GENE REGULATION BY X CHROMOSOME INACTIVATION

雌性真兽目哺乳动物已经进化出一种机制， 每个体细胞中的功能性X连锁基因与 男性。这种剂量补偿是通过转录来实现的。 使女性两条X染色体之一上的基因失活。因此 女性体细胞中的大多数X连锁基因，一个活跃和不活跃的等位基因 存在于同一个细胞核内，但受到不同的调节， 表达。本项目的长期目标是确定机制 X染色体失活。这份提案将专门调查 维持基因差异表达的分子基础 对体细胞中活跃和不活跃的X染色体的影响， 研究DNA-蛋白质相互作用在特异性X连锁 基因.完整细胞的体内足迹法将用于鉴定 序列特异性DNA结合蛋白，其与 人类和小鼠HPRT基因的活性或非活性等位基因。等 DNA结合蛋白可能在分化中起直接作用。 这些基因在活跃和不活跃的X染色体上的表达。的5' 将在杂交细胞系中研究每个基因的区域， 分别分析活性、非活性和再活化的等位基因。类似 还将对正常的人类和小鼠细胞进行研究。在 此外，来自两个基因的5'区域中的胞嘧啶甲基化将被抑制。 通过基因组测序在体内测定。这些研究将 在体内检查序列特异性结合之间的相关性， 蛋白质、DNA甲基化和基因转录活性， 活跃和不活跃的X染色体。特异性DNA蛋白相互作用 HPRT基因的活性或非活性等位基因将被进一步 通过凝胶迁移率变化测定，DNA酶I足迹法， 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 其他哺乳动物启动子（常染色体和X连锁），以及分析 在异源细胞中结合活性的进化保守性 哺乳动物提取物。DNA-蛋白质相互作用的研究 特异性的活性和非活性等位基因的差异表达 X连锁基因应该提供显着的洞察染色体范围内的 通过X染色体失活协调基因调控的机制。

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.