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Volume 270, Number 12, Issue of March 24, 1995 pp. 6619-6627
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Erythropoietin-induced Transcription at the Murine -Globin Promoter
A CENTRAL ROLE FOR GATA-1 (*)

(Received for publication, September 15, 1994; and in revised form, January 19, 1995)

Debra J. Taxman ,  Don M. Wojchowski (§)

From the Departments of Biochemistry and Molecular Biology, Pathobiology and Veterinary Science and the Center for Gene Regulation, the Pennsylvania State University, University Park, Pennsylvania 16802

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Using J2E cells and the murine beta-globin promoter as a model, we have performed the first direct analyses of erythropoietin (EPO)-activated transcription from defined templates. The -346 to +26 beta promoter was shown to comprise a target for maximal activation. This included a positive role for a -346 to -107-base pair (bp) domain in J2E cells, but not in F-MEL cells. Mutagenesis of a -215-bp AGATAA element within this domain showed that this effect did not require GATA-1 binding. In contrast, a critical role for GATA-1 at a -60-bp (G)GATAG element was defined by mutagenesis (GGgTAG and TGATAG), complementation with a synthetic TGATAA element, and the demonstrated specific binding of GATA-1. Proximal CCAAT(-75) and CACCC(-90) elements also were shown to contribute to transcriptional activation in J2E cells, yet exerted quantitatively distinct effects in the F-MEL system. Based on these results, minimal [TGATAA](4)-TATA and TGATAA-CACCC-TATA promoters were constructed and assayed in each system. Remarkably, the [TGATAA](4)-TATA promoter, but not the TGATAA-CACCC-TATA promoter, was induced efficiently by EPO in J2E cells, whereas the TGATAA-CACCC-TATA promoter was highly induced by Me(2)SO in F-MEL cells. These findings suggest that mechanisms of EPO-induced transcription in J2E cells involve GATA-1 and differ from chemically activated mechanisms studied previously in F-MEL cells. Globin induction in J2E cells was not associated with effects of EPO on levels or nuclear translocation of GATA-1. However, hemoglobinization was induced by okadaic acid, 8-Br-cAMP, and forskolin, a finding consistent with induction mechanisms that may involve modulated serine/threonine phosphorylation.

INTRODUCTION

Erythrocyte production in marrow, spleen, and fetal liver depends upon the exposure of pro-erythroblasts to the glycoprotein hormone erythropoietin (EPO). (^1)EPO is known to exert two general effects on late BFU-e and CFU-e. As a mitogen, EPO promotes the rapid expansion of CFU-e while inhibiting apoptosis(1, 2) ; and as a differentiation factor, EPO is required for the subsequent expression of late erythroid genes including alpha- and beta-globins(3) , the anion transporter band 3, and the cytoskeletal protein 4.1(4) . Through the cloning of the EPO receptor and the reconstitution of its mitogenic function in heterologous factor-dependent hematopoietic cell lines, significant progress recently has been made in identifying factors involved in proliferative signaling(5, 6) . This includes the recent identification of Jak2 as an essential receptor-associated protein tyrosine kinase (7) and the demonstrated association of p85/phosphatidylinositol 3-kinase (8) and Shc/Grb2/mSos-1 (9) with EPO receptor complexes. In contrast, comparably little is understood concerning mechanisms of EPO-induced late erythroid gene expression. This is due, in part, to limitations of model systems. Programs of late erythroid differentiation can be induced and have been widely studied in Friend virus-transformed murine erythroleukemia (F-MEL) cells (10) and human erythroleukemia K562 cells(11) . However, induction requires exposure to polar planar chemicals (Me(2)SO, hexamethylene-bisacetamide), butyrate, or hemin and is not activated by EPO. Among cell lines which are EPO-responsive, the primary response typically is proliferative(12, 13) , even among erythroid lines(14, 15, 16) . Notable exceptions are provided by SKT6, B6SUtA, and J2E cell lines. SKT6 cells were derived from splenocytes of mice infected with the anemia-inducing Friend leukemia virus complex and undergo efficient hemoglobinization in response to EPO(17) . A requirement for a transient decrease in c-myb expression during EPO-induced differentiation in SKT6 cells has been defined(18) , and tyrosine phosphorylation events associated with induced differentiation have been described(19) . B6SUtA cells were derived from an interleukin-3-dependent line(20) , and upon exposure to EPO (3-6 days) at least certain subclones express alpha-, beta-, beta-, and ^y-globin transcripts at ratios consistent with ^y to beta switching(21) . J2E cells were established via infection of murine fetal hepatocytes in vitro with the v-raf/v-myc-encoding virus, J2(22) , and respond to EPO with a rapid accumulation of beta-, beta-, and alpha-globin transcripts, hemoglobinization (30-40% of cells), enucleation, and differentiation to reticulocytes(23) . Transient decreases in c-myb transcript levels in J2E cells likewise have been associated with EPO-induced differentiation.

Using J2E cells as a model, in the present study we have tested whether EPO-induced expression of the murine beta-globin gene involves direct transcriptional activation at the proximal promoter, and if so, what cis-elements and trans-factors mediate induction. These analyses comprise the first study of regulated transcription from defined erythroid promoter constructs in an EPO-responsive cell line and were facilitated through our recent development of a novel transferrin/poly-L-lysine conjugate for receptor-mediated transfection(24) . The choice of the murine beta-globin promoter as a model was based on the rapid increase in beta transcript levels in J2E cells following EPO exposure(22, 23) . Also, regulatory elements of this promoter have been well studied in F-MEL cells, thus allowing for direct comparisons of beta promoter construct activities in an alternate, albeit chemically inducible, erythroid system.

Within the murine beta-globin promoter, a -106 to +26-bp region has been shown in F-MEL cells to be necessary and sufficient for regulated transcription(25) , and for enhancement by hypersensitive site 2 (HS2) of the murine beta locus control region (LCR)(26) . In this proximal promoter (and in the majority of beta-like globin promoters (27) ) TATA(-30), CCAAT(-75), and CACCC(-90) elements occur and contribute to beta promoter activity in erythroid and non-erythroid cells(25, 28) . Decreased activity in F-MEL cells of beta promoters mutated at the CACCC element correlates with loss of binding of both Sp1 and a 44-kDa protein (29) with binding specificity of the recently cloned erythroid Krüppel-like factor, EKLF(30) . MeSO induction in F-MEL cells increases dimethyl sulfate protection both at the CACCC element (26) and at an imperfect direct repeat element (betaDRE; position -53 to -32 bp) which contributes to beta promoter inducibility in the F-MEL system(31, 32) . A positive role for a nonconsensus GATA-1 element, (G)GATAG at -60 bp, also has been implicated in the expression of a -346 to +26 beta promoter in F-MEL cells(33) . GATA-1 is an erythroid member of a Cys-2/Cys-2 zinc finger family of transcriptional activators, is required for red cell development in chimeric mice, and binds to cis-elements within essentially all erythroid genes studies to date (see (34) for review).

The upstream region of the murine beta-globin promoter also influences transcriptional induction. In F-MEL cells an inhibitory domain at -100 to -250 bp represses transcription 6- and 4-fold following induction with hexamethylene-bisacetamide(33) . This repression involves a conserved element at -165 (BB1, which binds a ubiquitous factor in uninduced cells) and co-localized elements for GATA-1(-215) and NF1(-250).

Using wild type, mutated, and synthetic murine beta-globin promoter constructs, we presently demonstrate: (i) that the proximal -106 to +26 beta promoter is sufficient for activation by EPO in J2E cells; (ii) that maximal transcriptional activation by EPO requires the -346 to -107-bp promoter domain, as well as intact proximal CACCC(-90), CCAAT(-75), and GGATAG(-60) elements; (iii) that mechanisms of EPO-induced transcription in J2E cells from the distal beta promoter, the proximal beta promoter, and synthetic promoters comprised of TGATAA and CACCC elements differ significantly from Me(2)SO-dependent mechanisms in F-MEL cells; and (iv) that GATA-1 (or a direct co-factor) may play a central role in mediating EPO-induced expression of the beta-globin gene and possibly additional late erythroid genes. Levels and cytosolic versus nuclear distributions of GATA-1 remained constant during EPO-induced differentiation. However, J2E cells were shown to differentiate in response to okadaic acid and cAMP-elevating agents, suggesting that serine and/or threonine phosphorylation events may mediate induction.

MATERIALS AND METHODS

Cell Lines, Cell Culture, and Analyses of Induced Hemoglobinization

EPO-responsive J2E (22, 23) and Me(2)SO-responsive F-MEL (10) murine erythroid cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM), 10% fetal calf serum, 10M 2-mercaptoethanol at 1-9 times 10^5 cells/ml. Differentiation of J2E cells was induced by exposure to EPO (5 units/ml), and differentiation of F-MEL cells was induced by exposure to Me(2)SO (1.65%). Diaminofluorine staining of hemoglobin-positive cells was performed as described by Kaiho and Mizuro(35) . COS cells were maintained in DMEM, 8% calf serum.

Reporter Plasmids

Murine beta-Globin transcriptional reporter plasmids were constructed initially in pBluescriptII SK(+) (Stratagene) using parent 372-bp (-346 HindIII to +26 BglII) or 132-bp (ClaI to BglII) promoter fragments(25) . Constructs were excised (HindIII SpeI) and cloned 5` to the chloramphenicol acetyltransferase (CAT) gene (HindIII and XbaI sites) in pCAT-basic (Promega Corp.), yielding the plasmids pbeta106-CAT and pbeta340-CAT. Cis-elements of the beta proximal promoter and introduced point-mutations are summarized below (see Fig. 1). The -215 AGATAA element within pbeta340-CAT was mutated via polymerase chain reaction unique site elimination (36) (mutagenic oligomer 5`-TTGTCC[TTAcCT]GTGCAG-3`) to yield AGgTAA (mutation 1). Constructs mutated within the -90 CACCC, -75 CCAAT, and -60 (G)GATAG elements were prepared using the dut/ung method and the following oligonucleotides: 5`-GTAGAGCCA[9]0[aACCC]TGAAGGGC-3` (mutation 2); 5`-CTGAAGGG-75[CagAT] CTGCTCAC-3`(mutation 3); 5`-GCTCACACA-60[TGATAG]AGAGG-3`(mutation 4); and 5`-GCTCACACA-60[GGgTAG]AGAGG-3` (mutation 5). A TGATAA-CACCC-TATA promoter construct was prepared using the oligonucleotide 5`-[GAGCCACACCCTG][CAGAGCATATAAG]-3` to excise nucleotides -83 to -38 from the GATA106 promoter (PstI site introduced at -41). Following excision of the CACCC site from this construct (PstI site) a [GATA](4)-TATA promoter was prepared via insertion of a multimerized synthetic TGATAA element (5`-GATCCGGGCAACTGATAAGGATTCCCA-3`; and 5`-GATCTGGGAATCCTTATCAGTTGCCCG -3`)(37) . Promoter fragments (5`-XmnI BglII-3`) then were subcloned to pCAT-basic. The plasmid pGATA106-CAT was prepared by cloning a synthetic TGATAA element (see above) 5` to the beta106 promoter (BamHI site). All constructs were confirmed by dideoxy sequencing.

Figure 1: The murine wild type beta-globin proximal promoter and derived mutant constructs. cis-Elements of the murine beta-globin promoter are diagrammed together with point mutations prepared for present transcriptional analyses. (See ``Materials and Methods'' for construction of reporter plasmids with mutations 1-5.)


Transfections and CAT Assays

Transfection of reporter constructs was accomplished by receptor-mediated transferrinfection (38) using a novel transferrin/poly-L-lysine conjugate (24) . Briefly, plasmid DNA (80 µg) was incubated with transferrin/poly-L-lysine conjugate for 30 min at 37 °C in 20 mM Hepes, 150 mM NaCl, pH 7.4, and the resulting complexes then were added to exponentially growing J2E or F-MEL cells (4 ml; 7 times 10^5 cells/ml). Prior to DNA complex additions, chloroquine was added to final concentrations of 75 µM (J2E cells) or 100 µM (F-MEL cells). J2E cells were pretreated with desferral (20 h, 20 µM) to increase transfer in receptor densities. Transferrinfected cells (5 h, J2E; 8 h, F-MEL) were washed in DMEM and cultured in 10 ml of DMEM, 10% fetal bovine serum, 10M 2-mercaptoethanol for 40 h prior to CAT assays. Exposure to EPO (5 units/ml) or Me(2)SO (1.65%) was for 52 and 40 h, respectively. Transfections were performed in triplicate and were repeated using independent plasmid DNA preparations to confirm reproducibility. In all comparisons of promoter activities in J2E versus MEL cells identical plasmid preparations were used. To account for possible variability in transfections, in preliminary experiments pRSV/betagal and pSV40/luciferase plasmids were co-transfected. However, each affected transcription from beta-globin promoter constructs. Extracts therefore were standardized by protein concentration (BCA assay; Pierce). Also, in control experiments using a reporter containing the -1102 to +495-bp promoter of the rabbit alpha-globin gene (pBSalphaluc), activity was essentially equivalent (i.e. noninducible) in control versus EPO-exposed J2E cells. This control promoter is noninducible in K562 cells and fully active in non-erythroid HeLa cells (39) .

COS cells were transfected using Lipofectin reagent (Life Technologies, Inc.) as described previously(7) . Chloramphenicol acetyltransferase assays were performed using [^3H]chloramphenicol and butyryl-CoA (40) with the following modifications: freeze/thaw extracts were heated for 10 min at 60 °C; butyryl-CoA concentrations were increased to 0.3 µg/µl; incubations with substrates were extended to 16 h; and following TMPD/xylenes extractions, samples were back-extracted with an equal volume of 0.25 M Tris, pH 7.5.

GATA-1 Binding to -106 Promoter Constructs

In assays of GATA-1 binding at the proximal beta promoter, -106 to +26 BamHI XbaI fragments from wild-type and -60 GGgTAG mutant promoters were isolated and labeled using [alphaP]dATP and Klenow. Nuclear extracts from EPO-induced J2E cells (5 units/ml for 40 h) were prepared according to Dignam et al.(41) . Binding reactions (50 µl total) contained: 10 µl J2E extract, 300 µg/ml bovine serum albumin, 3 µg sheared calf thymus DNA, 3 µg of poly(dI-dC)bulletpoly(dI-dC), 1-2 µg of monoclonal antibody N-6(42) , and 40,000 cpm of wild type beta or GGgTAG mutant promoter fragment in 1 times binding buffer (60 mM KCl, 1 mM EDTA, 5 mM MgCl(2), 1 µM ZnCl(2), 12% glycerol, 1 mM dithiothreitol, 4 mM Tris, and 12 mM Hepes, pH 7.9). Where indicated the following annealed oligonucleotide pairs (40 ng/µl) also were included as competitors: GATA-1, 5`-TCGAGCTTGATAAGGCGACTGATAAGGCCAGCA-3` and 5`- TGCTGGCCTTATCAGTCGCCTTATCAAGCTCGA-3`; AP1/NF-E2, 5`-TCGACTCAAGCACAGCAATGCTGAGTCATGATGAGTCATGCTGAGGCTTA-3` and 5`-GATCTAAGCCTCAGAATGACTCATCATGACTCAGCAATGCTGTGCTTGAG-3` (Operon, Inc.). Samples were incubated for 10 min on ice and microcentrifuged prior to immunoprecipitation with 10 µl of anti-rat IgG agarose and 10 µl of protein A-Sepharose gel (Sigma) (30 min at 4 °C and 30 min at 23 °C). Gels were washed at 0 °C with binding buffer (above), and bound fractions were assayed by Cerenkov counting.

Analyses of GATA-1 Expression Levels

Northern analyses were performed using formaldehyde-agarose gels, Magnagraph nylon membranes (MSI), and the following probes (labeled using [alpha-P]dCTP, Klenow, and random hexameric primers): a 0.35-kb EcoRI fragment of the murine ^y-globin gene(43) ; the 0.8-kb HindIII fragment of the murine beta-globin gene(44) ; a 1-kb EcoRI fragment of a human GATA-1 cDNA (45) in pBluescript SK(+); and the 1.1-kb PstI fragment of a bovine actin cDNA in pBA1(46) . GATA-1 protein levels in whole cells were assayed by lysing washed cells directly in sample buffer for SDS-PAGE (10 min at 100 °C), and Western blotting (2.5 times 10^5 cell equivalents/lane) with mAb N-6. In analyses of cytosolic versus nuclear GATA-1 levels, cytosol was prepared by incubating cells for 30 min at 4 °C in 0.8% Triton X-100, 0.1% bovine serum albumin, 140 mM NaCl, 15 mM Tris, pH 7.6, 50 µg/ml phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 0.2 µg/ml aprotinin, 0.7 µg/ml pepstatin A, and 15 µg/ml benzamidine; removing nuclei by centrifugation (10 min at 400 times g); and adding SDS (0.1%), sodium deoxycholate (0.1%), and EDTA (1 mM) to cytosolic supernatants. Isolated nuclei then were washed and lysed directly in sample buffer (100 °C) (2.5 times 10^5 cell equivalents/lane; mAb N-6). Efficient fractionation of cytosol versus nuclei was confirmed by Western blotting using polyclonal antibodies against the following markers: p85 phosphatidylinositol-3 kinase (cytosol) (Upstate Biotechnology, Inc.); lactate dehydrogenase (cytosol) (Ventrex Corp.); and topoisomerase II (nuclei) (a generous gift from Leroy Liu).

Analyses of GATA-1 Nuclear Translocation

In analyses of GATA-1 nuclear translocation rates, J2E cells were cultured for 30 min in methionine- and cysteine-deficient DMEM (ICN), 10% fetal bovine serum, 10M 2-mercaptoethanol (5 times 10^6 cells/5 ml), and were labeled for 3 h using TransS-label (100 µCi/ml; ICN). Label then was chased (30 min to 4 h) in media containing methionine (30 mg/liter) and cystine (111 mg/liter). EPO exposure (5 units/ml) was for 42 h prior to labeling and was continued during labeling and chase intervals. Cytosol was prepared as described above, and nuclei were lysed by sonication in 0.4% SDS, 0.8% Triton X-100, 0.1% deoxycholate, 0.1% bovine serum albumin, 440 mM NaCl, 15 mM Tris, 1 mM EDTA, pH 7.6, 50 µg/ml phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 0.2 µg/ml aprotinin, 0.7 µg/ml pepstatin A, and 15 µg/ml benzamidine and then were diluted 3-fold to reduce SDS and NaCl concentrations. Following centrifugation, extracts were standardized for S incorporation by trichloroacetic acid precipitation to account for limited variability in labeling among samples. S-GATA-1 then was recovered by incubation of cytosolic or nuclear preparations with mAb N-6 (14 h at 4 °C), and immunoadsorption of GATA-1/N-6 complexes to anti-rat IgG-agarose (Sigma). Complexes were eluted by boiling in SDS-PAGE buffer and were analyzed by SDS-PAGE and autoradiography.

RESULTS

EPO-induced Transcription at the Murine beta-globin Promoter

Wild type, point-mutated, and synthetic constructs for use in assays of EPO-induced transcription were derived from the murine beta-globin promoter (Fig. 1). Transfection of J2E cells by standard procedures was inefficient, and it was necessary to develop a transferrin/poly-L-lysine conjugate for transferrinfection(24) . This procedure was nontoxic and allowed for the induction of differentiation by EPO. All reporter constructs (identical plasmid preparations) also were assayed in Me(2)SO-responsive F-MEL cells. Studies by Cowie and Myers have shown that the -106 to +26-bp proximal promoter is sufficient for regulated transcription in F-MEL cells(25) , and this construct (pbeta106-CAT) was used as a reference standard in all experiments. Control experiments in F-MEL cells confirmed the high activity of pbeta106-CAT, as well as the more recent finding that elements within the -346 to -107-bp promoter domain (pbeta340-CAT construct) repress transcription in F-MEL cells (33) (Fig. 2A). In contrast, in J2E cells this distal promoter domain activated transcription 2-3-fold (relative to pbeta106-CAT) both in EPO-induced, and uninduced J2E cells (Fig. 2B). Levels of induction of the -106 promoter in J2E cells (38.6-fold) also were significantly higher than in F-MEL cells (8-fold). Since EPO has been reported to nominally stimulate J2E cells growth(23) , the activity of a control template, pCAT-basic, was assayed to account for possible growth effects. Induction of differentiation increased activity of pCAT-basic 2.1-fold in J2E cells and 1.6-fold in F-MEL cells. When corrected for this effect, EPO-induced transcription of the -106 beta promoter in J2E cells was 18.4-fold, and Me(2)SO-induced transcription of this promoter in F-MEL cells was 5.0-fold. Also, in J2E cells little or no EPO-dependent induction of an alternate, transcriptionally competent control reporter, pBSalphaluc, was detectable (see ``Materials and Methods'').

Figure 2: Distal sequences of the murine beta-globin promoter increase EPO-induced transcription in J2E cells. A, for comparison and as a control, activities of pbeta106, pbeta340, and pbetaDeltaG215 promoter/CAT reporter constructs initially were assessed in F-MEL cells (uninduced, solid bars; Me(2)SO-induced, fill patterns). Activities are standardized versus the activity of the pbeta106 promoter in uninduced F-MEL cells (1.0 unit = 360 cpm). B, activities of the pbeta106, pbeta340, and pbetaDeltaG215 promoters in J2E cells (uninduced, solid bars; EPO-induced, fill patterns). Activities are standardized versus the activity of the pbeta106 promoter in uninduced J2E cells (1.0 unit = 28 cpm). t test values: pbeta340 versus pbeta106, p = 0.07; pbeta340 versus pbetaDeltaG215, p = 0.40. Results are representative of geq three independent experiments.


Footprinting of the above -346 to +426 beta promoter domain using extracts from J2E cells demonstrated specific factor binding to a naturally occurring AGATAA site (consensus GATA-1 element) at -215 (not shown). Based on this observation, this potential activating element was mutated within the -346 beta promoter and the resulting construct (pbetaDelta215-CAT) was assayed for transcriptional activity. However, no effect of this mutation was detectable in either J2E or F-MEL cells, indicating that alternate distal elements mediate activation in J2E cells of the -346 beta promoter.

In contrast, analyses of the proximal -106 to +26-bp promoter domain indicated an essential role for GATA-1 at a -60-bp (G)GATAG element. First, mutation of this element to GGgTAG (pbeta106DeltaG60-CAT) resulted in a dramatic loss of EPO-induced activity in J2E cells, whereas mutation to a consensus TGATAG element (pbeta106+G60-CAT) increased activity approximately 1.9-fold (Fig. 3A). Second, a synthetic consensus TGATAA element cloned immediately 5` to the -106 promoter was shown to increase promoter activity approximately 5-fold and to functionally complement the -60 GGgTAG mutant (Fig. 3B). Third, in non-erythroid COS cells, mutation of this -60 (G)GATAG element had little or no effect on beta106 promoter activity, indicating that activation at this element is erythroid-specific (Table 1). Finally, while GATA-1 binding at the -60 element was not detected by footprinting, or by gel mobility shift using a -72 to -48-bp promoter fragment, sequence-specific binding of GATA-1 (from J2E cell extracts) to a P-labeled -106 to +26 promoter was demonstrated via its co-immunoprecipitation with GATA-1/N-6 complexes (Fig. 3C). This -60 (G)GATAG element does not overlap with any previously identified beta-globin element (including the adjacent betaDRE at -53 to -32)(31, 32) , and corresponds in sequence to an element defined by polymerase chain reaction binding site selection and transactivation assays in NIH 3T3 cells as a functional GATA-1 element(47) . In F-MEL cells a requirement for this -60 GGATAG element recently has been demonstrated within a -346 beta promoter construct (33) and is supported further by activity data in Me(2)SO-induced F-MEL cells for these five presently studied constructs (Table 2).

Figure 3: Requirement for the -60 GGATAG element in EPO-induced transcription at the -106 beta promoter. A, effects on transcriptional activity of mutating the -60 GGATAG element within the -106 beta promoter were assayed in EPO-induced J2E cells: pbeta106DeltaG60, GGgTAG mutant; pbeta106+G60, TGATAG mutant. B, placement of a synthetic TGATAA element for GATA-1 5` to the above -106 promoter increased activity approximately 5-fold, and functionally complemented the GGgTAG mutant (pGATA106DeltaG60 versus pGATA106). Activities are standardized versus the activity of pbeta106-CAT in EPO-induced J2E cells. C, binding of GATA-1 to the -60 GGATAG element of the wild type -106 beta promoter. Specific binding of GATA-1 to the wild type (WT) versus a control -60 GGgTAG mutant promoter (DeltaG) was assayed by co-immunoprecipitation of P-end labeled promoter fragments with mAb N-6 and GATA-1 from nuclear extracts of J2E cells. Specificity of binding was assessed further by competition with TGATAA versus AP-1 oligonucleotides. Values represent averages minus background (i.e.P-promoter binding in the absence of antibody) and are representative of five independent experiments.


Using the above GATA106 beta-globin promoter construct, relative contributions of the proximal -90 CACCC and -75 CCAAT elements to transcription as induced by EPO in J2E cells versus Me(2)SO in F-MEL cells were assessed. Consistent with previous studies(25, 28) , mutation of either element markedly decreased levels of Me(2)SO-induced transcription in F-MEL cells ( Fig. 4and Table 2). In J2E cells, however, loss of EPO-induced activity was considerably greater for the -75 CagAT mutant (25-fold decrease) than for the -90 aACCC mutant (6-fold decrease). Thus, these results are consistent with differences in the roles for CCAAT and CACCC binding factors in EPO- versus Me(2)SO-induced transcription.

Figure 4: Activities of -90 CACCC and -75 CCAAT GATA106 beta promoter mutants. Activities of -90 aACCC (pGATA106DeltaCACCC) versus -75 CagAT (pGATA106DeltaCCAAT) mutants within the beta GATA106 promoter were assessed in Me(2)SO-induced F-MEL cells (A) and EPO-induced J2E cells (B). For each cell line, the activity of pGATA106 also was assayed.


EPO Activation of a [GATA](4)TATA Promoter Construct in J2E Cells

To further define factors which mediate EPO-induced transcriptional activation, synthetic promoters comprised of TGATAA-CACCC-TATA and [TGATAA](4)-TATA elements were constructed and assayed in J2E and MEL cells. Consistent with studies by Walters and Martin(48) , the TGATAA-CACCC-TATA promoter was 7-fold more active than the wild-type beta106 promoter in Me(2)SO-induced F-MEL cells, and induction was geq 65-fold. The [TGATAA](4)-TATA promoter was significantly less active in this system (Fig. 5A). In marked contrast, in EPO-induced J2E cells, transcription of the TGATAA-CACCC-TATA promoter was modest (2-3-fold below the -106 promoter), whereas activity of the [TGATAA](4)-TATA promoter was equivalent to the wild-type beta106 promoter with an induction of geq 75-fold (Fig. 5B). Efficient activation of this [TGATAA](4)-TATA promoter by EPO in J2E cells interestingly indicates that GATA-1 (or possibly a direct co-factor) can mediate induced transcription; and suggests that this mechanism may contribute to EPO-dependent expression of the murine beta-globin gene and potentially additional late erythroid genes.

Figure 5: EPO-induced transcription in J2E cells from a [GATA](4)-TATA promoter. A, activities of minimal GATA-CACCC-TATA and [GATA](4)-TATA promoters relative to the wild type beta106 promoter in uninduced (solid bars) and Me(2)SO-induced F-MEL cells (fill patterns). Induction (activities in induced versus uninduced cells) also is indexed for each construct. Levels of induction are standardized versus values for a control template, pCAT-basic. B, activities and induction (versus pCAT-basic) of GATA-CACCC-TATA and [GATA](4)-TATA promoters in control (solid bars) and EPO-exposed J2E cells (fill patterns). (t testing for p[GATA](4)-TATA versus pGATA-CACCC-TATA, p = 0.03.)


Levels of GATA-1 Transcripts, Protein, and Rates of Nuclear Translocation Are Not Modulated by EPO in J2E Cells

Based on studies in SKT6 cells in which EPO was reported to induce an increase in levels of GATA-1 transcripts(49) , levels of GATA-1 mRNA and protein in control versus EPO-induced J2E cells were analyzed. Levels of beta- and ^y-globin transcripts increased detectably at 8 h of exposure to EPO, with maximal levels observed at 32 to 64 h (Fig. 6A). In contrast, GATA-1 transcript levels were not affected significantly during this interval. Also, GATA-1 protein levels were not modulated detectably by EPO (5 units/ml, 0-8 days exposure) (Fig. 6B). Western analyses of cytosolic versus nuclear fractions from J2E cells indicated that GATA-1 was retained in cytosol at significant levels. However, EPO did not detectably alter its subcellular distribution (Fig. 6C). Based on the observed levels of cytosolic GATA-1, possible effects of EPO on nuclear translocation rates were assessed through S pulse/chase labeling experiments. Translocation in control versus EPO-exposed J2E cells was monitored via immunoadsorption, SDS-PAGE, and autoradiography of S-GATA-1 from cytosol and nuclei following 30 min to 4 h of chase (Fig. 7). A small, yet reproducible, increase in the apparent rate of GATA-1 translocation was detected in EPO-exposed cells (leq1.5-fold; five independent experiments). However, this modest effect required prolonged exposure to EPO (48 h) and was not observed upon exposure for shorter intervals (14 and 20 h; data not shown). Thus, increased translocation of GATA-1 is not considered to account for the observed comparably rapid effect of EPO on globin gene induction.

Figure 6: EPO-induced globin expression in J2E cells is not associated with increased expression or enhanced nuclear localization of GATA-1. A, levels of GATA-1 and beta- and ^y-globin transcripts in J2E cells were assayed by Northern blotting following exposure to EPO (5 units/ml; 0-64 h). Actin mRNA was assayed as a control. B, GATA-1 protein levels in J2E cells (5 units/ml EPO; 0-8 days) were assayed by Western blotting (whole cell lysates). C, GATA-1 levels in J2E cell cytosol versus nuclei were assayed by Western blotting. Efficient separation of cytosolic versus nuclear fractions was confirmed by Western blotting with antisera to p85/phosphatidylinositol 3-kinase and lactate dehydrogenase (cytosolic markers) and topoisomerase II (nuclear marker). M(r) standards are indexed in the left margin (B and C).


Figure 7: Rates of nuclear translocation of GATA-1 in control versus EPO-induced J2E cells. Following exposure to EPO (-/+ EPO), J2E cells were labeled metabolically with TranS-label and were chased with methionine and cystine for 4 h in the presence (or absence) of EPO. S-GATA-1 then was immunoprecipitated from cytosol and nuclei using mAb N-6 and was assayed by SDS-PAGE and autoradiography. EPO exposure (+ lanes) resulted in a reproducible 1.2-1.5-fold increase in ratios of nuclear:cytosolic S-GATA-1 (range of five independent experiments; beta-scope analysis). This effect required 42-h EPO exposure and was independent of the length of chase (0.5-4 h). M(r) standards are indexed in the right margin.


Induced Hemoglobinization of J2E Cells by Forskolin, 8-Br-cAMP, and Okadaic Acid

Based on the above-defined requirement for the -60-bp (G)GATAG element of the beta promoter for EPO-induced transcription (Fig. 3), the demonstrated EPO-dependent transcriptional activation of a [TGATAA](4)-TATA promoter construct in J2E cells (Fig. 5), and the recent observation that GATA-1 in MEL cells and K562 cells (and as a recombinant factor from SF9 cells) is a complex phosphoprotein(50, 51) , the possibility that globin gene expression might be activated in J2E cells by agents which modulate serine and threonine phosphorylation was tested. Interestingly, okadaic acid (an inhibitor of phosphatases 1 and 2A), 8-Br-cAMP (a nonhydrolyzable cAMP analogue), and forskolin (an activator of adenylate cyclase) each induced J2E cell hemoglobinization (Fig. 8). At high concentrations, the phosphodiesterase inhibitor isobutylmethylxanthine also was shown to affect a small increase in hemoglobin levels. While these agents possibly may act via EPO-independent pathways, these results at least are consistent with a role for modulated serine/threonine phosphorylation events in globin gene induction. This potentially includes the modulated phosphorylation of GATA-1.

Figure 8: Hemoglobinization of J2E cells in response to okadaic acid, forskolin, and 8-Br-cAMP. Hemoglobinization of J2E cells (2,7-diaminofluorine (DAF) positive cells, percent of total) was measured following 60-h exposure to the following agents: A, okadaic acid, EPO; B, isobutylmethylxanthine (IBMX), 8-Br-cAMP, forskolin, EPO. Values represent means ± S.D. for 200 cells and are representative of three independent experiments.


DISCUSSION

Mechanisms by which the glycopeptide hormone erythropoietin (EPO) induces the coordinate expression of late erythroid genes in pro-erythroblasts are poorly understood. Using EPO-responsive J2E cells (22, 23) and the murine beta-globin promoter as a model, we presently have examined mechanisms of induction by this hematopoietin. These studies comprise the first direct analyses of cis-elements which mediate induction by EPO and involved the development of a novel nontoxic conjugate for transferrinfection(24) . In this system, the proximal -106 to +26 beta promoter is shown to comprise a direct target for activation by EPO, with a requirement for the -346 to -107 distal domain for maximal activity. Elements within the distal -346 to -107-bp beta domain; the proximal -106 to +26-bp domain; and simple synthetic constructs that mediate EPO-induced transcription in J2E cells are defined and are shown to differ significantly from those which mediate Me(2)SO-induced transcription in F-MEL cells. Also, an important role for GATA-1 in EPO-induced beta transcription at a -60 (G)GATAG element is indicated, and efficient activation by EPO of a [TGATAA]-(4)TATA promoter in J2E cells is demonstrated. These latter findings suggest that EPO-induced transcription at the beta promoter, and potentially other late erythroid genes, may involve the activation of GATA-1 (or possibly a direct co-activator). EPO-induced differentiation in J2E cells is not associated with significant effects on GATA-1 levels, nuclear versus cytosolic compartmentalization, or rates of nuclear translocation. However, the observed induction of hemoglobinization in J2E cells by forskolin, 8-Br-cAMP, and okadaic acid is consistent with the possible involvement of serine/threonine phosphorylation pathways, potentially including the modulated phosphorylation of GATA-1.

Given the complex sets of cis- and trans-factors known to coordinately regulate globin gene expression (52) together with the diverse sets of pathways known to be activated by EPO(5, 6) , it is perhaps remarkable that the minimal -106 to +26 beta promoter per se is activated efficiently by EPO in J2E cells. The further positive effect exerted by the upstream -346 to -107 bp region of this promoter in J2E cells also is of interest in that cis-elements within this promoter domain, including a BB1 element at -165, previously have been shown to repress transcription in F-MEL cells(33) . In J2E cells, specific positively acting elements within the -346 to -107-bp region have not yet been identified. Comparisons with activating sequences in the human beta-globin promoter in F-MEL cells (53) suggest that this effect may be exerted by a consensus -250-bp NF1 site or a conserved -165-bp element (with activation by CP1 versus BB1 repression), whereas a possible role for a consensus AGATAA element at -215 (which footprints using nuclear extract from J2E cells) was excluded via mutagenesis studies (Fig. 2). In the human epsilon globin promoter a -165-bp consensus GATA element which lacks proximal promoter function has been demonstrated to mediate enhancer activity(54) , and the -215-bp AGATAA element of the murine beta-globin promoter might be speculated to serve a similar function.

In contrast, GATA-1 is indicated to play an essential role in EPO-induced transcription at the proximal beta-globin promoter via a nonconsensus (G)GATAG element at -60 bp. Mutation of this element to GGgTAG dramatically inhibited activity in EPO-induced J2E cells (but not in non-erythroid cells), whereas mutation to TGATAG (GATA-1 consensus) increased activity 1.9-fold. Also, an upstream synthetic TGATAA element (pGATA106-CAT) complemented this -60 GGgTAG mutation; and specific binding of GATA-1 to the wild type GGATAG element was demonstrated (Fig. 3). Notably, a promoter comprised of (G)GATAG elements and a TATA box recently has been demonstrated to be efficiently transactivated in fibroblasts by GATA-1 (but not GATA-3)(47) . The present studies demonstrate activation from this nonconsensus (G)GATAG element within a wild type promoter in response to EPO. Transcriptional assays in F-MEL cells of presently constructed GGgTAG and TGATAG mutants and previously of a GGATAGAG GctgcagG mutant within the -346 to +26 promoter (33) further demonstrate a requirement for the -60-bp (G)GATAG element. Cowie and Myers (25) and Charnay et al.(28) , however, failed to detect effects of either a GaATAG point mutation (25) or a linker scanning mutation within this -60 (G)GATAG element (CACAGGATAG CcagatcTgG)(28) . In these studies a 300-bp Friend virus long terminal repeat or the 3` human beta-globin gene enhancer was included to increase transcriptional activity. Thus, effects of these mutations may have been complemented by functional GATA elements within these enhancers (as illustrated presently by the ability of an upstream TGATAA element to complement a -60 GGgTAG mutation).

In the course of this study we also have defined several apparent differences in cis-elements which mediate murine beta promoter activation in J2E/EPO versus F-MEL/Me(2)SO differentiation pathways. First, as mentioned above, the distal -340 to -107 domain was found to increase promoter activity in J2E cells, yet repressed transcription in F-MEL cells (Fig. 2). This distinction may be due to differential levels and/or activities of CP1, NF1, or the BB1 repressor protein. Second, within the proximal -106 to +26-bp beta promoter, CACCC(-90) and CCAAT(-75) elements each contributed to the activity of a GATA106 promoter in J2E and F-MEL cells. However, in J2E cells CCAAT mutation (CagAT) inhibited EPO-induced transcription more markedly than CACCC mutation (aACCC) ( Fig. 4and Table 1). Thus, the -90 CACCC element appears less important for EPO-induced beta transcription in J2E cells than for Me(2)SO-induced transcription in F-MEL cells. Third, this finding is substantiated by the efficient activation of a minimal synthetic GATA-CACCC-TATA promoter in F-MEL cells versus nominal activation of this promoter by EPO in J2E cells (Fig. 5). Although these effects are consistent with direct action at the GATA106 and TGATAA-CACCC-TATA promoters of a dominant CACCC binding factor in F-MEL cells (i.e. Sp1 or EKLF(29, 30) ), a CACCC-TATA promoter was found to be insufficient for Me(2)SO activation (48) . (^2)By comparison with the porphobilinogen promoter, the spacing of TGATAA and CACCC elements in the GATA106 and GATA-CACCC-TATA promoters (i.e. 35 bp) is optimal for co-activation(55) . Thus, a possible explanation for differences in activities of these promoter constructs in J2E versus F-MEL cells is that GATA-1 may function predominately as a co-factor (e.g. EKLF/Sp1)-dependent activator in F-MEL cells and as a direct activator in the J2E/EPO pathway. The finding that a [GATA](4)-TATA promoter is efficiently induced (>75-fold) in J2E cells following EPO exposure, yet is less highly induced (approximately 10-fold) by Me(2)SO in F-MEL cells (Fig. 5) is consistent with this model and, importantly, indicates that transcriptional activation by GATA-1 is induced directly and efficiently by EPO.

EPO-induced transcription of beta-globin promoter constructs, however, was not explained by induced increases in levels of GATA-1 in J2E cells (Fig. 6), as has been suggested for responsive SKT6 cells(49) . Enriched early progenitor cells have been shown to accumulate GATA-1 transcripts upon exposure to interleukin-3 as well as EPO, yet require exposure to EPO for subsequent terminal differentiation(56) . This observation in normal pro-erythroblasts likewise suggests that possible effects of EPO on GATA-1 levels are not sufficient to promote late erythroid gene expression.

For certain alternate trans-factors which mediate the action of extracellular growth and differentiation signals, including ISGF-3, NFkappaB/rel, and Swi-5, regulated nuclear translocation (especially via regulated phosphorylation) recently has emerged as a common alternate activating mechanism(57) . Because GATA-1 is present in cytosol at significant levels, possible effects of EPO on GATA-1 translocation in J2E cells were assessed. However, nuclear and cytosolic distributions of GATA-1 were not affected by EPO, and rates of nuclear translocation were enhanced at most 1.5-fold following 48 h of exposure (Fig. 6C and Fig. 7).

In J2E cells, F-MEL cells, and human erythroleukemia K562 cells, GATA-1 occurs as a phosphoprotein(50, 51) . (^3)Thus, modulated phosphorylation (in the absence of induced increases in GATA-1 levels or nuclear translocation) represents an alternate possible mechanism for direct induction of the [TGATAA](4)-TATA promoter by EPO in J2E cells. Using the monoclonal antibody N-6, attempts were made to map P-phosphotryptic GATA-1 peptides from control versus EPO-exposed J2E cells. However, this was complicated by the poor solubility and limited recovery of purified GATA-1. Consequently, activators of serine/threonine phosphorylation pathways were tested for the ability to activate globin expression in J2E cells. Forskolin, 8-Br-cAMP, okadaic acid, and to a lesser extent isobutylmethylxanthine each were shown to induce hemoglobinization (Fig. 8). Induced increases in cAMP previously have been shown to promote hemoglobinization in EPO-responsive SKT6 cells (58) and to enhance EPO-dependent activation in committed TSA8 cells(59) . While these agents possibly may act via EPO-independent pathways, these results are at least consistent with a role for modulated serine/threonine phosphorylation, potentially including the regulated phosphorylation of GATA-1 by EPO. Possible significance of GATA-1 phosphorylation at conserved sites presently is being addressed via directed mutagenesis. Also, a recently developed cell-free transactivation system for the assay of direct and co-factor-dependent GATA-1 transactivation should facilitate these studies(51) . Notably, EPO-induced hemoglobinization of J2E cells, as well as normal murine CFU-e, occurs over an extended time course (4-48 h). Therefore, mechanisms beyond the possible modulated phosphorylation of primary effectors also likely contribute to the regulated expression of late erythroid genes.

FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL44491 and KO4HL203042 (to D. M. W.) and by a Sigma Xi grant-in-aid of research (to D. J. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 115 Henning Laboratory, University Park, PA 16802. Tel.: 814-865-0657; Fax: 814-863-6140.

(^1)
The abbreviations used are: EPO, erythropoietin; bp, base pair(s); kb, kilobase(s); DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; CAT, chloramphenicol acetyltransferase; mAb, monoclonal antibody.

(^2)
D. J. Taxman and D. M. Wojchowski, unpublished results.

(^3)
D. J. Taxman and D. M. Wojchowski, unpublished observations.

ACKNOWLEDGEMENTS

We thank Sherrill K. Sonsteby for expert technical assistance; Dr. S. P. Klinken for provision of J2E cells; Drs. J. D. Engel and S. H. Orkin for provision of mAb N-6; Dr. R. C. Hardison for provision of pBSalphaluc; Dr. K. J. Lynch for footprinting of the beta promoter; R. Burkert-Smith for data on induced hemoglobinization of J2E cells; Amgen Corp. for provision of recombinant human erythropoietin; and Drs. Ross Hardison, David Gilmour, Jerry Workman, and Peter Emanuel for helpful discussions.

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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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