Regulation of gene expression and chromatin dynamics in living cells

在真核生物的细胞核中，DNA被一个包含两个核心组蛋白拷贝H2A、H2B、H3和H4的八聚体包裹，形成核小体，核小体是染色质的基本单位。一系列证据表明，活细胞中的核小体不是静态的，它们的组成与染色质功能如基因激活和着丝粒形成有关。然而，调控组蛋白动力学的机制尚不清楚。我们首先研究了过表达的组蛋白伴侣如Nap1和Asf1对绿色荧光蛋白（GFP）标记的组蛋白迁移的影响；然而，观察到的影响很小。然后，我们建立了一个体外系统，允许使用通透化细胞在完整的染色质中组装和交换组蛋白。GFP-H2A和H2B-GFP通过独立于DNA复制的交换被整合到常染色质中，H3-GFP被组装成复制的染色质，这在活细胞中发现。纯化了协助H2A/H2B交换的细胞因子，我们鉴定出蛋白磷酸酶2Cγ -亚型（PP2Cγ/PPM1G）是一种特异性结合H2A/H2B二聚体的组蛋白伴侣。当使用RNAi抑制PP2Cγ的表达至正常水平的5%以下时，GFP-H2A和H2B- gfp的迁移率明显降低，提示PP2Cγ是介导H2A/H2B交换的因子。相比之下，nap1特异性siRNA对移动性的影响很小；然而，这可能是因为低水平的抑制仅达到10 ~ 20%，而Napi在细胞中大量存在。我们还发现细菌在体外表达和纯化的PP2Cγ去磷酸化组蛋白。这些结果表明，交换与单一多肽介导的去磷酸化之间存在联系，可能抵消参与基因表达调节的磷酸化。

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.