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，在相间，非活性X染色体（XI）采用特定的3维 与活性X染色体（XA）不同的构象。但是我们对如何了解 安排是实现的，也不是该组织维持表观基因组变化的关键 XI的特征。人xi至少排列成两种不同类型的兼性异染色质 沿着染色体长度占据大约十二个替代性多兆库（MB）域， 通过免疫荧光使中期染色体具有条纹外观。在相间 相同类型的聚结的异染色质，导致XI分为两个隔室，导致A 双方外观。几个大的（40-400千座）串联重复（TR）DNA位于边界 在X染色体上的这些异染色质带中的一些之间。这些TR采用XI特定的 由结构蛋白CCCTC结合因子（CTCF）绑定的白染色质组织。尽管 居住在15-60 MB的情况下，TRS经常使XI特异性 - 染色体内接触。鉴于这种行为， TR元素可能充当表观遗传调节的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中删除为 检验该假设的最直接方法。在XA和雄性X染色体上，DXZ4包装到 组成型异染色质，这是其他常染色体TR元素共有的排列。但是，在 一些雄性癌细胞，发现DXZ4将其染色质状态“翻转”到类似于看到的染色质状态 在XI上。此外，已经描述了类似现象的几个常染色体TR元素 转化的细胞以及一个常染色体TR，该TR是导致一种肌肉营养不良形式的常染色体TR 此配置。大型TR DNA在两种不同的配置之间过渡的能力，事实 一种形式可以介导新的远程染色质接触使您了解染色质状态 这些序列对表观遗传的调节很重要。该提案中描述的实验试图 地址，（1）如何调节TRS的染色质状态，重点是 随着染色质状态改变表达的相关长期非编码RNA（LNCRNA），（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触点。预计 这些研究将评估TR DNA在X染色体上的功能，并提供机械洞察力 进入XI领域的组织和维护以及LNCRNA在确定染色质中的作用 复杂基因组中的TR DNA。