Biotin sensing and chromatin remodeling by holocarboxylase synthetase

描述（由申请方提供）：细胞通过增加生物素转运蛋白SMVT和MCT 1的表达对生物素缺乏做出反应，SMVT和MCT 1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蛋白的调节尚不清楚。已经提供的证据表明，生物素直接在染色质水平上调节基因表达。以前的研究表明，全羧化酶合成酶（HCS）介导生物素与组蛋白（DNA结合蛋白）H2 A，H3和H4的结合，并且组蛋白的生物素化导致基因抑制。我们的研究是一致的假设，生物素依赖性核转位的HCS作为一个生物素传感器，生物素与组蛋白的结合HCS与染色质重塑事件，调节生物素转运基因的转录。长期目标：我们的长期目标是阐明人体生物素稳态的机制。我们试图识别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MCT 1。我们还试图通过鉴定和表征HCS结合蛋白来阐明人类HCS调节的机制。具体目标：确定HCS的生物素依赖性核转位机制，并表征影响生物素转运蛋白基因座基因转录的HCS依赖性染色质重塑事件。这一目标将检验下列假设。(1)HCS在人细胞质中充当生物素传感器。增加的细胞生物素浓度与HCS-结合蛋白的HCS-介导的生物素化相关，触发HCS的核转位。(2)核HCS结合蛋白将HCS募集到染色质的特定区域，包括SMVT和MCT 1位点。(3)HCS催化靶基因座组蛋白的生物素化;响应于生物素补充，SMVT和MCT 1基因座组蛋白的生物素化增加降低了生物素转运蛋白基因SMVT和MCT 1的转录。(4)总的来说，细胞内生物素直接控制生物素转运蛋白的表达，由HCS依赖性染色质重塑介导。研究方法：细胞质和细胞核中的HCS结合蛋白将通过使用诸如酵母双杂交测定、计算机域搜索、免疫共沉淀和转基因细胞系等技术来鉴定。在人生物素补充研究和人细胞系中，将通过染色质免疫沉淀试验和实时PCR定量生物素转运蛋白基因座处HCS和生物素化组蛋白的相对富集。在人生物素补充研究和转基因细胞系中，将使用实时PCR和转录因子基因构建体定量SMVT和MCT 1的转录。公共卫生相关性：与公共卫生的相关性：组蛋白的生物素化是一种独特的表观遗传标记，因为它依赖于膳食中必需维生素生物素的摄入。生物素缺乏症在美国人中很普遍，在高达50%的孕妇中观察到中度生物素缺乏症。先前的研究表明，组蛋白的生物素化在基因调控和基因组稳定性中起着关键作用，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.