|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 2003 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 2002 : ¥1,900,000 (Direct Cost : ¥1,900,000)
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.