Spatial separation of Xist RNA and polycomb proteins revealed by superresolution microscopy
Spatial separation of Xist RNA and polycomb proteins revealed by superresolution microscopy
复制标题
DOI：
10.1073/pnas.1312951111
复制
发表时间：
2014-02-11
影响因子：
11.1
通讯作者：
Brockdorff, Neil
中科院分区：
文献类型：
--
作者：
Cerase, Andrea;Smeets, Daniel;Brockdorff, Neil
在雌性哺乳动物中，两条X染色体中的一条在转录上被沉默，以相对于XY雄性平衡X连锁基因剂量，这一过程称为X染色体失活。从机制上讲，这被认为是通过主调节因子X失活特异性转录物（Xist）RNA定向募集染色质修饰因子而发生的，所述主调节因子X失活特异性转录物（Xist）RNA沿染色体的整个长度顺式沿着定位。一个充分研究的例子是多梳阻遏复合物2（PRC 2）的募集，有证据表明PRC 2蛋白质zeste 2的增强子（Ezh 2）和zeste 12的抑制子（Suz 12）与位于Xist RNA 5'端的A-重复区直接相互作用。在这项研究中，我们分析了Xist介导的招聘PRC 2使用两种方法，基于微阵列的表观基因组定位和超分辨率三维结构照明显微镜。利用胚胎干细胞系携带诱导Xist转基因位于小鼠17号染色体上，我们表明，24小时后同步诱导Xist表达，收购PRC 2结合位点地图主要generich地区，特别是在基因体内。奇怪的是，PRC 2沉积的这些新位点与Xist介导的基因沉默无关。进行3D结构照明显微镜以评估PRC 2蛋白和Xist RNA的相对定位。出乎意料的是，我们在诱导型Xist转基因ES细胞系和正常XX体细胞中都观察到显著的空间分离和共定位的缺乏。我们的观察反对Xist RNA和PRC 2蛋白之间的直接相互作用，因此，促使重新评估X染色体失活中PRC 2招募的机制。
In female mammals, one of the two X chromosomes is transcriptionally silenced to equalize X-linked gene dosage relative to XY males, a process termed X chromosome inactivation. Mechanistically, this is thought to occur via directed recruitment of chromatin modifying factors by the master regulator, X-inactive specific transcript (Xist) RNA, which localizes in cis along the entire length of the chromosome. A well-studied example is the recruitment of polycomb repressive complex 2 (PRC2), for which there is evidence of a direct interaction involving the PRC2 proteins Enhancer of zeste 2 (Ezh2) and Supressor of zeste 12 (Suz12) and the A-repeat region located at the 5' end of Xist RNA. In this study, we have analyzed Xist-mediated recruitment of PRC2 using two approaches, microarray-based epigenomic mapping and superresolution 3D structured illumination microscopy. Making use of an ES cell line carrying an inducible Xist transgene located on mouse chromosome 17, we show that 24 h after synchronous induction of Xist expression, acquired PRC2 binding sites map predominantly to generich regions, notably within gene bodies. Paradoxically, these new sites of PRC2 deposition do not correlate with Xist-mediated gene silencing. The 3D structured illumination microscopy was performed to assess the relative localization of PRC2 proteins and Xist RNA. Unexpectedly, we observed significant spatial separation and absence of colocalization both in the inducible Xist transgene ES cell line and in normal XX somatic cells. Our observations argue against direct interaction between Xist RNA and PRC2 proteins and, as such, prompt a reappraisal of the mechanism for PRC2 recruitment in X chromosome inactivation.