| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2004: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
The mechanism of imprinting in chromosome 15q11-q13, AS/PWS domain has been analyzed continuously from several years ago. We have previously identified AS and PWS imprinting control center (IC) located at SNURF-SNRPN upstream region. The bipartite IC is supposed to have a function to establish maternal epigenotype on maternal chromosome in female germ line and early embryogenesis with AS-IC, and maintenance of the paternal epigenotype on paternal chromosome in somatic cells with PWS-IC. This function was predicted from inherited pattern of several familial AS or PWS patients with imprinting defect, AS/PWS model mouse analysis, and transgenic mouse analysis. Th elucidate the IC function, we analyzed the PWS-IC region with identification of some trans-acting factor binding sites. Since the human PWS-IC and mouse Snurf-Snrpn promoter are ortholog region based on several evidence of the knock out mouse, phylogenetic analysis was performed, and several conserved sequence were found in the PWS-IC region.
In H19/Igf2 region, CTCF binding sites were found in the H19 promoter region. These sites are working as an insulator to prevent enhancer activity to the Igf2promoter.
In the phylogenetic analysis, there are conserved sequences that have similar with the CTCF binding site, and some transcription binding sites in the PWS-IC as well as the H 19 promoter. To confirm the function of the transcription binding sites, transient transfection assay was performed with several kind of DNA construct including mutation at the binding site. In the assay, CTCF like binding site didn't show any insulator activity, and other transcription factor binding site show a positive activity for the expression. Therefore, the CTCF like binding site may not have a function of the insulator, and other transcription-binding site could have some function for imprinting maintenance. This research is currently ongoing. Furthermore, several imprinting genes were also analyzed.
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