Favaedi Raha, Shahhoseini Maryam, Pakzad Mahammad, Mollamohammadi Sepideh, Baharvand Hossein
Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Int J Dev Biol. 2016;60(4-6):103-10. doi: 10.1387/ijdb.140332ms.
Histone H3 lysine 9 methylation has been shown to be a critical barrier to efficient cell reprogramming. This discovery allows the assessment of the cell pluripotency state by considering the extent of H3K9 methylation vs. acetylation at the same position. A set of pluripotent and differentiated human cells including embryonic stem cells, their differentiated and reprogrammed counterparts, along with human fibroblasts and their derived reprogrammed cells, were used to evaluate the ratio of total H3K9 methylation over acetylation using a quantitative ELISA-based approach. Also, the occurrence of the H3K4me3 and H3K27me3 bivalent marks was evaluated. Additionally, using ChIP-qPCR the occurrence of these histone marks on the regulatory regions of stemness genes (Nanog, Oct4 and Sox2) as well as on genes indicating fibroblast differentiation (Vim, COL1A1 and THY1) was evaluated. We evidence remarkably high ratios of H3K9ac/K9me2 in ES and iPS cells vs. differentiated cells. In iPSCs, a direct relationship between the ratios of total H3K9ac/H3K9me2 and the ratios of these marks on pluripotency gene regulatory regions and their expression was observed. In differentiated cells, in contrast, the ratios of global H3K9ac/K9me2 is low but the active genes escape this general situation and bear higher amounts of H3K9ac vs. H3K9me. Total H3K4me3/K27me3 ratios presented the same trends, but with reduced amplitudes. We propose that the rapid quantitative measurements of relative amounts of H3K9ac and K9me2 in iPS cells compared to the parental differentiated cells constitute a reliable and convenient criterion to rapidly assess the cell pluripotency potentials and the efficiency of cell reprogramming.
组蛋白H3赖氨酸9甲基化已被证明是高效细胞重编程的关键障碍。这一发现使得通过考虑同一位置H3K9甲基化与乙酰化的程度来评估细胞多能性状态成为可能。一组多能和分化的人类细胞，包括胚胎干细胞、其分化和重编程的对应细胞，以及人类成纤维细胞及其衍生的重编程细胞，被用于采用基于定量ELISA的方法评估总H3K9甲基化与乙酰化的比率。此外，还评估了H3K4me3和H3K27me3双价标记的出现情况。另外，使用ChIP-qPCR评估了这些组蛋白标记在干性基因（Nanog、Oct4和Sox2）的调控区域以及指示成纤维细胞分化的基因（Vim、COL1A1和THY1）上的出现情况。我们证明，与分化细胞相比，ES细胞和iPS细胞中H3K9ac/K9me2的比率显著较高。在iPSCs中，观察到总H3K9ac/H3K9me2的比率与这些标记在多能性基因调控区域上的比率及其表达之间存在直接关系。相比之下，在分化细胞中，全局H3K9ac/K9me2的比率较低，但活跃基因不受这种普遍情况的影响，与H3K9me相比，其H3K9ac的含量更高。总H3K4me3/K27me3的比率呈现相同趋势，但幅度较小。我们提出，与亲代分化细胞相比，快速定量测量iPS细胞中H3K9ac和K9me2的相对量构成了一个可靠且方便的标准，可用于快速评估细胞的多能性潜力和细胞重编程的效率。
