Leukemia (1997) 11, 273–280 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 NF-E2p18/mafK is required in DMSO-induced differentiation of Friend erythroleukemia cells by enhancing NF-E2 activity C Francastel, V Poindessous-Jazat, Y Augery-Bourget and J Robert-Le´ze´ne`s INSERM U268, Hoˆpital Paul Brousse, 94800 Villejuif, France When Friend murine erythroleukemia (F-MEL) cells are induced (LCR).6 The b-globin complex LCR (b-LCR) include four to differentiate by dimethylsulfoxide (DMSO), erythroid-specific erythroid-specific DNasel hypersensitive sites (designated HS- genes are transcriptionally activated. The erythroid transcrip- 1 to HS-4) and acts as a powerful enhancer of transcription tion factor NF-E2 is essential for enhancer activity of the globin 7,8 locus control regions. NF-E2 functions as a heterocomplex of globin promoters. While all four HS have LCR activity in consisting of a 45-kDa subunit (NF-E2p45) and a 18-kDa sub- transgenic mice, only one region (HS-2) is crucial for unit (NF-E2p18). The larger subunit NF-E2p45 is tissue-restric- enhancer activity when assayed in transient transfection ted and is believed to play a role in globin gene expression in systems.9–11 Within HS-2, the enhancer activity for globin F-MEL cells. The expression of the smaller subunit NF-E2p18, expression requires a tandem repeat of AP-1-like which is a Maf family member (MafK), is cell type- and develop- 12,13 mental stage-specific. We have investigated the possible role elements. This tandem motif was shown to bind two tran- of NF-E2p18 in Friend erythroid differentiation by stably trans- scription factors: the ubiquitous AP-1 protein and the lineage fecting either sense and antisense p18 constructs into differen- limited NF-E2 protein.14,15 The NF-E2 complex is a hetero- tiation-sensitive 745A and partially defective-differentiation dimer of two b-Zip proteins of 45 and 18 kDa.16,17 While NF- TFP10 cell lines. Overexpression of NF-E2p18 induced E2p45 expression is restricted to cells of the erythroid megaka- expression of globin transcripts in both cell lines and increased ryocyte and mast cell lineage, the expression of the smaller their sensitivity to erythroid differentiation when exposed to subunit NF-E2p18 is cell type- and developmental stage-spe- DMSO. Conversely, inhibition of p18 expression by antisense 18 transcripts resulted in the inhibition of DMSO-induced differen- cific. Since p45 homodimers bind very poorly to NF-E2 tiation in both cell lines. These results indicate that NF-E2p18 motifs, NF-E2p45 protein binds as an obligate heterodimer is necessary for globin expression in F-MEL cells and that it is with NF-E2p18 protein,16 to form the NF-E2 complex which the predominant gene of the Maf family involved in DMSO- has been described as the transcriptional activator of NF-E2 induced erythroid differentiation. Moreover F-MEL clones over- enhancer elements.19 The smaller subunit NF-E2p18 protein expressing NF-E2p18 showed an increase in specific NF-E2 DNA-binding activity whereas this activity was decreased in is encoded by a Maf family gene, the mafK gene, and lacks 20,21 clones expressing antisense p18. Finally, studies using transi- the putative trans-activator domain. Enhancer activity of ent transfection assays showed that p18 activated NF-E2 site- the a-globin locus HS-40 element is also dependent on NF- dependent transcription in F-MEL cells. These data suggest E2/AP-1 sequences.22 Beside a- and b-globin LCR, NF-E2/AP- that NF-E2p18 can participate in DMSO-induced erythroid 1 binding sites are also present in the promoters of three genes differentiation of F-MEL cells by enhancing NF-E2 activity. encoding heme biosynthetic enzymes, porphobilinogen Keywords: Friend erythroleukemia; NF-E2p18; Maf protein; 14 23 erythroid differentiation; globin expression deaminase, 5-aminolevulinate synthetase and ferrochelat- ase24 suggesting a broad role for the protein NF-E2 in erythroid-specific gene expression. The role of the NF-E2 complex and its subunits p45 and Introduction p18 in the DMSO- or HMBA-induced erythroid differentiation of F-MEL cells is unclear. It was reported that both NF-E2 DNA Friend murine erythroleukemia (F-MEL) cells are virus-trans- binding and NF-E2 site-dependent transcriptional activities formed erythroid precursors cells blocked at an early stage increase during the induction of MEL differentiation.13,14,25,26 in erythroid differentiation. This arrest can be overcome by Recent studies on one Friend erythroleukemia cell line, which exposure to a variety of chemical inducers, including DMSO lacks NF-E2 p45 due to Friend virus integration, showed a and HMBA.1,2 Variants of F-MEL cells have been obtained drastic reduction in expression of the a- and b-globin genes.27 which differ in their response to chemical inducers. These cell Reintroduction of the NF-E2p45 gene into this cell line par- models are useful for studying the mechanism of erythroid dif- tially restores globin RNA expression suggesting that it may ferentiation since induced F-MEL cells undergo phenotypic be critical for differentiation of F-MEL cells. The presence of changes that resemble the final changes of normal erythropo- NF-E2p18 in the NF-E2 complex increases binding specificity iesis. Induction of erythroid differentiation of MEL cells by for the bases lying outside the AP-1 core of the NF-E2 consen- DMSO or HMBA is characterized by the cessation of DNA sus binding site, suggesting its possible role in transcription synthesis and by the expression of hemoglobin in the cells.3 activation of the globin gene.17 Igarashi et al28 have recently The most striking feature during the process of induced differ- reported that overexpression of mafK induces spontaneous entiation of F-MEL cells is the increased synthesis of globin erythroid differentiation of a Friend cell line. However, the chains which results from the activation of a and b globin molecular basis of this induction of differentiation by gene transcription.4,5 p18/mafK, specially the involvement of NF-E2 activity, has not Expression of mammalian globin genes depends on been clarified. upstream regulated elements, termed locus control regions In order to gain better understanding of the function of NF- E2p18/MafK during the DMSO-induced differentiation of F-MEL cell, we have introduced NF-E2p18 expression vectors Correspondence: J Robert-Le´ze´ne`s INSERM U268, Hoˆpital Paul- Brousse, 14 avenue Paul Vaillant Couturier, 94807 Villejuif cedex, in both sense and antisense orientations into differentiation- France sensitive 745A and partially defective TFP10 Friend cell lines. Received 26 July 1996; accepted 24 October 1996 Sense p18 vectors increase the sensitivity to DMSO-induced NF-E2p18 and erythroid differentiation C Francastel et al 274 Figure 1 Expression of NF-E2p45 and NF-E2p18 mRNAs in F-MEL cells (strains 745A and TFP10), following stimulation with DMSO. Poly(A)+ mRNA (5 mg) was isolated at the time points indicated (in hours) after addition of 2% (v/v) DMSO. Northern blots were hybridized sequentially to probes homologous to NF-E2p45, NF-E2p18 and GAPDH followed by autoradiography. The different transcripts migrate as follows: NF- E2p45 1.8 kb; NF-E2p18 3.1 kb; GAPDH 1.4 kb. Figure 2 NF-E2p45 protein expression and NF-E2 DNA complex formation in F-MEL cells following stimulation with 2% of DMSO. (a) Western blot analysis of total proteins using a NF-E2p45-specific antibody. Total protein 50 mg were separated by SDS-PAGE and blotted onto nitrocellulose. The blots were developed using the alkaline phosphatase system. The time in hours of DMSO treatment is indicated. (b) DNA- binding activity of NF-E2 in nuclear extracts proteins from F-MEL cells before DMSO treatment. The oligonucleotide probe containing NF-E2 consensus sequence was incubated with 10 mg of nuclear extract protein and EMSA were performed as described in Materials and methods. Specificity of binding was confirmed by competition with excess unlabeled oligonucleotide probe (not shown). Where indicated, 1 ml of anti- NF-E2p45 antiserum was added before addition of probe. (c) DNA-binding activity of NF-E2 in nuclear extracts proteins from F-MEL cells after DMSO treatment (4 days). erythroid differentiation of both cell lines. Conversely, trans- increasing both NF-E2 DNA-binding and NF-E2 transcrip- fection with antisense p18 vectors inhibits the induction of tional activities. F-MEL erythroid differentiation by the DMSO suggesting that NFE2p18/MafK is necessary to induce globin expression in F- MEL cells during the process of differentiation. In an effort to Materials and methods elucidate the molecular mechanism that underlie the relation- ship between p18/MafK and F-MEL differentiation, we show a Plasmid constructs specific increase of the NF-E2 DNA-binding activity in clones overexpressing p18. In addition, we demonstrate that NF- The expression vector used for the construct was pcDNA3 E2p18 induces an increase of NF-E2 site-dependent transcrip- (Invitrogen, San Diego, CA, USA). Murine NF-E2p18 cDNA tional activity in both F-MEL cell lines suggesting that it can containing one ORF (open reading frame) was excised from be a positive regulator in erythroid cells which contain NF- its plasmid vector (gift from N Andrews, Dana-Farber Cancer E2p45. Taken together, these findings indicate that p18/MafK Institute, Boston, MA, USA) as 0.51-kb EcoRI fragment and can participate in erythroid differentiation of F-MEL cells by ligated into EcoRI-digested pcDNA3 with the sense and anti- NF-E2p18 and erythroid differentiation C Francastel et al 275 Figure 3 Construction of NF-E2p18 sense and antisense vectors (pcDNA3-p18S and pcDNA3-p18AS). A 510-bp EcoRI/EcoRI cDNA fragment encompassing the entire coding region was inserted into the corresponding sites of the expression pcDNA3 vector (Invitrogen).