The molecular basis of endothelial cell (EC)-specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5'-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed in non-ECs. In this study, we tested the hypothesis that epigenetic mechanisms contribute to EC-specific Robo4 gene expression.
Protein-tyrosine phosphatase non-receptor type 23 (PTPN23) is a candidate tumor suppressor involved in the tumorigenesis of various organs. However, its physiological role(s) and detailed expression profile(s) have not yet been elucidated. We investigated the function and regulation of PTPN23 in the formation of testicular germ cell tumors (TGCTs). Expression of PTPN23 in human TGCT cell lines was significantly lower than that in spermatogonial stem cells in mice. Overexpression of PTPN23 in NEC8, a human TGCT cell line, suppressed soft agar colony formation in vitro and tumor formation in nude mice in vivo. These data indicate that PTPN23 functions as a tumor suppressor in TGCTs. Multiple computational algorithms predicted that the 3 UTR of human PTPN23 is a target for miR-142-3p. A luciferase reporter assay confirmed that miR-142-3p bound directly to the 3 UTR of PTPN23. Introduction of pre-miR-142 in the PTPN23 transfectant of NEC8 led to suppressed expression of PTPN23 and increased soft agar colony formation. Quantitative RT-PCR data revealed a significantly higher expression of miR-142-3p in human seminomas compared with normal testes. No difference in mRNA expression between seminoma and non-seminoma samples was detected by in situ hybridization. Both quantitative RT-PCR and immunohistochemical analyses revealed that PTPN23 expression was significantly lower in TGCTs than in normal testicular tissues. Finally, a lack of PTPN23 protein expression in human TGCTs correlated with a relatively higher miR-142-3p expression. These data suggest that PTPN23 is a tumor suppressor and that repression of PTPN23 expression by miR-142-3p plays an important role in the pathogenesis of TGCTs.
We previously demonstrated that hematopoietic stem cell (HSC)-like cells are robustly expanded from mouse embryonic stem (ES) cells by enforced expression of Lhx2, a LIM-homeobox domain (LIM-HD) transcription factor. In this study, we analyzed the functions of Lhx2 in that process using an ES cell line harboring an inducible Lhx2 gene cassette. When ES cells are cultured on OP9 stromal cells, hematopoietic progenitor cells (HPCs) are differentiated and these HPCs are prone to undergo rapid differentiation into mature hematopoietic cells. Lhx2 inhibited differentiation of HPCs into mature hematopoietic cells and this effect would lead to accumulation of HSC-like cells. LIM-HD factors interact with LIM domain binding (Ldb) protein and this interaction abrogates binding of LIM-only (Lmo) protein to Ldb. We found that one of Lmo protein, Lmo2, was unstable due to dissociation of Lmo2 from Ldb1 in the presence of Lhx2. This effect of Lhx2 on the amount of Lmo2 contributed into accumulation of HSC-like cells, since enforced expression of Lmo2 into HSC-like cells inhibited their self-renewal. Expression of Gata3 and Tal1/Scl was increased in HSC-like cells and enforced expression of Lmo2 reduced expression of Gata3 but not Tal1/Scl. Enforced expression of Gata3 into HPCs inhibited mature hematopoietic cell differentiation, whereas Gata3-knockdown abrogated the Lhx2-mediated expansion of HPCs. We propose that multiple transcription factors/co-factors are involved in the Lhx2-mediated expansion of HSC-like cells from ES cells. Lhx2 appears to fine-tune the balance between self-renewal and differentiation of HSC-like cells. Stem Cells 2013.
In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4) is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.
Identification of genes involved in in vitro differentiation induction of embryonic stem cells (ESCs) into hematopoietic stem cells (HSCs) has been challenged during last decade. To date, a homeobox transcription factor Hoxb4 has been only demonstrated to possess such an effect in mice. Here, we show that HSC-like cells were efficiently induced from mouse ESCs by enforced expression of Lhx2, a LIM-homeobox transcription factor. Transduction of Lhx2 into ESC-derived mesodermal cells resulted in robust differentiation of c-Kit(+)/Sca-1(+)/Lineage(-) (KSL) cells in vitro. The KSL cell induction frequency was superior to the case of Hoxb4. Furthermore, transplantation of Lhx2-transduced hematopoietic cells into lethally irradiated mice resulted in multilineage repopulation of hematopoietic cells over 4 months. Transduction of Lhx2 into induced pluripotent stem cells (iPSCs) was also effective in generating KSL cells in vitro, as well as HSC-like activities in vivo. These results demonstrate that ectopic expression of Lhx2 confers an in vivo engrafting capacity to ESC/iPSC-derived hematopoietic cells and in vivo behavior of iPSC-derived hematopoietic cells is almost identical to that of ESC-derived cells.
A variety of signaling networks are implicated in the control of mesoderm differentiation. Previous studies demonstrated that Disabled-2 (DAB2) is a multifunctional protein involved in growth factor signaling and embryonic development. In this study, we investigated DAB2 expression and function during in vitro mesoderm differentiation of murine embryonic stem cells (ESCs). We found that DAB2 was up-regulated when ESCs were co-cultured with OP9 stromal cells for mesoderm differentiation. DAB2 was also up-regulated when ESCs were induced for embryoid body formation. Expression of DAB2 short hairpin small interfering RNA (shDAB2) did not alter the puripotency of ESCs. However, shDAB2 disrupted ESCs cell-cell adhesion and affected embryoid body and colony formation that subsequently impeded mesoderm differentiation of ESCs. Immunofluorescent staining revealed that disorganization of beta-catenin and plakoglobin cellular distribution may account for the aberrant cell-cell adhesion in DAB2-deficient cells. Accordingly, DAB2 was identified as a plakoglobin-binding partner with the interaction mediated by the phosphotyrosine binding domain of DAB2 and the Asn-Pro-Asp-Tyr (NPDY) motif of plakoglobin. Molecular analysis and transcriptome profiling also revealed that DAB2 was involved in the regulation of insulin-like growth factor 2-mediated signaling and in the expression of p53, asparagine synthetase and glutathione peroxidase 2. Expression screening of 52 ESCs-related miRNAs further unveiled the interplay between DAB2 and the signaling networks associated with cell death, differentiation and development. This study thereby defines a role of DAB2 in fate determination of ESCs and suggests the presence of a DAB2-associated regulatory circuit in the control of mesoderm differentiation.
Capillary morphogenesis gene (CMG)-1 is a mammalian homologue of the intraflagellar transport protein IFT-74/72 of Chlamydomonas. CMG-1 is abundantly expressed in immature stages of male germ-line cells of the adult mouse testis and is required for the expression of cyclin-D2 in GC-2, a mouse premeiotic spermatocyte-derived cell line. In this study, we show that the knockdown of CMG-1 in GC-2 cells leads to down-regulation of E-cadherin, integrin-alpha1, alpha2, alpha10, and alpha11 expression. The ability of the CMG-1-knockdown GC-2 cells to adhere to type-I collagen-coated plates was consequently impaired. Inducible expression of an siRNA-resistant CMG-1 cDNA in these cells rescued the expression of E-cadherin and the integrin-alpha family genes and partially restored adherence to type-I collagen. CMG-1 participates in the transcriptional regulation of cyclin-D2 via a genomic DNA region between -250 and -216 of the mouse cyclin-D2 gene. Closely related sequences were found in the enhancer/promotor regions of E-cadherin and the four integrin-alpha family genes. Based on these data, we propose that CMG-1 serves as a transcriptional regulator of proliferation and adhesion-associated genes in early stage male germ-line cells in the testis.
Runx1 is essential for both the establishment of hematopoiesis during development and maintenance of adult hematopoiesis. To reveal the roles of Runx1, we examined how and when Runx1 functions during development of hematopoiesis, and revealed the genes controlled by Runx1.
Megakaryocytic differentiation is accompanied by marked morphological changes induced by endomitosis and proplatelet formation. Molecular mechanisms underlying this unique cell differentiation process have been investigated by gain/loss-of-function studies using leukemic cell lines. However, these cell lines cannot completely mimic physiological megakaryocytic differentiation, including the morphological changes, and sometimes lead to contradictory results between cell lines. The goal of this study was to establish a novel cell differentiation system that completely mimics physiological megakaryocytic differentiation for analyzing gene function. To that end, we used homologous recombination to prepare an embryonic stem (ES) cell line containing a GFP-transgene driven by the PF4 promoter at the Hprt locus. Differentiation of these cells resulted in megakaryocytes and proplatelets, suggesting physiological megakaryocytic differentiation. However, the number of GFP-expressing cells was low (1.7% GFP(+) cells among CD41(+) cells). Insertion of full-length or small core ?-globin insulators on either side of the transgene significantly increased the number of GFP-expressing cells (?60% GFP(+) cells among CD41(+) cells), and GFP-expression was specifically observed in megakaryocytic cells. Similar results were obtained with other ES cells containing a GPIIb-GFP transgene. Altogether, we have succeeded in efficiently expressing exogenous genes specifically in differentiating megakaryocytes and in establishing a novel ES cell differentiation system for analyzing gene function involved in physiological megakaryocytic differentiation.
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