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

近年来，人们逐渐接受了从时间概念的角度来理解转录的机制。细胞的发育、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，我们重点研究了组织特异性转录因子和启动子DNA在成肌细胞分化系统中的相互作用。系统由1组成。小鼠10T1/2成纤维细胞稳定转化ER-MyoD （ER为诱导信号，MyoD为肌肉分化的主控基因）。2. 转录因子为MyoD、CBP、p300和Six家族蛋白。3. 在肌肉分化过程中协同起作用的启动子有肌酸激酶、肌原蛋白、desmin、p21、肌球蛋白轻链、tublin α。我们通过Northern blot分析发现这些基因在不同的分化时间点表达。接下来，我们研究了各种转录因子与其启动子区域的结合，发现首先MyoD与启动子的结合与转录表达平行。其他的根据启动子是可变的。我们使用三维荧光原位杂交（3D FISH）技术（用BAC探针可视化染色体区域和感兴趣的基因）进一步检查了基因动力学，并观察了MyoD结合前放松的启动子区域。这些结果表明，基因转录不仅是由特定的转录因子结合而开启的，而且在转录因子结合之前，染色质填充的DNA（包含要转录的启动子区域）也被放松。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.