| Budget Amount *help |
¥15,500,000 (Direct Cost: ¥15,500,000)
Fiscal Year 2004: ¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 2003: ¥11,100,000 (Direct Cost: ¥11,100,000)
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| Research Abstract |
In eukaryotic nuclei, DNA is wrapped around an octamer containing two copies of core histones, H2A, H2B, H3, and H4, making nucleosomes, the fundamental units of chromatin. Lines of evidence indicate that nucleosomes in living cells are not static but their components undergo exchanges associated with the chromatin functions like gene activation and centromere formation. However, the mechanisms regulating the histone dynamics are not well understood. We first investigated the effects of overexpressed histone chaperones like Nap1 and Asf1 on the mobility of hisones tagged with the green fluorescent protein(GFP) ; however, only little effects were observed. We then established an in vitro system that allows the assembly and exchange of histones in intact chromatin using permeabilized cells. GFP-H2A and H2B-GFP are incorporated into euchromatin by exchange independently of DNA replication, and H3-GFP is assembled into replicated chromatin, as found in living cells. Purifying the cellular factors assisting the exchange of H2A/H2B, we identified protein phosphatase 2C γ-subtype (PP2Cγ/PPM1G) as a histone chaperone that binds specifically to the H2A/H2B dimer. When the expression of PP2Cγ was suppressed to <5% of the normal level using RNAi, the mobility of GFP-H2A and H2B-GFP was significantly reduced, suggesting the function of PP2Cγ as a factor mediating H2A/H2B exchange. In contrast, Nap1-specific siRNA had little effects on the mobility ; however, this may be because the low level of suppression was only achieved down to 10〜20% and Napi is abundantly present in cells. We also found that the bacterially-expressed and purified PP2Cγ dephosphorylated histones in vitro. These results suggest a link between the exchange and dephosphorylation mediated by a single polypeptide, possibly counteracting the phosphorylation involved in the regulation of gene expression.
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