Biotin sensing and chromatin remodeling by holocarboxylase synthetase

描述（由申请人提供）：细胞通过增加生物素转运体SMVT和MCT1的表达来应对生物素缺乏，它们作为生物素进入细胞的“检查点”。然而，细胞如何感知生物素状态以及哪些机制介导生物素转运体的调节尚不清楚。已有证据表明，生物素在染色质水平上直接调控基因表达。先前的研究表明，全新羧化酶合成酶（HCS）介导生物素与组蛋白（dna结合蛋白）H2A、H3和H4的结合，组蛋白的生物素化导致基因抑制。我们的研究与HCS的生物素依赖核易位作为生物素传感器的假设是一致的，并且HCS将生物素与组蛋白结合与调节生物素转运基因转录的染色质重塑事件有关。长期目标：我们的长期目标是阐明人体内生物素稳态的机制。我们试图确定细胞生物素的传感器和调节生物素进入细胞的“检查点”的机制，SMVT和MCT1。我们还试图通过鉴定和表征HCS结合蛋白来阐明人类HCS调控的机制。具体目的：确定HCS依赖生物素的核易位机制，并表征影响生物素转运位点基因转录的HCS依赖染色质重塑事件。这一目标将检验以下假设。(1) HCS在人细胞质中作为生物素传感器。细胞中生物素浓度的增加与HCS介导的HCS结合蛋白的生物素化有关，从而引发HCS的核易位。(2)核HCS结合蛋白将HCS招募到染色质的特定区域，包括SMVT和MCT1位点。(3) HCS在靶位点催化组蛋白的生物素化；生物素补充后，SMVT和MCT1位点组蛋白的生物素化增加，降低了生物素转运基因SMVT和MCT1的转录。(4)总的来说，细胞内生物素通过hcs依赖性染色质重塑介导直接控制生物素转运体的表达。方法：细胞质和细胞核中的hcs结合蛋白将通过酵母双杂交测定、硅结构域搜索、共免疫沉淀和转基因细胞系等技术进行鉴定。在人类生物素补充研究和人类细胞系中，HCS和生物素化组蛋白在生物素转运位点的相对富集将通过染色质免疫沉淀测定和实时PCR来定量。在人类生物素补充研究和转基因细胞系中，SMVT和MCT1的转录将通过实时荧光定量PCR和报告基因构建进行定量。公共卫生相关性：与公共卫生相关：组蛋白的生物素化是一种独特的表观遗传标记，因为它取决于必需维生素生物素的饮食摄入量。生物素缺乏在美国人中很普遍，在高达50%的孕妇中观察到中度生物素缺乏。以往的研究表明，组蛋白的生物素化在基因调控和基因组稳定性中起着至关重要的作用，从而降低染色体异常和癌症的风险。

DESCRIPTION (provided by applicant): Cells respond to biotin deficiency by increasing the expression of the biotin transporters SMVT and MCT1, which serve as "checkpoints" for biotin entry into cells. It is unknown, however, how cells sense biotin status and which mechanisms mediate regulation of biotin transporters. Evidence has been provided that biotin regulates gene expression directly at the chromatin level. Previous studies suggested that holocarboxylase synthetase (HCS) mediates the binding of biotin to histones (DNA-binding proteins) H2A, H3, and H4, and that biotinylation of histones causes gene repression. Our studies are consistent with the hypotheses that biotin-dependent nuclear translocation of HCS serves as a biotin sensor, and that binding of biotin to histones by HCS is associated with chromatin remodeling events that regulate the transcription of biotin transporter genes. Long-term objective: Our long-term objective is to elucidate mechanisms of biotin homeostasis in humans. We seek to identify both sensors of cellular biotin and mechanisms that regulate the "checkpoints" for biotin entry into cells, SMVT and MCT1. We also seek to elucidate mechanisms of HCS regulation in humans by identifying and characterizing HCS-binding proteins. Specific aim: To identify mechanisms of biotin-dependent nuclear translocation of HCS, and to characterize HCS-dependent chromatin remodeling events that affect gene transcription at biotin transporter loci. This aim will test the following hypotheses. (1) HCS serves as a biotin sensor in human cytoplasm. Increased cellular concentrations of biotin are associated with HCS-mediated biotinylation of HCS-binding proteins, triggering nuclear translocation of HCS. (2) Nuclear HCS-binding proteins recruit HCS to specific regions in chromatin, including SMVT and MCT1 loci. (3) HCS catalyzes biotinylation of histones at target loci; the increased biotinylation of histones at SMVT and MCT1 loci in response to biotin supplementation decreases the transcription of biotin transporter genes SMVT and MCT1. (4) Collectively, intracellular biotin directly controls the expression of biotin transporters, mediated by HCS-dependent chromatin remodeling. Methods: HCS-binding proteins in cytoplasm and nucleus will be identified by using techniques such as yeast-two-hybrid assays, in silico domain searches, co-immunoprecipitations, and transgenic cell lines. The relative enrichment of HCS and biotinylated histones at biotin transporter loci will be quantified by chromatin immunoprecipitation assays and real-time PCR in both human biotin supplementation studies and human cell lines. Transcription of SMVT and MCT1 will be quantified by using real-time PCR and reporter-gene constructs in both human biotin supplementation studies and transgenic cell lines. PUBLIC HEALTH RELEVANCE: Relevance to public health: Biotinylation of histones is a unique epigenetic mark because it depends on the dietary intake of the essential vitamin biotin. Biotin deficiency is prevalent among Americans, and moderate biotin deficiency has been observed in up to 50% of pregnant women. Previous studies suggest that biotinylation of histones plays a critical role in gene regulation and genomic stability, thereby decreasing the risk for chromosomal abnormalities and cancer.