Investigating the nuclear organization of the inactive X in female lymphocytes

研究女性淋巴细胞中失活 X 的核组织

• 批准号: 9907389
• 负责人: Isabel Sierra
• 金额: $ 4.55万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907389
• 关键词: Address; Affect; Alleles; Autoimmune Diseases; Autoimmunity; B-Lymphocytes; Binding Proteins; Caenorhabditis elegans; Cell Nucleus; Cells; Chromatin; Chromatin Structure; Chromosomes; DNA; Data; Defect; Development; Disease; Dosage Compensation (Genetics); Epigenetic Process; Failure; Female; Fluorescent in Situ Hybridization; Gene Dosage; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic Transcription; Heterochromatin; Hybrids; Image; Immune; Immunofluorescence Immunologic; Individual; Knock-out; Knockout Mice; Knowledge; Light; Link; LoxP-flanked allele; Lymphocyte; Maintenance; Malignant Neoplasms; Microscopy; Modeling; Movement; Mus; Nuclear; Nuclear Structure; Organizational Change; Pattern; Process; Proteins; RNA; RNA Interference; Regulation; Role; Somatic Cell; Structural Protein; Structure; System; Untranslated RNA; Up-Regulation; Variant; X Chromosome; X Inactivation; YY1 Transcription Factor; condensin; conditional knockout; dosage; experimental study; genomic locus; male; mouse model; multiplexed imaging; novel; prevent; programs; recruit

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.

项目摘要 细胞核中的染色质组织受到高度调控，以提供对基因表达的精确控制。这个 染色质结构和基因调控之间关系最显著的例子是X染色体 失活(XCI)。XCI表观遗传学将一条女性X染色体转化为转录沉默的不活跃染色体 X(Xi)使男性和女性的剂量相等。XCI是由长的非编码RNA的传播启动的 XIST在独联体中将XI重新组织成紧凑的两党结构，并将XI定位于核外围。 必须保持XI，以防止X连锁基因的异常表达，这与 患有自身免疫性疾病和癌症。然而，对XI的沉默或核组织是如何监管的 XCI的维护过程仍有待深入了解。目前的维护模式是稳定的 所有雌性体细胞中Xist RNA与XI的相关性。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。在幼稚的女性B细胞中，Xist RNA不定位于XI，但 令人惊讶的是，在刺激后，Xist RNA强健地返回到了Xi。XIST本地化的一个必备因素 B细胞刺激后对XI的RNA是转录因子YY1，因为YY1的缺失使其定位丧失。 Xist RNA对刺激的B细胞中的XI。YY1在刺激后与多种结构蛋白相互作用 女性B细胞，但这些因素是否协同调节XCI尚不清楚。此外，动态的 Xist RNA的运动对XI核组织的影响尚不清楚。使用女性卵泡B细胞，我们 将询问XI的核组织，并确定YY1与结构蛋白如何相互作用 有助于动态XCI维护。在目标1中，我们将对幼稚的XI进行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领土的组织。我们 将考察习的紧密性、XI领土在核心内的定位以及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，并且 将利用这一点来产生基因特异性探针，以检查这些基因组座位在XI中的空间定位 领地。我们假设Xist RNA在B细胞刺激下的动态运动将改变 习领地的组织。在目标2中，我们将确定三个YY1相互作用的结构蛋白的作用， XCI中的LaminB1、Satb1和凝集素。利用漂浮的小鼠对每个基因进行单独的体外删除 将决定他们对Xist RNA定位的要求，以XI，丰富规范的Xi异染色质 标记H3K27me3和H2AK119Ub，X连锁基因表达，以及XI地区的核组织。我们 假设这些蛋白通过调节Xist RNA定位与YY1协同维持XCI 在刺激的女性卵泡B细胞中有核组织。这些目标加在一起，将扩大我们的 了解XCI维护的调节机制，有助于我们对核的理解 组织和基因调控。