Analysis of the state of binding of tissue specific and general transcription factors to the promoter in vivo.

近年来，人们逐渐接受用时间概念的观点来理解转录的机制。发育、细胞分化和细胞对环境刺激的反应可以解释为DNA和转录因子随时间相互作用的过程。在本项目中，我们利用成肌细胞分化系统研究了组织特异性转录因子与启动子DNA之间的相互作用。该系统由1.稳定转化ER-MyoD(ER代表诱导信号，MyoD是肌肉分化的主控基因)的10T1/2小鼠成纤维细胞组成。2.转录因子有MyoD、CBP、p300和6个家族蛋白。3.已被认为在肌肉分化过程中起协同作用的启动子的基因有：肌酸激酶、肌生成素、结蛋白、p21、肌球蛋白轻链、微管蛋白α。Northern印迹分析发现，这些基因在不同的分化时间点都有表达。接下来，我们检查了各种转录因子与它们的启动子区域的结合，发现第一个MyoD结合到它们的启动子上，在Parrell中有转录表达。其他的则根据发起人的不同而不同。我们进一步利用三向荧光原位杂交(3D FISH)技术(用BAC探针显示染色体区域和感兴趣的基因)进一步检测了基因动力学，并观察到MyoD结合前的松弛启动子区域。这些结果表明，基因的转录不仅通过特定的转录因子结合来进行，而且含有要转录的启动子区域的染色质填充的DNA在转录因子结合之前被松弛。后者的分子机制目前正在研究中。

Recently, it has been gradually accepted that the mechanism of transcription should be understood with time concept viewpoint. Development, cell differentiation, and cell response to environmental stimuli can be interpreted to the process of the interaction between DNA and transcription factors with time. In this project, we focused on the interaction between tissue specific transcription factors and promoter DNA using myoblast differentiation system. The system was consist of 1. Mouse fibloblast of 10T1/2 which is stably transformed with ER-MyoD (ER for induction signal and MyoD is the master gene for muscle differentiation). 2. Transcription factors are MyoD, CBP, p300, and Six family protein. 3. Genes whose promoters have been suggested to cordinately function during muscle differentiation are muscle creatine kinase, myogenin, desmin, p21, myosin light chain, tublin alpha. We found that these genes are expressed at different time point of differentiation by Northern blot analysis. We next examined various transcription factor bindings to their promoter regions and found that first MyoD binds to their promoter in pararell with the transcriptional expression. Others are variable depending on the promoters. We further examined the gene dynamics using three dimentinal fluorescence in situ hybridization (3D FISH) technique (visualization of chromosomal territories and gene of interest with BAC probes) and observed the relaxing promoter region before MyoD binding. These results suggest that gene transcription is swithed on not only by specific transcription factor binding, but also chromatin-packed DNA, which contains promoter regions to be transcribed, is relaxed just before transcription factor binding. The molecular mechanism of the latter is now under investigation.