GATA-1 and C-Myb Crosstalk During Red Blood Cell Differentiation Through GATA-1 Binding Sites in the C-Myb Promoter

GATA-1 and c-myb crosstalk during red blood cell differentiation through GATA-1 binding sites in the c-myb promoter

Petr Bartu˚ neˇ k1,2, Jarmila Kra´ lova´ 2, Gitta Blendinger1, Michal Dvorˇ a´ k2 and Martin Zenke*,1

1Max-Delbru¨ck-Center for Molecular Medicine, Robert-Ro¨ssle Str. 10, D-13092 Berlin, Germany; 2Institute of Molecular Genetics, Flemingovo nam. 2, 16637, Prague 6, Czech Republic

GATA-1 and c-Myb transcription factors represent key transcriptional regulators involved in erythropoiesis regulators of red blood cell development. GATA-1 is includes nuclear receptors such as the thyroid hormone upregulated and c-myb proto-oncogene expression is receptor TRa, the retinoic acid receptors RAR and downregulated when red cell progenitors differentiate into RXR, and the steroid hormone receptors estrogen erythrocytes. Here we have employed a culture system, receptor and glucocorticoid receptor (ER and GR) that faithfully recapitulates red blood cell differentiation respectively; Wessely et al., 1997; Bartunek and Zenke, in vitro,to follow the kinetics of GATA-1 and c- myb 1998; Bauer et al., 1998; von Lindern et al., 1999; and expression. We show that c-myb proto-oncogene expres- references therein). sion is high in progenitors and effectively downregulated GATA-1, the first major erythroid lineage specific at the time when nuclear GATA-1 accumulates and cells transcription factor identified, represents one of the differentiate into erythrocytes. Additionally,we identified major regulators of erythroid cell development as two GATA-1 binding sites within the c-myb promoter and demonstrated by gene inactivation and rescue experi- demonstrate that GATA-1 protein binds to these sites in ments in vivo and in vitro (Evans and Felsenfeld, 1989; vitro. Furthermore,GATA-1 represses c- myb expression Tsai et al., 1989; Pevny et al., 1991; Simon et al., 1992). through one of the GATA-1 binding sites in transient GATA-1 protein binds to the consensus DNA motif transfection experiments and this requires FOG-1. Thus, WGATAR that is contained in virtually all erythroid our study provides evidence for a direct molecular link specific genes (Weiss and Orkin, 1995). During erythroid between GATA-1 activity and c-myb proto-oncogene cell differentiation GATA-1 is post-translationally expression during terminal red cell differentiation. regulated: in immature progenitors GATA-1 protein is Oncogene (2003) 22, 1927–1935. doi:10.1038/sj.onc.1206281 confined to the cytoplasm, while induction of differentiation causes GATA-1 to translocate into the nucleus Keywords: c-myb; proto-oncogene; GATA-1; GATA (Briegel et al., 1996). Additionally, GATA-1 protein factor; erythropoiesis; red blood cell becomes post-translationally modified by phosphorylation in response to external signals and by acetylation (Crossley and Orkin, 1994; Boyes et al., 1998). Finally, Introduction GATA-1 activity requires association with Friend of GATA-1 (FOG-1; Tsang et al., 1997), and gene Red blood cells represent one of the most abundant inactivation of FOG-1 in mice generates a phenotype specialized cell types in vertebrate organisms. They that is similar to that observed for GATA-1 null mice develop from bone marrow stem cells through successive although more pronounced in megakaryocytes (Tsang stages of differentiation including progenitors that et al., 1998). are already committed to the erythroid lineage Erythroid progenitors abundantly express the proto- referred to as burst forming unit erythroid (BFU-E) oncogene c-myb, and gene inactivation of c-myb in mice and colony forming unit erythroid (CFU-E). Prolifera- leads to embryonic lethality because of the absence of tion of erythroid progenitor cells and their differentia- adult erythropoiesis, while the development of the fetal tion into fully mature red cells are controlled by erythroid compartment is not compromised (Mucenski different sets of transcription factors such as the c- et al., 1991). These findings unequivocally demonstrate Myb proto-oncoprotein, GATA-1 and GATA-2, ery- that both GATA-1 and c-myb are absolutely required throid kruppel-like factor (EKLF), the leucine zipper for development of red blood cells. protein NF-E2 and the helix–loop–helix transcription During the onset of differentiation c-myb expression is factor Tal-1/SCL and the LIM domain containing downregulated in various hematopoietic cell types, and factor LMO2/Rbtn2 (Ness and Engel, 1994; Orkin, downregulation of c-myb appears to be required for 1995; Graf, 2002). Another important family of their terminal differentiation (Cuddihy et al., 1993; Lyon and Watson, 1995). Specific factors have been impli- *Correspondence: M Zenke; E-mail: [email protected] cated in direct regulation of c-myb transcription such as Received 2 August 2002; revised 28 November 2002; accepted 28 E2F, various Ets and AP-1 family members, MZF1, November 2002 WT1 and the respective binding sites have been GATA-1 and c-myb crosstalk in erythroid differentiation P Bartu˚neˇk et al 1928 identified within the c-myb promoter (Nicolaides et al., 1992; McCann et al., 1995; Perrotti et al., 1995; Bellon et al., 1997; Sullivan et al., 1997; Campanero et al., 1999). Yet, the factors that are responsible for c-myb downregulation during erythroid cell differentiation have remained elusive. In the past several years in vitro culture systems have been developed that faithfully recapitulate red blood cell development in vitro employing various types of hematopoietic progenitor cells and culture conditions (Fibach et al., 1991; Dolznig et al., 1995, 2001, 2002; Wessely et al., 1997; Bartunek and Zenke, 1998; Panzenbock et al., 1998; von Lindern et al., 1999; Zhang et al., 2001; Bartunek et al., 2002). Such in vitro differentiation systems have allowed the study of the molecular mechanisms that underlie the erythroid cell differentiation program. Erythroid progenitor cells can be isolated for example from human, mouse and chicken bone marrow and amplified in vitro in the presence of cytokines and steroid hormones as homogenous cell Figure 1 c-myb and GATA-1 expression during red cell differ- populations to large cell numbers. Additionally, cells entiation. Northern blot analysis of RNA from SCF progenitors at various time points of differentiation as indicated. Blots were can be induced to effectively undergo normal terminal hybridized with c-myb and GATA-1 probes, respectively. 28S RNA differentiation in the presence of specific differentiation stained with methylene blue is shown to demonstrate equal RNA factors, like erythropoietin and insulin, and yield fully loading mature red cells. Since well-defined experimental conditions are employed, such in vitro systems are particularly well suited to study the molecular events ongoing when cells differentiate. Using stem cell factor (SCF)-dependent erythroid progenitor cells (in the following referred to as SCF progenitors) we have now investigated the direct interaction of the GATA-1 protein with the c-myb locus and have identified GATA-1 binding sites in the c-myb promoter. We demonstrate that GATA-1 negatively regulates c-myb promoter activity and this requires FOG-1.

Results

c-myb and GATA-1 expression during red blood cell differentiation SCF progenitors were generated from bone marrow and induced to differentiate in vitro. RNA was isolated after various periods of time and subjected to Northern blot analysis. It was found that SCF progenitors expressed high levels of c-myb proto-oncogene mRNA that Figure 2 GATA-1 protein expression and DNA-binding activity remained unchanged for 16 h after differentiation during red blood cell differentiation. (a) Western blot analysis with induction (Figure 1). At 20 h of differentiation c-myb GATA-1 speciﬁc antibody of total cell lysates (total) from expression started to decline and c-myb mRNA was differentiating SCF progenitors (same samples as in Figure 1) barely detectable at the late stages of differentiation and from cytoplasmic (cyto) or nuclear (nuclear) extracts after cell when terminally differentiated cells accumulated (72 and fractionation. (b) EMSA with nuclear extracts (same as in a) using 32P-labelled MaP and HS77 (À1040 GATA-1 binding site) 96 h). Conversely, GATA-1 mRNA was low in progeni- oligonucleotide probes as indicated. Only DNA–protein complexes tor cells and accumulated during differentiation starting are shown at 8 h and reaching maximal levels at 16 h (Figure 1). In terminally differentiated cells GATA-1 expression was low because of shut down of overall production of RNA. (Figure 2a). Additionally, in accord with our previous GATA-1 protein expression followed similar kinetics studies, GATA-1 protein in SCF progenitors was as GATA-1 mRNA starting at 8 h of differentiation cytoplasmic, while in differentiating cells GATA-1

Oncogene GATA-1 and c-myb crosstalk in erythroid differentiation P Bartu˚neˇk et al 1929 protein was increasingly abundant in the nuclear fraction from 20 h onwards (Figure 2a and Briegel et al., 1996). Furthermore, multiple GATA-1 bands were observed, which most likely represent different phosphorylated GATA-1 isoforms (Crossley and Orkin, 1994; Briegel et al., 1996). The GATA-1 protein effectively bound to MaP oligonucleotide containing a GATA consensus site of the mouse a1-globin promoter (Tsai et al., 1989; Briegel et al., 1996) already 2 h after the induction of differentiation. Interestingly, the binding afﬁnity of GATA-1 protein to HS77 oligonucleotide containing the À1040 GATA site of the chicken c-myb promoter (see below) started at 4 h, remained low to moderate until 16 h of differentiation, and then increased thereby following nuclear GATA-1 protein levels (Figure 2b).

Identification of GATA-1 binding sites in the c-myb promoter The efficient downregulation of c-myb transcription concomitantly with GATA-1 upregulation suggested to us that GATA-1 might be directly involved in the regulation of the c-myb proto-oncogene. Such an idea would also be in line with our previous observation that c-myb upstream sequences bind erythroid specific factors (P Bartunek, M Dvorak and M Zenke, unpublished results). Furthermore, GATA-1 protein represents one of the major regulators in erythroid cells and downregulation of c-myb expression was suggested to be required for effective differentiation of red blood cells (Cuddihy et al., 1993; Lyon and Watson, 1995). Figure 3 GATA-1 binding to c-myb promoter. (a) Schematic representation of the 50 end of the chicken c-myb locus with À1040 Therefore we set up experiments to identify GATA-1 and À30/+1 GATA-1 binding sites. Exons 1–5 (el–e5; filled boxes binding sites in the c-myb promoter by employing represent coding sequences) and selected restriction endonuclease multiple band shift assay (Kozmik and Paces, 1990). cleavage sites (BstEII, NotI, EcoRI and HindIII) are shown. The 1.4 kb c-myb promoter fragment (Figure 3a) was 3 Â MBS, cluster of three Myb binding sites (MBS); EC, evolutionary conserved element shaded area CPG island (Dvorak et al., digested with HaeIII and SacII, respectively, incubated 1989) (b) Identification of GATA-1 binding sites in the c-myb with recombinant GATA-1 protein and subjected to promoter. Multiple band shift assay employing 1.4 kb BstEII-NotI multiple band shift assay. In this assay the disappear- fragment (see a) digested with HaeIII and SacII, respectively. ance of bands with increasing GATA-1 concentrations is Increasing amounts of GST-GATA-1 protein were added as indicated (see also Materials and methods) and fragments were indicative for protein binding to the respective DNA resolved in polyacrylamide gels. Fragments that bind GATA-1 fragment(s). This analysis identified two fragments of with high affinity are depicted (arrows) and their size is indicated 240 and 307 bp in the HaeIII digest and two fragments of 124 and 477 bp in the SacII digest that bound GATA- 1 protein (Figure 3b). Interestingly, the 240 and 124 bp vertebrate species therefore referred to as evolutionary fragments comprise the same DNA element, while the conserved (EC) element (Urbanek et al., 1988). Inspec- 307 and 477 bp fragments comprise yet a second DNA tion of the EC nucleotide sequence identified a potential element (see below). GATA-1 binding site located at position +1 with Inspection of the nucleotide sequence of the 307 bp respect to cap site region I (CSR I, Dvorak et al., 1989) HaeIII fragment revealed a potential GATA-1 binding and corresponding to position À30 with respect to c- site at position À1040 (in the following referred to as myb cap site region II (CSR II; Figure 5a). This À1040 GATA-1 binding site; Figures 3a and 4a). This sequence element is in the following referred to as finding is in accord with our previous observation that (À30/+1) GATA-1 binding site. this c-myb promoter fragment binds an erythroid specific factor (P Bartunek, M Dvorak and M Zenke, GATA-1 binding to À1040 and À30/+1 of the c-myb unpublished results). Interestingly, the À1040 GATA-1 promoter site overlaps with one of three myb binding sites (MBS) that were identified before (Figures 3a and 4a; Dvorak To demonstrate directly GATA-1 binding to the À1040 et al., 1989). GATA-1 binding site, a HpaII–SmaI fragment compris- The 124 bp SacII fragment comprises c-myb promoter ing this site (in the following referred to as HS77) was sequences that are highly conserved among various subjected to EMSA analysis (Figure 4a and b). As

Oncogene GATA-1 and c-myb crosstalk in erythroid differentiation P Bartu˚neˇk et al 1930

Figure 4 Characterization of the À1040 GATA-1 binding site. (a) Nucleotide sequence of the HpaII–SmaI DNA fragment (HS77) containing the high afﬁnity À1040 GATA-1 site and overlapping MBS (arrows). (b) EMSA with HS77 probe, and oligonucleotide competition and antibody supershift experiments (polyclonal anti- GATA-1 #180) with nuclear extracts from SCF progenitors. MaP, mMaP and MYB, wild-type MaP, mutated MaP and MYB oligonucleotides, respectively, used in oligocompetition experiments. a-GATA-1, a-GATA-2, a-GATA-3 and a-MYB, GATA-1, GATA-2, GATA-3 and Myb speciﬁc antisera, respectively, employed in supershifts

expected GATA-1 protein present in extracts of SCF progenitors was effectively bound to HS77 fragment, and complex formation was competed with MaP oligonucleotide harboring a functional GATA-1 site but not with an oligonucleotide containing a mutated GATA-1 site (mMaP), or a myb binding site. Addition- ally, GATA-1 specific antibodies abolished GATA-1 binding, while GATA-2 and GATA-3, and Myb specific Figure 5 Characterization of À30/+l GATA-1 binding site. (a) Nucleotide sequence of chicken EC element (open arrowheads; antibodies did not. Note that with the conditions Urbanek et al., 1988) containing GATA/TATA-like element, and employed there is no c-myb binding to the HS77 CSRI and CSRII, respectively (Dvorak et al., 1989). E2F, PEA3 fragment, since the extracts used were prepared accord- and Sp1 binding sites, and SacII endonuclease cleavage sites are ing to standard methods (Dignam et al., 1983) that do indicated. Transcriptional start sites, filled triangles. (b)EMSA not contain c-myb protein (P Bartunek, M Dvorak and using 124 bp long SacII fragment of the EC element and nuclear extracts from chicken HD3 erythroleukemia cells (ch) and mouse M Zenke, unpublished results). We emphasize, however, MEL erythroleukemia cells (mu). Arrows indicate chicken (ch) and that the À1040 GATA-1 site can bind both c-Myb and mouse (m) DNA–GATA-1 complex, respectively. Vertical bar v-Myb proteins (see below). indicates multiple complexes binding to EC element (e.g. to E2F GATA-1 binding to the À1040 GATA-1 binding site and Sp1 sites). MaP and Sp1, competition with MaP and Sp1 oligonucleotide, respectively; a-GATA-1, addition of pan-anti- is intriguing since this site overlaps with a cluster of myb GATA-1 antibody #H2 (a-GATA-1). (c) ZOO band shift with binding sites (MBS; Figures 3a and 4a) suggesting that nuclear extracts from chicken (ch), mouse (mu) and human (hu) mutually exclusive binding of these protein factors erythroleukemia cell lines (HD3, MEL and K562), respectively, might occur. Additionally, GATA-1 binding to the and chicken CSR I oligonucleotide as a probe. Bandshift reactions À1040 binding site was moderate to low during the were done in the absence (À) or presence (+) of polyclonal pan- anti-GATA-1 antibody #H2 (a-GATA-1) to supershift DNA– initial phase of differentiation but effectively increased GATA-1 complexes. Arrows indicate GATA complexes, star after 20 h and thus correlated with c-myb downregula- depicts the migration of ternary DNA–protein–antibody complexes tion (Figures 1b and 2b). and bracket indicates non–specific binding

Oncogene GATA-1 and c-myb crosstalk in erythroid differentiation P Bartu˚neˇk et al 1931 GATA-1 also bound to the À30/+1 GATA-1 binding site in EC as demonstrated by EMSA (Figure 5 a–c). Addition of an oligonucleotide bearing a consensus GATA site (MaP) effectively competed for binding while an oligonucleotide with a mutated GATA-1 or Sp1 site did not (Figure 5b and data not shown). Furthermore GATA-1 specific antibody supershifted the DNA–protein complex. Interestingly, the addition of Sp1 oligonucleotide diminished the high molecular weight complexes bound to EC element and enhanced the GATA-1 specific band. This was observed both for chicken and mouse GATA-1 proteins present in HD3 and MEL nuclear extracts, respectively (Figure 5b). The EC region was then scanned with a panel of oligonucleotides for GATA-1 binding and only the oligonucleotide bearing the À30/+1 GATA-1 site efficiently bound GATA-1 protein (Figure 5c and data not shown; see Materials and methods for chCSR I). GATA-1 binding to the À30/+1 GATA-1 site is interesting since this GATA-1 site overlaps with the cap site CSR I or the À30 position of CSR II. It is demonstrated here that chicken, mouse and human GATA-1 bind to this element and that DNA–GATA-1 protein complexes are supershifted with GATA-1 specific antibody (Figure 5c). Additionally, an oligonucleotide corresponding to the respective human sequence element also bound chicken, mouse, human and even Xenopus GATA-1, albeit with somewhat different preferences (data not shown).

GATA-1 regulates c-myb promoter activity in vivo To determine whether the À1040 and À30/+1 GATA-1 sites are active in vivo, transient cotransfection experiments were performed. A reporter plasmid with elevated basal luciferase expression (pTL109; Nordeen, 1988) was selected and À1040 or À30/+1 GATA-1 sites were cloned upstream of the HSV-TK promoter that drives luciferase expression. GATA-1 and FOG-1 expression Figure 6 c-myb repression by GATA-1 requires FOG-1 (a) vector were cotransfected, both individually and simul- Transcriptional activity of pTL109 luciferase reporter containing taneously. Repression of reporter gene activity via the the À1040 GATA-1 binding site (wt) or the respective mutated 1040 GATA-1 binding site required both GATA-1 GATA-1 site (mut) was analysed in transient cotransfection À experiments in CEF. GATA-1 and FOG-1 expression vectors were and FOG-1, while GATA-1 or FOG-1 alone were cotransfected as indicated and luciferase activity was determined at inactive (Figure 6a). Additionally, there was no repres- day 2 after transfection. Reporter activity is shown as fold sion by a mutated À1040 GATA-1 site as expected. To repression with basal reporter activity set to 1. Average values of further extend these observations, a reporter plasmid four independent experiments with standard deviation are shown. (b) Transcriptional activity of pLucbTATA luciferase reporter pLucbTATA with low basal activity was used (Bartunek containing the À1040 GATA-1 binding site. C-Myb, GATA-1 and et al., 1997). In this vector the composite À1040 GATA- FOG-1 expression vectors were transfected into CEF as indicated 1/Myb binding site was activated by c-Myb by ﬁvefold (+, ++ represents 0.5 and 1 mg of the expression vector, (Figure 6b). Transfection of GATA-1 resulted in only a respectively). Reporter activity is shown as fold activation with marginal increase of reporter activity while there was no basal reporter activity set to 1. Average values of three independent experiments with standard deviation are shown. (c) Northern blot effect by FOG-1. Transfection of c-Myb and increasing analysis of FOG-1 expression in SCF progenitors at various time amounts of GATA-1 enhanced reporter activity to levels points of differentiation. 28S RNA, methylene blue staining of 28S higher than seen for c-Myb alone. Importantly however, ribosomal RNA in the presence of FOG-1 increasing amounts of GATA- 1 repressed reporter activity close to basal levels (Figure 6b). À1040 and À30/+1 GATA-1 binding sites within the Transfection experiments with the À30/+1 GATA-1 context of the bona ﬁde c-myb promoter was not possible luciferase reporter and the respective mutated GATA-1 since reporter constructs containing even several kilo- site gave luciferase values similar to empty vector bases of c-myb upstream sequence were essentially control (data not shown). So far the analysis of the inactive employing a variety of different cell types

Oncogene GATA-1 and c-myb crosstalk in erythroid differentiation P Bartu˚neˇk et al 1932 including, for example fibroblasts, erythroblasts and T binding to the À1040 site in vitro was more selective than cells (Sobieszczuk et al., 1989; P Bartunek, M Dvorak binding to the MaP element of the mouse al-globin and M Zenke, unpublished results). promoter, which might be because of the sequence Finally, since c-myb repression by GATA-1 required context of the À1040 site and/or specific post-transla- FOG-1 and the GATA-l/FOG-1 cooperation was demon- tional modifications of the GATA-1 protein such as strated in several systems (Tsang et al., 1997, 1998), we phosphorylation. analysed the pattern of FOG-1 expression during terminal The second more proximal À30/+1 GATA-1 binding red cell differentiation. SCF progenitors were prepared site is contained in a sequence that is highly conserved and induced to differentiate, RNA was isolated after between different organisms, referred to as EC element various periods of time and analysed for FOG-1 expres- (Urbanek et al., 1988). In addition, this GATA-1 site sion by Northern blotting. It was found that erythroid overlaps with the upstream transcriptional start site progenitors express 4.0 kb FOG-1 mRNA which is further (CSR I) which most interestingly also represents the À30 increased during differentiation (Figure 6b). position of the downstream transcriptional start site (CSR II). Here we show that GATA-1 protein binding to CSR I is evolutionary conserved from Xenopus to man. GATA-1 did however not repress expression of Discussion reporter genes via this À30/+1 GATA-1 site in transient cotransfection assays. This was observed irrespective of GATA-1 and c-Myb transcription factors represent key the presence or absence of FOG-1 and the reasons for regulators of red cell development as demonstrated by this are not clear but might be because of inherent gene inactivation experiments in mice (Mucenski et al., limitations in this type of assay. In this context we note 1991; Pevny et al., 1995). Here we followed the kinetics that similar to the c-myb gene, a number of known of GATA-1 and c-myb proto-oncogene expression GATA-1 target genes have a GATA site at position during red cell differentiation by employing a culture À30, including genes for platelet factor 4 (PF4), system that recapitulates red cell development in vitro. erythropoietin and b-globin (Chiba et al., 1991; Fong We show that GATA-1 is effectively upregulated during and Emerson, 1992; Aird et al., 1994). The model differentiation and nuclear GATA-1 protein starts to proposed for the regulation of PF4 gene in megakar- accumulate at 20 h postdifferentiation induction, and at yopoiesis was that GATA proteins compete with TFIID the same time c-myb expression is downregulated. for core promoter binding (Aird et al., 1994). Thus, it is Additionally, we have identified two GATA-1 binding very well possible that GATA-1 and factors of the basal sites within the c-myb promoter and demonstrate that transcription machinery compete for binding to the very GATA-1 protein binds to these sites in vitro. Further- same sequence element. This might be particularly more, GATA-1 represses c-myb expression through the relevant late in red cell differentiation when GATA-1 À1040 GATA-1 binding site in transient cotransfection protein becomes very abundant and c-myb transcription experiments and this requires FOG-1. is downregulated. Such a mechanism is further sup- The more distal GATA-1 binding site identified in this ported by our initial experiments with TATA-binding study is located at position À1040 with respect to the c- protein (TBP) showing that His-huTBP binds to CSR I myb transcriptional start site. Interestingly, this GATA in vitro (data not shown). site overlaps with a cluster of three Myb binding sites All this is in line with the observation that during that are also present in an analogous position in the terminal differentiation of red cells, c-myb transcription human c-myb promoter. These Myb sites were shown is downregulated concomitantly with the upregulation before to bind c-Myb protein and mediate transactiva- of GATA-1 expression. Thus, increased GATA-1 level tion of the c-myb promoter by c-Myb (Nicolaides et al., might well be the trigger that causes downregulation of 1991). Importantly, as demonstrated here increasing c-myb transcription. This idea is also supported by amounts of GATA-1 repressed gene expression via this experiments on in vitro differentiation of GATA-1À ES À1040 GATA-1 site in transient cotransfection experi- cells. GATA-1À erythroid cells show higher levels of ments and this required FOG-1. Thus, GATA-1/FOG-1 c-myb expression than erythroid cells derived from wild- and Myb might bind to these overlapping sequence type ES cells (Weiss et al., 1994) suggesting that GATA- elements in a mutual exclusive fashion. This idea is also 1 is important for c-myb downregulation in vivo. supported by in vitro binding studies demonstrating that Furthermore, in previous work we demonstrated that GATA-1 and c-Myb proteins effectively competed for the hormone inducible GATA-1 estrogen receptor binding to the À1040 site in EMSA (data not shown). fusion GATA-1/ER caused c-myb downregulation in Therefore, a model presents itself where Myb induces response to estrogen (Briegel et al., 1996). The present c-myb transcription via this composite À1040 GATA- study extends these findings in that GATA-1 protein 1/Myb binding site, while increasing amounts of GATA- (and probably also GATA-1/ER protein) directly 1 downregulate c-myb expression through the very same interact with c-myb promoter sequences. sequence element and this requires FOG-1. In support GATA-1 action requires association with the cofactor of this model GATA-1 binding to the À1040 GATA-1 FOG-1 (Tsang et al., 1997, 1998). Accordingly, GATA- binding site was low from 2 to 16 h of differentiation, 1 and FOG-1 synergize both in transient transactivation but effectively increased after 20 h and thus correlated assays and in inducing terminal erythroid differentiation with c-myb downregulation (Figures 1 and 2b). GATA-1 of G1E cells (Tsang et al., 1997). Here we show that

Oncogene GATA-1 and c-myb crosstalk in erythroid differentiation P Bartu˚neˇk et al 1933 FOG-1 increases when progenitor cells differentiate into chCSR I-2: 50-TCGACGGTTGAGATTAAAGAGT- mature red cells. Furthermore, in Xenopus FOG was AAACTCTG-30 shown to act as a repressor of red cell development in huCSR I-1: 50-TCGACAGAGTTTACACTTTAATA- vivo, which led to the hypothesis that in addition to its TCAACCG-30 coactivator function FOG might act as a corepressor for huCSR I-2: 50-TCGACGGTTGATATTAAAGTGTA- some GATA target genes, such as GATA-2 (Deconinck AACTCTG-30 et al., 2000). This mode of action would be in line with HX45-1: 50-AGCTTACCCTAACGGGGCTGC- the results reported here that FOG is required for the AGTTATCATCCCCGTTACTTGTAC-30 repression of c-myb transcription. Additionally, both HX45-2: 50-TCGAGTACAAGTAACGGGGAT- GATA-2 and c-myb have an important function in GATAACTGCAGCCCCGTTAGGG- proliferating erythroid progenitor cells and their down- TA-30 regulation is required for terminal differentiation mHX45-l: 50-AGCTTACCCTAACGGGGCTGCA- (Briegel et al., 1993; Cuddihy et al., 1993; Lyon and GTTAGAAAGCCCGTTACTTGTAC-30 Watson, 1995). mHX45-2: 50-TCGAGTACAAGTAACGGGCTT- In summary, while previous studies demonstrated the TCTAACTGCAGCCCCGTTAGGGTA- impact of GATA-1 and Myb for red cell development, 30 this work extends these findings and provides evidence for a direct molecular link between these transcription factors. In addition, GATA-1 and c-myb appear to be Cells and tissue culture important both for establishing the red cell lineage and SCF progenitors were generated by cultivating bone marrow for orchestrating the terminal red cell differentiation cells from 3 to 7-day-old SPAFAS chicks in modified CFU-E program. medium supplemented with 100 ng/ml of recombinant avian stem cell factor (SCF; Bartunek et al., 1996; Bartunek and Materials and methods Zenke, 1998). Homogeneous cell populations were obtained after 3–4 days (Dolznig et al., 1995; Bartunek and Zenke, Plasmids and oligonucleotides 1998). Luciferase reporters contain HX45 or mHX45 oligonucleo- To induce erythroid differentiation, cultures of SCF tides (see below) harboring the wildtype À1040 GATA-1 progenitors were incubated in CFU-E medium without binding site or a mutated GATA-1 binding site, respectively, chicken serum (referred to as differentiation medium; Zenke between HindIII and XhoI of pTL109 vector (Nordeen, 1988). et al., 1990; Beug et al., 1995) supplemented with 3% anemic pLuc bTATA is a derivative of pGLBasic and contains a chicken serum (as a source for erythropoietin) plus 100 ng/ml rabbit b-globin TATA sequence element upstream of the insulin (Novo Nordisk). Effective erythroid differentiation was luciferase gene. Chicken GATA-1 was expressed from TFAneo monitored by assessing hemoglobinization of cells in cytospin preparations with neutral benzidine staining and by measuring (Yamamoto et al., 1990) and chicken c-myb from pNeo-CCd 3 vector (Grasser et al., 1991). Human FOG-1 cDNA (Tsang [ H]thymidine incorporation and hemoglobin accumulation et al., 1997) was cloned into pcDNAl and used in transient (Beug et al., 1995; Dolznig et al., 1995; Bartunek et al., 2002). transfection assays. Chicken 0.29 kb FOG-1 specific probe was Cell numbers and cell size were determined by employing the obtained by RT–PCR from SCF progenitors using specific CASY-1 Cell Counter and Analyzer System (Scha¨ rfe System, primers FOG-1 and FOG-2 (50-CCAGGGCAGCATCCA- Reutlingen, Germany). CAGTG-30 and 50-CGGGTAGGGCGAGTCTCCAG-30, respectively) that were designed based on chicken EST Gen Cell fractionation Bank Accession number BG710230, and the FOG-1 specific fragment was subcloned in pCR2.1 (Invitrogen). SCF progenitors, prior to and at various time points post In EMSA the following oligonucleotides were used (Tsai induction of differentiation, were harvested, washed with PBS et al., 1989; Briegel et al., 1993, 1996; P Bartunek, M Dvorak and fractionated as described (Briegel et al., 1996). Briefly, and M Zenke, unpublished; the GATA and Myb sequence 1 Â l07 cells were lysed in 1 ml lysis buffer (50 mm Tris-HCl pH elements and the respective mutated sequences are underlined): 7.5, 1 mm MgCl2, 0.5% Triton X-100, 10 mm DTT, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mm PMSF) for 10 min on ice. Cytoplasmic and nuclear fractions were separated by centri- MaP-1: 50-GATCTCCGGCAACTGATAAGGA- fugation and cytoplasmic supernatants were recovered. Nuclei TTCCCTG-30 were resuspended in 0.2 m NaCl, 30 mm Tris-HCl pH 7.5, 5 mm 0 MaP-2: 5 -GATCCAGGGAATCCTTATCAGTT- MgCl2, 10% glycerol and protease inhibitors as above, and GCCGGA-30 proteins were extracted by adjusting NaCl to 0.4 m final mMaP-1: 50-GATCTCCGGCAACTGGCAAGGA- concentration (20 min on ice), and both cytoplasmic and TTCCCTG-30 nuclear extracts were used for Western blot analysis and electrophoretic mobility shift assays. mMaP-2: 50-GATCCAGGGAATCCTTGCCA- GTTGCCGG A-30 MYB-1: 50-GATCCGTTATAACGGTTAGTAA- Northern blot analysis CGGTTTCG-30 0 RNA was isolated from SCF progenitors and analysed by MYB-2: 5 -GATCCGAAACCGTTACTAACCGT- Northern blotting as described (Briegel et al., 1996; Bartunek 0 TATAACG-3 and Zenke, 1998). Probes were full-length cDNA of chicken chCSR I-1: 50-TCGACAGAGTTTACTCTTTAATC- GATA-1 (Yamamoto et al., 1990), EcoRI–XbaI fragment of TCAACCG -30 v-myb (Bartunek et al., 1992) and a 290 bp chicken FOG-1

Oncogene GATA-1 and c-myb crosstalk in erythroid differentiation P Bartu˚neˇk et al 1934 fragment. Blots were hybridized and washed at moderate restriction enzymes and end-labelled with [a32P]dCTP by stringency and exposed to film. Klenow DNA polymerase. Increasing amounts of recombinant GST-GATA-1 fusion protein (Ko and Engel, 1993) was added Western blot analysis to EMSA reactions and protein–DNA complexes were resolved on 5% nondenaturing polyacrylamide gels (Kozmik Total, cytoplasmic and nuclear extracts from SCF progenitors and Paces, 1990). were separated on 10% SDS–PAGE gels and blotted onto nitrocellulose membranes (BA85, Schleicher & Schuell). Cotransfection transactivation assay Membranes were blocked overnight in TBS and reacted with polyclonal GATA-1 specific anti-peptide antibody #180 Chicken embryo fibroblasts (CEF; 0.6 Â l06 cells) or QT6 cells (Briegel et al., 1993, 1996) or pan-GATA-1 #H2 polyclonal were seeded onto a 60 mm Petri dish in DMEM medium, 5% antibody (a gift from R. H. Nicolas, Institute Cancer fetal calf serum, 2% chicken serum, 20 mm HEPES pH 7.5 and Research, London, UK) in TBS supplemented with 5% nonfat transfected with 5 mg of DNA (1 mg of pNeo-CCd, 0.5–1 mgof milk powder for 1 h. Subsequently, blots were washed and GATA-1 TFAneo and/or pcDNA–FOG-1 expression vector reacted with the respective secondary antibodies (ECL kit, plus 1 mg of pTL109 or pLucbTATA based luciferase reporter Amersham Pharmacia Biotech) in TBS supplemented with 5% vector, 0.5 mg of pRSV-bGal, and 2.5 mg of pBluescript carrier non fat milk powder (45 min), developed in ECL reagents and DNA) using calcium phosphate transfection (Disela et al., exposed to film. 1991; Briegel et al., 1993). After 16 h cells were rinsed and incubated with fresh growth medium. At day 2 after Electrophoretic mobility shift assay (EMSA) transfection cells were harvested and cell extracts were prepared and analysed for luciferase activity and normalized 32 EMSA was performed using P-radiolabelled oligonucleotides for b-galactosidase expression and/or protein concentration or DNA restriction fragments in the absence or presence of (Disela et al., 1991). 200-fold excess of unlabelled oligonucleotide (30 min on ice). For supershifts, 0.5 ml of polyclonal GATA-1 antibody #180 or #H2 was added to the reactions after 10 min of preincubation. Acknowledgements Protein–DNA complexes were resolved on a 5% nondenatur- We thank SH Orkin and H Wolfes for FOG-1 cDNA and ing polyacrylamide gel in 0.25 Â TBE buffer and exposed to expression plasmid, H Stunnenberg for His-huTBP, JD Engel film (Briegel et al., 1996). for GST-GATA-1 and pLucbTATA, RH Nicolas for GATA-1 specific antibody and P Podlatis for expert secretarial assistance. Part of this work was funded by grants of the Multiple band shift assay Grant Agency of the Czech Republic (301/98/K042) to MD, The 1.4 kb BstEII–NotI fragment of chicken c-myb promoter the Fonds der Chemischen Industrie to MZ and Volkswagen (Dvorak et al., 1989) was digested with HaeIII or SacII Foundation to PB and MZ.

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