GENE REGULATION BY X CHROMOSOME INACTIVATION

雌性真兽类进化出一种机制，通过这种机制， 每个体细胞中的功能X连锁基因与 男性。这种剂量补偿是通过转录完成的 女性两条X染色体中的一条染色体上的基因失活。因此，对于 雌性体细胞中大多数X连锁基因，一种活跃和不活跃的等位基因 驻留在同一个核内，但受到不同的调节和 表达。这个项目的长期目标是确定机制 X染色体失活。这项提案将专门调查 维持基因差异表达的分子基础 体细胞中活性与非活性X染色体的比较 检测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被用来识别 序列特异的DNA结合蛋白，与 人类和小鼠HPRT基因的活性或非活性等位基因。是这样的 DNA结合蛋白可能在这种差异中起直接作用。 这些基因在活跃和非活跃的X染色体上的表达。5英尺 每个基因的区域将在允许的杂交细胞系中进行研究 对激活、非激活和重新激活的等位基因进行单独分析。类似 研究还将在正常的人类和小鼠细胞上进行。在……里面 此外，这两个基因5‘端的胞嘧啶甲基化将是 通过基因组测序在体内确定。总而言之，这些研究将 体内检测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 活跃和非活跃的X染色体。DNA与蛋白质之间的相互作用 HPRT基因的活跃或非活跃等位基因将进一步 通过凝胶迁移率-位移分析，DNA酶I足迹， 结合蛋白的部分纯化(S)。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连锁的)，以及一项分析 异源植物中结合活性的进化保守性(S) 哺乳动物提取物。DNA-蛋白质相互作用的相关研究 特定基因活性等位基因和非活性等位基因的差异表达 X连锁基因应该提供对整个染色体范围的重要洞察力 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.