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

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凝聚是基因组准确传递到子细胞的必要条件。尽管在模拟高阶染色体凝聚方面取得了进展，但高度凝聚中期染色体中染色质的位点特异性可及性尚不清楚。此外，人们认为，沿着染色体长度的凝聚模式在同源染色体对之间是不同的，但一对染色体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探针的研究表明，情况并非如此（Khan等人，Mol Cytogen 7:70, 2014）。通过对特定基因或基因间区域的短DNA序列进行颜色标记，并使用荧光原位杂交（FISH）将其标记到染色体对上的互补序列上，我们已经确定了单个染色体对彼此不同的区域。这些差异不是发生在所有的染色体区域，而是发生在大多数（如果不是全部的话）染色体上。这些差异是非随机的，可复制的，并且在许多无关个体的染色体上共享相同的模式。DA区域的DNA更紧密地集中在一对染色体的一个成员上，这个成员更开放（称为差异可及性或DA）。我们还能够用染色质冷凝抑制剂修饰中期染色体结构，通过放松染色体来消除这些结构差异（Khan等人，Mol Cytogen, 8:65, 2015）。我们研究了来自血液组织中期染色体的14个DNA探针（9个显示差异可及性[DA]， 5个不显示差异可及性的对照组）。这些发现提出了许多问题。在本研究计划中，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)是否可遗传，是否与亲本有关；C)研究更多的基因座是否会揭示DA区域的共同序列性或结构性；D) DA是否在基因组中广泛存在；E)表征DA的超螺旋差异是否直接或间接来自于拓扑异构酶与DNA的结合；F) DA是否在开发或分化过程中建立；G)如果它是一个早期事件，一旦发生就保持稳定。DA可能是染色质调控的一种结构编码，它在同源中期染色体上的出现可能是细胞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来解决这些问题。

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.