Biotin sensing and chromatin remodeling by holocarboxylase synthetase

描述(申请人提供)：细胞对生物素缺乏的反应是通过增加生物素转运体SMVT和MCT1的表达，这两个转运体是生物素进入细胞的“检查点”。然而，目前尚不清楚细胞如何感知生物素状态，以及哪些机制介导了生物素转运体的调节。已有证据表明，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，全羧酶合成酶(HCS)介导生物素与组蛋白(DNA结合蛋白)H_2A、H_3和H_4的结合，组蛋白的生物素化导致基因抑制。我们的研究与生物素依赖的HCS核转位作为生物素感受器的假设是一致的，并且HCS与组蛋白的结合与调节生物素转运蛋白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类体内生物素动态平衡的机制。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hcs在人类中的调节机制。目的：探讨生物素依赖的人巨噬细胞核转位的机制，以及影响生物素转运蛋白基因转录的染色质重塑事件。这一目标将检验以下假设。(1)HCS作为生物素感受器存在于人的细胞质中。细胞内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，从而触发了HCS的核移位。(2)核内HCS结合蛋白将HCS募集到染色质的特定区域，包括SMVT和MCT1基因座。(3)HCS催化靶基因座上组蛋白的生物素化；补充生物素后，SMVT和MCT1基因座上组蛋白的生物素化增加，降低了生物素转运蛋白基因SMVT和MCT1的转录。(4)总的来说，细胞内生物素通过HCS依赖的染色质重塑直接调控生物素转运体的表达。方法：采用酵母双杂交、硅胶结构域搜索、免疫共沉淀和转基因细胞系等技术鉴定胞浆和胞核中的HCS结合蛋白。在人类生物素补充研究和人类细胞系中，将通过染色质免疫沉淀分析和实时定量聚合酶链式反应来定量生物素转运蛋白基因座上HCS和生物素化组蛋白的相对富集量。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聚合酶链式反应和报告基因构建进行量化。公共卫生相关性：与公共健康相关：组蛋白的生物素化是一个独特的表观遗传学标志，因为它取决于膳食中必需的维生素生物素的摄入量。生物素缺乏症在美国人中很普遍，高达50%的孕妇出现了中度生物素缺乏症。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中发挥着关键作用，从而降低了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.