MECHANISM OF 5-AZACR-MEDIATED ALTERATION IN GENE ACT.

这些研究的目标是了解分子 基因表达的可遗传变化的机制 发生在分化和肿瘤性转化过程中 实现并保持。5-氮胞苷(5-azaCR)和诱导剂 各种细胞类型的可遗传表型变化的 这是一个很有价值的工具，可以用来研究 建议的调控机制、DNA甲基化和 基因表达。我们发现DNA甲基转移酶(MTase) 与DNA中的5-azaC残基形成高亲和力的络合物 导致其失活。这导致了新的低甲基化 合成DNA，随后激活特定基因。 我们的研究导致了额外的非组蛋白的鉴定 缺乏DNA MTase活性但具有较高活性的核蛋白 对DNA中的5-azaC具有亲和力。如果他们的因果关系 与DNA中5-azaC残基的结合类似于DNA的结合 MTase，可以预测1)这些细胞的正常功能 蛋白质需要与C或5-甲基C(5mC)的特定相互作用 DNA中的残基和2)与5-azaC结合的残基干扰了这一点 功能。报告显示5-azaCR治疗导致 在没有遗传基因的生物体中基因表达的可遗传变化 它们DNA中可检测到的5mC表明，一些蛋白质 与DNA中的5-azaC结合可能参与调控基因 通过不涉及DNA变化的过程进行表达 甲基化。 因此，我们的具体目标是： 一、确定非组蛋白核蛋白是否具有高水平 DNA中5-azaC的亲和力直接调控转录，如 反式作用因子或通过对DNA的影响间接作用 甲基化。与5-azaC残基具有高亲和力的蛋白质 DNA将被提纯并检测a)序列特异性 约束性，b)改变启动的速度和/或特异性的能力 在定义的模板上合成RNA的能力以及c)影响 确定底物的DNA甲基化的速率或位置特异性。 II.确定特定基因中的5mC和5-azaC残留量 区域影响基因表达的调节。瞬时表达 通过区域或位点特异性掺入修饰的基因 5mC或5azaC将与未修饰的基因进行比较。 同样的基因将被用来研究5mC或 5azaC残基在特定基因区域的结合特异性 识别已知调控序列的蛋白质。克隆的 乙肝病毒DNA将作为所有人的底物 因为病毒基因的表达受到 甲基化和5azaCR治疗。

The goal of these studies is to understand the molecular mechanisms by which heritable alterations in gene expression that occur during differentiation and neoplastic transformation are achieved and maintained. 5-azacytidine (5-azaCR), and inducer of heritable phenotypic changes in a variety of cell types has been a valuable tool for examining the relationship between one proposed regulatory mechanism, DNA methylation, and changes in gene expression. We found that DNA methyltransferase (MTase) forms high affinity complexes with 5-azaC residues in DNA which causes its inactivation. This leads to hypomethylation of newly synthesized DNA and subsequently to activation of specific genes. Our studies led to the identification of additional non-histone nuclear proteins that lacked DNA MTase activity but had high affinity for 5-azaC in DNA. If the cause and effect of their binding to 5-azaC residues in DNA is analogous to that of DNA MTase, it can be predicted 1) that the normal function of these proteins requires specific interactions with C or 5-methylC (5mC) residues in DNA and 2) that binding to 5-azaC interferes with this function. Reports demonstrating that 5-azaCR treatment causes heritable changes in gene expression in organisms without detectable 5mC in their DNA suggest that some of the proteins that bind to 5-azaC in DNA may be involved in regulating gene expression through processes that do not involve changes in DNA methylation. Thus, our specific aims are: I. To determine whether non-histone nuclear proteins with high affinity for 5-azaC in DNA regulate transcription directly as trans-acting factors or indirectly through effects on DNA methylation. Proteins with high affinity for 5-azaC residues in DNA will be purified and tested for a) sequence specificity of binding, b) ability to alter the rate and/or specificity of initiation of RNA synthesis on defined templates and c) ability to affect the rate or site specificity of DNA methylation of defined substrates. II. To determine how 5mC and 5-azaC residues in specific gene regions affect regulation of gene expression. Transient expression of genes modified by regional or site specific incorporation of 5mC or 5azaC will be compared with that of unmodified genes. The same genes will be utilized to study the effect of 5mC or 5azaC residues in specific gene regions on the binding specificity of proteins that recognize known regulatory sequences. Cloned hepatitis B virus DNA will be used as the substrate for all experiments since the expression of viral genes is affected by methylation and 5azaCR treatment.