Induced pluripotent stem (iPS) cells derived from terminally differentiated human fibroblasts are re-programmed to possess stem cell like properties. However, the extent to which iPS cells exhibit unique properties of the human embryonic stem (hES) cell cycle remains to be established. Human ES cells are characterized by an abbreviated G1 phase ( approximately 2.5 h) and accelerated organization of subnuclear domains that mediate the assembly of regulatory machinery for histone gene expression [i.e., histone locus bodies (HLBs)]. We therefore examined cell cycle parameters of iPS cells in comparison to hES cells. Analysis of DNA synthesis (BrdU incorporation), cell cycle distribution (FACS analysis and Ki67 staining) and subnuclear organization of HLBs [immuno-fluorescence microscopy and fluorescence in situ hybridization (FISH)] revealed that human iPS cells have a short G1 phase ( approximately 2.5 h) and an abbreviated cell cycle (16-18 h). Furthermore, HLBs are formed and reorganized rapidly after mitosis (within 0.5 to 1.5 h). Thus, reprogrammed iPS cells have cell cycle kinetics and dynamic subnuclear organization of regulatory machinery that are principal properties of pluripotent hES cells. Our findings support the concept that the abbreviated cell cycle of hES and iPS cells is functionally linked to pluripotency. (c) 2010 Wiley-Liss, Inc.

Human histone gene expression is controlled at the level of transcription initiation and subsequent 3'end processing to generate non-polyadenylated stem-loop containing histone mRNAs. Transcription is controlled at the G1/S phase transition by the Cyclin E/CDK2 mediated induction of p220(NPAT)/HiNF-P complexes at subnuclear domains designated Histone Locus Bodies (HLBs) that associate with histone gene clusters. Histone mRNA maturation is mediated by Lsm10 containing U7snRNP complexes. In normal human somatic and embryonic stem cells, the 6p histone locus, the transcription marker p220(NPAT) and the 3'end processing marker Lsm10 (but not the Cajal Body marker coilin) co-localize, reflecting the assembly of an integrated factory for histone gene expression. Using in situ immuno-fluorescence microscopy and fluorescence in situ hybridization (FISH), we show that this subnuclear organization is compromised in some cancer cell lines. In aneuploid cells, the presence of HLBs correlates with the number of histone gene loci. More importantly, the in situ co-localization of p220(NPAT) and Lsm10 is disrupted in HeLa S3 cervical carcinoma cells and MCF7 breast adenocarcinoma cells, with most Lsm10 residing in Cajal Bodies. The finding that the subnuclear integration of transcriptional initiation and 3'end processing of histone gene transcripts is deregulated may be causally linked to tumor-related modifications in molecular pathways controlling histone gene expression during the cell cycle.

Human embryonic stem (hES) cells have an abbreviated G(1) phase of the cell cycle. How cells expedite G(1) events that are required for the initiation of S phase has not been resolved. One key regulatory pathway that controls G(1)/S-phase transition is the cyclin E/CDK2-dependent activation of the coactivator protein nuclear protein, ataxia-telangiectasia locus/histone nuclear factor-P (p220(NPAT)/HiNF-P) complex that induces histone gene transcription. In this study, we use the subnuclear organization of factors controlling histone gene expression to define mechanistic differences in the G(1) phase of hES and somatic cells using in situ immunofluorescence microscopy and fluorescence in situ hybridization (FISH). We show that histone gene expression is supported by the staged assembly and modification of a unique subnuclear structure that coordinates initiation and processing of transcripts originating from histone gene loci. Our results demonstrate that regulatory complexes that mediate transcriptional initiation (e.g., p220(NPAT)) and 3'-end processing (e.g., Lsm10, Lsm11, and SLBP) of histone gene transcripts colocalize at histone gene loci in dedicated subnuclear foci (histone locus bodies) that are distinct from Cajal bodies. Although appearance of CDK2-phosphorylated p220(NPAT) in these domains occurs at the time of S-phase entry, histone locus bodies are formed approximately 1 to 2 h before S phase in embryonic cells but 6 h before S phase in somatic cells. These temporal differences in the formation of histone locus bodies suggest that the G(1) phase of the cell cycle in hES cells is abbreviated in part by contraction of late G(1).