Global Gene Silencing is Caused by the Dissociation of RNA Polymerase II from DNA in Mouse Oocytes
Global Gene Silencing is Caused by the Dissociation of RNA Polymerase II from DNA in Mouse Oocytes
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DOI：
10.1262/jrd.10-068a
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发表时间：
2010-10-01
影响因子：
1.8
通讯作者：
Aoki, Fugaku
中科院分区：
文献类型：
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作者：
Abe, Ken-ichiro;Inoue, Azusa;Aoki, Fugaku
随着小鼠卵母细胞接近成熟，基因转录发生全面抑制。在这里，我们调查的参与RPB 1，RNA聚合酶II（RNAP II）的最大亚基，在这种转录沉默机制的调节。使用BrUTP在体外运行试验中转录后，我们观察到当卵母细胞达到其完整大小（约80 μ m）时转录活性突然下降。使用特异性磷酸化和非磷酸化形式的RPB 1的抗体的免疫印迹显示，RPB 1是磷酸化的丝氨酸-2和丝氨酸-5在小的生长中的卵母细胞中，在活跃的转录发生。相比之下，在转录不活跃的完全生长的卵母细胞中，RPB 1主要是未磷酸化的。当我们在免疫细胞化学固定过程中使用Triton X-100透化核膜时，未磷酸化形式的RPB 1在完全生长的卵母细胞中扩散出核，但在小的生长卵母细胞中仍然保留在那里，表明RPB 1不与完全生长的卵母细胞中的DNA结合。这些结果表明，全球转录沉默的直接原因是RNAP IT从DNA上解离。我们还观察到，在完全生长的卵母细胞中，RPB 1从用抑制素A处理的DNA中解离，以使其染色质去致密，这表明染色质浓缩不是卵母细胞生长过程中基因沉默的必要过程。
As mouse oocytes approach maturity, a global repression of gene transcription occurs. Here, we investigated the involvement of RPB1, the largest subunit of RNA polymerase II (RNAP II), in the regulation of this transcriptional silencing mechanism. Using BrUTP to follow transcription in an in vitro run-on assay, we observed an abrupt decrease in transcriptional activity when oocytes reached their full size (approximately 80 mu m). Immunoblotting using antibodies specific for the phosphorylated and unphosphorylated forms of RPB1 revealed that RPB1 is phosphorylated at Ser-2 and Ser-5 in the small growing oocytes in which active transcription occurs. By contrast, in transcriptionally inactive, full-grown oocytes, RPB1 is predominantly unphosphorylated. When we permeabilized the nuclear membrane using Triton X-100 during fixation for immunocytochemistry, the unphosphorylated form of RPB1 diffused out of the nucleus in the full-grown oocytes but still remained there in the small growing oocytes, indicating that RPB1 is not bound to DNA in full-grown oocytes. These results suggest that the immediate cause of global transcriptional silencing is the dissociation of RNAP IT from the DNA. We also observed dissociation of RPB1 from the DNA in full-grown oocytes treated with trichostatin A to decondense their chromatin, suggesting that chromatin condensation is not an essential process in gene silencing during oocyte growth.