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.

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.

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.

Riboflavin deficiency causes protein and DNA damage in HepG2 cells, triggering arrest in G1 phase of the cell cycle.

Eukaryotes convert riboflavin to flavin adenine dinucleotide, which serves as a coenzyme for glutathione reductase and other enzymes. Glutathione reductase mediates the regeneration of reduced glutathione, which plays an important role in scavenging free radicals and reactive oxygen species. Here we tested the hypothesis that riboflavin deficiency decreases glutathione reductase activity in HepG2 liver cells, causing oxidative damage to proteins and DNA, and cell cycle arrest. As a secondary goal, we determined whether riboflavin deficiency is associated with gene expression patterns indicating cell stress. Cells were cultured in riboflavin-deficient and riboflavin-supplemented media for 4 days. Activity of glutathione reductase was not detectable in cells cultured in riboflavin-deficient medium. Riboflavin deficiency was associated with an increase in the abundance of damaged (carbonylated) proteins and with increased incidence of DNA strand breaks. Damage to proteins and DNA was paralleled by increased abundance of the stress-related transcription factor GADD153. Riboflavin-deficient cells arrested in G1 phase of the cell cycle. Moreover, oxidative stress caused by riboflavin deficiency was associated with increased expression of clusters of genes that play roles in cell stress and apoptosis. For example, the abundance of the pro-apoptotic pleiomorphic adenoma gene-like 1 gene was 183% greater in riboflavin-deficient cells compared with riboflavin-sufficient controls. We conclude that riboflavin deficiency is associated with oxidative damage to proteins and DNA in liver cells, leading to cell stress and G1 phase arrest.

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.

Jurkat cells respond to biotin deficiency with increased nuclear translocation of NF-kappaB, mediating cell survival.

Members of the NF-kappaB family of transcription factors cause transcriptional activation of anti-apoptotic genes. Here we determined whether survival of biotin-deficient cells is mediated by nuclear translocation of NF-kappaB. Human T (Jurkat) cells were cultured in biotin-deficient or biotin-supplemented media; nuclear translocation of NF-kappaB was stimulated with phytohemagglutinin and phorbol-12-myristate-13-acetate. Nuclear abundance of two members (p50 and p65) of the NF-kappaB family was greater in biotin-deficient compared to biotin-supplemented cells; this effect was mediated by phosphorylation of IkappaBalpha. The nuclear enrichment of p50 and p65 in biotin-deficient cells was associated with transcriptional activation of NF-kappaB-depedent genes such as the tumor suppressor gene p53 and the anti-apoptotic gene Bfl-1/A1. Biotin-deficient cells exhibited smaller activities of the apoptotic enzyme caspase-3 in response to treatment with tumor necrosis factor alpha, and decreased cell death in response to serum starvation compared to biotin-supplemented cells. These findings suggest that NF-kappaB mediates survival of biotin-deficient cells.

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.

Clusters of biotin-responsive genes in human peripheral blood mononuclear cells.

Effects of biotin in cell signaling are mediated by transcription factors such as nuclear factor-kappa B (NF-kappa B) and Sp1/Sp3 as well as by posttranslational modifications of DNA-binding proteins. These signaling pathways play roles in the transcriptional regulation of numerous genes. Here we tested the hypothesis that biotin-dependent genes are not randomly distributed in the human genome but are arranged in clusters. Peripheral blood mononuclear cells were isolated from healthy adults before and after supplementation with 8.8 micromol/day biotin for 21 days. Cells were cultured ex vivo with concanavalin A for 3 hours to stimulate gene expression. Abundances of mRNA encoding approximately 14,000 genes were quantified by both DNA microarray and reverse transcriptase-polymerase chain reaction. The expression of 139 genes increased by at least 40% in response to biotin supplementation, whereas the expression of 131 genes decreased by at least 40% in response to biotin supplementation. The following clusters of biotin-responsive genes were identified: 1) 16% of biotin-responsive gene products localized to the cell nucleus; at least 28% of biotin-responsive genes play roles in signal transduction (these findings are consistent with a role for biotin in cell signaling); and 2) of the biotin-responsive genes, 54% clustered on chromosomes 1, 2, 3, 11, 12, and 19, whereas no biotin-responsive genes were found on chromosomes 10, 16, 18, 21, and heterosomes. This suggests that position effects play a role in biotin-dependent gene expression. Collectively, these findings suggest that the human genome contains clusters of biotin-dependent genes.

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.

Diaminobiotin and desthiobiotin have biotin-like activities in Jurkat cells.

In mammals, biotin serves as a coenzyme for carboxylases such as propionyl-CoA carboxylase. The expression of genes encoding interleukin-2 (IL-2) and IL-2 receptor (IL-2R)gamma also depends on biotin. Biotin metabolites are structurally similar to biotin, and their concentrations in tissues are quantitatively important. Here, the hypothesis was tested that biotin metabolites can mimic the effects of biotin on gene expression and thus have biotin-like activities. A human T-cell line (Jurkat cells) was used to model effects of biotin and synthetic metabolites (diaminobiotin and desthiobiotin) on the expression of genes encoding IL-2 and IL-2Rgamma. Cells were cultured in biotin-deficient medium (0.025 nmol/L biotin) for 35 d; controls were cultured in medium containing 10 nmol/L biotin. The biotin-deficient medium was supplemented with 10 nmol/L of diaminobiotin, desthiobiotin, biotin or no biotin 24 h before gene expression analyses. Transcriptional activities of genes encoding IL-2 and IL-2Rgamma were increased up to 43% in cells supplemented with diaminobiotin, desthiobiotin or biotin compared with biotin-deficient cells, as judged by luciferase activities after transfection with reporter-gene constructs. These findings are consistent with the hypothesis that diaminobiotin and desthiobiotin mimic the effects of biotin on gene expression and thus have biotin-like activities. Supplementation of cells with diaminobiotin and desthiobiotin did not affect abundances of holocarboxylases and activities of propionyl-CoA carboxylase, suggesting that effects of synthetic biotin metabolites on gene expression are not mediated by carboxylase-dependent pathways. It is not known whether naturally occurring biotin metabolites also have biotin-like activities.

Interleukin-2 receptor-gamma -dependent endocytosis depends on biotin in Jurkat cells.

Biotin has been credited with having beneficial effects on immune function despite observations that biotin supplementation causes decreased secretion of interleukin-2. Here this paradox was addressed by determining whether receptor-dependent internalization of interleukin-2 by immune cells depends on biotin. Theoretically, this would be consistent with both decreased net secretion of interleukin-2 by biotin-supplemented cells (causing increased endocytosis) and beneficial effects of biotin on immune function (causing increased receptor signaling). Jurkat cells were cultured in biotin-defined media (25, 250, or 10,000 pM). Secretion of interleukin-2 correlated negatively with biotin supply, but transcriptional activity of the interleukin-2 gene correlated positively with biotin supply, suggesting that decreased secretion of interleukin-2 by biotin-supplemented cells was not caused by decreased gene expression. Expression of the interleukin-2 receptor-gamma gene was greater at 10,000 pM than 25 pM biotin, mediating increased endocytosis of interleukin-2 in biotin-supplemented medium. Inhibition of endocytosis by genistein and overexpression of interleukin-2 receptor-gamma abolished the effect of biotin. These findings suggest that endocytosis of interleukin-2 depends on biotin.