Cell cycle dependent phosphorylation and subnuclear organization of the histone gene regulator p220(NPAT) in human embryonic stem cells.

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.