Prachi N. Ghule

Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase

Competency for self‐renewal of human embryonic stem (ES) cells is linked to pluripotency. However, there is a critical paucity of fundamental parameters of human ES cell division. In this study we show that human ES cells (H1 and H9; NIH‐designated WA01 and WA09) rapidly proliferate due to a very short overall cell cycle (15–16 h) compared to somatic cells (e.g., normal diploid IMR90 fibroblasts and NT‐2 teratocarcinoma cells). The human ES cell cycle maintains the four canonical cell cycle stages G1, S, G2, and M, but the duration of G1 is dramatically shortened. Bromodeoxyuridine (BrdU) incorporation and FACS analysis demonstrated that 65% of asynchronously growing human ES cells are in S phase. Immunofluorescence microscopy studies detecting BrdU labeled mitotic chromosomes, Ki67 domains, and p220NPAT containing Cajal bodies revealed that the durations of the S (∼8 h), G2 (∼4 h), and M phases (∼1 h) are similar in ES and somatic cells. We determined that human ES cells remain viable after synchronization with either nocodazole or the anti‐tumor drug Paclitaxel (taxol) and have an abbreviated G1 phase of only 2.5–3 h that is significantly shorter than in somatic cells. Molecular analyses using quantitative RT‐PCR demonstrate that human ES cells and somatic cells express similar cell cycle markers. However, among cyclins and cyclin‐dependent kinases (CDKs), we observed high mRNA levels for the G1‐related CDK4 and cyclin D2 genes. We conclude that human ES cells exhibit unique G1 cell cycle kinetics and use CDK4/cyclin D2 related mechanisms to attain competency for DNA replication. J. Cell. Physiol. 209: 883–893, 2006.

Survival Responses of Human Embryonic Stem Cells to DNA Damage

Pluripotent human embryonic stem (hES) cells require mechanisms to maintain genomic integrity in response to DNA damage that could compromise competency for lineage‐commitment, development, and tissue‐renewal. The mechanisms that protect the genome in rapidly proliferating hES cells are minimally understood. Human ES cells have an abbreviated cell cycle with a very brief G1 period suggesting that mechanisms mediating responsiveness to DNA damage may deviate from those in somatic cells. Here, we investigated how hES cells react to DNA damage induced by ionizing radiation (IR) and whether genomic insult evokes DNA repair pathways and/or cell death. We find that hES cells respond to DNA damage by rapidly inducing Caspase‐3 and ‐8, phospho‐H2AX foci, phosphorylation of p53 on Ser15 and p21 mRNA levels, as well as concomitant cell cycle arrest in G2 based on Ki67 staining and FACS analysis. Unlike normal somatic cells, hES cells and cancer cells robustly express the anti‐apoptotic protein Survivin, consistent with the immortal growth phenotype. SiRNA depletion of Survivin diminishes hES survival post‐irradiation. Thus, our findings provide insight into pathways and processes that are activated in human embryonic stem cells upon DNA insult to support development and tissue regeneration. J. Cell. Physiol. 220: 586–592, 2009.

The abbreviated pluripotent cell cycle.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells proliferate rapidly and divide symmetrically producing equivalent progeny cells. In contrast, lineage committed cells acquire an extended symmetrical cell cycle. Self‐renewal of tissue‐specific stem cells is sustained by asymmetric cell division where one progeny cell remains a progenitor while the partner progeny cell exits the cell cycle and differentiates. There are three principal contexts for considering the operation and regulation of the pluripotent cell cycle: temporal, regulatory, and structural. The primary temporal context that the pluripotent self‐renewal cell cycle of hESCs is a short G1 period without reducing periods of time allocated to S phase, G2, and mitosis. The rules that govern proliferation in hESCs remain to be comprehensively established. However, several lines of evidence suggest a key role for the naïve transcriptome of hESCs, which is competent to stringently regulate the embryonic stem cell (ESC) cell cycle. This supports the requirements of pluripotent cells to self‐propagate while suppressing expression of genes that confer lineage commitment and/or tissue specificity. However, for the first time, we consider unique dimensions to the architectural organization and assembly of regulatory machinery for gene expression in nuclear microenviornments that define parameters of pluripotency. From both fundamental biological and clinical perspectives, understanding control of the abbreviated ESC cycle can provide options to coordinate control of proliferation versus differentiation. Wound healing, tissue engineering, and cell‐based therapy to mitigate developmental aberrations illustrate applications that benefit from knowledge of the biology of the pluripotent cell cycle. J. Cell. Physiol. 228: 9–20, 2013.

Staged assembly of histone gene expression machinery at subnuclear foci in the abbreviated cell cycle of human embryonic stem cells

Human embryonic stem (hES) cells have an abbreviated G1 phase of the cell cycle. How cells expedite G1 events that are required for the initiation of S phase has not been resolved. One key regulatory pathway that controls G1/S-phase transition is the cyclin E/CDK2-dependent activation of the coactivator protein nuclear protein, ataxia–telangiectasia locus/histone nuclear factor-P (p220NPAT/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 G1 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., p220NPAT) 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 p220NPAT in these domains occurs at the time of S-phase entry, histone locus bodies are formed ≈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 G1 phase of the cell cycle in hES cells is abbreviated in part by contraction of late G1.

Cell cycle dependent phosphorylation and subnuclear organization of the histone gene regulator p220NPAT in human embryonic stem cells

Human embryonic stem (ES) cells have an expedited cell cycle (∼15 h) due to an abbreviated G1 phase (∼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 p220NPAT and localization of HiNF‐P/p220NPAT 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 p220NPAT foci in G1 reflecting the assembly of histone gene regulatory complexes in situ. In contrast to somatic cells where duplication of p220NPAT foci is evident in S phase, the increase in the number of p220NPAT foci in ES cells appears to precede the onset of DNA synthesis as measured by BrdU incorporation. Phosphorylation of p220NPAT 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/p220NPAT pathway is rapidly established as ES cells emerge from mitosis and that p220NPAT is subsequently phosphorylated in situ. Our findings establish that the HiNF‐P/p220NPAT 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. J. Cell. Physiol. 213: 9–17, 2007.

Reprogramming the pluripotent cell cycle: restoration of an abbreviated G1 phase in human induced pluripotent stem (iPS) cells.

Induced pluripotent stem (iPS) cells derived from terminally differentiated human fibroblasts are reprogrammed 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. hES cells are characterized by an abbreviated G1 phase (∼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 [5‐bromo‐2′‐deoxy‐uridine (BrdU) incorporation], cell cycle distribution (FACS analysis and Ki67 staining) and subnuclear organization of HLBs [immunofluorescence microscopy and fluorescence in situ hybridization (FISH)] revealed that human iPS cells have a short G1 phase (∼2.5 h) and an abbreviated cell cycle (16–18 h). Furthermore, HLBs are formed and reorganized rapidly after mitosis (within 1.5–2 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. J. Cell. Physiol. 226: 1149–1156, 2011.

Cyclin D2 and the CDK substrate p220NPAT are required for self‐renewal of human embryonic stem cells

Self‐renewal of pluripotent human embryonic stem (hES) cells utilizes an abbreviated cell cycle that bypasses E2F/pRB‐dependent growth control. We investigated whether self‐renewal is alternatively regulated by cyclin/CDK phosphorylation of the p220NPAT/HiNF‐P complex to activate histone gene expression at the G1/S phase transition. We show that cyclin D2 is prominently expressed in pluripotent hES cells, but cyclin D1 eclipses cyclin D2 during differentiation. Depletion of cyclin D2 or p220NPAT causes a cell cycle defect in G1 reflected by diminished phosphorylation of p220NPAT, decreased cell cycle dependent histone H4 expression and reduced S phase progression. Thus, cyclin D2 and p220NPAT are principal cell cycle regulators that determine competency for self‐renewal in pluripotent hES cells. While pRB/E2F checkpoint control is relinquished in human ES cells, fidelity of physiological regulation is secured by cyclin D2 dependent activation of the p220NPAT/HiNF‐P mechanism that may explain perpetual proliferation of hES cells without transformation or tumorigenesis. J. Cell. Physiol. 222: 456–464, 2010.

The subnuclear organization of histone gene regulatory proteins and 3′ end processing factors of normal somatic and embryonic stem cells is compromised in selected human cancer cell types

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 p220NPAT/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 p220NPAT 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 p220NPAT 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. J. Cell. Physiol. 220: 129–135, 2009.

Epigenetic control of cell cycle-dependent histone gene expression is a principal component of the abbreviated pluripotent cell cycle

ABSTRACT Self-renewal of human pluripotent embryonic stem cells proceeds via an abbreviated cell cycle with a shortened G1 phase. We examined which genes are modulated in this abbreviated period and the epigenetic mechanisms that control their expression. Accelerated upregulation of genes encoding histone proteins that support DNA replication is the most prominent gene regulatory program at the G1/S-phase transition in pluripotent cells. Expedited expression of histone genes is mediated by a unique chromatin architecture reflected by major nuclease hypersensitive sites, atypical distribution of epigenetic histone marks, and a region devoid of histone octamers. We observed remarkable differences in chromatin structure—hypersensitivity and histone protein modifications—between human embryonic stem (hES) and normal diploid cells. Cell cycle-dependent transcription factor binding permits dynamic three-dimensional interactions between transcript initiating and processing factors at 5′ and 3′ regions of the gene. Thus, progression through the abbreviated G1 phase involves cell cycle stage-specific chromatin-remodeling events and rapid assembly of subnuclear microenvironments that activate histone gene transcription to promote nucleosomal packaging of newly replicated DNA during stem cell renewal.

The histone gene activator HINFP is a nonredundant cyclin E/CDK2 effector during early embryonic cell cycles

Competency for DNA replication is functionally coupled to the activation of histone gene expression at the onset of S phase to form chromatin. Human histone nuclear factor P (HiNF-P; gene symbol HINFP) bound to its cyclin E/cyclin-dependent kinase 2 (CDK2) responsive coactivator p220NPAT is a key regulator of multiple human histone H4 genes that encode a major subunit of the nucleosome. Induction of the histone H4 transcription factor (HINFP)/p220NPAT coactivation complex occurs in parallel with the CDK-dependent release of pRB from E2F at the restriction point. Here, we show that the downstream CDK-dependent cell cycle effector HINFP is genetically required and, in contrast to the CDK2/cyclin E complex, cannot be compensated. We constructed a mouse Hinfp-null mutation and found that heterozygous Hinfp mice survive, indicating that 1 allele suffices for embryogenesis. Homozygous loss-of-function causes embryonic lethality: No homozygous Hinfp-null mice are obtained at or beyond embryonic day (E) 6.5. In blastocyst cultures, Hinfp-null embryos exhibit a delay in hatching, abnormal growth, and loss of histone H4 gene expression. Our data indicate that the CDK2/cyclin E/p220NPAT/HINFP/histone gene signaling pathway at the G1/S phase transition is an essential, nonredundant cell cycle regulatory mechanism that is established early in embryogenesis.