Exploring the contribution of large tandem repeat DNA to the organization and maintenance of the inactive X chromosome

探索大串联重复 DNA 对失活 X 染色体的组织和维护的贡献

• 批准号: 9324288
• 负责人: Brian P. Chadwick
• 金额: $ 28.58万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9324288
• 关键词: 3-Dimensional; Address; Adopted; Affect; Alleles; Alpha Cell; Appearance; Architecture; Bacterial Artificial Chromosomes; Behavior; CCCTC-binding factor; Cell Cycle; Cell Differentiation process; Cell Nucleus; Cell division; Cells; Characteristics; Chromatin; Chromosomal Instability; Chromosome Structures; Chromosome Territory; Chromosomes; Complex; DNA; Data; Development; Diploidy; Disease; Disease susceptibility; Elements; Epigenetic Process; Euchromatin; Failure; Female; Future; Gene Expression; Gene Silencing; Genes; Genome; Genome engineering; Goals; Heterochromatin; Human; Immunofluorescence Immunologic; Interphase; Length; Link; Location; Macaca; Maintenance; Malignant Epithelial Cell; Malignant Neoplasms; Mediating; Metaphase; Modeling; Molecular Conformation; Mus; Muscular Dystrophies; Pathway interactions; Process; Proteins; Role; Side; Somatic Cell; Tandem Repeat Sequences; Testing; Untranslated RNA; X Chromosome; X Inactivation; base; cell transformation; embryonic stem cell; epigenetic regulation; epigenomics; experimental study; human female; insight; male; tool

It is well established that at interphase the inactive X chromosome (Xi) adopts a specific 3-dimensional conformation that differs from that of the active X chromosome (Xa). Yet we know very little about how this arrangement is achieved, nor how critical this organization is to maintaining the epigenomic changes that are characteristic of Xi. Human Xi is arranged into at least two distinct types of facultative heterochromatin that occupy approximately a dozen alternating multi-megabase (Mb) domains along the length of the chromosome, that by immunofluorescence gives a striped appearance to the chromosome at metaphase. At interphase heterochromatin of the same type coalesce resulting in Xi arranging itself into two compartments resulting in a bi-partite appearance. Several large (40-400 kilobase) tandem repeat (TR) DNA reside at the boundary between some of these heterochromatin bands on the X chromosome. These TRs adopt an Xi-specific euchromatin organization that is bound by the architectural protein CCCTC-binding factor (CTCF). Despite residing 15-60 Mb apart, the TRs make frequent Xi-specific intra-chromosomal contact. Given this behavior, the TR elements may function as epigenetically regulated Xi-specific chromosome folding elements. Using cutting edge genome engineering tools, the largest of these TRs (DXZ4), has been removed from Xi as the most direct way to test this hypothesis. On the Xa and male X chromosome, DXZ4 is packaged into constitutive heterochromatin, an arrangement that is common to other autosomal TR elements. However, in some male carcinoma cells, DXZ4 has been found to “flip” its chromatin state to one that resembles that seen on Xi. Furthermore, a similar phenomena has been described for several autosomal TR elements in transformed cells as well as at one autosomal TR that is responsible for a form of muscular dystrophy when in this configuration. The ability of large TR DNA to transition between two distinct configurations, and the fact that one form can mediate new long-range chromatin contacts makes understanding how chromatin states are epigenetically regulated at these sequences important. Experiments described in this proposal seek to address, (1) how chromatin states at the TRs are regulated, with an emphasis on the potential role of associated long noncoding RNAs (lncRNAs) that alter in expression as chromatin states change, (2) assess the impact of DXZ4 loss on the maintenance and organization of Xi, and (3) determine if the introduction of TR elements at different locations on the X is sufficient to establish new long-range contacts. It is anticipated that these studies will assess the function of TR DNA on the X chromosome, as well as provide mechanistic insight into the organization and maintenance of Xi territory and the role of lncRNAs in regulating chromatin at large TR DNA in complex genomes.

众所周知，在间期，失活的 X 染色体 (Xi) 采用特定的 3 维结构 与活性 X 染色体 (Xa) 的构象不同。然而我们对如何做到这一点知之甚少 安排是否已实现，也不是该组织对于维持表观基因组变化有多重要 习的特点。人类 Xi 被排列成至少两种不同类型的兼性异染色质， 沿着染色体的长度占据大约十几个交替的多兆碱基（Mb）结构域， 通过免疫荧光显示中期染色体呈现条纹外观。在间期 相同类型的异染色质合并，导致 Xi 排列成两个区室，从而形成 两部分的外观。几个大的（40-400 kilobase）串联重复（TR）DNA位于边界处 X 染色体上的一些异染色质带之间。这些 TR 采用 Xi 特定的 由结构蛋白 CCCTC 结合因子 (CTCF) 结合的常染色质组织。尽管 TR 相距 15-60 Mb，频繁地进行 Xi 特异性染色体内接触。鉴于这种行为， TR元件可能作为表观遗传调控的Xi特异性染色体折叠元件。使用 尖端基因组工程工具，其中最大的 TR（DXZ4），已从 Xi 中删除，因为 检验这个假设的最直接的方法。在 Xa 和男性 X 染色体上，DXZ4 被包装成 组成型异染色质，是其他常染色体 TR 元件常见的排列。然而，在 在某些男性癌细胞中，DXZ4 被发现可以将其染色质状态“翻转”到与所看到的类似的状态 关于习。此外，在一些常染色体 TR 元件中也描述了类似的现象。 转化细胞以及一种常染色体TR，该常染色体TR在处于 这个配置。大 TR DNA 在两种不同构型之间转变的能力，以及事实 一种形式可以介导新的远程染色质接触，这有助于理解染色质状态是如何的 这些序列的表观遗传调控很重要。本提案中描述的实验旨在 解决，(1) TR 处的染色质状态如何受到调节，重点是 TR 的潜在作用 相关的长非编码 RNA (lncRNA) 随着染色质状态的变化而改变表达，(2) 评估 DXZ4损失对Xi的维护和组织的影响，以及（3）确定是否引入TR X 上不同位置的元素足以建立新的远程接触。预计 这些研究将评估 X 染色体上 TR DNA 的功能，并提供机制见解 Xi 区域的组织和维护以及 lncRNA 在调节整个染色质中的作用 复杂基因组中的 TR DNA。