Biotin sensing and chromatin remodeling by holocarboxylase synthetase

全羧化酶合成酶的生物素传感和染色质重塑

• 批准号: 8007021
• 负责人: JANOS ZEMPLENI
• 金额: $ 6万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-07 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8007021
• 关键词: Acetyl-CoA Carboxylase; Address; Affect; Affinity; American; Amides; Binding; Binding Proteins; Biological Assay; Biotin; Biotinylation; Cell Line; Cell Nucleus; Cell Proliferation; Cell surface; Cells; Chromatin; Chromosome abnormality; Co-Immunoprecipitations; Coenzymes; Computer Simulation; Culture Media; Cytoplasm; DNA Binding Domain; DNA Repair; DNA-Binding Proteins; Dietary intake; Epigenetic Process; Event; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome Stability; Genomics; Goals; Health; Heterochromatin; Histone H2A; Histone H3; Histone H4; Histones; Holocarboxylase Synthetase Deficiency; Homeostasis; Human; Human Cell Line; K-18 conjugate; Ligase; Link; Lymphoid Cell; Lysine; Malignant Neoplasms; Mediating; Messenger RNA; Methods; Molecular Chaperones; Multivitamin; Nuclear; Nuclear Translocation; Nutrient; Overdose; Play; Pregnant Women; Process; Proteins; Public Health; Pyruvate Carboxylase; Recruitment Activity; Regulation; Relative (related person); Reporter Genes; Repression; Retrotransposon; Risk; Role; SECTM1 gene; Site; Sodium; Structure; Supplementation; Techniques; Testing; Time; Transgenic Organisms; Two-Hybrid System Techniques; Vitamins; Yeasts; biotin transporter; cancer risk; chromatin immunoprecipitation; chromatin remodeling; gene repression; holocarboxylase synthetases; meetings; methylcrotonyl coA carboxylase; methylmalonyl-CoA decarboxylase; public health relevance; research study; response; sensor; uptake

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.

描述（由申请人提供）：细胞通过增加生物素转运蛋白 SMVT 和 MCT1 的表达来应对生物素缺乏，它们充当生物素进入细胞的“检查点”。然而，尚不清楚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。有证据表明生物素直接在染色质水平调节基因表达。先前的研究表明，全羧化酶合成酶 (HCS) 介导生物素与组蛋白（DNA 结合蛋白）H2A、H3 和 H4 的结合，并且组蛋白的生物素化会导致基因抑制。我们的研究与以下假设一致：HCS 的生物素依赖性核易位充当生物素传感器，并且 HCS 生物素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相关。长期目标：我们的长期目标是阐明人类生物素稳态的机制。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“检查点”的机制，即 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 调节机制。具体目的：确定 HCS 生物素依赖性核易位的机制，并表征影响生物素转运蛋白位点基因转录的 HCS 依赖性染色质重塑事件。该目标将检验以下假设。 (1) HCS 作为人体细胞质中的生物素传感器。生物素细胞浓度的增加与 HCS 介导的 HCS 结合蛋白的生物素化有关，从而触发 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在目标位点催化组蛋白的生物素化；补充生物素后，SMVT 和 MCT1 位点组蛋白的生物素化增加，从而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。方法：细胞质和细胞核中的 HCS 结合蛋白将通过使用酵母双杂交测定、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测定和实时 PCR 来量化生物素转运蛋白位点上 HCS 和生物素化组蛋白的相对富集度。 SMVT 和 MCT1 的转录将通过在人类生物素补充研究和转基因细胞系中使用实时 PCR 和报告基因构建体进行量化。公共健康相关性： 与公共健康的相关性：组蛋白的生物素化是一种独特的表观遗传标记，因为它取决于必需维生素生物素的膳食摄入量。生物素缺乏症在美国人中很普遍，高达 50% 的孕妇存在中度生物素缺乏症。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发挥着关键作用，从而降低染色体异常和癌症的风险。