DNA CONFORMATION DEPENDENT REGULATOR OF GENE EXPRESSION

一种新型的 c-myc 反式激活蛋白已被克隆。这种蛋白质结合 c-myc 的远上游元件（FUSE）可刺激启动子活性。 由于 FBP 仅结合远上游的非编码链 (NCS) c-myc 的元件（FUSE）以序列特异性方式，而不是双 链状 (ds) DNA，蛋白质-DNA 复合物的形成，在体内，首先 需要解开 DNA 螺旋。 FBP 表现出独特的 dsDNA 熔化活动。在负超螺旋 DNA 中，目标链 通过 FBP 分离 DNA，位于 NCS FBP 结合位点旁边 一个富含 AT 的螺旋不稳定区域，使这种反式作用成为可能 蛋白质选择性暴露并结合其 NCS 同源序列。这些 研究结果提出了一个模型，其中 FBP 1) 扫描 dsDNA 2) 熔化单个 螺旋不稳定区域中的绞合 DNA，3) 暴露并结合 FUSE 中的单链同源序列和 4) 刺激启动子 活动。在使用突变 FUSE 报告基因和 FBP 的转染研究中 表达载体、螺旋不稳定性和超螺旋的影响 1) FBP 靶向 FUSE 和 2) 启动子反式激活将 已测试。为了实现这一点，包含拓扑异构体的 FUSE 的结合 结合FBP亲和柱将被表征。此外，还有一系列 改变 A T 螺旋不稳定性程度的突变 丰富的区域，但不会改变 NCS 与 FBP 的结合，将是 执行。此时是否存在非 B 构象变化 通过用 MBN 和/或溴乙醛探测来确定位点 (BAA) 并与 FBP 靶向的敏感性相关。 至 确定 FBP 的 FUSE 靶向是否与转录相关 组装染色质时诱导扭转，相反的两个启动子 极性将链接到一个共同的 c-myc 上游片段，其中包含 调控元件并稳定整合到基因组 DNA 中。由 核酸酶和化学裂解分析，其他非 FUSE 的层次结构 c-myc 上游序列中形成螺旋到卷曲的位点 超螺旋存在下的转变也将被识别。 FBP表达对c-myc表达调控的影响 细胞分化将通过选择性去除来确定 来自未分化细胞的核酶和寡核苷酸。这些 研究将阐明一类有趣的新类的功能 基因表达的调节因子。

A novel transactivator of c-myc has been cloned. This protein binds a far upstream element (FUSE) of c-myc to stimulate promoter activity. Since FBP binds only the non-coding strand (NCS) of a far upstream elements (FUSE) of c-myc in a sequence-specific manner, and not double- stranded (ds) DNA, formation of the protein-DNA complex, in vivo, first requires unwinding of the DNA helix. FBP manifests a distinct dsDNA melting activity. In negatively supercoiled DNA, the targeted strand separation of DNA by FBP, in the NCS FBP binding site that is next to an A + T rich region of helical instability, enables this trans-acting protein to selectively expose and bind its NCS cognate sequence. These findings suggest a model in which FBP 1) scans dsDNA 2) melts single stranded DNA in regions of helical instability, 3) exposes and binds the single-stranded cognate sequence in FUSE and 4) stimulates promoter activity. In transfection studies with mutated FUSE-reporter and FBP expression vectors, the impact of helical instability and supercoiling on 1) FBP targeting to FUSE and 2) promoter transactivation will be tested. To achieve this, binding of FUSE containing topoisomers which bind a FBP affinity column will be characterized. In addition, a series of mutations that alter the degree of helical instability in the A +T rich region, but that do not alter NCS binding to FBP, will be performed. The presence/absence of non-B conformational changes at this site will be determined by probing with MBN and/or bromoacetaldehyde (BAA) and correlated with susceptibility to targeting by FBP. To ascertain whether FUSE targeting by FBP is linked to transcriptionally induced torsion in assemble chromatin, two promoters of opposite polarity will be linked to a common c-myc upstream segment containing the regulatory element and stably integrated into genomic DNA. By nuclease and chemical cleavage analysis, a hierarchy of other non FUSE sites in the upstream sequences of c-myc which form helix to coil transitions in the presence of supercoiling will also be identified. The impact of FBP expression on the regulation of c-myc expression and cellular differentiation will be determined, by selective removal with ribozymes and oligonucleotides from undifferentiated cells. These studies will elucidate the function(s) of an interesting new class of regulators of gene expression.