Regulation of gene expression and chromatin dynamics in living cells

在真核细胞核中，DNA被包裹在一个八聚体周围，该八聚体含有两个核心组蛋白H2 A、H2 B、H3和H4的拷贝，从而形成核小体，即染色质的基本单位。一系列的证据表明，活细胞中的核小体不是静止的，而是它们的组分经历与染色质功能相关的交换，如基因激活和着丝粒形成。然而，调节组蛋白动力学的机制还没有很好地理解。我们首先研究了过表达的组蛋白伴侣如Nap 1和Asf 1对用绿色荧光蛋白（GFP）标记的组蛋白迁移率的影响;然而，只观察到很少的影响。然后，我们建立了一个体外系统，允许组装和交换组蛋白在完整的染色质使用透化细胞。GFP-H2 A和H2 B-GFP通过独立于DNA复制的交换掺入常染色质中，并且H3-GFP组装成复制的染色质，如在活细胞中发现的。纯化了促进H2 A/H2 B交换的细胞因子，我们鉴定了蛋白磷酸酶2C γ-亚型（PP 2C γ/PPM 1G）作为特异性结合H2 A/H2 B二聚体的组蛋白伴侣。当PP 2C γ的表达被RNAi抑制到正常水平的5%以下时，GFP-H2 A和H2 B-GFP的迁移率显著降低，表明PP 2C γ作为介导H2 A/H2 B交换的因子的功能。相比之下，Nap 1特异性siRNA对迁移率几乎没有影响;然而，这可能是因为低水平的抑制仅达到10 - 20%，并且Napi大量存在于细胞中。我们还发现，细菌表达和纯化的PP 2C γ在体外去磷酸化组蛋白。这些结果表明，由单一多肽介导的交换和去磷酸化之间的联系，可能会抵消参与基因表达调控的磷酸化。

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.