权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

基本信息

批准号：
10213675
负责人：
Isabel Sierra
金额：
$ 4.6万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
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/10213675
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

项目摘要

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.

项目概要细胞核中的染色质组织受到高度调控，以精确控制基因表达。这染色质结构与基因调控之间关系最引人注目的例子是 X 染色体失活（XCI）。 XCI 通过表观遗传将一条女性 X 染色体转化为转录沉默的非活性染色体 X (Xi) 使男性和女性之间的剂量相等。 XCI 是通过长链非编码 RNA 的扩散启动的 Xist in cis 将 Xi 重组为紧凑的二分结构，并将 Xi 瞄准核外围。必须实现 Xi 的维持，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，如何规范习近平的沉默或核组织 XCI 维护期间的情况仍有待很好地理解。目前的维护模式是稳定的 Xist RNA 与所有女性体细胞中 Xi 的关联。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机制。在幼稚雌性 B 细胞中，Xist RNA 并不定位于 Xi，但令人惊讶的是，刺激后 Xist RNA 强劲地返回 Xi。 Xist 本地化的必要因素之一 B 细胞刺激后 Xi 的 RNA 是转录因子 Yy1，因为 Yy1 的丢失消除了 Xist RNA 位于受刺激的 B 细胞中的 Xi 上。 Yy1被发现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。另外，动态如何 Xist RNA 的运动对 Xi 核组织的影响尚不清楚。使用女性滤泡 B 细胞，我们将询问 Xi 的核组织，并确定 Yy1 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 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 区域的组织。我们将研究 Xi 的压缩、Xi 领土在核心内的定位以及 Xi 的组织二分结构。我们最近在雌性 B 细胞中发现了来自 Xi 的新型 X 连锁基因转录，并且将使用它来生成基因特异性探针，以检查 Xi 内这些基因组位点的空间定位领土。我们假设 Xist RNA 在 B 细胞刺激后的动态运动将改变 Xi领土的组织。在目标 2 中，我们将确定三种 Yy1 相互作用结构蛋白的作用， XCI 中的 LaminB1、Satb1 和凝缩蛋白。使用 floxed 小鼠对每个基因进行单独的离体删除将确定 Xist RNA 定位到 Xi、富集典型 Xi 异染色质的要求标记 H3K27me3 和 H2AK119Ub、X 连锁基因表达以及 Xi 区域的核组织。我们假设这些蛋白质与 Yy1 合作通过调节 Xist RNA 定位来维持 XCI 受刺激的雌性滤泡 B 细胞中的 Xi 核组织。这些目标共同将扩大我们的了解调节 XCI 维护的机制，并有助于我们理解核组织和基因调控。