Unravelling the metaphase epigenome: Differences in DNA accessibility between homologous chromosomes

揭示中期表观基因组：同源染色体之间 DNA 可及性的差异

• 批准号: RGPIN-2016-05419
• 负责人: Knoll, Joan
• 金额: $ 2.4万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=685126
• 关键词: Unravelling; metaphase; epigenome; Differences; DNA

My research focuses on understanding the structure and organization of metaphase chromosomes in mitosis. High fidelity mitotic metaphase chromosome condensation is essential for accurate transmission of the genome into daughter cells. Despite advances in modeling higher order chromosome condensation, locus-specific accessibility of chromatin within highly condensed metaphase chromosomes is not well understood. Furthermore, it is believed that the pattern of condensation along the chromosome's length differs between homologous chromosome pairs but that individual chromosomes in a pair share the same pattern. Our recent studies using 3D super-resolution microscopy and unique sequence DNA FISH probes developed from our genomic technology demonstrate that this is not the case (Khan et al, Mol Cytogen 7:70, 2014). By color labeling short DNA sequences from parts of specific genes or intergenic regions and tagging them to their complementary sequence on the chromosome pair using fluorescence in situ hybridization (FISH), we have identified regions of individual chromosome pairs that differ from each other. These differences do not occur on all chromosome regions but occur on most if not all chromosomes. These differences are nonrandom, reproducible and share the same pattern on chromosomes from many unrelated individuals. The DNA in DA regions is more compacted on one member of the chromosome pair which is more open (referred to as differential accessibility or DA). We have also been able to modify metaphase chromosome structure with a chromatin condensation inhibitor to remove these structural differences by relaxing the chromosome (Khan et al, Mol Cytogen, 8:65, 2015). We have studied 14 DNA probes (9 that show differential accessibility [DA] and 5 controls that do not) on metaphase chromosomes from blood tissue. These findings have raised many questions. In this research program, we want to understand the significance of DA by broadening our studies to include: A) whether DA occurs in other tissues and is identical between tissues; B) whether it is heritable and related to parental origin; C) whether studying a larger number of loci will reveal common sequence-based or structural properties of DA regions; D) if DA is widespread DA in the genome; E) whether the super-helical differences that characterize DA arise directly or indirectly from topoisomerase binding to DNA; F) whether DA is established during development or differentiation; and G) if it is an early event that remains stable once it occurs. DA may represent a structural code of chromatin regulation, and its occurrence on homologous metaphase chromosomes may be a way for the cell to re-establish identity after mitosis. We propose a laboratory based program that addresses each of these questions.

我的研究重点是了解有丝分裂中期染色体的结构和组织。高保真度有丝分裂中期染色体浓缩对于将基因组准确传输到子细胞中至关重要。尽管在模拟高阶染色体浓缩方面取得了进展，但高度浓缩的中期染色体内染色质的位点特异性可及性尚不清楚。此外，据信，同源染色体对之间沿染色体长度的凝聚模式不同，但一对中的各个染色体共享相同的模式。我们最近使用 3D 超分辨率显微镜和从我们的基因组技术开发的独特序列 DNA FISH 探针进行的研究表明，情况并非如此（Khan 等人，Mol Cytogen 7:70, 2014）。通过对特定基因部分或基因间区域的短 DNA 序列进行颜色标记，并使用荧光原位杂交 (FISH) 将它们标记到染色体对上的互补序列，我们已经识别出各个染色体对中彼此不同的区域。这些差异并不发生在所有染色体区域上，但发生在大多数（如果不是全部）染色体上。这些差异是非随机的、可重复的，并且在许多不相关个体的染色体上具有相同的模式。 DA 区域中的 DNA 在染色体对的一个更开放的成员上更加紧密（称为差异可及性或 DA）。我们还能够使用染色质缩合抑制剂修饰中期染色体结构，通过松弛染色体来消除这些结构差异（Khan 等人，Mol Cytogen，8:65，2015）。我们研究了血液组织中期染色体上的 14 个 DNA 探针（9 个显示出差异可及性 [DA]，5 个对照则没有）。这些发现提出了许多问题。 在本研究项目中，我们希望通过扩大研究范围来了解 DA 的重要性：A) DA 是否出现在其他组织中并且在组织之间是否相同； B) 是否具有遗传性，是否与父母血统有关； C) 研究大量基因座是否会揭示 DA 区域基于序列或结构的共同特性； D) DA是否是基因组中广泛存在的DA； E) 表征 DA 的超螺旋差异是否直接或间接由拓扑异构酶与 DNA 的结合引起； F) DA是否在发育或分化过程中建立； G) 如果是早期事件，一旦发生就保持稳定。 DA可能代表染色质调控的结构密码，其在同源中期染色体上的出现可能是细胞在有丝分裂后重新建立身份的一种方式。我们提出了一个基于实验室的计划来解决这些问题。