GENE REGULATION BY X CHROMOSOME INACTIVATION

X 染色体失活的基因调控

• 批准号: 2182462
• 负责人: THOMAS P YANG
• 金额: $ 13.49万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182462
• 关键词: DNA binding protein; DNA footprinting; DNA methylation; DNA replication; alleles; azacitidine; biochemical evolution; cell transformation; chromosome aberrations; gel mobility shift assay; gene interaction; genetic promoter element; genetic regulatory element; genetic transcription; genome; hybrid cells; hypoxanthine phosphoribosyltransferase; laboratory mouse; linkage mapping; nucleic acid sequence; sex chromosomes

Female eutherian mammals have evolved a mechanism by which the dosage of functional Xlinked genes in each somatic cell is equalized to that of males. This dosage compensation is accomplished by transcriptionally inactivating genes on one of the two X chromosomes in females. Thus, for most X-linked genes in female somatic cells, an active and inactive allele reside within the same nucleus but are differentially regulated and expressed. The long-term goal of this project is to determine the mechanism of X chromosome inactivation. This proposal will specifically investigate the molecular basis for maintaining the differential expression of genes on the active versus the inactive X chromosome in somatic cells by examining the role of DNA-protein interactions within specific X-linked genes. In vivo footprinting of intact cells will be used to identify sequence-specific DNA-binding proteins which interact with either the active or inactive allele of the human and mouse HPRT genes. Such DNA-binding proteins are likely to have a direct role in the differential expression of these genes on the active and inactive X chromosomes. The 5' region of each gene will be studied in hybrid cell lines which permit separate analysis of the active, inactive, and reactivated alleles. Similar studies will also be carried out on normal human and mouse cells. In addition, cytosine methylation in the 5' region from both genes will be determined in vivo by genomic sequencing. Together, these studies will examine in vivo the correlation between binding of sequence-specific proteins, DNA methylation, and transcriptional activity of genes on the active and inactive X chromosomes. DNAprotein interactions specific to either the active or inactive allele of the HPRT genes will be further characterized in vitro by gel mobility-shift assays, DNase I footprinting, and partial purification of the binding protein(s). These in vitro studies will include an examination of the interaction of the binding proteins with other mammalian promoters (both autosomal and X-linked), and an analysis of the evolutionary conservation of the binding activity(s) in heterologous mammalian extracts. Studies of DNA-protein interactions correlated with differential expression of the active and inactive alleles of specific X-linked genes should provide significant insight into the chromosome-wide mechanism for coordinate gene regulation by X chromosome inactivation.

雌性eutherian哺乳动物已经发展了一种机制 每个体细胞中的功能性Xlink基因均等 男性。该剂量补偿是通过转录完成的 在女性的两个X染色体之一上灭活基因。因此， 雌性体细胞中的大多数X连锁基因，一个活性和非活性等位基因 驻留在同一核内，但受到差异调节，并且 表达。该项目的长期目标是确定机制 X染色体灭活的含量。该建议将专门调查 维持基因差异表达的分子基础 在活动细胞中的活性与无活性X染色体上 检查DNA-​​蛋白质相互作用在特定X连锁中的作用 基因。完整细胞的体内足迹将用于识别 序列特异性的DNA结合蛋白与任何一种相互作用 人和小鼠HPRT基因的活性或非活性等位基因。这样的 DNA结合蛋白可能在差异中具有直接作用 这些基因在活性X染色体上的表达。 5' 每个基因的区域将在允许的杂化细胞系中进行研究 对活性，无活性和重新激活等位基因的单独分析。相似的 研究还将对正常的人类和小鼠细胞进行。在 另外，两个基因的5'区域中的胞嘧啶甲基化将是 通过基因组测序在体内确定。这些研究将在一起 在体内检查序列特异性的结合之间的相关性 基因上蛋白质，DNA甲基化和转录活性 活性和非活性X染色体。 dnaprotein相互作用特定于 HPRT基因的主动等位基因将进一步 通过凝胶移动迁移测定，DNase I足迹在体外表征， 和结合蛋白的部分纯化。这些体外研究 将包括检查结合蛋白与 其他哺乳动物的启动子（均常染色体和X连锁）和分析 在异源中的结合活性的进化保守 哺乳动物提取物。 DNA蛋白相互作用的研究与 特定的活性和非活性等位基因的差异表达 X连锁基因应为整个染色体的基因提供重要的见解 通过X染色体灭活的坐标基因调节的机制。