Ghule Prachi N, Becker Klaus A, Harper J Wade, Lian Jane B, Stein Janet L, van Wijnen Andre J, Stein Gary S
Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
J Cell Physiol. 2007 Oct;213(1):9-17. doi: 10.1002/jcp.21119.
Human embryonic stem (ES) cells have an expedited cell cycle ( approximately 15 h) due to an abbreviated G1 phase ( approximately 2.5 h) relative to somatic cells. One principal regulatory event during cell cycle progression is the G1/S phase induction of histone biosynthesis to package newly replicated DNA. In somatic cells, histone H4 gene expression is controlled by CDK2 phosphorylation of p220(NPAT) and localization of HiNF-P/p220(NPAT) complexes with histone genes at Cajal body related subnuclear foci. Here we show that this 'S point' pathway is operative in situ in human ES cells (H9 cells; NIH-designated WA09). Immunofluorescence microscopy shows an increase in p220(NPAT) foci in G1 reflecting the assembly of histone gene regulatory complexes in situ. In contrast to somatic cells where duplication of p220(NPAT) foci is evident in S phase, the increase in the number of p220(NPAT) foci in ES cells appears to precede the onset of DNA synthesis as measured by BrdU incorporation. Phosphorylation of p220(NPAT) at CDK dependent epitopes is most pronounced in S phase when cells exhibit elevated levels of cyclins E and A. Our data indicate that subnuclear organization of the HiNF-P/p220(NPAT) pathway is rapidly established as ES cells emerge from mitosis and that p220(NPAT) is subsequently phosphorylated in situ. Our findings establish that the HiNF-P/p220(NPAT) gene regulatory pathway operates in a cell cycle dependent microenvironment that supports expression of DNA replication-linked histone genes and chromatin assembly to accommodate human stem cell self-renewal.
与体细胞相比，人类胚胎干细胞（ES细胞）由于G1期缩短（约2.5小时）而具有更快的细胞周期（约15小时）。细胞周期进程中的一个主要调节事件是在G1/S期诱导组蛋白生物合成以包装新复制的DNA。在体细胞中，组蛋白H4基因表达受p220（NPAT）的CDK2磷酸化以及HiNF-P/p220（NPAT）复合物与组蛋白基因在与卡哈尔体相关的核内亚灶处的定位控制。在这里，我们表明这种“S点”途径在人类ES细胞（H9细胞；美国国立卫生研究院指定的WA09）中在原位起作用。免疫荧光显微镜显示G1期p220（NPAT）灶增加，反映了组蛋白基因调节复合物在原位的组装。与体细胞中p220（NPAT）灶在S期明显复制不同，ES细胞中p220（NPAT）灶数量的增加似乎在通过BrdU掺入测量的DNA合成开始之前。当细胞中细胞周期蛋白E和A水平升高时，p220（NPAT）在CDK依赖性表位处的磷酸化在S期最为明显。我们的数据表明，当ES细胞从有丝分裂中出现时，HiNF-P/p220（NPAT）途径的核内亚组织迅速建立，随后p220（NPAT）在原位被磷酸化。我们的研究结果表明，HiNF-P/p220（NPAT）基因调节途径在支持与DNA复制相关组蛋白基因表达和染色质组装以适应人类干细胞自我更新的细胞周期依赖性微环境中起作用。
