JOURNAL OF VIROLOGY, May 1992, p. 3056-3061 Vol. 66, No. 5 0022-538X/92/053056-06$02.00/0 Copyright ©) 1992, American Society for Microbiology Transactivation of Erythroid Transcription Factor GATA-1 by a myb-ets-Containing Retrovirus ROSEMARIE E. AURIGEMMA, DONALD G. BLAIR, AND SANDRA K. RUSCETFI* Laboratory of Molecular Oncology, National Cancer Institute, Frederick, Matyland 21702-1201 Received 26 December 1991/Accepted 13 February 1992 ME26 virus is a recombinant mouse retrovirus construct homologous to the avian E26 virus. Both encode a 135-kDa gag-myb-ets fusion protein which is localized in the nucleus. We have recently shown that ME26 virus can induce erythropoietin (Epo) responsiveness in hematopoietic cells. Mice infected with ME26 virus develop a hyperplasia of Epo-dependent hematopoietic precursor cells from which permanent cell lines can be established. In vitro, ME26 virus specifically induces Epo responsiveness in the interleukin-3-dependent myeloid cell line FDC-P2 by enhancing expression of the Epo receptor (EpoR). In the present study we demonstrate that ME26 virus infection of FDC-P2 cells also results in enhanced expression of 13-globin and the erythroid-specific transcription factor GATA-1, a protein which can transactivate both the EpoR promoter and globin genes. In addition, these cells exhibit a down-regulation of c-myb expression similar to that seen in differentiating erythroid cells. To determine the molecular basis for activation of erythroid genes in ME26 virus-infected cells, we carried out transient expression assays with DNA constructs of either the EpoR promoter or the GATA-1 promoter linked to reporter genes. Our results indicate that while ME26 virus did not directly enhance expression from the EpoR promoter, both it and its avian parent, E26, transactivated the GATA-1 promoter. Furthermore, ME26 virus cooperates with the GATA-1 protein to enhance expression of the EpoR gene. We propose that the mechanism by which ME26 virus induces erythroleukemia involves transactivation of the GATA-1 gene, thus positively regulating the expression of the EpoR and leading to the proliferation of a unique population of Epo-responsive cells. By specifically inducing Epo responsiveness in hematopoietic cells via transactivation of a transcription factor, ME26 virus utilizes a novel mechanism for retrovirus pathogenesis. ME26 virus (46), the mouse equivalent of the avian E26 other gene involved in EpoR regulation. A good candidate virus (26, 33), encodes a 135-kDa gag-myb-ets fusion protein for such a gene is GA TA-1, a major regulator of genes which is localized in the nucleus of infected cells. Injection expressed in erythroid cells (9, 31). GATA-1 is a 413-amino- of an amphotrophic murine leukemia virus pseudotype of acid DNA-binding protein that recognizes the consensus ME26 virus into newborn NFS/N mice causes a high inci- sequence (A/T)GATA(A/G) (11, 41). It was first shown to dence of leukemia within 2 to 4 months (46). Spleen cells bind to regulatory sequences in globin genes (9, 13), but from the majority of these mice proliferate to high levels in GATA-1 binding motifs are also present in the regulatory the presence of the erythroid hormone erythropoietin (Epo) regions of other erythroid-specific genes (4, 14, 32), includ- and can easily and reproducibly be established as permanent ing the EpoR gene (45). Previous studies with transient Epo-dependent cell lines (38). Unlike erythroleukemia cell expression systems have shown that GATA-1 can transacti- lines derived from mice infected with Friend murine leuke- vate its own promoter (16, 42) as well the promoters of ox- mia virus or spleen focus-forming virus, the ME26 virus- and I-globin (9, 12, 19, 20) and the EpoR gene (5, 47). induced Epo-dependent cell lines appear more immature and Since binding motifs for the myb and ets oncogenes are more closely resemble hematopoietic precursor cells, sug- present in the regulatory regions of both the EpoR and gesting that the virus activates the Epo receptor (EpoR) in GATA-1 genes, the purpose of this study was to determine hematopoietic cells that normally do not express it. Consis- whether either of the latter genes could be transactivated by tent with this idea, we have been able to convert an the ME26 virus. Our results indicate that ME26 virus can interleukin-3-dependent myeloid cell line, FDC-P2, to Epo transactivate the GA TA-1 gene and cooperates with the responsiveness after infection with ME26 virus (38). GATA-1 protein to enhance the expression of the EpoR The mechanism by which ME26 virus activates the EpoR gene. is not known. The EpoR gene from the virus-infected cell lines is not rearranged (38), indicating that the virus is inducing Epo responsiveness by a mechanism other than MATERUILS AND METHODS retrovirus promoter/enhancer insertion. Since the ME26 viral protein, p135, is localized in the nucleus (46) and Cell lines. The interleukin-3-dependent myeloid cell line contains fused portions of the DNA-binding, trans-acting FDC-P2 (10) was grown in RPMI 1640 medium supple- mented with 15% fetal calf serum and 5% WEHI-3-condi- factors myb and ets-1 (29, 39, 40, 43), it is possible that p135 tioned medium as a source of interleukin-3. FDC-P2 cells may be transactivating the EpoR gene by binding to its infected with an amphotropic pseudotype of ME26 virus (38) promoter. Alternatively, the ME26 virus may indirectly were grown in RPMI 1640 medium supplemented with 15% activate the EpoR gene by transcriptionally activating an- fetal calf serum and 0.3 U of Epo per ml. NIH 3T3 cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum. * Corresponding author. RNA preparation and analysis. Total RNA was purified 3056 VOL. 66, 1992 TRANSACTIVATION OF GATA-1 3057 from cells by using RNAzol (6). RNAs were denatured with various intervals for a period of 12 h. All lysates within a 50% formamide-2.2 M formaldehyde, separated electro- given experiment were found to contain equally high levels phoretically on 1.2% agarose gels containing 2.2 M formal- of CAT activity. dehyde, and transferred to nitrocellulose filters by following Experiments designed to test the cooperative effects of standard protocols (1). mRNA representing the EpoR gene ME26 and GATA-1 on expression of the EpoR promoter was identified by hybridization with a probe prepared from a were also performed as described above, except that sam- 1.9-kb KpnI fragment of the mouse EpoR cDNA from ples were tested in quadruplicate in two separate experi- plasmid pXM(ER) (8). GATA-1 mRNA was identified by ments. The DNA concentration was maintained at 20 ,ug per hybridization with a probe prepared from an 870-bp fragment plate except for plates receiving pO.45GH, pGF-1, and prepared by StuI-PstI digestion of the mouse GATA-1 pME26, to which 30 ,ug of DNA was added. Also, plates cDNA from plasmid pGF-1 (41). Hybridizations were also were not transfected with pRSVCAT. carried out with a v-ets probe (E1.28) (44) to detect expres- Transient expression analysis of the GATA-1 promoter. sion of the ME26 viral transcripts, and a probe for ,B-actin Plasmid pGATP1, which contains the chicken GATA-1 was utilized to determine comparative levels of RNA per promoter placed in the vector pCAT-Basic (16), was the lane. The probe used to detect c-myb mRNA was a 2-kb transient expression vector used. Plasmids pRSV20-2 (16) BamHI fragment of the mouse c-myb cDNA clone (3). and pGF-1 (41) express the chicken and the mouse GATA-1 P-Globin mRNA sequences were detected by hybridization protein, respectively. pME26 (46) and pE26 (29) express the with a 600-bp HincII-Hindlll fragment of the mouse 1majr mouse homolog of E26 virus and the parent E26 virus, globin cDNA. All probes were prepared by using a random respectively. NIH 3T3 cells were plated and transfected as priming kit (GIBCO-BRL) with [32P]dCTP (Amersham). described above with 20 ,ug of DNA per plate. In addition, 7 Hybridizations were performed at 42°C for 24 h in 50% jig of plasmid pXGH5 per plate was cotransfected as a formamide-5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M means of assaying transfection efficiency. Cell lysates were sodium citrate). Filters were washed twice at room temper- collected and assayed as described above. Culture mediums ature with 2x SSC-0.1% sodium dodecyl sulfate (SDS) and were harvested at the same time as cell lysates, and hGH then washed twice at 420C with 0.2x SSC-0.1% SDS. Filters concentrations were determined. were prepared for rehybridization by immersion in boiling distilled water to remove probes. RESULTS Transient expression analysis of the EpoR promoter. NIH 3T3 cells were seeded at 4 x 105 cells per 60-mm dish 24 h Effects of ME26 virus on expression of the EpoR gene. As prior to transfection. Twenty micrograms of test DNA per previously shown (38), FDC-P2 cells infected with ME26 plate was transfected by using the Ca2PO4 precipitation virus show a greatly enhanced amount of EpoR-specific method. Each plasmid utilized was present at a concentra- RNA compared with that in their uninfected counterpart tion of 10 ,ug per plate. If only one plasmid was being tested, (Fig. 1A). In contrast, NIH 3T3 cells either uninfected or 10 ,ug of calf thymus DNA was added prior to precipitation. infected with ME26 virus do not express transcripts for the The reporter plasmid pO.45GH, containing the EpoR pro- EpoR. Both virus-infected cell lines expressed high levels of moter placed 5' to the human growth hormone gene (hGH) ME26 viral RNA (Fig.