DNA CONFORMATION DEPENDENT REGULATOR OF GENE EXPRESSION

克隆了一种新的c-myc反式激活因子。这种蛋白质结合了一种 C-myc远上游元件(FUSE)刺激启动子活性。 由于FBP仅与远上游的非编码链(NC)结合 C-myc元件(融合)以序列特异的方式，而不是双重- DNA链，蛋白质-DNA复合体的形成，在体内，首先 需要解开DNA螺旋。FBP表现出明显的dsDNA 熔化活动。在负超螺旋DNA中，靶向链 在NCS FBP结合部位用FBP分离DNA 富含A-T的螺旋不稳定区域，使这种反式作用成为可能 选择性地暴露和结合其NCS同源序列的蛋白质。这些 研究结果表明，在一种模式中，FBP1)扫描dsDNA 螺旋不稳定区域中的滞留DNA，3)暴露和结合 FUSE中的单链同源序列和4)刺激启动子 活动。用突变的融合报告基因和FBP进行的转基因研究 表达载体，螺旋不稳定性和超螺旋的影响 1)靶向融合的FBP和2)启动子反式激活 测试过。为了实现这一点，含有拓扑异构体的熔丝结合 结合一根FBP亲和层析柱将被表征。此外，一系列 改变A-T中螺旋不稳定程度的突变 丰富区域，但不改变NCS与FBP的结合，将是 已执行。在这里存在/不存在非B构象变化 将通过用MBN和/或溴乙醛探测来确定位置 (BAA)，并与FBP靶向的易感性相关。至 确定FBP的FUSE靶向是否与转录相关 两个相对的启动子--染色质组装过程中的诱导扭转 极性将链接到包含以下内容的公共c-myc上游片段 该调控元件稳定地整合到基因组DNA中。通过 核酸酶和化学裂解分析，其他非融合的层次结构 C-myc上游序列中形成螺旋到螺旋的位点 还将识别在存在超级卷曲的情况下的转变。 FBP基因表达对c-myc基因表达调控的影响 细胞分化将通过选择性去除来确定 未分化细胞中的核酶和寡核苷酸。这些 研究将阐明一个有趣的新班级(S)的作用 基因表达的调节者。

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.