Investigating the nuclear organization of the inactive X in female lymphocytes

项目摘要 细胞核中的染色质组织受到高度调节，以提供对基因表达的精确控制。的 染色质结构和基因调控之间关系的最生动的例子是X染色体 灭活（XCI）。XCI表观遗传学将一个女性X染色体转化为转录沉默的非活性 X（Xi）以均衡雄性和雌性之间的剂量。XCI由长的非编码RNA的扩散启动， 顺式Xist将Xi重新组织成一个紧凑的二分结构，并将Xi瞄准核外围。 必须维持Xi，以防止X连锁基因的异常表达， 自身免疫性疾病和癌症然而，Xi的沉默或核组织是如何被调节的， 在维护XCI的过程中，仍然需要很好地理解。当前的维护范式是稳定的 在所有雌性体细胞中Xist RNA与Xi的结合。最近，安格拉实验室发现了一本小说 XCI在女性淋巴细胞中的作用机制。在幼稚的雌性B细胞中，Xist RNA不定位于Xi，但 令人惊讶的是，在刺激后，Xist RNA强烈地返回到Xi。Xist本地化的一个必要因素是 在B细胞刺激后，与Xi结合的RNA是转录因子Yy 1，因为Yy 1的缺失消除了 Xist RNA与刺激的B细胞中的Xi的结合。在受刺激的细胞中，发现YY 1与多种结构蛋白相互作用。 女性B细胞，但这些因素是否合作，以调节XCI尚不清楚。此外，动态 Xist RNA的运动影响Xi的核组织尚不清楚。使用女性卵泡B细胞，我们 将询问Xi的核组织，并确定Yy 1相互作用的结构蛋白 有助于动态XCI维护。在目标1中，我们将在未接受过治疗的患者和未接受过治疗的患者中进行Xi的等位基因特异性成像， 刺激B细胞，以确定Xist RNA的动态运动如何影响Xi区域的组织。我们 我将研究Xi的压缩，核心内Xi领土的定位，以及Xi的组织 二分结构我们最近在女性B细胞中发现了来自Xi的新的X连锁基因转录， 我将用它来产生基因特异性探针，以检查这些基因组位点在Xi中的空间定位 领土我们假设Xist RNA在B细胞刺激后的动态运动将改变Xist RNA的表达。 组织Xi领地。在目标2中，我们将确定三个与YY 1相互作用的结构蛋白的作用， LaminB1、Satb1和XCI中的缩合物。使用floxed小鼠进行每个基因的单独离体缺失 将确定它们对Xist RNA定位于Xi的需要，富集典型的Xi异染色质 标记H3 K27 me3和H2 AK 119 Ub、X连锁基因表达和Xi域的核组织。我们 假设这些蛋白与Yy 1合作，通过调节Xist RNA定位来维持XCI 和Xi核组织在刺激的女性卵泡B细胞。总之，这些目标将扩大我们的 调节XCI维持机制的知识，并有助于我们对核动力学的理解。 组织和基因调控。

Project Summary Chromatin organization in the nucleus is highly regulated to provide precise control over gene expression. The most dramatic example of the relationship between chromatin structure and gene regulation is X Chromosome Inactivation (XCI). XCI epigenetically converts one female X chromosome into a transcriptionally silent inactive X (Xi) to equalize dosage between males and females. XCI is initiated by spreading of the long non-coding RNA Xist in cis which re-organizes the Xi into a compact, bipartite structure, and targets the Xi to the nuclear periphery. Maintenance of the Xi must be achieved to prevent aberrant expression of X-linked genes, which is associated with autoimmune diseases and cancers. However, how silencing or nuclear organization of the Xi is regulated during maintenance of XCI remains to be well understood. The current paradigm of maintenance is stable association of Xist RNA with the Xi in all female somatic cells. Recently, the Anguera lab found a novel mechanism of XCI in female lymphocytes. In naïve female B cells, Xist RNA is not localized to the Xi, but surprisingly, after stimulation Xist RNA robustly returns to the Xi. One factor necessary for the localization of Xist RNA to the Xi after B cell stimulation is the transcription factor Yy1, as loss of Yy1 abrogates the localization of Xist RNA to the Xi in stimulated B cells. Yy1 was found to interact with multiple structural proteins in stimulated female B cells, but whether these factors cooperate to regulate XCI is unclear. Additionally, how the dynamic movement of Xist RNA affects the nuclear organization of the Xi is unknown. Using female follicular B cells, we will interrogate the nuclear organization of the Xi, and determine how Yy1-interacting structural proteins contribute to dynamic XCI maintenance. In Aim 1 we will perform allele-specific imaging of the Xi in naïve and stimulated B cells to determine how dynamic movement of Xist RNA impacts organization of the Xi territory. We will examine compaction of the Xi, localization of the Xi territory within the nucleus, and organization of the Xi bipartite structure. We have recently found novel X-linked gene transcription from the Xi in female B cells, and will use this to generate gene-specific probes to examine spatial localization of these genomic loci within the Xi territory. We hypothesize that the dynamic movement of Xist RNA upon B cell stimulation will change the organization of the Xi territory. In Aim 2, we will determine the role of three Yy1-interacting structural proteins, LaminB1, Satb1, and condensins in XCI. Use of floxed mice to perform individual ex vivo deletions of each gene will determine their requirement for Xist RNA localization to the Xi, enrichment of canonical Xi heterochromatin marks H3K27me3 and H2AK119Ub, X-linked gene expression, and nuclear organization of the Xi territory. We hypothesize that these proteins cooperate with Yy1 to maintain XCI through regulation of Xist RNA localization and Xi nuclear organization in stimulated female follicular B cells. Together, these aims will expand our knowledge of the mechanisms regulating XCI maintenance, and contribute to our understanding of nuclear organization and gene regulation.