Differential requirement for nucleostemin in embryonic stem cell and neural stem cell viability.

Stem cells have the remarkable ability to self-renew and to generate multiple cell types. Nucleostemin is one of proteins that are enriched in many types of stem cells. Targeted deletion of nucleostemin in the mouse results in developmental arrest at the implantation stage, indicating that nucleostemin is crucial for early embryogenesis. However, the molecular basis of nucleostemin function in early mouse embryos remains largely unknown, and the role of nucleostemin in tissue stem cells has not been examined by gene targeting analyses due to the early embryonic lethality of nucleostemin null animals. To address these questions, we generated inducible nucleostemin null embryonic stem (ES) cells in which both alleles of nucleostemin are disrupted, but nucleostemin cDNA under the control of a tetracycline-responsive transcriptional activator is introduced into the Rosa26 locus. We show that loss of nucleostemin results in reduced cell proliferation and increased apoptosis in both ES cells and ES cell-derived neural stem/progenitor cells. The reduction in cell viability is much more profound in ES cells than in neural stem/progenitor cells, an effect that is mediated at least in part by increased induction and accumulation of p53 and/or activated caspase-3 in ES cells than in neural stem/progenitor cells.

Identification of an ES cell pluripotent state-specific DUSP6 enhancer.

To identify genes with pluripotent state-specific expression in embryonic stem (ES) cells, we compared gene expression profiles between undifferentiated and differentiated mouse ES cells using DNA microarrays. Among the numerous genes identified, we focused on dual specificity phosphatase 6 (DUSP6), which had previously been shown to be expressed in undifferentiated human ES cells. We have identified and characterized a regulatory enhancer that we have termed PEDRE that controls pluripotent state-specific expression of DUSP6. This 82-base pair enhancer overlaps with, but is distinct from, a recently identified regulatory element that is regulated by the FGF-ERK pathway. The sequence of PEDRE is 100% identical between mouse and human DUSP6, suggesting that the molecular basis of DUSP6 gene expression in undifferentiated state of ES cells is highly conserved during evolution.

miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation.

Although various microRNAs regulate cell differentiation and proliferation, no miRNA has been reported so far to play an important role in the regulation of osteoblast differentiation. Here we describe the role of miR-125b in osteoblastic differentiation in mouse mesenchymal stem cells, ST2, by regulating cell proliferation. The expression of miR-125b was time-dependently increased in ST2 cells, and the increase in miR-125b expression was attenuated in osteoblastic-differentiated ST2 cells induced by BMP-4. The transfection of exogenous miR-125b inhibited proliferation of ST2 cells and caused inhibition of osteoblastic differentiation. In contrast, when the endogenous miR-125b was blocked by transfection of its antisense RNA molecule, alkaline phosphatase activity after BMP-4 treatment was elevated. These results strongly suggest that miR-125b is involved in osteoblastic differentiation through the regulation of cell proliferation.

Utilization of digital differential display to identify novel targets of Oct3/4.

Embryonic stem (ES) cells proliferate infinitely while maintaining pluripotency. The POU family transcription factor Oct3/4 is specifically expressed in ES cells and early embryos and plays a critical role in self-renewal of ES cells. However, only a few examples of Oct3/4 target genes have been identified. In this chapter, we describe our strategy to isolate novel Oct3/4 target genes. We first identify genes that are specifically expressed in ES cells by means of digital differential display of expressed sequence tag databases. Reporter gene and gel mobility shift assays are used to confirm the role of Oct3/4. Identification of novel Oct3/4 targets will facilitate our understanding of pluripotency.

Identification and targeted disruption of the mouse gene encoding ESG1 (PH34/ECAT2/DPPA5).

BACKGROUND: Embryonic stem cell-specific gene (ESG) 1, which encodes a KH-domain containing protein, is specifically expressed in early embryos, germ cells, and embryonic stem (ES) cells. Previous studies identified genomic clones containing the mouse ESG1 gene and five pseudogenes. However, their chromosomal localizations or physiological functions have not been determined. RESULTS: A Blast search of mouse genomic databases failed to locate the ESG1 gene. We identified several bacterial artificial clones containing the mouse ESG1 gene and an additional ESG1-like sequence with a similar gene structure from chromosome 9. The ESG1-like sequence contained a multiple critical mutations, indicating that it was a duplicated pseudogene. The 5' flanking region of the ESG1 gene, but not that of the pseudogene, exhibited strong enhancer and promoter activity in undifferentiated ES cells by luciferase reporter assay. To study the physiological functions of the ESG1 gene, we replaced this sequence in ES cells with a beta-geo cassette by homologous recombination. Despite specific expression in early embryos and germ cells, ESG1-/- mice developed normally and were fertile. We also generated ESG1-/- ES cells both by a second independent homologous recombination and directly from blastocysts derived from heterozygous intercrosses. Northern blot and western blot analyses confirmed the absence of ESG1 in these cells. These ES cells demonstrated normal morphology, proliferation, and differentiation. CONCLUSION: The mouse ESG1 gene, together with a duplicated pseudogene, is located on chromosome 9. Despite its specific expression in pluripotent cells and germ cells, ESG1 is dispensable for self-renewal of ES cells and establishment of germcells.

Differential roles for Sox15 and Sox2 in transcriptional control in mouse embryonic stem cells.

Sox family transcription factors play essential roles in cell differentiation, development, and sex determination. Sox2 was previously thought to be the sole Sox protein expressed in mouse embryonic stem (ES) cells. Sox2 associates with Oct3/4 to maintain self-renewal of ES cells. In the current study, digital differential display identified transcripts for an additional Sox family member, Sox15, enriched in mouse ES cells. Reverse transcription-PCR confirmed that Sox15 expression is highest in undifferentiated ES cells and repressed upon differentiation. Sox15 is expressed at low levels in several tissues, including testis and muscle. In vitro studies showed that Sox15, like Sox2, associated with Oct3/4 on DNA sequences containing the octamer motif and Sox-binding site. Gel mobility shift assays and SELEX analyses showed that Sox15 binds similar DNA sequences as Sox2 but with weaker affinity. In contrast to the early embryonic lethality observed in Sox2-null mice, Sox15-null ES cells and mice were grossly normal. DNA microarray analyses revealed that Otx2, Ctgf, Ebaf, and Hrc are dysregulated in Sox15-null ES cells, however. Chromatin immunoprecipitation showed that Sox15, but not Sox2, bound to a Sox consensus binding site within the Hrc gene. Taken together, these data demonstrate differential roles for Sox15 and Sox2 in transcriptional control in mouse ES cells.

Evolutionarily conserved non-AUG translation initiation in NAT1/p97/DAP5 (EIF4G2).

Only a few cases of exclusive translation initiation at non-AUG codons have been reported. We recently demonstrated that mammalian NAT1 mRNA, encoded by EIF4G2, uses GUG as its only translation initiation codon. In this study, we identified NAT1 orthologs from chicken, Xenopus, and zebrafish and found that in all species, the GUG codon also serves as the initiation codon. In all species, the GUG codon fulfilled the reported requirements for non-AUG initiation: an optimal Kozak motif and a downstream hairpin structure. Site-directed mutagenesis showed that nucleotides at positions -3 and +4 are critical for the GUG-mediated translation initiation in vitro. We found that NAT1 orthologs in Drosophila melanogaster and Halocynthia roretzi also use non-AUG start codons, demonstrating evolutionary conservation of the noncanonical translation initiation.

The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.

Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts grow infinitely while maintaining pluripotency. Leukemia inhibitory factor (LIF) can maintain self-renewal of mouse ES cells through activation of Stat3. However, LIF/Stat3 is dispensable for maintenance of ICM and human ES cells, suggesting that the pathway is not fundamental for pluripotency. In search of a critical factor(s) that underlies pluripotency in both ICM and ES cells, we performed in silico differential display and identified several genes specifically expressed in mouse ES cells and preimplantation embryos. We found that one of them, encoding the homeoprotein Nanog, was capable of maintaining ES cell self-renewal independently of LIF/Stat3. nanog-deficient ICM failed to generate epiblast and only produced parietal endoderm-like cells. nanog-deficient ES cells lost pluripotency and differentiated into extraembryonic endoderm lineage. These data demonstrate that Nanog is a critical factor underlying pluripotency in both ICM and ES cells.

Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development.

Embryonic stem (ES) cells are immortal and pluripotent cells derived from early mammalian embryos. Transcription factor Oct3/4 is essential for self-renewal of ES cells and early mouse development. However, only a few Oct3/4 target genes have been identified. In this study, we found that F-box-containing protein Fbx15 was expressed predominantly in mouse undifferentiated ES cells. Inactivation of Oct3/4 in ES cells led to rapid extinction of Fbx15 expression. Reporter gene analyses demonstrated that this ES cell-specific expression required an 18-bp enhancer element located approximately 500 nucleotides upstream from the transcription initiation site. The enhancer contained an octamer-like motif and an adjacent Sox-binding motif. Deletion or point mutation of either motif abolished the enhancer activity. The 18-bp fragment became active in NIH 3T3 cells when Oct3/4 and Sox2 were coexpressed. A gel mobility shift assay demonstrated cooperative binding of Oct3/4 and Sox2 to the enhancer sequence. In mice having a beta-galactosidase gene knocked into the Fbx15 locus, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining was detected in ES cells, early embryos (two-cell to blastocyst stages), and testis tissue. Despite such specific expression of Fbx15, homozygous mutant mice showed no gross developmental defects and were fertile. Fbx15-null ES cells were normal in morphology, proliferation, and differentiation. These data demonstrate that Fbx15 is a novel target of Oct3/4 but is dispensable for ES cell self-renewal, development, and fertility.