Transactivation of Erythroid Transcription Factor GATA-1 by a Myb-Ets-Containing Retrovirus ROSEMARIE E

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 supplemented 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. 1C). To determine whether ME26 (45) was transfected into NIH 3T3 cells alone, with ME26 viral DNA could directly transactivate the EpoR promoter, DNA, or with a mouse GATA-1 expression vector (pGF-1) reporter plasmid pO.45GH, containing the EpoR promoter (41). Culture supernatant was assayed for hGH produced 72 linked to the hGH gene (45), was utilized. This promoter h posttransfection by using a radioimmunoassay (Nichols construct, which is normally inactive in NIH 3T3 cells (45), Institute Diagnostics, San Juan Capistrano, Calif.). hGH was cotransfected with either ME26 viral DNA or GATA-1 concentrations were obtained from a standard curve gener- DNA, which has recently been shown to transcriptionally ated with hGH solutions provided by the manufacturer. activate the EpoR gene (5, 47). As shown in Table 1, Control plasmids, also provided by the manufacturer, were cotransfection of pO.45GH with ME26 viral DNA does not pOGH, an hGH expression vector which lacks a eukaryotic result in production of hGH over that seen in cells trans- promoter, and pXGH5, a metallothionein promoter-driven fected with pO.45GH alone. In contrast, cells cotransfected hGH construct. Relative hGH concentrations were deter- with pO.45GH and GATA-1 DNA produce 20 times more mined by using duplicate samples in three separate experi- hGH than cells transfected with only pO.45GH. These results ments, with the exception of the GATA-1 cotransfection, indicate that ME26 virus is not exerting its effect directly which was only present in one of the three experiments. upon the EpoR gene. All plates also received 7 ,ug of pRSVCAT as a means for Effects of ME26 virus on expression of the GATA-1 gene. determining transfection efficiency. Cell lysates were ob- Since GATA-1 has been shown to transactivate the EpoR tained at the time of culture medium harvest and were gene, we carried out studies to determine if ME26 virus assayed for chloramphenicol acetyl transferase (CAT) activ- could be indirectly activating the EpoR gene by transacti- ity by a modification of the method of Neumann et al. (27). vating the GA TA-I gene. To evaluate whether GATA-1 Briefly, cells were rinsed with cold phosphate-buffered sa- levels were affected by ME26 virus infection, Northern line, scraped into test tubes, and centrifuged. Cell pellets (RNA) blots were prepared from RNA isolated from ME26 were resuspended in 250 mM Tris (pH 7.8) and lysed by virus-infected and uninfected FDC-P2 and NIH 3T3 cells three cycles of freeze-thaw in liquid nitrogen. Lysates were and hybridized with a GATA-1-specific probe. GATA-1 has collected after centrifugation, and 25-,ul samples were mixed previously been shown to be expressed only in erythroid in scintillation vials with 100 ,ul of a reaction mixture cells, megakaryocytes, and bone marrow-derived mast cells containing 12.5 mM Tris (pH 7.8), 2.5 mM chloramphenicol, (21, 37), all of which are believed to be descended from a and 2.5 ,uCi of [3H]acetyl coenzyme A (200 mCi/mmol; New common progenitor cell (30). As shown in Fig. 1B, FDC-P2 England Nuclear). After thorough mixing, reactions were cells infected with the ME26 virus express GATA-1 at a overlaid with 3 ml of Econoflour (New England Nuclear), much higher level than uninfected cells. Epo-dependent cell and vials were counted in a liquid scintillation counter at lines derived from ME26 virus-infected mice also express 3058 AURIGEMMA ET AL. J. VIROL.

A B C D 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 .. .:. *.sh"

4.9m w ~~~~~~28S

W.. W f .~ ~~~0im-:_ 2.0 k " ... , w

FIG. 1. Northern analysis of expression of the EpoR and GATA-1 genes in uninfected and ME26 virus-infected cells. Total RNA was extracted from uninfected FDC-P2 cells (lanes 1), ME26 virus-infected FDC-P2 cells (lanes 2), NIH 3T3 cells (lanes 3), or ME26 virus-infected NIH 3T3 cells (lanes 4). A single Northern blot was prepared by using 10 p.g of RNA per lane. The filter was hybridized with the following probes: EpoR cDNA (A), GATA-1 cDNA (B), v-ets (C), and ,-actin (D). The EpoR transcript appears as a band of approximately 2.0 kb, the GATA-1 transcript is approximately 1.8 kb, and the ME26 transcript is approximately 4.9 kb. The positions of the 18S and 28S rRNAs are shown.

high levels of GATA-1 mRNA (data not shown). In contrast, effect on the EpoR promoter in the presence of GATA-1, we uninfected or ME26 virus-infected NIH 3T3 cells do not cotransfected the EpoR promoter-hGH construct (pO.45GH) express GATA-1 mRNA. Thus, activation of the EpoR gene into NIH 3T3 cells with both ME26 and GATA-1 expression by ME26 virus is positively correlated with a high level of vectors. As shown in Table 3, these cells produced almost expression of the GATA-1 gene. twice as much hGH as cells transfected with only pO.45GH To establish whether ME26 virus can transactivate the and GATA-1 DNA. As shown before, cells transfected with GATA-1 gene, we utilized the reporter construct pGATP1, pO.45GH and ME26 DNA produced no more hGH than cells which contains the chicken GATA-1 promoter linked to the transfected with pO.45GH alone, indicating that the effect of CAT gene (16). pGATP1 was cotransfected into NIH 3T3 ME26 virus on the EpoR promoter occurred only in the cells with pME26 DNA, pE26 DNA, mouse GATA-1 (pGF- presence of GATA-1 protein. NIH 3T3 cells persistently 1), or chicken GATA-1 (pRSV20-2). As shown in Table 2, infected with ME26 virus also expressed twofold the amount the relative CAT activity of cells cotransfected either with of hGH as uninfected cells when cotransfected with pGATP1 and pME26 or with pE26 virus is 28.7- and 12.9- pO.45GH and GATA-1 DNA (data not shown). fold higher, respectively, than that of cells transfected with Effects of ME26 virus on expression of other genes involved the promoterless CAT constructs pSVOCAT or pCATBasic. in erythropoiesis. Because ME26 virus infection caused a A comparison of relative CAT activities indicates that the dramatic change in GATA-1 and EpoR gene expression in ME26 virus is as potent a transactivator of the GATA-1 FDC-P2 cells, studies were carried out to determine whether promoter as the mouse GATA-1 protein. Identical results other genes involved in erythropoiesis were affected as well. were obtained when transient expression assays were car- As shown in Fig. 2B, FDC-P2 cells are induced by ME26 ried out with the mouse GATA-1 promoter linked to the virus infection to express large amounts of 3-globin mRNA hGH gene (42) (data not shown). (lane 2), whose expression has been shown to be regulated Cooperative effects of ME26 virus and GATA-1 on expres- by the GATA-1 protein (9, 13, 20, 32). Similar high levels of sion of the EpoR gene. Our earlier data indicated that ME26 ,-globin mRNA were also detected in Epo-dependent cell virus could not transactivate the EpoR promoter in NIH 3T3 lines derived from ME26 virus-infected mice (data not cells. However, unlike hematopoietic cells infected with shown). No globin mRNA could be detected in uninfected ME26 virus, virus-infected NIH 3T3 cells do not express FDC-P2 cells (lane 1). As previously shown in Fig. 1B, GATA-1 mRNA. To determine whether ME26 virus has an ME26 virus-infected FDC-P2 cells express a greatly en-

TABLE 1. Transactivation studies of the EpoR promoter in a TABLE 2. Transactivation studies of the GATA-1 promoter in a transient expression assay transient expression assay Relative level of Promoter tested DNA cotransfected Relative CAT activity' Promoter tested DNA cotransfected hGH production' pSVOCAT or pCATBasic 1 pGATP1 3.7 pOGH 1 pGATP1 pGF-1 27.5 pO.45GH 1.7 pGATP1 pRSV20-2 15.3 pO.45GH pGF-1 20 pGATP1 pME26 28.7 pO.45GH pME26 1.7 pGATP1 pE26 12.9 aValues are expressed as levels of hGH produced relative to that of the a Values are expressed as levels of CAT activity relative to those of the promoterless hGH construct, pOGH. Data were obtained from duplicate promoterless CAT constructs pSVOCAT or pCATBasic. Data were obtained samples in three separate experiments. from duplicate or triplicate samples in three separate experiments. VOL. 66, 1992 TRANSACTIVATION OF GATA-1 3059

TABLE 3. Cooperative transactivation of the EpoR promoter by transactivation of the EpoR gene. Although ME26 virus, in GATA-1 and ME26 the absence of GATA-1 expression, cannot directly transac- level tivate the EpoR gene, it can enhance the ability of the PromoterPromoter DNA cotransfected ~~~~~~~~~~~~Relativeof hGH GATA-1 protein to transactivate the EpoR. It is not known productiona whether this enhancement is due to the ME26 viral gene pOGH 1 product binding to the EpoR promoter or to some interaction pO.45GH 1 between the ME26 and GATA-1 proteins which results in pO.45GH pME26 1 more efficient binding to or transactivation of the EpoR pO.45GH pGF-1 8 promoter. We are currently carrying out studies to deter- pO.45GH pME26 + pGF-1 20 mine where the viral protein binds to the GATA-1 promoter and whether it can also bind to the EpoR promoter. a Values are expressed as levels of hGH produced relative to that by the promoterless hGH construct pOGH. Data were obtained from quadruplicate The role of particular segments of the ME26 virus-en- samples in two separate experiments. coded gag-myb-ets fusion protein in transactivation of the GA TA-I gene and in cooperating with the GATA-1 protein to transactivate the EpoR gene is not known. Recently, it has hanced level of GATA-1 mRNA compared with that of their been demonstrated that human c-myb protein can interact uninfected counterpart (Fig. 2A). synergistically with the Epstein-Barr virus transcription fac- Virus-infected FDC-P2 cells were also examined for tor BZLF1 to transactivate both the Epstein-Barr virus early c-myb expression, which decreases as erythroid cells begin promoter, BMRF1, and the simian virus 40 early promoter, expressing globin during the process of differentiation (34, although c-myb, by itself, has no effect on these promoters 36). As shown in Fig. 2C, infection of FDC-P2 cells with (17). Therefore, it is possible that the p135 fusion protein of ME26 virus results in a similar down-modulation of c-myb. ME26 virus interacts with transcription factors such as Epo-dependent cell lines from ME26 virus-infected mice GATA-1 via its myb sequences. However, both the myb and also express extremely low levels of c-myb transcripts (data ets segments of the avian E26 virus are required for leuke- not shown). mogenesis in chickens, and the two segments must be expressed as a fusion protein (23, 24). It is possible that different regions of the ME26 virus-encoded protein are DISCUSSION needed for binding to the GATA-1 promoter and for cooperating with the GATA-1 protein to transactivate the EpoR Our data indicate that the induction of Epo responsiveness gene. Mutants of ME26 virus that contain changes in the by ME26 virus is due to transactivation of the GA TA-1 gene gag, myb, and ets segments of the virus are currently being by the ME26 viral gene product, which in turn results in analyzed in transactivation assays and for their abilities to induce Epo responsiveness in FDC-P2 cells. GATA-1 is known to transcriptionally activate its own A B c promoter (16, 42), the ot- and 0-globin genes (9, 12, 19), and 2 1 2 1 2 the EpoR gene (5, 47). Our results clearly show that ,-globin is induced in ME26 virus-infected hematopoietic cells. Since preliminary data suggest that ME26 virus has no direct effect on transcription from the chicken a-globin promoter (data not shown), we believe that induction of globin in virus- 28S infected cells results from high levels of GATA-1 expression. ,B-Globin is generally expressed late in erythropoiesis, after cells have become irreversibly committed to differentiation (18). However, both ME26 virus-infected FDC-P2 cells and Epo-dependent cell lines derived from ME26 virus-infected mice are blocked from differentiating into mature erythro- -18S cytes (38). This suggests that induction of GATA-1 by ME26 virus infection in hematopoietic cells results in only some of the phenotypic changes which occur during erythropoiesis, such as induction of the EpoR and globin genes. Presum- ably, some additional signal is required before the cells can proceed toward terminal differentiation. Previous studies have shown that there is a marked decrease in the levels of c-myb when murine erythroleuke- FIG. 2. Northern analysis of expression of GATA-1, ,B-globin, mia cell lines are induced to differentiate (34, 36), and and c-myb in uninfected and ME26 virus-infected FDC-P2 cells. constitutive expression of a c-myb cDNA blocks erythroleu- Total RNA was extracted from uninfected (lanes 1) or ME26 kemia cell differentiation (7). Furthermore, it has been virus-infected (lanes 2) FDC-P2 cells. A single Northern blot was reported that erythroleukemia cells can be induced to ex- prepared by using 10 ,ug of RNA per lane. Equal quantities of RNA press at- and ,-globin genes after stimulation with hexa- per lane were verified by staining with ethidium bromide. The filter methylenebisacetamide, but these cells will not terminally was hybridized with the following probes: GATA-1 cDNA (A), differentiate if c-myb is constitutively expressed (22). Thus, 0raJ-globin DNA (B), and c-myb DNA (C). The 3-globin transcript high levels of c-myb expression appear to be appears as a band of approximately 600 bp, and the c-myb transcript incompatible is approximately 3.4 kb. Also visible in panel C, lane 2, is the with erythroid cell differentiation. Although our results presence of the ME26 viral transcript (4.9 kb) which cross-hybrid- indicate that c-myb decreases markedly after ME26 virus izes with the c-myb probe. The positions of the 18S (1.9 kb) and 28S infection, the cells are clearly blocked in differentiation (38). (4.8 kb) rRNAs are shown. The failure of the virus-infected cells to differentiate may be 3060 AURIGEMMA ET AL. J. VIROL.

due to the small amount of c-myb remaining in the cells, cellular oncogene c-myb and members of the c-ets family of since previous experiments on differentiation of erythroleu- oncogenes in the regulation of normal erythropoiesis. kemic cells demonstrated that c-myb transcripts virtually disappear during terminal differentiation (22, 34, 36). Alter- ACKNOWLEDGMENTS natively, the gag-myb-ets fusion protein encoded by ME26 We thank A. D'Andrea for providing the EpoR cDNA, H. may be replacing c-myb in blocking terminal differentiation. Youssoufian for providing the EpoR promoter-hGH contruct, S. It has been reported that levels of murine c-myb may be Orkin for providing the murine GATA-1 cDNA, G. Felsenfeld for controlled by premature termination of transcription at a providing the chicken GATA-1 cDNA and the GATA-1 promoter- transcription pause site within the first intron of the murine CAT construct, G. Shen-Ong for providing c-myb cDNA, and M. c-myb gene (40). In addition, this gene may be regulated by Eglitis for providing the mouse `aJ-globin DNA. We also thank binding of several nuclear factors within this T. S. Papas and M. Masuda for critical reading of the manuscript differential and K. Cannon for assistance with its preparation. same region (35). Examination of the sequence of intron 1 (35) of murine c-myb reveals the presence of several REFERENCES GATA-1 consensus binding sites. Our observation that 1. Ausubel, F. M., R. Brent, R. W. Kingston, D. D. Moore, J. G. c-myb levels decline in ME26 virus-infected FDC-P2 cells Seidman, J. A. Smith, and K. Struhl (ed.). 1989. Current suggests that the induction of GATA-1 in these cells results protocols in molecular biology. John Wiley & Sons, Inc., New in direct negative regulation of the c-myb gene, perhaps by York. GATA-1 competing with transcription factors that normally 2. Ben-David, Y., E. B. Giddens, K. Letwin, and A. Bernstein. keep c-myb expressed at constitutive levels. 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