DNA CONFORMATION DEPENDENT REGULATOR OF GENE EXPRESSION

克隆了一种新的c-myc反式激活因子。这种蛋白质结合一种 c-myc的远上游元件（FUSE）以刺激启动子活性。 由于FBP仅结合远上游的非编码链（NCS）， 元件（FUSE）的c-myc在序列特异性的方式，而不是双- 在体内，首先，蛋白质-DNA复合物的形成 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，靶链 DNA被FBP分离，在NCS FBP结合位点， 一个富含A + T的螺旋不稳定区域，使这种反式作用 蛋白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）单解链 螺旋不稳定区域中的DNA链，3）暴露并结合 FUSE中单链同源序列和4）刺激启动子 活动在使用突变的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）靶向FUSE的FBP和2）启动子反式激活将被激活。 测试.为了实现这一点，结合含有拓扑异构体的FUSE， 将表征结合FBP亲和柱。此外，A系列 改变A +T中螺旋不稳定程度的突变 富含区域，但不改变NCS与FBP的结合， 执行。在此条件下非B构象变化的存在/不存在 将通过用MBN和/或溴乙醛探测来确定位点 (BAA)并与FBP靶向敏感性相关。 到 确定FBP的FUSE靶向是否与转录相关 在装配染色质中诱导扭转，两个相反的启动子 极性将连接到一个共同的c-myc上游片段， 调控元件并稳定整合到基因组DNA中。通过 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 还将鉴定在超螺旋存在下的转变。 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 研究将阐明一个有趣的新类别的功能 基因表达的调节器。

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.