Cooperative interaction between ETS1 and GFI1 transcription factors in the repression of Bax gene expression.

The proto-oncoproteins ETS1 and growth factor independent-1 (GFI1) are implicated in cell growth and differentiation in various types of cells, and their deregulated expression is involved in malignant transformation. Here, we report that ETS1 and GFI1 interact and affect gene expression through their cross-talk. Co-immunoprecipitation analyses and glutathione-S-transferase pull-down assays revealed that ETS1 bound directly to GFI1 via its Ets domain, and GFI1 bound to ETS1 via its zinc-finger domain. Luciferase (Luc) assays using artificial reporters showed that GFI1 repressed ETS1-mediated transcriptional activation and ETS1 repressed GFI1-mediated transcriptional activation, in a dose-dependent manner. However, in the Bax promoter where the Ets- and Gfi-binding sites (EBS and GBS) are adjacent, ETS1 and GFI1 cooperatively reduced activation. Site-directed mutagenesis on the EBS and GBS of the Bax promoter showed that both binding sites were necessary for full repression. Chromatin immunoprecipitation analyses confirmed that an ETS1-GFI1 complex formed on the Bax promoter even when either EBS or GBS was mutated. Introduction of small interfering RNA against ETS1 and/or GFI1 enhanced endogenous Bax gene expression. Our results suggest that the interaction between ETS1 and GFI1 facilitates their binding to specific sites on the Bax promoter and represses Bax expression in vivo.

Site-specific DNA methylation by a complex of PU.1 and Dnmt3a/b.

The Ets transcription factor PU.1 is a hematopoietic master regulator essential for the development of myeloid and B-cell lineages. As we previously reported, PU.1 sometimes represses transcription on forming a complex with mSin3A-histone deacetyl transferase-MeCP2. Here, we show an interaction between PU.1 and DNA methyltransferases, DNA methyltransferase (Dnmt)3a and Dnmt3b (Dnmt3s). Glutathione-S-transferase pulldown assay revealed that PU.1 directly interacted with the ATRX domain of Dnmt3s through the ETS domain. Dnmt3s repressed the transcriptional activity of PU.1 on a reporter construct with trimerized PU.1-binding sites. The repression was recovered by addition of 5-aza-deoxycitidine, a DNA methyltransferase inhibitor, but not trichostatin A, a histone deacetylase inhibitor. Bisulfite sequence analysis revealed that several CpG sites in the promoter region neighboring the PU.1-binding sites were methylated when Dnmt3s were coexpressed with PU.1. We also showed that the CpG sites in the p16(INK4A) promoter were methylated by overexpression of PU.1 in NIH3T3 cells, accompanied by a downregulation of p16(INK4A) gene expression. These results suggest that PU.1 may downregulate its target genes through an epigenetic modification such as DNA methylation.

In vivo complex formation of PU.1 with HDAC1 associated with PU.1-mediated transcriptional repression.

We previously reported that overexpression of PU.1, a member of the Ets family of transcription factors, induces differentiation inhibition and apoptosis associated with c-Myc down-regulation in murine erythroleukemia (MEL) cells. To understand the molecular mechanism by which c-Myc is down-regulated due to overexpression of PU.1, we performed luciferase reporter assays using the mouse c-myc promoter. PU.1 repressed the activities of not only the c-myc promoter but also several other promoters. Experiments with deletion mutants of PU.1 revealed that the C-terminal region spanning amino acids (aa) 123-272 including the PEST and ETS domains but not the activation domain was sufficient for this transcriptional repression. It was unlikely that the repression was due to sequestration of a limited amount of CBP/p300 nor pCAF, because overexpression of these co-activators did not relieve PU.1-mediated transcriptional repression. Instead, it was found that the C-terminal aa 101-272 of PU.1 formed a complex with mSin3A and HDAC1 in vivo, which was speculated to be associated with the repression. The C-terminal region of PU.1 also formed a complex with the basic transcription factor TBP in vitro and in vivo. Our results suggest that overexpression of PU.1 induces transcriptional repression in several gene promoters including the c-myc promoter which may be mediated by its complex formation with HDACs.

Lineage switch induced by overexpression of Ets family transcription factor PU.1 in murine erythroleukemia cells.

PU.1 is an Ets family transcription factor essential for myelomonocyte and B-cell development. We previously showed that overexpression of PU.1 in murine erythroleukemia (MEL) cells inhibits growth and erythroid differentiation and induces apoptosis of the cells. In an effort to identify target genes of PU.1 concerning these phenomena by using a messenger RNA differential display strategy, we found that some myeloid-specific and lymphoid-specific genes, such as the osteopontin gene, are transcriptionally up-regulated in MEL cells after overexpression of PU.1. We then found that expression of several myelomonocyte-specific genes, including the CAAT-enhancer-binding protein-alpha and granulocyte-macrophage colony-stimulating factor receptor genes, was induced in MEL cells after overexpression of PU.1. B-cell-specific genes were also examined, and expression of the CD19 gene was found to be induced. Expression of the myelomonocyte-specific proteins CD11b and F4/80 antigen but not the B-cell-specific proteins B220 and CD19 was also induced. After overexpression of PU.1, MEL cells became adherent and phagocytic and showed enhanced nitroblue tetrazolium reduction activity. Expression of myelomonocyte-specific and B-cell-specific genes was not induced when a mutant PU.1 with part of the activation domain deleted (a change found to inhibit erythroid differentiation of MEL cells) was expressed. These results indicate that PU.1 induces a lineage switch in MEL cells toward myelomonocytic cells and that its activation domain is essential for this effect. The results also suggest that the pathway of the lineage switch is distinct from that of inhibition of erythroid differentiation in MEL cells.

The role of Ets family transcription factor PU.1 in hematopoietic cell differentiation, proliferation and apoptosis.

The PU.1 gene encodes an Ets family transcription factor which controls expression of many B cell- and macrophage-specific genes. Expression of the gene is critical for development of lymphoid and myeloid cell lineages, since PU.1-deficient mice exhibit defects in the development of these cell lineages. The PU.1 gene is identical to the Spi-1 gene isolated from common proviral integration sites in Friend virus-induced murine erythroleukemia (MEL), and deregulated expression of the gene is believed to be an essential step of the disease. We recently demonstrated that overexpression of PU.1 inhibits erythroid differentiation of MEL cells induced with the differentiating agent DMSO. We also noticed unexpectedly that overexpression of PU.1 together with DMSO induces marked growth arrest and apoptosis in MEL cells, supporting the notion that some oncogenes induce growth inhibition and apoptosis rather than cell proliferation and transformation under specific circumstances as shown with the c-myc gene. In this review, the role of PU.1 in hematopoietic cell differentiation, proliferation and apoptosis is described and the possible molecular mechanisms of PU.1-induced effects in MEL cells are discussed.

Physical and functional interactions between the transcription factor PU.1 and the coactivator CBP.

Yeast two-hybrid system was employed to isolate novel proteins that physically interact with PU.1, a member of Ets family transcription factors. Sequence analyses of several isolated clones positive for beta-galactosidase activity revealed that one of these clones was confirmed to encode a transcriptional coactivator, CREB binding protein (CBP). GST binding assay showed that the interacting sites were located at the transcriptional activation domain of PU.1 through 74-122 and the region spanning residues 1283-1915 of CBP. CBP potentiated PU.1-mediated transcription of the reporter gene driven by the multimerized PU.1-binding sites, suggesting that CBP functions as a coactivator for PU.1. Considering that CBP is a limited cellular component to function as a coactivator for several transcription factors, CBP may mediate synergistic and antagonistic interactions between PU.1 and other transcription factors during the process of hematopoietic cell differentiation.

Reduction of DNA binding activity of the GATA-1 transcription factor in the apoptotic process induced by overexpression of PU.1 in murine erythroleukemia cells.

Previously we have shown that overexpression of PU.1, an Ets family transcription factor, in murine erythroleukemia (MEL) cells results in apoptotic cell death in the presence of the differentiation-inducing reagent dimethyl sulfoxide (DMSO). In this study, we examined the dynamics of GATA-1 and NF-E2 hematopoietic transcription factors during the induction of apoptosis, because GATA-1 has been shown to be implicated in survival of erythroid cells. Formation of the GATA-1-DNA complex as judged by EMSA was markedly reduced when apoptosis was induced, although subcellular localization of the GATA-1 protein and expression levels of the GATA-1 mRNA and protein were not changed during the apoptotic process. Complex formation was not reduced when apoptosis was avoided by adding 30% serum in culture medium and when mutant PU.1 proteins with the deletion of the DNA-binding (Ets) or transactivation domain were expressed. Complex formation in nuclear extracts of parental MEL cells was reduced when they were mixed with those of apoptotic cells, suggesting that apoptotic cells may contain a factor(s) preventing GATA-1 from binding to DNA. In contrast to GATA-1, formation of the NF-E2-DNA complex was not changed during the process of apoptosis, although the expression level of the NF-E2 p45 gene was reduced in the process. These results suggest that reduction of the DNA-binding activity of GATA-1 may partly account for PU.1-mediated apoptosis in MEL cells.

Enhanced expression of the urokinase-type plasminogen activator gene and reduced colony formation in soft agar by ectopic expression of PU.1 in HT1080 human fibrosarcoma cells.

To investigate the cell biological function of PU.1, a member of the Ets family of transcription factors, a vector capable of expressing the protein was transfected into HT1080 human fibrosarcoma cells. Exogenous expression of PU.1 in HT1080 cells reduced colony-forming efficiency but stimulated cell migration in soft agar, although it did not affect cell growth in adherent culture. Expression of the urokinase-type plasminogen activator (uPA) mRNA, which is known to be correlated with cell migration and invasion, was enhanced in PU.1 transfectants compared with mock transfectants. Run-on analysis demonstrated that uPA transcription was unaffected by PU.1, suggesting that this enhancement mainly occurs at a post-transcriptional level. On the other hand, treatment of HT1080 cells with the synthetic glucocorticoid dexamethasone (DEX; 10(-7) M) significantly reduced uPA gene expression at a transcriptional level. Furthermore, DEX inhibited cell migration in soft agar without affecting cell growth. These negative effects of DEX on uPA expression and cell migration were alleviated by the expression of PU.1 in HT1080 cells, whereas expression of the N-ras oncogene, which is responsible for maintenance of the transformed phenotypes in HT1080 cells, was unaffected by PU.1 expression or DEX treatment in the cells. Our results suggest that expression of PU.1 can stimulate uPA gene expression at the post-transcriptional level, which may subsequently lead to activation of cell motility and/or reduced cell-cell adhesion, but reduces anchorage-independent growth of HT1080 cells.

Down-regulation of c-myc and bcl-2 gene expression in PU.1-induced apoptosis in murine erythroleukemia cells.

We found that over-expression of PU.1, a member of the ets family of transcription factors, induces apoptotic cell death along with differentiation of DMSO stimulation in murine erythroleukemia (MEL) cells. To elucidate the molecular mechanisms of apoptosis, cell-cycle distribution and expression of several genes encoding apoptosis-promoting and -inhibiting factors were analyzed during the process of PU.1-induced apoptosis. FACS analysis revealed that cells were accumulated in the G0/G1 phase of the cell cycle before apoptosis. Morphological analysis of PI-stained nuclei of the apoptotic cells sorted by a FACScan showed 22.6% in G0/G1, 35.8% in S and 8.5% in G2/M phase by fluorescent microscopy after cell sorting, suggesting that PU.1-induced apoptosis in MEL cells occurs in G0/G1 through S phases. Semi-quantitative RT-PCR revealed that expression of c-myc and bcl-2 genes was reduced during the apoptotic process, while expression of bax and bcl-X(L) genes was not changed. Expression of the p53 gene was reduced rather than enhanced, suggesting that PU.1-induced apoptosis in MEL cells is p53-independent. Apoptosis was inhibited by adding 30% serum in culture, while no reduction of c-myc and bcl-2 gene expression was observed. Forced expression of the c-myc, bcl-2 and bcl-X(L) genes protected MEL cells from apoptosis. Our results suggest that a reduction of at least 2 important apoptosis-inhibiting factors, c-Myc and Bcl-2, is involved in PU.1-induced apoptosis in MEL cells.

Localization, expression, and the role in fertilization of spermosin, an ascidian sperm trypsin-like protease.

In order to elucidate the role of spermosin (a novel sperm trypsin-like protease) in ascidian fertilization, we developed an antibody against the ascidian spermosin: the antibody appears specific to the spermosin but not to the acrosin. By Western blot analysis, spermosin was found to be expressed just before and during the spawning season. Immunocytochemical studies have shown that spermosin is localized on the sperm head. It was found that the spermosin was released from the sperm during the sperm reaction, and the anti-spermosin antibody, but not control antibody, inhibited the fertilization in a concentration-dependent manner. These results indicate that spermosin plays an essential role in ascidian fertilization.

Molecular cloning of the complementary DNA for the mouse pyruvate kinase M-2 gene whose expression is dependent upon cell differentiation.

A cDNA encoding M2-type pyruvate kinase from mouse was cloned and its nucleotide sequence was determined. The cDNA encoded a protein containing 531 amino acids, the nucleotide and amino acid sequences of which were 93.2% and 98.1%, respectively, homologous to those of rat M2 pyruvate kinase. Expression of the pyruvate kinase in mouse embryonal carcinoma P19 cells altered according to differentiation stages; high at undifferentiated and low at differentiated stages.

BOX DNA: a novel regulatory element related to embryonal carcinoma cell differentiation.

BOX DNA was previously isolated from the DNA sequence inserted in the enhancer B domain of mutant polyomavirus (fPyF9) DNA. We also reported that BOX DNA functioned negatively on DNA replication and transcription of another polyomavirus mutant (PyhrN2) in F9-28 cells, a subclone of mouse F9 embryonal carcinoma (EC) cells expressing the polyomavirus large T antigen. In this study, we demonstrate that BOX DNA enhances transcription from the thymidine kinase (TK) promoter in various EC cells. One or three copies of BOX DNA, linked to the bacterial chloramphenicol acetyltransferase gene under the control of the herpes simplex virus TK promoter, activated promoter activity in F9, P19, and ECA2 cells. Band shift assays using BOX DNA as a probe revealed that specific binding proteins were present in all EC cells examined; the patterns of BOX DNA-protein complexes were the same among them. A mutation introduced within BOX DNA abolished enhancer activity as well as the formation of specific DNA-protein complexes. In non-EC cells, including L and BALB/3T3 cells, the enhancer activity of BOX DNA on the TK promoter was not observed, although binding proteins specific to the sequence exist. In band shift assays, the patterns of the DNA-protein complexes of either L or BALB/3T3 cells were different from those of EC cells. Furthermore, the enhancer activity of BOX DNA decreased upon differentiation induction in all EC cells examined, of different origins and distinct differentiation ability. In parallel with the loss of enhancer activity, the binding proteins specific for BOX DNA decreased in these cells. Moreover, we cloned a genomic DNA of F9, termed BOXF1, containing BOX DNA sequence approximately 400 bp upstream from the RNA start site of the gene. BOXF1, containing a TATA-like motif and the binding elements for Sp1 and Oct in addition to BOX DNA, possessed promoter activity deduced by a BOXF1-chloramphenicol acetyltransferase construct. Deletion analyses of the construct revealed that the transcription of BOXF1 gene is regulated by BOX DNA, preferentially in undifferentiated EC cells versus differentiated cells. Hence, BOX DNA is probably a novel transcriptional element related to EC cell differentiation.