| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥300,000)
Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2006: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
Polycomb Group (PcG) proteins play a central role in epigenetic regulations of a verity of genes related to differentiation, proliferation, and sternness. Generally, PcG proteins function as large complexes on their target loci, which execute gene silencing. To better understand mechanisms of PcG-repressive functions, we have been trying to visualize in vivo PcG complexes and analyze them. Here we report generations of several kinds of PcG protein-GFP targeted mice (Me118-GFP, Ring1B-YFP, Edr2-Celurean etc.), in which every fusion protein is properly expressed from their original locus and then the dynamics of the PcG-GFP fusions and their nuclear structures.
Me118-EGFP and Rnfl-EYFP form hundreds of distinct speckled bodies (PcG bodies) in their MEF nuclei. Single molecular analysis revealed that PcG bodies include Me118 and Rnf2 at a given ratio of 1:3. Immunostaining and subsequent 3-D analysis showed that PcG bodies located specifically on trimethylated histone H3K27 (H3K27me3)-rich regions, indicating that PcG bodies were chromatin-associated structures. We further found that Me118-GFP moved around three times slower on. H3K27-rich regions than on H3K27-poor ones. The lower kinetics might make it easy for PcG proteins to accumulate on H3K27-rich regions. Most importantly, we succeeded in generating PcG body-lacking cells by Edr2-Cerulean that lost an intrinsic function of self-polymerization, and then observed the derepressed expression of Ink4a-Arf as a PcG target in the cells and skeletal abnormalities typical to PcG mutated phenotypes in mice. These results suggest that PcG body is a functional repressive domain and its assemble is dependent on the Edr2 self-polymerization function. In conclusion, PcG proteins need to form PcG bodies so that they can repress genes.
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