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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=615682
• 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。高保真有丝分裂中期染色体浓缩对于将基因组准确传递到子细胞中是必不可少的。尽管在高阶染色体凝聚模型方面取得了进展，但高度凝聚的中期染色体内染色质的位点特异性可及性还没有得到很好的理解。此外，据信同源染色体对之间沿着染色体长度的浓缩模式不同，但一对中的单个染色体具有相同的模式。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探针的研究证明情况并非如此（Khan等人，Mol Cytogen 7：70，2014）。通过颜色标记短DNA序列的特定基因或基因间区域的一部分，并标记它们的互补序列的染色体对使用荧光原位杂交（FISH），我们已经确定了区域的个别染色体对，彼此不同。这些差异并不发生在所有的染色体区域，但发生在大多数，如果不是所有的染色体。这些差异是非随机的，可重复的，并且在许多无关个体的染色体上具有相同的模式。DA区域中的DNA在染色体对的一个成员上更紧密，该成员更开放（称为差异可及性或DA）。我们还能够用染色质凝聚抑制剂修饰中期染色体结构，以通过松弛染色体来去除这些结构差异（Khan等人，Mol Cytogen，8：65，2015）。我们已经研究了14个DNA探针（9个显示差异可及性[DA]和5个控制，不）中期染色体从血液组织。这些发现引发了许多问题。在这项研究计划中，我们希望通过扩大我们的研究范围来了解DA的意义，包括：A）DA是否发生在其他组织中，并且在组织之间是相同的; B）它是否是可遗传的，并且与亲本起源有关; C）研究大量的基因座是否会揭示DA区域的共同序列或结构特性; D）DA是否是基因组中广泛分布的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结合引起; F）DA是否在发育或分化期间建立;以及G）它是否是一旦发生就保持稳定的早期事件。DA可能代表染色质调节的结构密码，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方案，解决这些问题。