MECHANISM OF 5-AZACR-MEDIATED ALTERATION--GENE ACTIVITY

这些研究的目的是了解分子 基因表达中的遗传性改变， 发生在分化和肿瘤转化过程中， 实现并保持。 5-氮杂胞苷（5-azaCR）和诱导剂 在各种细胞类型的遗传表型变化已经被 一个有价值的工具，用于检查一个人与另一个人之间的关系， 提出的调控机制，DNA甲基化，以及 基因表达。 我们发现DNA甲基转移酶（MTase） 与DNA中的5-azaC残基形成高亲和力复合物， 导致其失活。 这会导致新的 合成的DNA和随后的特定基因的激活。 我们的研究导致了额外的非组蛋白的鉴定 缺乏DNA MTase活性但具有高活性的核蛋白 对DNA中5-azaC的亲和力。 如果他们之间的因果关系 与DNA中的5-azaC残基的结合类似于与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（5 mC）特异性相互作用 2）与5-azaC的结合会干扰DNA中的氨基酸残基， 功能 证明5-azaCR治疗导致 基因表达的遗传性变化 DNA中可检测到的5 mC表明， 与DNA中5-azaC结合可能参与调节基因 通过不涉及DNA变化的过程表达 甲基化 因此，我们的具体目标是： I. 为了确定非组蛋白核蛋白是否具有高表达， DNA中5-azaC的亲和力直接调节转录， 反式作用因子或间接通过对DNA的影响 甲基化 对5-azaC残基具有高亲和力的蛋白质 将纯化DNA并测试a）以下的序列特异性： 结合，B）改变起始速率和/或特异性的能力 c）影响RNA合成的能力， 确定的底物的DNA甲基化的速率或位点特异性。 二. 为了确定特定基因中的5 mC和5-azaC残基 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 将5 mC或5azaC与未修饰的基因进行比较。 将利用相同的基因来研究5 mC或 5azaC残基在特定基因区域上的结合特异性 识别已知调节序列的蛋白质。 克隆 将使用B型肝炎病毒DNA作为所有 由于病毒基因的表达受到 甲基化和5azaCR处理。

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.