cell survival was monitored at timed intervals. Biotin deficiency was associated with enhanced survival of cells treated with doxorubicin and vinblastine, but did not affect survival of cells treated with taxol. Collectively, these observations suggest that biotin deficiency may enhance resistance of cancer cells to antineoplastic agents.

Biotin supplementation increases expression of the cytochrome P450 1B1 gene in Jurkat cells, increasing the occurrence of single-stranded DNA breaks.

DNA microarray studies provided evidence that biotin supplementation increases the abundance of mRNA encoding cytochrome P(450) 1B1 (CYP1B1) in human lymphocytes. CYP1B1 hydroxylates procarcinogens, generating electrophilic mutagens. Here, we sought to identify the signaling pathways that increase the expression of CYP1B1 in biotin-supplemented human T (Jurkat) cells and to determine whether activation of the CYP1B1 gene is associated with increased occurrence of single-stranded DNA breaks. Jurkat cells were cultured in biotin-deficient (0.025 nmol/L) and biotin-supplemented (10 nmol/L) media. The transcriptional activity of a CYP1B1 reporter gene construct was 24% greater in biotin-supplemented compared with biotin-deficient cells (P < 0.01). Similarly, the abundance of CYP1B1 mRNA was 72% greater in biotin-supplemented than in biotin-deficient cells (P < 0.05). Electrophoretic mobility shift assays suggested that Sp1 sites in the regulatory region of the CYP1B1 gene play important roles in transcriptional activation by biotin. The abundance of CYP1B1 protein and activity of CYP1B1 were 124 and 35% greater, respectively, in biotin-supplemented compared with biotin-deficient cells (P < 0.05). The increased expression of CYP1B1 in biotin-supplemented cells was associated with an increase in the occurrence of single-stranded DNA breaks compared with biotin-deficient cells; synthetic inhibitors of CYP1B1 prevented strand breaks, suggesting that the effects of biotin were specific for CYP1B1. These studies provide evidence that transcription factors with an affinity for Sp1 sites mediate transcriptional activation of the CYP1B1 gene in biotin-supplemented T cells, increasing the occurrence of single-stranded DNA breaks.

Biotin supply affects rates of cell proliferation, biotinylation of carboxylases and histones, and expression of the gene encoding the sodium-dependent multivitamin transporter in JAr choriocarcinoma cells.

BACKGROUND: Placental transfer of nutrients and secretion of hormones is essential for normal fetal development. AIM OF THE STUDY: To determine whether biotin supply affects biotin homeostasis, proliferation rates, and progesterone secretion in placenta cells. METHODS: JAr choriocarcinoma cells were cultured in media containing deficient (25 pmol/L), physiological (250 pmol/L), or pharmacological concentrations (10,000 pmol/L) of biotin for three weeks; markers for biotin homeostasis, proliferation, and hormone secretion were quantified. RESULTS: Biotin concentrations in culture media correlated negatively with expression of the biotin transporter SMVT, as judged by cellular transport rates of biotin, abundance of SMVT protein, and transcriptional activity of SMVT reporter-gene constructs. Notwithstanding this homeostatic mechanism, biotin concentrations in media correlated positively with activities of biotin-dependent propionyl-CoA carboxylase, abundance of biotinylated carboxylases, and with biotinylation of histones. Biotin deficiency was associated with decreased rates of thymidine uptake into JAr cells [pmol thymidine/( 10(6) cells x 24 h)]: 1.6 +/- 0.1 (25 pmol/L biotin) versus 2.3 +/- 0.2 (250 pmol/L biotin) versus 3.7 +/- 0.4 (10,000 pmol/L biotin), suggesting that cell proliferation depends on biotin. Secretion of progesterone was reduced in biotin-deficient cells; this effect was caused by reduced generation of new cells in deficient media rather than by an immediate effect of biotin on progesterone secretion at the singlecell-level. CONCLUSIONS: This study provides evidence that choriocarcinoma cells cannot maintain normal activities of biotin-dependent metabolic pathways if biotin concentrations in culture media are low. It is uncertain whether activities of biotin-dependent pathways in placenta affect fetal development in vivo.

The nuclear abundance of transcription factors Sp1 and Sp3 depends on biotin in Jurkat cells.

Biotin affects gene expression in mammals; however, the signaling pathways leading to biotin-dependent transcriptional activation and inactivation of genes are largely unknown. Members of the Sp/Krüppel-like factor family of transcription factors (e.g., the ubiquitous Sp1 and Sp3) play important roles in the expression of numerous mammalian genes. We tested the hypothesis that the nuclear abundance of Sp1 and Sp3 depends on biotin in human T cells (Jurkat cells) mediating biotin-dependent gene expression. Jurkat cells were cultured in biotin-deficient (0.025 nmol/L) and biotin-supplemented (10 nmol/L) media for 5 wk prior to transcription factor analysis. The association of Sp1 and Sp3 with DNA-binding sites (GC box and CACCC box) was 76-149% greater in nuclear extracts from biotin-supplemented cells compared with biotin-deficient cells, as determined by electrophoretic mobility shift assays. The increased DNA-binding activity observed in biotin-supplemented cells was caused by increased transcription of genes encoding Sp1 and Sp3, as shown by mRNA levels and reporter-gene activities; increased transcription of Sp1 and Sp3 genes was associated with the increased abundance of Sp1 and Sp3 protein in nuclei. Notwithstanding the important role for phosphorylation of Sp1 and Sp3 in regulating DNA-binding activity, the present study suggests that the effects of biotin on phosphorylation of Sp1 and Sp3 are minor. The increased nuclear abundance of Sp1 and Sp3 in biotin-supplemented cells was associated with increased transcriptional activity of 5'-flanking regions in Sp1/Sp3-dependent genes in reporter-gene assays. This study provides evidence that some effects of biotin on gene expression might be mediated by the nuclear abundance of Sp1 and Sp3.