From the Department of Adult Oncology, Dana-Farber
Cancer Institute, ¶ Children's Hospital, Boston,
Massachusetts 02115 and the
Department of Medicine,
Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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Biochemical analysis of megakaryocytes, the precursors of blood platelets, is limited by their rarity in vivo, and studies on lineage-specific gene expression have been conducted exclusively in cell lines with limited megakaryocytic potential. Mice lacking the transcription factor NF-E2 display arrested megakaryocyte differentiation and profound thrombocytopenia. To study the heterodimeric NF-E2 protein in primary cells, we cultured mouse fetal livers with the c-Mpl ligand, obtained highly enriched megakaryocyte populations, and readily detected NF-E2 activity in nuclear extracts. As in erythroid cells, p45 NF-E2 is the only large subunit in primary megakaryocytes that dimerizes with distinct small Maf proteins to constitute a heterogeneous NF-E2 complex. Whereas p18/MafK is the predominant small Maf protein in erythroid cells, the related polypeptides MafG and/or MafF predominate in megakaryocytes. Although this represents the first example of differential small Maf protein expression among closely related blood lineages, the DNA-binding specificity of NF-E2 is similar in both cell types. Although the megakaryocyte protein preferentially binds an asymmetric AP-1-related motif, it also recognizes cAMP-responsive element-related sequences, albeit with lower affinity, and nucleotides outside the core sequence influence the DNA-protein interaction. These results demonstrate the feasibility of biochemical studies on primary murine megakaryocytes and provide a basis to dissect the critical functions of NF-E2 in megakaryocyte differentiation.
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INTRODUCTION |
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Megakaryocytes, which give rise to blood platelets, constitute an extremely rare subpopulation of cells in vivo. Therefore, despite the importance attached to the process by which megakaryocytes form and release platelets, molecular aspects of their development are poorly understood and have largely been studied in a few multipotential hematopoietic cell lines with limited megakaryocyte differentiation. The c-Mpl ligand (also known as thrombopoietin or the megakaryocyte growth and development factor) is the major cytokine that regulates megakaryocyte proliferation and differentiation and raises platelet counts substantially in vivo (reviewed in Ref. 1). Recombinant c-Mpl ligand has facilitated in vitro culture of sufficient numbers of primary megakaryocytes to permit biochemical studies (2), partially relieving the dependence on transformed cell lines. However, studies on gene expression and transcription factors have not been reported in primary cells.
Studies in immortalized cell lines suggest that several genes expressed specifically within megakaryocytes are regulated in part by a combination of transcription factors belonging to the GATA and Ets protein families (3-5). Indeed, mice with megakaryocyte-selective loss of GATA-1 activity are severely thrombocytopenic as a result of arrested megakaryocyte differentiation (6). Analysis of megakaryocyte promoters per se has not consistently pointed to other proteins that may mediate lineage-specific gene expression and participate in platelet biogenesis.
Like GATA-1, the transcription factor NF-E2 was originally identified
through its interaction with critical cis-elements within the -globin locus control region (7, 8). NF-E2 is an obligate heterodimer between a 45-kDa hematopoietic restricted polypeptide (p45
NF-E2) and widely expressed ~18-kDa proteins related to the avian
oncogene v-maf; both subunits belong to the bZip
(basic leucine zipper) family of
transcriptional regulators (9-11). Although several lines of evidence
point to NF-E2 as the major enhancer protein acting at the
-globin
gene locus in developing erythroid cells, mice lacking p45 NF-E2
display only mild, albeit consistent, red blood cell abnormalities
(12). The most notable feature of these knockout mice is the virtual
absence of circulating blood platelets, associated with arrested
megakaryocyte maturation (13). Thus, two distinct "erythroid"
transcription factors play critical roles in proper megakaryocyte
development and are required to generate normal platelets in
vivo.
In contrast to the biochemical basis of interactions between the monomeric GATA factors and their cognate DNA-binding sites (14, 15), our knowledge of NF-E2 interactions with DNA is more limited and is based exclusively on studies in erythroid cells (7-9) or with purified recombinant proteins (16, 17). Several aspects of the NF-E2 protein lend complexity to this question. First, at least in erythroid cells, NF-E2 recognizes the extended DNA sequence GCTGA(G/C)TCA, which lacks dyad symmetry but includes the core symmetric AP-1 motif (underlined) known to bind dimers between the Jun and Fos subfamilies of bZip proteins. Second, there is the potential for variable dimerization between p45 NF-E2 and a number of small Maf proteins, which appear to dictate the binding site preference on DNA (10, 16, 18). Finally, a number of p45 NF-E2-related polypeptides have been identified through molecular cloning (19-23), although their interactions with specific DNA sequences have not been defined completely. These considerations raise the possibility that NF-E2·DNA complexes distinct from the one characterized in erythroleukemia cells may function within megakaryocytes in vivo. As attention turns to the mechanisms responsible for thrombocytopenia and arrested megakaryocyte differentiation in the absence of NF-E2, it is particularly important to establish the nature of this transcription factor in bona fide megakaryocytes. Here we report on our studies characterizing NF-E2 in primary murine megakaryocytes.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- Whole livers were recovered from mouse fetuses between embryonic days 13 and 15, and single cell suspensions were prepared by successive passage through 22- and 25-gauge needles. Fetal liver and mouse erythroleukemia (MEL) cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, and 0.1 mM nonessential amino acids. Fetal liver cultures were further supplemented with 0.1 µg/ml polyethylene glycol-conjugated recombinant human c-Mpl ligand (Amgen Inc., Thousand Oaks, CA) or 1% tissue culture supernatant from a murine c-Mpl ligand producer cell line (generously provided by Dr. J.-L. Villeval) (24). 75% of the cultured megakaryocytes were harvested on the fifth day of culture; with further expansion of the remaining cells, almost the same number of cells were harvested on the eighth day, after which the culture was terminated.
Purification of Primary Megakaryocytes--
For most
experiments, the entire culture of fetal liver cells was used without
additional purification of megakaryocytes. To further enrich for
megakaryocytes (see Fig. 2), fetal liver cultures containing 50-60%
acetylcholinesterase-positive cells were depleted of non-megakaryocytic
cells by incubation for 30 min at 4 °C with the monoclonal
antibodies Mac-1, GR-1, and TER-119 (Pharmingen, Los Angeles, CA),
followed by addition of magnetic beads coated with sheep anti-rat IgG
(Dynal A. S., Oslo, Norway) for 30 min at 4 °C. The immunodepleted
cell fraction contained 90% large acetylcholinesterase-positive
cells. Acetylcholinesterase activity was detected as described
previously (25).
Electrophoretic Mobility Shift Assay
(EMSA)1--
Nuclear
extracts from native or cultured fetal liver and MEL cells were
prepared according to a modification of previously described techniques
(26). Cells were lysed in 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM EDTA, and 0.5 mM dithiothreitol for 10 min
at 4 °C, and nuclei were pelleted by centrifugation at 13,500 × g for 10 s at 4 °C. Nuclear proteins were
extracted over 20 min in 20 mM HEPES, pH 7.9, 25%
glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, and 0.5 mM
dithiothreitol at 4 °C. Both incubations included protease
inhibitors (1 mM phenylmethylsulfonyl fluoride, 50 µg/ml
leupeptin, 5 µg/ml pepstatin, 10 µg/ml aprotinin, and 10 mM antipain; all from Sigma). The nuclear residue was
eliminated by centrifugation at 13,500 × g for 2 min
at 4 °C, and nuclear extracts were stored at 80 °C.
NF-E2 site | |
AP-1 core | |
NF-E2: | TGGGGAACCTGT GCTGAGTCA CTGGAG |
NF-E2 M1: | TGGGGAACCTGT TCTGAGTCA CTGGAG |
NF-E2 M2: | TGGGGAACCTGT GCTTAGTCA CTGGAG |
NF-E2 M3: | TGGGGAACCTGT ACTGAGTCA CTGGAG |
NF-E2 M4: | TGGGGAACCTGT GCTGAGTAA CTGGAG |
HS2NFE2X2: | AGCACAGCAAT GCTGAGTCATCATGAGTCA TGCTGAGCC |
NA.CRE: | TGGGGAACCTGT GCTGACGTCACTGGAG |
MAF: | TGGGGAACCTGT GCTGACGTCAGCAGAG |
Immunoblot Analysis-- Immunoblots were performed according to standard protocols (28). 20-40 µg of nuclear extract from MEL cells or primary cultured megakaryocytes was resolved by SDS-polyacrylamide gel electrophoresis; transferred to nitrocellulose; and incubated with a 1:1000 dilution of anti-p45 NF-E2, anti-MafG, or anti-p18/MafK antiserum for 1 h at room temperature. After five washes, incubation with a 1:1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham International, Buckinghamshire, United Kingdom) for 1 h, and five additional washes, the bound antibodies were detected using an enzymatic chemiluminescence kit (Amersham International) and exposure to Reflections autoradiography film (NEN Life Science Products). The anti-MafK blot was exposed approximately five times longer than the anti-MafG and anti-p45 NF-E2 blots.
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RESULTS |
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Detection of Transcription Factor DNA-binding Activity in Primary Murine Megakaryocytes-- To date, knowledge about gene regulation in megakaryocytes has derived almost exclusively from experiments conducted in immortalized cell lines. To initiate studies on transcription factor structure and function in primary murine megakaryocytes, we cultivated fetal liver cells in the presence of fetal calf serum and recombinant c-Mpl ligand for several days. Within 3-4 days into the culture, megakaryocytes, identified by their large size and acetylcholinesterase activity, were the predominant differentiated cell; although at 5 days, they constituted 50-60% of all viable nonadherent cells (Fig. 1, A and B), their substantially increased DNA content suggested that nuclear extracts from the mixed culture probably contained >90% megakaryocyte-derived proteins. Nuclear extracts prepared from nonadherent cells at 5 or 8 days included an abundant quantity of the erythroid-megakaryocytic transcription factor GATA-1, as detected by binding of this monomeric zinc finger protein to a radiolabeled specific probe in EMSAs (Fig. 1C). Hence, megakaryocytes cultured directly from mouse fetal livers constitute a useful source of nuclear proteins for biochemical and functional studies.
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Nature of the NF-E2 Complex in Primary Megakaryocytes--
In
erythroid cells, the NF-E2 heterodimer is composed of p45 and p18
subunits. Three distinct genes that are homologous to p45 NF-E2,
Nrf1 (also known as LCR-F1), Nrf2 (for
NF-E2-related factors 1 and 2) and
Ech, have been isolated (19-23), but their functions in
hematopoiesis remain uncertain (29, 30). Furthermore, the Maf family
encompasses at least three small proteins that can heterodimerize with
p45 NF-E2 and related proteins in vitro and in
vivo, including MafK (the avian homologue of p18 NF-E2), MafG, and
MafF (31, 32). Thus, a heterogeneity of DNA-protein complexes is
possible at relevant cis-elements. To investigate the
composition of the NF-E2 complex in primary megakaryocytes, we included
a set of antibodies directed against p45 NF-E2 and the different Maf
proteins in the EMSA reactions. Addition of two distinct anti-p45
antisera abolished the NF-E2 DNA-protein complex in both MEL cells and
primary megakaryocytes (Fig.
4A, lanes 3,
4, 10, and 11). Together with the
finding obtained with p45 NF-E2/
megakaryocytes (Fig.
3), these results establish that p45 NF-E2 is the only large subunit
contributing to the dominant complex in primary megakaryocytes.
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DNA-binding Specificity of NF-E2 in Primary Megakaryocytes-- The specificity of DNA binding by the NF-E2 heterodimer depends not only on p45, which recognizes the (C/G)TCA half-site, but also on the small Maf subunit, which binds to the larger GCTGA(C/G) half-site (10, 16, 18). To establish the binding specificity of the NF-E2 complex in primary megakaryocytes, we tested the ability of various modified oligonucleotides to compete for binding between the standard probe and nuclear extracts from these cells. With four different oligonucleotides modified either within the AP-1 core sequence (M2 and M4) or in the extended half-site that distinguishes NF-E2 binding from AP-1 (M1 and M3), the pattern of competition was similar between MEL cells and primary megakaryocytes (Fig. 5). Notably, however, oligonucleotide M3, which competes effectively for binding to the p45·p18 complex purified from MEL cells (9), consistently competed less well when crude nuclear extracts from either MEL cells or primary megakaryocytes were used in the EMSA (Fig. 5, lanes 5 and 11). To identify any potential differences in DNA-binding specificity between erythroid cells and megakaryocytes, we further used an extended panel of 22 oligonucleotides. Again, no significant differences in NF-E2 binding were seen in nuclear extracts from MEL cells, uncultured fetal liver (FL, representing primary erythroid cells), and primary megakaryocytes (Fig. 6). Hence, the observed difference in small Maf subunit abundance between MEL cells and primary megakaryocytes (Fig. 4) does not translate into discernible differences in DNA-binding specificity between erythroid cells and megakaryocytes. The present resolution of the NF-E2·DNA complex therefore suggests that the various Maf homologues impart overlapping rather than exclusive DNA-binding specificities. Finally, oligonucleotides 6, 8, and 17 (Fig. 6), which have an intact NF-E2 core recognition sequence, competed only partially for binding to the NF-E2 protein, indicating that residues outside the previously established consensus sequence (9, 27) can also influence the DNA-protein interaction.
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Interaction of NF-E2 with Non-consensus DNA Sequences-- The demonstrated importance of NF-E2 in megakaryocyte maturation has spurred interest in identifying its direct transcriptional targets. Because one criterion that candidate target genes must fulfill is the presence of an NF-E2-responsive site in a cis-regulatory element, it is important to define the spectrum of DNA sequences that can bind the NF-E2 protein complex found in primary megakaryocytes. AP-1-related bZip proteins can recognize both AP-1 sites and the cyclic AMP-responsive element (CRE), and NF-E2 heterodimers assembled from bacterially expressed recombinant proteins in vitro can bind a CRE sequence (GCTGAGNTCA) distinct from the NF-E2 consensus site (17). Indeed, a CRE-like DNA sequence represents the only potential NF-E2-responsive cis-element in the human thromboxane synthase gene, a candidate transcriptional target of NF-E2 in megakaryocytes (33). Additionally, symmetric sequences that preferentially bind dimers of small Maf proteins could potentially interact with the heterodimeric NF-E2 complex and mediate lineage-specific gene expression. We addressed each of these possibilities.
Although a CRE oligonucleotide (oligonucleotide 14 in Fig. 6) competed partially for binding of the NF-E2 protein to the porphobilinogen deaminase promoter probe, we did not detect direct binding of the NF-E2 protein in megakaryocyte nuclear extracts to a radiolabeled CRE probe (data not shown); other nuclear proteins dominated binding to this sequence in vitro. Furthermore, the relative affinity of the megakaryocyte NF-E2 protein for this selected CRE sequence was lower than that for the porphobilinogen deaminase promoter sequence; complete inhibition by unlabeled oligonucleotide required a
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DISCUSSION |
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Transcription factors regulate many aspects of cell differentiation. Our current appreciation of this process is based on the premise that lineage-restricted and ubiquitous transcription factors integrate the various signals that impinge upon immature cells and cooperate to establish or maintain lineage-specific programs of gene expression. The study of transcription factor functions within individual cell lineages thus greatly improves our understanding of differentiation.
Study of the transcriptional regulation of megakaryocyte maturation and platelet production has been particularly hampered by the rarity of this cell type in vivo and by the limited megakaryocyte differentiation potential of established cell lines. Targeted disruption of the p45 NF-E2 gene provided the first in vivo demonstration of the critical role of a transcription factor in megakaryocyte development: absence of p45 NF-E2 leads to arrested megakaryocyte maturation, a uniformly profound platelet deficit, and frequent death due to hemorrhage (13). As with other key lineage-restricted transcription factors, however, the mechanism of NF-E2 action in megakaryocytes is not known, making it important to study its functions within the natural context of primary cells. To this end, we have used recombinant c-Mpl ligand to culture adequate numbers of mature megakaryocytes and thus obtain materials from a physiologic source. Although the c-Mpl ligand promotes proliferation of multiple immature blood cell lineages (34), its most potent effects are exerted through all stages of megakaryocyte maturation, including platelet differentiation in vivo (35-37) and in vitro (38, 39). This relative specificity of the c-Mpl ligand on murine hematopoietic cells makes it possible to achieve significant expansion of mature megakaryocytes at the expense of other lineages (2); in our experience, the fetal liver is a superior source of megakaryocytes relative to the bone marrow, probably because of considerably greater proliferation potential of its megakaryocyte progenitors. In both cases, a large number of primary megakaryocytes serve as an excellent source of proteins and nucleic acids for biochemical analysis of NF-E2 and other transcription factors.
At least four potential large subunits (p45 NF-E2, Nrf1/LCR-F1/TCF11, Nrf2, and Ech) and three potential small subunits (p18/MafK, MafG, and MafF) of an NF-E2 or NF-E2-like protein have been identified by molecular cloning in chicken, mouse, and man (9-11, 19-23, 27, 31, 32). In addition, heterodimers of Jun- and Fos-related bZip proteins also bind the same DNA sites in vitro. While the enormous potential for heterodimerization inherent in this system suggests a complex circuitry regulating cell differentiation, such models are difficult to test in vivo. To date, only p45 NF-E2 has a clear and nonredundant role in megakaryocyte development; for example, mice lacking p18/MafK or Nrf2 show no hematologic or other detectable abnormalities (29, 40), and embryonic stem cells lacking Nrf1/LCR-F1 contribute normally to blood cell lineages in chimeric mice (30). In erythroid cells, binding to an NF-E2 site is represented by an AP-1-related complex as well as a mixture of heterodimers between p45 and two or three of the known small Maf proteins (10, 11, 40). In contrast, the nuclei of highly differentiated normal megakaryocytes harbor little AP-1 activity, and p45-containing heterodimers account for almost all of the binding to an NF-E2 site probe. This AP-1 deficit may simply reflect the lower proliferation of differentiated megakaryocytes relative to maturing erythrocytes; alternately, this might account, at least in part, for the megakaryocyte-restricted severity in the phenotype of p45 NF-E2 knockout mice (12, 13).
Many conclusions regarding structural and functional interactions between p45 NF-E2 or related proteins and the small Maf proteins are based on in vitro studies using modified recombinant proteins (16, 18). For example, MafG and MafF bind an NF-E2 probe as heterodimers with p45, Ech, c-Fos, or Nrf1/LCR-F1/TCF11, with each other, or as homodimers, with different affinities. Primary megakaryocytes do not reveal significant binding of Maf homodimers to an NF-E2 probe. However, as in erythroid cells, at least two (and possibly all three) of the known small Maf proteins are present in the dominant p45-containing complex. More interesting is the clear heterogeneity of small Maf protein expression between primary or transformed erythroid cells and primary megakaryocytes (Fig. 4): p18/MafK is under-represented in the latter relative to either whole fetal livers or MEL cells, from which the p45·p18 complex was originally isolated (9, 10). Previous studies have suggested that the expression patterns of the small Maf proteins, although wide, overlap significantly (31, 32). Our demonstration of heterogeneity in small Maf protein expression in two closely related hematopoietic lineages in which NF-E2 fulfills distinct functions emphasizes the potential significance of these differences in vivo. Again, the possibility that this disparity is partially responsible for the lack of megakaryocyte abnormalities in p18 knockout mice (40) suggests itself, but remains unproven.
Indeed, the extent to which the small Maf proteins, which lack canonical transactivation domains, might compensate for each other functionally is presently unclear. Experimental evidence indicates that these subunits recognize the extended portion of the NF-E2 consensus sequence that distinguishes it from an AP-1 site and thus establish the DNA-binding specificity of the heterodimer (10). Although this raises the possibility that the various small Maf proteins manifest distinct DNA-binding preferences, our data rather suggest that the DNA sequence preferences overlap. Despite the differences between erythroid and megakaryocytic cells in the contribution of individual small Maf proteins to the NF-E2 complex (Fig. 4), the profile of competition by unlabeled oligonucleotides with either source of nuclear extract is substantially similar (Fig. 6). Nevertheless, the heterogeneity in small Maf proteins among closely related hematopoietic lineages leaves open the possibility that these proteins, which appear to lack a transactivation domain, fulfill important functions other than determining DNA-binding site preferences, such as interacting with other transcriptional regulators. This hypothesis may be tested directly once megakaryocyte promoters with functional NF-E2-binding sites are identified and characterized.
In conclusion, our studies provide the first example of detailed characterization of a transcription factor in primary megakaryocytes rather than immortalized multipotential cell lines. Further analysis of NF-E2 and other important lineage-restricted transcription factors in primary megakaryocytes should continue to shed light on the transcriptional control of platelet biogenesis.
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ACKNOWLEDGEMENTS |
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We are grateful to Bethany Swencki for outstanding technical assistance; to Nancy Andrews, and Paul Ney for generously providing antisera and selected oligonucleotide competitors; to Amgen, Inc. and Jean-Luc Villeval for providing recombinant c-Mpl ligand; and to Paresh Vyas and Stuart Orkin for helpful comments on the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a fellowship from the Association pour la Recherche contre le Cancer (France).
** Supported in part by Grant HL03290 from the National Institutes of Health. To whom correspondence should be addressed: Dana-Farber Cancer Inst., 44 Binney St., Boston, MA 02115. Tel.: 617-632-5746; Fax: 617-632-5739; E-mail: ramesh_shivdasani{at}dfci.harvard.edu.
1 The abbreviations used are: EMSA, electrophoretic mobility shift assay; CRE, cyclic AMP-responsive element.
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REFERENCES |
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