Yan Yitang, Cummings Connie A, Sutton Deloris, Yu Linda, Castro Lysandra, Moore Alicia B, Gao Xiaohua, Dixon Darlene
National Toxicology Program Laboratory (NTPL), Molecular Pathogenesis Group, Division of the NTP (DNTP), National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), Research Triangle Park, NC, 27709.
Cellular and Molecular Pathology Branch, Pathology Support Group, DNTP, NIEHS, NIH, Research Triangle Park, NC, 27709.
Genes Chromosomes Cancer. 2016 Apr;55(4):397-406. doi: 10.1002/gcc.22343. Epub 2016 Jan 22.
Histone phosphorylation has a profound impact on epigenetic regulation of gene expression, chromosome condensation and segregation, and maintenance of genome integrity. Histone H3 Serine 10 is evolutionally conserved and heavily phosphorylated during mitosis. To examine Histone H3 Serine 10 phosphorylation (H3S10ph) dynamics in mitosis, we applied immunogold labeling and confocal microscopy to visualize H3S10ph expression in MCF-7 cells. Confocal observations showed that MCF-7 cells had abundant H3S10ph expression in prophase and metaphase. In anaphase, the H3S10ph expression was significantly decreased and displayed only sparsely localized staining that mainly associated with the chromatid tips. We showed that immunogold bead density distribution followed the H3S10ph expression patterns observed in confocal analysis. At a higher magnification in metaphase, the immunogold beads were readily visible and the bead distribution along the condensed chromosomes was distinctive, indicating the specificity and reliability of the immunogold staining procedure. In anaphase, the beads were found to distribute focally in specific regions of chromatids, reinforcing the confocal observations of differential H3 phosphorylation. To our knowledge, this is the first report to show the specific H3S10ph expression with an immunogold technique and transmission electron microscopy. Additionally, with confocal microscopy, we analyzed H3S10ph expression in an immortalized cell line derived from benign uterine smooth muscle tumor cells. H3S10ph epitope was expressed more abundantly during anaphase in the benign tumor cells, and there was no dramatic differential expression within the condensed chromatid clusters as observed in MCF-7 cells. The differences in H3S10ph expression pattern and dynamics may contribute to the differential proliferative potential between benign tumor cells and MCF-7 cells.
组蛋白磷酸化对基因表达的表观遗传调控、染色体凝聚与分离以及基因组完整性的维持有着深远影响。组蛋白H3丝氨酸10在进化上保守，且在有丝分裂期间高度磷酸化。为了检测有丝分裂过程中组蛋白H3丝氨酸10磷酸化（H3S10ph）的动态变化，我们应用免疫金标记和共聚焦显微镜来观察MCF-7细胞中H3S10ph的表达。共聚焦观察显示，MCF-7细胞在前期和中期有丰富的H3S10ph表达。在后期，H3S10ph表达显著降低，仅显示稀疏的局部染色，主要与染色单体末端相关。我们发现免疫金珠密度分布遵循共聚焦分析中观察到的H3S10ph表达模式。在中期更高放大倍数下，免疫金珠清晰可见，且沿凝聚染色体的珠子分布具有特异性，表明免疫金染色程序的特异性和可靠性。在后期，发现珠子集中分布在染色单体的特定区域，进一步证实了共聚焦观察到的H3磷酸化差异。据我们所知，这是首次使用免疫金技术和透射电子显微镜展示特定的H3S10ph表达。此外，通过共聚焦显微镜，我们分析了源自良性子宫平滑肌肿瘤细胞的永生化细胞系中的H3S10ph表达。在良性肿瘤细胞的后期，H3S10ph表位表达更为丰富，并且在凝聚的染色单体簇内没有观察到如MCF-7细胞中那样显著的差异表达。H3S10ph表达模式和动态变化的差异可能导致良性肿瘤细胞与MCF-7细胞之间增殖潜能的差异。
