Khan Wahab A, Rogan Peter K, Knoll Joan Hm
Department of Pathology and Laboratory Medicine, University of Western Ontario, London, ON N6A 5C1 Canada ; Cytognomix, Inc, London, ON N6G 4X8 Canada.
Departments of Biochemistry and Computer Science, University of Western Ontario, London, ON N6A 5C1 Canada ; Cytognomix, Inc, London, ON N6G 4X8 Canada.
Mol Cytogenet. 2014 Nov 19;7(1):70. doi: 10.1186/s13039-014-0070-y. eCollection 2014.
Condensation differences along the lengths of homologous, mitotic metaphase chromosomes are well known. This study reports molecular cytogenetic data showing quantifiable localized differences in condensation between homologs that are related to differences in accessibility (DA) of associated DNA probe targets. Reproducible DA was observed for ~10% of locus-specific, short (1.5-5 kb) single copy DNA probes used in fluorescence in situ hybridization.
Fourteen probes (from chromosomes 1, 5, 9, 11, 15, 17, 22) targeting genic and intergenic regions were developed and hybridized to cells from 10 individuals with cytogenetically-distinguishable homologs. Differences in hybridization between homologs were non-random for 8 genomic regions (RGS7, CACNA1B, GABRA5, SNRPN, HERC2, PMP22:IVS3, ADORA2B:IVS1, ACR) and were not unique to known imprinted domains or specific chromosomes. DNA probes within CCNB1, C9orf66, ADORA2B:Promoter-Ex1, PMP22:IVS4-Ex 5, and intergenic region 1p36.3 showed no DA (equivalent accessibility), while OPCML showed unbiased DA. To pinpoint probe locations, we performed 3D-structured illumination microscopy (3D-SIM). This showed that genomic regions with DA had 3.3-fold greater volumetric, integrated probe intensities and broad distributions of probe depths along axial and lateral axes of the 2 homologs, compared to a low copy probe target (NOMO1) with equivalent accessibility. Genomic regions with equivalent accessibility were also enriched for epigenetic marks of open interphase chromatin (DNase I HS, H3K27Ac, H3K4me1) to a greater extent than regions with DA.
This study provides evidence that DA is non-random and reproducible; it is locus specific, but not unique to known imprinted regions or specific chromosomes. Non-random DA was also shown to be heritable within a 2 generation family. DNA probe volume and depth measurements of hybridized metaphase chromosomes further show locus-specific chromatin accessibility differences by super-resolution 3D-SIM. Based on these data and the analysis of interphase epigenetic marks of genomic intervals with DA, we conclude that there are localized differences in compaction of homologs during mitotic metaphase and that these differences may arise during or preceding metaphase chromosome compaction. Our results suggest new directions for locus-specific structural analysis of metaphase chromosomes, motivated by the potential relationship of these findings to underlying epigenetic changes established during interphase.
沿同源有丝分裂中期染色体长度的凝聚差异是众所周知的。本研究报告了分子细胞遗传学数据，显示同源染色体之间在凝聚方面存在可量化的局部差异，这些差异与相关DNA探针靶标的可及性差异（DA）有关。在荧光原位杂交中使用的约10%的基因座特异性短（1.5 - 5 kb）单拷贝DNA探针观察到了可重复的DA。
开发了针对基因和基因间区域的14个探针（来自染色体1、5、9、11、15、17、22），并与来自10个具有细胞遗传学上可区分同源染色体的个体的细胞进行杂交。8个基因组区域（RGS7、CACNA1B、GABRA5、SNRPN、HERC2、PMP22:IVS3、ADORA2B:IVS1、ACR）的同源染色体之间杂交差异是非随机的，且并非已知印记区域或特定染色体所特有。CCNB1、C9orf66、ADORA2B:启动子 - 外显子1、PMP22:IVS4 - 外显子5以及基因间区域1p36.3内的DNA探针未显示DA（等效可及性），而OPCML显示无偏DA。为了精确定位探针位置，我们进行了三维结构光照显微镜（3D - SIM）。这表明与具有等效可及性的低拷贝探针靶标（NOMO1）相比，具有DA的基因组区域在体积、整合探针强度方面大3.3倍，并且沿着两个同源染色体的轴和横轴具有广泛的探针深度分布。具有等效可及性的基因组区域也比具有DA的区域更富集开放间期染色质的表观遗传标记（DNase I HS、H3K27Ac、H3K4me1）。
本研究提供了证据表明DA是非随机且可重复的；它是基因座特异性的，但并非已知印记区域或特定染色体所特有。非随机DA在一个两代家族中也显示出可遗传性。杂交中期染色体的DNA探针体积和深度测量通过超分辨率3D - SIM进一步显示了基因座特异性的染色质可及性差异。基于这些数据以及对具有DA的基因组区间的间期表观遗传标记的分析，我们得出结论，在有丝分裂中期同源染色体的压缩存在局部差异，并且这些差异可能在中期染色体压缩期间或之前出现。我们的结果为由这些发现与间期建立的潜在表观遗传变化的关系所推动的中期染色体基因座特异性结构分析提出了新方向。
