The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, biotin is covalently linked to histones in a reaction catalyzed by holocarboxylase synthetase (HCS); biotinylation of lysine 12-biotinylated histone H4 (K12Bio H4) causes gene silencing. Here, we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesized that nuclear translocation of HCS increases in response to biotin supplementation; HCS then biotinylates histone H4 at SMVT promoters, silencing biotin transporter genes. Jurkat lymphoma cells were cultured in media containing 0.025, 0.25, or 10 nmol/l biotin. The nuclear translocation of HCS correlated with biotin concentrations in media; the relative enrichment of both HCS and K12Bio H4 at SMVT promoter 1 (but not promoter 2) increased by 91% in cells cultured in medium containing 10 nmol/l biotin compared with 0.25 nmol/ l biotin. This increase of K12Bio H4 at the SMVT promoter decreased SMVT expression by up to 86%. Biotin homeostasis by HCS-dependent chromatin remodeling at the SMVT promoter 1 locus was disrupted in HCS knockdown cells, as evidenced by abnormal chromatin structure (K12Bio H4 abundance) and increased SMVT expression. The findings from this study are consistent with the theory that HCS senses biotin, and that biotin regulates its own cellular uptake by participating in HCS-dependent chromatin remodeling events at the SMVT promoter 1 locus in Jurkat cells.

SummaryAnimals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/vHL pathway to accelerate HIF-mediated metabolic responses to hypoxia.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is known as a mediator of toxic responses. Recently, it was shown that the AhR has dual functions. Besides being a transcription factor, it also possesses an intrinsic E3 ubiquitin ligase function that targets, e.g., the steroid receptors for proteasomal degradation. The aim of this study was to identify the molecular switch that determines whether the AhR acts as a transcription factor or an E3 ubiquitin ligase. To do this, we used the breast cancer cell line MCF7, which expresses a functional estrogen receptor alpha (ER␣) signaling pathway. Our data suggest that aryl hydrocarbon receptor nuclear translocator (ARNT) plays an important role in the modulation of the dual functions of the AhR. ARNT knockdown dramatically impaired the transcriptional activation properties of the ligand-activated AhR but did not affect its E3 ubiquitin ligase function. The availability of ARNT itself is modulated by another basic helix-loop-helix (bHLH)-Per-ARNT-SIM (PAS) protein, the repressor of AhR function (AhRR). MCF7 cells overexpressing the AhRR showed lower ER␣ protein levels, reduced responsiveness to estradiol, and reduced growth rates. Importantly, when these cells were used to produce estrogendependent xenograft tumors in SCID mice, we also observed lower ER␣ protein levels and a reduced tumor mass, implying a tumor-suppressive-like function of the AhR in MCF7 xenograft tumors.KEYWORDS aryl hydrocarbon receptor, molecular switch, E3 ubiquitin ligase, transcription factor, aryl hydrocarbon receptor nuclear translocator, aryl hydrocarbon receptor repressor B asic helix-loop-helix (bHLH)-Per-aryl hydrocarbon receptor (AhR) nuclear translocator (ARNT)-SIM (PAS) proteins function as biological sensors that respond to physiological stimuli and environmental signals to mediate adaptive cellular responses. In general, these proteins form heterodimers that consist of a signal-induced subunit and an unregulated, ubiquitously expressed subunit (1). A member of the family of bHLH-PAS proteins is the AhR. Initially identified by Poland and Knutson (2) as a binding site for planar, nonhalogenated, and halogenated xenobiotics, in particular the highly toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the AhR was viewed for a long time as a mediator of xenobiotic-induced toxicity and carcinogenesis. However, mouse knockdown studies have revealed a role for the AhR beyond xenobiotic-induced toxicity and carcinogenesis. For example, the AhR has been shown to be involved in fetal liver development (3), immune cell modulation and differentiation (4-6), and gastrointestinal homeostasis (7, 8). The developmental defects observed in AhR Ϫ/Ϫ mice and the strong conservation of the AhR throughout evolution (for a review, see reference 9)

The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, holocarboxylase synthetase (HCS) mediates covalent binding of biotin to histones; biotinylation of lysine-12 in histone H4 (K12BioH4) causes gene repression. Here we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesize that nuclear translocation of HCS increases in response to biotin supplementation; HCS then biotinylates histone H4 at SMVT promoters, silencing biotin transporter genes. We show that nuclear translocation of HCS is a biotin-dependent process that might involve tyrosine kinases, histone deacetylases, and histone methyltransferases in human lymphoid (Jurkat) cells. The nuclear translocation of HCS correlated with biotin concentrations in cell culture media; the relative enrichment of both HCS and K12BioH4 at SMVT promoter 1 (but not promoter 2) increased by 91% in cells cultured in medium containing 10 nmol/L biotin compared with 0.25 nmol/L biotin. This increase of K12BioH4 at the SMVT promoter was inversely linked to SMVT expression. Biotin homeostasis by HCS-dependent chromatin remodeling at the SMVT promoter 1 locus was disrupted in HCS knockdown cells, as evidenced by abnormal chromatin structure (K12BioH4 abundance) and increased SMVT expression. The findings from this study are consistent with the theory that HCS senses biotin, and that biotin regulates its own cellular uptake by participating in HCS-dependent chromatin remodeling events at the SMVT promoter 1 locus in Jurkat cells.

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