©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activity and Expression of Murine Small Maf Family Protein MafK (*)

(Received for publication, September 8, 1994; and in revised form, November 30, 1994)

Kazuhiko Igarashi (1) Ken Itoh (1) Hozumi Motohashi (1) Norio Hayashi (1) Yumi Matuzaki (2) Hiromitu Nakauchi (2) Makoto Nishizawa (3) Masayuki Yamamoto (1)

From the  (1)Department of Biochemistry, Tohoku University School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai 980-77, the (2)Department of Immunology, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, and the (3)Department of Molecular Oncology, Kyoto University Faculty of Medicine, Yoshida-Konoecho, Sakyo-ku, Kyoto 606, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transcription factor NF-E2 is believed to be crucial for the regulation of erythroid-specific gene transcription. The three small Maf family proteins (MafF, MafG, and MafK), which are closely related to c-Maf proto-oncoprotein, constitute half of NF-E2 activity by virtue of forming heterodimers with the large, tissue-restricted subunit of NF-E2 (p45). We isolated cDNA clones encoding the murine small Maf family protein MafK and characterized the structure, activity, and expression profile of MafK mRNA. Functional analyses demonstrate that MafK binds to consensus NF-E2 sites in the absence of p45 in vitro and represses transcription of NF-E2 site-dependent reporter genes in transient transfection assays, while p45 introduced into cells alone does not effectively bind to DNA and does not affect transcription. In the presence of p45, MafK confers site-specific DNA binding activity to p45, and p45 in turn mediates transcriptional activation with its amino-terminal proline-rich domain. mRNA for MafK is expressed in fractions enriched for hematopoietic stem cells as well as erythroid cells, suggesting that MafK plays an important regulatory role in hematopoiesis.


INTRODUCTION

Six members of the maf proto-oncogene (1) family have been identified. The translation products of the family genes possess a conserved basic region-leucine zipper (b-zip) (^1)domain that mediates dimer formation and DNA binding(2) . While chicken v-Maf(3) , MafB(55) , and human NRL (4) contain putative transcription activation domains, chicken MafF and MafK (5) and MafG (56) lack any canonical trans-activation domains. MafF, MafG, and MafK are essentially composed of b-zip domains and are collectively referred to as the small Maf family proteins. mRNAs for these small Maf family proteins are expressed in a wide range of tissues in chicken(5) . The homodimer of each Maf family member binds to either 13- or 14-bp palindromic sequences that contain 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE) (TGA(C/G)TCA) and cAMP-responsive element (TGACGTCA), respectively(2) . The 13-bp sequence is TGCTGAGTCAGCA (named T-MARE for TRE-type Maf recognition element), whereas the 14-bp sequence is TGCTGACGTCAGCA (named C-MARE).

Various cellular genes have been shown to harbor T-MARE- or C-MARE-like sequences in their transcriptional regulatory regions(2) . Specifically, T-MARE resembles the recognition element for the NF-E2, which was originally described as an erythroid-restricted DNA binding activity(6) . The consensus binding sequence for NF-E2 is TGCTGAGTCAT (7) , hence 10 out of the 11 nucleotides of the NF-E2 site are identical to that of T-MARE. Although the NF-E2 binding site contains TRE, sequences outside of the TRE are also essential for the binding of NF-E2(6, 7, 8) .

The recent molecular description of the large subunit (p45) of mouse NF-E2 showed that the p45 protein also possesses a b-zip domain (7) but that p45 alone cannot bind to an NF-E2 site. p45 was therefore suggested to comprise one chain of a heterodimer with another unknown polypeptide to constitute NF-E2 activity(7, 9) . Andrews et al.(10) subsequently isolated a cDNA clone encoding an 18-kDa polypeptide, which copurified with p45 from murine erythroleukemia (MEL) cells. They found that the 18-kDa polypeptide formed a heterodimer with p45 and that its primary structure shows a significant similarity to that of the chicken v-Maf protein. Consistent with these observations, we found that the chicken small Maf family proteins, but not other members of the Maf family, can specifically form heterodimers with mouse p45(11) . We also found that the small Maf family proteins function as efficient transcription repressors in a transient transfection assay and that p45 antagonizes the repressor function of the small Maf family proteins.

Here, we report a cloning and characterization of mouse cDNAs encoding the small Maf family protein MafK. Three issues were then addressed in this study. First, we examined the interaction between murine MafK and p45 and their transcriptional regulatory properties. These analyses, with aid of anti-p45 antiserum, verified the results previously obtained with mouse p45 and chicken MafK or other small Maf family proteins. Second, we identified a domain that mediates transcription activation by the p45bulletMafK heterodimer by introducing a mutation into the p45 molecule, which does not affect the heterodimer formation or binding to the NF-E2 sites. Third, to evaluate a potential regulatory role of MafK, we analyzed the expression profile of the mRNA encoding MafK in various mouse tissues and primary bone marrow cells after separation by flow cytometry. Results of these analyses indicate that a p45bulletMafK heterodimer directly activates transcription from NF-E2 sites using the amino-terminal proline-rich domain of p45 and suggest that both MafK and p45 function from early stages of hematopoiesis.


MATERIALS AND METHODS

Library Screening

A mouse fetal liver gt11 cDNA library (generous gift from Drs. Minetaro Ogawa and Shin-ichi Nishikawa, Kyoto University) was plated on 150-mm Petri dishes at the density of 6 times 10^4 plaque-forming units per plate. Duplicate plaque lifts were made from each plate and processed for hybridization as previously described(12) . A total of 2 times 10^6 plaques were screened by using either individual or a mixture of cDNA and genomic DNA fragments derived from chicken mafF, mafG, and mafK as probes. Hybridization was carried out at 42 °C in 5 times SSC, 5 times Denhardt's solution, 1% SDS, 10 mM Tris-HCl (pH 7.4), 25% formamide, and 0.2 mg/ml denatured salmon sperm DNA. The membranes were washed two times each for 30 min at 50 °C in 2 times SSC and 0.1% SDS, and the hybridization signals were visualized by autoradiography. Positive plaques were isolated and purified by three additional rounds of plaque hybridization screening.

Four phage clones were isolated through these screenings. All four clones were judged to be derived from the same gene based on the results of restriction enzyme mapping. The longest cDNA insert in clone 9 was subcloned into pBluescript (Stratagene), and its sequence was determined on both strands using deletion subclones as templates. Sequencing reaction was carried out using Taq DyeDeoxy cycle sequencing system (ABI Japan, Tokyo), and sequence data were collected with an automated DNA sequence analyzer (ABI model 373A).

Construction of Plasmids

Prokaryotic expression plasmids for mouse MafK were constructed as follows. The phage clone 4 encodes a segment from the polyadenylation consensus sequence up to the second codon of the mouse mafK mRNA and carries EcoRI adapter sequence instead of the first ATG codon. The 0.75-kb EcoRI-PstI fragment of the insert of 4 clone was subcloned into the HindIII site of the pMALc2 vector (New England Biolabs) after filling in the relevant DNA ends with T4 DNA polymerase. The resulting plasmid encodes a fusion protein of maltose binding protein (MBP) and the entire mouse MafK except the first methionine (MBP-MafK). The plasmid that expresses a fusion protein of MBP and p45 (MBP-p45) was previously described(11) . To construct a plasmid that expresses p45 fused with glutathione S-transferase, the NcoI-HindIII fragment of the p45 cDNA (11) was inserted into the BamHI site of pGEX2T vector (Pharmacia Biotech Inc.) after filling in the DNA ends involved.

The eukaryotic expression plasmid of mouse MafK (pEFmMafK) was constructed by inserting the 0.76-kb SmaI-PstI fragment (see Fig. 1A) of 9 clone into the BssHII site of pEF-BssHII, a modified version of pEF-BOS(13) , after filling in of the DNA ends involved. The pEFp45, which expresses p45 NF-E2, was previously described(11) . The amino-terminal truncation mutation of p45 was created by deleting a region between the NcoI site and HpaI site of the p45 cDNA(11) . The resulting cDNA encodes a mutant p45 (p45DeltaNd) whose first methionine is fused to the 244th asparagine and lacks amino acid residues between them. This mutant p45 cDNA was transferred to the pEF-BssHII vector, resulting in pEFp45DeltaNd.


Figure 1: Structure of mouse mafK cDNA. A, schematic representation of the structure of mouse mafK cDNA. The coding region is indicated by an arrow, and fragments used for RNA hybridization analysis and functional analyses are indicated by lines. Restriction enzyme sites indicated are EcoV, EcoRV; Nco, NcoI; Pst, PstI; Sac, SacI; and Xho, XhoI. B, nucleotide sequence of the mouse mafK cDNA clone. The deduced amino acid sequence is indicated below the line in the standard one-letter amino acid code. The termination codon is indicated by an asterisk. Polyadenylation consensus sequence (AATAAA) is shown in boldfacetype. Five ATTTA sequences that may be involved in the regulation of mRNA stability (30) are underlined. C, comparison of the amino acid sequences of chicken and mouse small Maf family proteins. Conserved amino acids among the four small Maf family proteins are boxed. A region conserved among Maf family members and the leucine repeats are indicated with arrows and dots above the line, respectively.



The luciferase reporter plasmids pRBGP2 and pRBGP4 were previously described(11) . The luciferase reporter plasmid pRBGP6 that carries the HS-2 region of mouse beta-LCR in front of a rabbit beta-globin TATA box was constructed as follows. A DNA fragment of the HS-2 containing the tandem NF-E2 sites (14) was PCR-amplified from mouse genomic DNA using a set of primers (5`-ATTATTGCAGTACCACTGTC-3` and 5`-CTTTTCACCTTCCCTGTGGA-3`) and cloned in the SmaI site of pRBGP3 that carries TATA-luciferase gene fusion, resulting in pRBGP6. The integrity of the HS-2 DNA inserted in pRBGP6 was verified by sequencing. The direction of the HS-2 in pRBGP6 was that GATA site was close to, and the tandem NF-E2 sites were far from, the TATA box (see Fig. 5A).


Figure 5: Regulation of transcription by MafK and p45 through beta-LCR HS-2. A, sequence of the mouse beta-LCR HS-2 inserted in the reporter plasmid pRBGP6. NF-E2 sites, GATA site, and CACCC element are indicated. B, results of transfection assays. Reporter gene activities in the presence of various combinations of the MafK and p45 expression plasmids (1.0 µg) are shown. Values are means of two independent transfections, each carried out in duplicate. Standard error is indicated with bars.



Bacterial Expression of Recombinant Transcription Factors

The plasmids that express either mouse MafK (above) or p45 (11) as fusions with MBP were introduced into the Escherichia coli strain SG12036, and the expressions of the fusion proteins were induced with 0.1 mM isopropyl-1-thio-beta-D-galactopyranoside. The fusion proteins were purified from the cell lysates by amylose resin affinity column chromatography as previously described(2) , stored on ice, and used within 3 weeks after preparation.

Preparation of Anti-p45 Antibody

Full-length p45 expressed in E. coli as a fusion protein with glutathione S-transferase (GST-p45, see above) was purified with glutathione affinity column (Sigma) and MonoQ (Pharmacia) column chromatography. Purified GST-p45 fusion protein was administered subcutaneously to a Japanese white rabbit as emulsions with RIBI adjuvant system (RIBI ImmunoChem, Hamilton, MT) after collecting the preimmune serum. The antiserum was collected after three successive immunizations following standard procedures(15) .

Transient Transfection Assays

Quail fibroblast cell line QT6 (16) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and plated 24 h before transfection. Cells were transfected by calcium phosphate precipitation (12) with 1.0 µg of reporter plasmids, 0.5 µg of pENL, which expresses beta-galactosidase as an internal control, various combinations of effector plasmids, e.g. pEFmMafK, pEFp45, or pEFp45DeltaNd, and pEF-BssHII to equalize the total amount of DNA (5.0 µg/60-mm dish) and the promoter DNA transfected. 12 h after transfection, cells were washed twice with phosphate-buffered saline, fed with fresh Dulbecco's modified Eagle's medium with 10% fetal bovine serum, and incubated for another 24 h. Preparation of cell lysates and luciferase assay of them were carried out utilizing the luciferase assay system (Promega) following the supplier's protocol and with Biolumat luminometer (Berthold Japan, Tokyo). beta-Galactosidase activities in the cell lysates were assayed as described(12) . Results are reported as mean of relative luciferase activities normalized for beta-galactosidase activities from two to four independent transfections each carried out in duplicate. To analyze DNA binding activities generated by the effector plasmids in transfected cells, transfection was carried out as described above except for the omission of the reporter and internal control plasmids.

Electrophoretic Gel Mobility Shift Analysis (EGMSA)

Whole cell extracts and nuclear extracts were prepared from QT6 cells using previously described protocols(17, 18) . Double-stranded oligonucleotide DNAs were labeled with [-P]ATP and T4 polynucleotide kinase. EGMSA with proteins purified from overexpressing E. coli strains were performed as previously described(2) . Assays with cell extracts prepared from transfected QT6 cells were carried out similarly except that incubation of the extracts with probe DNA was carried out at 25 °C for 10 min. Where indicated, the rabbit preimmune or the anti-p45 serum was added to the binding reactions at dilution, and the reactions were incubated for 10 min on ice before addition of probe DNAs. The reactions were electrophoresed at 4 °C and at 200 V for 2 h on a 4% polyacrylamide gel in 45 mM Tris borate buffer containing 1 mM EDTA.

RNA Blot Hybridization Analysis

Total RNAs were prepared from cultured cells or various mouse tissues with guanidine-acidified phenol chloroform method(19) , electrophoretically separated on 1.0% agarose gel containing 1.1 M formaldehyde, and transferred onto ZetaProbe membranes (Bio-Rad). Radiolabeled RNA probes specific for mouse mafK and p45 mRNAs were synthesized with T3 RNA polymerase and hybridized with the membranes as previously described(20) .

Bone Marrow Cell Sorting

Bone marrow mononuclear cells were obtained from 6-week-old female C57BL/6 mice from Clea Japan (Tokyo). For cell sorting, biotinylated rat monoclonal antibodies, RA-6B2 (anti-B220)(21) , M1/70 (anti-Mac-1)(22) , RA3-8C5 (anti-Gr-1) (23) , GK1.5 (anti-L3T4)(24) , 53-6.7 (anti-CD8alpha-chain)(25) , and TER119 (26) were used as lineage markers. Fluorescein (FITC)-labeled Sca-1 and allophycocyanin (APC)-conjugated ACK-2 (anti-c-Kit) were used as stem cell markers(27) . TER119-biotin was a generous gift from Dr. T. Kina (Kyoto University). All biotinylated reagents were visualized by using Streptoavidine-PE second-step reagent (Becton Dickinson Immunocytometry Systems, San Jose, CA). TER119 recognizes a glycophorin-like molecule whose expression is restricted to erythroid lineage cells, especially of erythroblast and later stage(26) . B220 is an isoform of CD45 that is expressed on B lymphocytes, and Gr-1 is expressed on myeloid lineage cells.

Mouse bone marrow cells (1 times 10^7) were reacted with a mixture of biotinylated rat monoclonal antibodies specific for mouse differentiation antigens Gr-1, Mac-1, B220, TER119, CD4, and CD8 (see above for antibody designations) for 30 min at 4 °C. After washing the cells three times with phosphate-buffered saline, cells were reacted with Sca-1-FITC, c-Kit-APC, and SAV-PE at 4 °C for another 30 min. After washing with phosphate-buffered saline again, the cells were resuspended in staining medium at the final concentration of 1 times 10^6 cells/ml supplemented with propidium iodide ( 1 µg/ml). Bone marrow cells were also stained with c-Kit-APC, TER119-FITC, Gr-1-PE, and B220-PE. The stained cells were analyzed by FACSterplus (Becton Dickinson) equipped with a 488-nm argon laser and a 599-nm dye laser. Data from 5 times 10^4 cells were collected and analyzed. After analysis, 1 times 10^4 cells in 1) Kit/Sca-1/Lin, 2) Kit/Sca-1/Lin, 3) Kit/TER119/Gr-1/B220 and 4) Kit/TER119/Gr-1/B220 fractions were sorted. Residual erythrocytes, debris, doublets, and dead cells were excluded by forward scatter, side scatter, and propidium iodide gating.

RT-PCR Analysis

Polyadenylated RNA was isolated from the sorted cells (1 times 10^4-10^5 cells per purification) with QuickPrep mRNA purification kit (Pharmacia). Semi-quantitative PCR analysis (28) was carried out as follows. cDNA was synthesized using the mRNAs as templates with Superscript II reverse transcriptase (Life Technologies, Inc.) and random hexamer oligonucleotide primers. First, PCR was carried out using a series of dilutions of the reverse transcribed cDNAs with a primer set specific for -actin cDNA(29) . (^3)The reaction products were electrophoresed on agarose gels, and relative amounts of specific PCR products were determined by staining of the gels with ethidium bromide. Based on these results, the relative yields of cDNAs synthesized from RNAs of various cell fractions were evaluated. Then, using defined amounts of cDNAs containing similar amounts of -actin cDNA as templates, PCR was carried out with specific primers for MafK, p45, and GATA-1 cDNAs. All PCR reactions were carried out in a 50-µl system using 0.5 units of Tth DNA polymerase (Toyobo, Osaka) and 100 pmol of each primer, with a thermal cycler (ASTEC, Shizuoka, Japan) in a profile of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min for 30 cycles. The products were resolved on 2% agarose gel, stained with ethidium bromide, and transferred onto ZetaProbe membranes. The filters were hybridized with radiolabeled DNA fragments specific for each target cDNA. The primer sets for amplification of -actin, MafK, p45, and GATA-1 cDNAs and the sizes of specific cDNA amplification products are summarized in Table 1.




RESULTS

Isolation of a Mouse mafK cDNA Clone

Four bacteriophage clones with similar restriction enzyme site patterns were isolated from a fetal mouse liver cDNA library by screening with mixture of chicken mafF, mafG, and mafK DNA fragments as probes under low stringent hybridization and washing conditions (Fig. 1A). Sequence analysis of the longest cDNA insert (2.8 kb in clone 9) revealed that it encoded an open reading frame (ORF) located at the 5`-side of the cDNA. This ORF was flanked by 224 and 2195 bp of 5`- and 3`-untranslated regions, respectively (Fig. 1B). There is a polyadenylation consensus sequence (AATAAA) before the poly(A) stretch at the 3`-end of the clone, indicating that the cDNA was probably primed from an authentic post-transcriptionally added poly(A) sequence in the mRNA. There are five copies of RNA destabilizing signal (ATTTA) (30) in the 3`-untranslated region, which may have some significance in regulation of the stability of this mRNA.

The deduced amino acid sequence of the ORF showed 96% identity with that of chicken MafK, indicating that this clone encodes a genuine mouse homologue of chicken MafK (Fig. 1C). Mouse MafK consists primarily of a b-zip coding domain, and the region is highly conserved between the mouse and chicken proteins. In addition, regions outside the b-zip domain are also highly conserved. Mouse MafK showed a high degree of similarity in primary structure with other members of the small Maf family proteins (Fig. 1C).

Andrews et al.(10) recently reported the cloning of the small subunit (p18) of mouse NF-E2 and found that the structure of the p18 protein was predicted to have high similarity to that of the oncoprotein, v-Maf. The reported cDNA clone is 516 bp long and contains one ORF. Although the cDNA corresponds to a significantly smaller region than that covered by the present mouse mafK clone (2.8 kb), the cDNA sequence corresponds almost precisely to that of the mouse MafK determined here, indicating that the two cDNAs are almost certainly derived from the same gene. Comparison of the two cDNA sequences shows two consecutive mismatches, resulting in two amino acid differences at the 36th and 37th positions (EL instead of DV) (Fig. 1B). Even though we have verified this sequence by comparison to the corresponding region of a mouse mafK genomic DNA clone, (^4)it remains possible that these differences are due to alleic variations.

MafK Binds to DNA by Forming a Homodimer or a Heterodimer with the Large Subunit of NF-E2 (p45)

We previously showed that chicken MafK can modulate the DNA binding activity of p45 subunit of NF-E2 in vitro. To examine the DNA binding property of mouse MafK, p45 and mouse MafK were expressed in E. coli as fusion proteins with the MBP. The overexpressed proteins were then purified and tested in EGMSA.

For this analysis, we examined three kinds of potential NF-E2 binding sites (Fig. 2A). Two of them contained the NF-E2 site of the porphobilinogen deaminase (PBGD) gene. The first probe is an authentic PBGD NF-E2 site originally described by Mignotte et al.(6) and contains the natural sequences of the NF-E2 site and its surrounding region (PBGD probe). The second probe is a modified version of the first, containing the NF-E2 site of the PBGD gene in a sequence context that was previously used to determine the binding sequence for the v-Maf homodimer (PBGD/M)(2) . The third probe contains the NF-E2 site found in the chicken beta-globin gene enhancer (CbetaE2 probe)(31) .


Figure 2: Interaction with p45 NF-E2 and DNA binding of mouse MafK. A, summary of oligonucleotide probes containing NF-E2 sites used for EGMSA. PBGD and PBGD/M probes represent the NF-E2 site of human porphobilinogen gene in native or modified context, respectively. CbetaE2 was from the NF-E2 site of chicken beta-globin enhancer. B, autoradiographic image of EGMSA with the authentic PBGD probe. Binding reactions were carried out with proteins expressed in and purified from E. coli as follows: 100 ng of MBP (lane1), 100 ng of MBP and 20 ng of MBP-p45 (lane2), 100 ng of MBP-MafK and 20 ng of MBP (lane3), or 100 ng of MBP-MafK and 20 ng of MBP-p45 (lanes4-6). Anti-p45 antiserum or preimmune serum was included in binding reactions in lanes5 and 6, respectively. C, autoradiographic image of EGMSA with the modified PBGD NF-E2 site probe. Binding reactions were carried out with 50 ng of MBP-p45 (lane1), 50 ng each of MBP-p45 and MBP-MafK (lane2), or 50 ng of MBP-MafK (lane3). D, autoradiographic image of EGMSA with the CbetaE probe. Binding reactions were carried out with 50 ng of MBP-MafK in the absence (lane1) or presence (lane2) of 50 ng of MBP-p45.



Fig. 2B shows the binding characteristics of the fusion proteins to the authentic PBGD probe. Mouse p45 homodimer bound to the authentic PBGD probe only very weakly (lane2). In the presence of both p45 and MafK, however, a strong signal that migrated much faster than the p45 homodimer complex appeared (lane4), and the weak p45 homodimer complex concomitantly disappeared. The new complex was shown to contain p45 by virtue of the fact that formation of this complex was completely abolished by the addition of anti-p45 antiserum (lane5) but not by the addition of preimmune serum (lane6). As is the case for chicken MafK(11) , mouse MafK did not bind to the authentic PBGD probe (lane3). However, when assayed with the PBGD/M probe, MafK clearly bound to the probe as a homodimer (Fig. 2C, lane3). As was the case for the PBGD probe, another nucleoprotein complex appeared when both p45 and MafK were included in the reaction (Fig. 2C, lane2). The mobility of this complex was slower than that of the MafK homodimer, indicating that this band represents a p45bulletMafK heterodimer. Based on these results, we concluded that MafK modulates the DNA binding activity of p45 (and vice versa) by heterodimer formation.

Consistent with the observations using the PBGD and PBGD/M probes, the mouse MafK protein bound to the NF-E2 site of the chicken beta-globin enhancer as either a homodimer (Fig. 2D, lane2) or as a heterodimer with NF-E2 p45 (lane1). As previously noted(11) , binding of the heterodimer to this CbetaE2 probe was more prominent than that of the MafK homodimer (see below).

Regulation of Transcription by MafK and p45 in Vivo

We next addressed the possible function of mouse MafK as a transcription regulator using a transient cotransfection assay. Various amounts of mouse MafK expression plasmid were transfected into a quail fibroblast QT6 cell line with reporter plasmids. We examined two different reporter genes in these assays. pRBGP2 contains three copies of the chicken beta-globin gene enhancer NF-E2 sites 5` to a rabbit beta-globin TATA box, which then directs transcription of the firefly luciferase gene. pRBGP4 is the same as pRBGP2 but contains mutated NF-E2 sites instead of the wild-type sequence; the mutated NF-E2 sites have previously been shown to fail to bind p45/small Maf heterodimers(11) . In the absence of effector plasmid, luciferase reporter activity generated from pRBGP2 within the transfected cells was roughly 300-fold greater than that from pRBGP4 (see Fig. 3legend). This endogenous cellular activity, which stimulates expression of the reporter plasmid through the NF-E2 sites was less evident when a reporter plasmid with TRE, which cannot be bound by Maf family proteins, was used. (^4)This observation suggests that, in addition to AP-1 family factors, c-Maf or its related proteins may also contribute to the endogenous activity stimulating transcription from NF-E2 sites.


Figure 3: Transcriptional regulation by MafK and p45 in QT6 cells. A and B, transcription repression by MafK. Various amounts of pEFmMafK were transfected into QT6 cells with 1.0 µg of either pRBGP2 (A) or pRBGP4 (B) reporter plasmids. Reporter gene activities, relative to that of pRBGP2 in the absence of pEFmMafK, are shown as a function of amount of the effector plasmid cotransfected. The results are the mean of three independent transfections, each carried out in duplicate, and the standard error is shown by bars. C, effect of p45 and p45DeltaNd on MafK-mediated transcription repression. Various amounts of pEFp45 or pEFp45DeltaNd plasmid were transfected into QT6 cells with fixed amounts of pRBGP2 (1.0 µg) and pEFmMafK (0.8 µg), and relative reporter gene activities are shown as a function of amounts of pEFp45 or pEFp45DeltaNd. The reporter activity in the absence of pEFmMafK was set as 100%. The results are the mean of four (pEFp45, closedtriangle) or two (pEFp45DeltaNd, opensquare) independent transfections, each carried out in duplicate, and a standard error is shown by bars.



Cotransfection of the MafK expression plasmid with pRBGP2 efficiently repressed reporter gene activity in a dose-dependent manner (Fig. 3A). In contrast, the same plasmid did not repress reporter activity from pRBGP4 (Fig. 3B). Thus, the ability of MafK to repress transcription was dependent on the presence of functional NF-E2 binding sites on the reporter gene plasmid. Transcription repression by MafK could be completely reversed by cotransfecting a p45 expression plasmid with the MafK expression vector (Fig. 3C). The antagonizing effect of the p45 on the repressor function of MafK was strictly dependent on the amount of cotransfected p45 expression vector.

Mutant p45 Defective in Transcription Activation

There are two possible mechanistic reasons for the antagonistic effect of p45 on MafK repressor activity. One is that a heterodimer formed between MafK and p45 directly activates transcription through binding to the NF-E2 sites; the other is that p45 displaces MafK from the NF-E2 sites by heterodimer formation with MafK and thereby reveals the NF-E2 sites, which are then activated by endogenous cellular factors. If the latter hypothesis is correct, the leucine zipper domain of p45 should alone be sufficient to confer the observed effect of p45 on MafK-mediated transcriptional repression. However, a plasmid that expresses a mutant p45 protein (p45DeltaNd), lacking the amino-terminal region but possessing an intact b-zip domain, fails to antagonize the repressing activity of MafK (Fig. 3C). This observation strongly argues that a p45bulletMafK heterodimer directly activates transcription from NF-E2 sites in DNA.

MafK and p45 Form Heterodimers in Vivo

Proper expression of MafK and either p45 or p45DeltaNd and formation of heterodimers between them within transfected cells were verified by EGMSA experiments using whole cell extracts prepared from transfected cells (Fig. 4A). In the absence of MafK, we did not detect specific DNA binding activity attributable to the p45 or to p45DeltaNd (lanes2 and 3). This observation was consistent with the poor DNA binding activity of the p45 bacterial fusion protein described above. Although mRNAs of MafF and MafK are expressed in QT6 cells(11) , the amounts of the small Maf proteins within QT6 cells are probably insufficient to form detectable heterodimers. In contrast, distinct complexes were detected in the presence of both MafK and p45 or MafK and p45DeltaNd (lanes4 and 5). The complexes are specific for NF-E2 sites, since complex formation was inhibited by adding excess unlabeled oligonucleotide (lanes7 and 8). Furthermore, the complex formed in the presence of both p45DeltaNd and MafK accumulated in nuclei inasmuch as this binding activity was detected in nuclear extracts from transfected cells (not shown). Although we could not detect the MafK homodimer complex in this analysis (probably because of overlapping mobility with a nonspecific band just below the specific p45DeltaNd/MafK band), the formation of a MafK homodimer complex was clearly identified in a similar analysis (10) .


Figure 4: Formation of heterodimers within transfected cells. A, DNA binding activities generated in QT6 cells by cotransfection of p45 and MafK expression plasmids. Whole cell extracts prepared from QT6 cells transfected with pEF-BssHII vector (lanes1 and 6), pEFp45 (lanes2, 4, and 7), or pEFp45DeltaNd (lanes3, 5, and 8) in the absence (lanes1-3) or presence (lanes4-8) of pEFmMafK were analyzed for NF-E2 site binding activity by EGMSA. EGMSA was carried out with the CbetaE probe in the absence (lanes1-6) or presence (lanes7 and 8) of 200-fold excess cold probe DNA. B, effect of anti-p45 antiserum on the DNA binding activities of MafK-p45 heterodimer. Whole cell extracts were prepared from QT6 cells transfected with pEFB-ssHII vector (lane1) or pEFp45 (lanes2-4) in the absence (lane1) or presence (lanes2-4) of pEFmMafK. The extracts were subjected to EGMSA with the CbetaE probe in the absence (lanes1 and 2) or presence (lane3) of anti-p45 antiserum. As a control for the specificity of the antiserum, preimmune serum was included in the binding reactions (lane4).



As shown in Fig. 4B, complex formation in the presence of both MafK and p45 was abolished by the addition of anti-p45 antiserum to the binding reaction but not by the addition of preimmune serum (compare lanes 2-4). In contrast, the complex formed in the presence of MafK plus p45DeltaNd was not affected by including the anti-p45 antiserum in the binding reaction (not shown). This finding suggests that the anti-p45 antiserum recognized the amino-terminal region of p45, which was missing in p45DeltaNd.

These results described above verified heterodimer complex formation between MafK and NF-E2 p45 or p45DeltaNd within transfected cells and thereby strongly suggested that the reporter gene activity, observed in the cells transfected with both MafK and p45 expression vector, reflected transcriptional activation by the MafKbulletp45 heterodimer. The p45bulletMafK heterodimer acts as a transcriptional activator through binding to NF-E2 sites, whereas the presence of MafK alone acts to repress transcription. Thus, the cis-regulatory function elicited through NF-E2 sites appears to be modulated by the relative balance of p45 NF-E2 and MafK within living cells.

In the presence of exogenous MafK, p45DeltaNd resulted in much stronger DNA binding in transfected cells than that elicited by wild-type p45 (e.g. compare lanes4 and 5, Fig. 4A). The amino-terminal region of p45 may negatively regulate either the heterodimeric association of p45 with MafK or the DNA binding activity of this heterodimer, but at the present time we cannot distinguish between these two possibilities.

Regulation of LCR Function by MafK and p45

The LCRs of the mammalian globin gene loci are essential for proper cell type-specific and developmental regulation of globin gene expression (14, 32, 33, 34) . The major erythroid-specific enhancer function of DNase I HS-2 of the human beta-LCR, which is induced upon erythroid differentiation, resides in tandem NF-E2 sites(8, 35, 36, 37) . To assess whether the p45bulletMafK heterodimer can function in vivo through a native cis-regulatory element, we examined the effect of p45 and MafK expression on the enhancer activity of murine HS-2. Reporter plasmid pRBGP6 contains a fragment of the mouse HS-2 placed 5` to a TATA box. The HS-2 fragment inserted in pRBGP6 contained binding sites for both GATA factors and CACCC binding factors in addition to the tandem NF-E2 sites (Fig. 5A). Various combinations of reporter and effector plasmids were then transfected into QT6 cells.

The sequences on the murine HS-2 enhancer fragment efficiently stimulated transcription, and reporter activity increased more than 100-fold in the presence of the HS-2 fragment when it was placed 5` to the TATA box (not shown). As was the case for the experiments using the reporter plasmid with synthetic triplicated NF-E2 sites, cotransfection of a MafK expression plasmid with pRBGP6 resulted in reduced reporter gene activity within the transfected cells (Fig. 5B), although the magnitude of repression was not so great as when multiple NF-E2 sites were used (see above). This may reflect residual transcription stimulation via sequences other than the NF-E2 sites. The inhibitory effect of the exogenous expression of MafK was again antagonized by the expression of p45. These results suggest that the enhancer function of the LCR HS-2 can be modulated by the relative level of MafK and p45 within cells, as that of the synthetic multiple NF-E2 sites. Because there is a synergistic transcription stimulatory effect between GATA sites and NF-E2 sites within erythroid cells(38) , the modest repression of transcription by MafK observed here would be expected to be augmented within erythroid cells.

Expression of mafK mRNA in Various Tissues and Fetal Liver of Mouse

To further elucidate presumptive regulatory roles of MafK and p45, the expression pattern of each mRNA was compared in various mouse tissues. For this purpose, total RNAs were electrophoretically separated on agarose gels, transferred to nylon membranes, and then hybridized with a mafK-specific probe. MafK mRNA was detectable in MEL cell lines DS-19 (Fig. 6A, lane8) and B8 (not shown). The size of the mafK mRNA was estimated to be 3.1 kb, very close to the size of the cDNA clone isolated in this study, suggesting that the mafK cDNA includes most of the mafK mRNA. The expression of mafK mRNA was detected in all tissues examined (Fig. 6A), although the level of expression varied significantly from tissue to tissue. Of the tissues examined, lung, kidney, and cardiac muscle expressed mafK mRNA more abundantly than liver, spleen, and skeletal muscle.


Figure 6: mafK mRNA expression in various tissues. Total RNAs (5 µg) were electrophoretically separated on denaturing agarose gels, transferred onto nylon membranes, and hybridized with either mafK- or p45-specific RNA probes. Integrity and equal loading of RNAs were verified by staining the gels before transfer with acrydine orange and examining ribosomal RNA. A, RNAs from brain (lane1), liver (lane2), spleen (lane3), lung (lane4), kidney (lane5), heart (lane6), skeletal muscle (lane7), and MEL DS-19 clone (lane8). Hybridization was carried out with the mafK-specific probe. B, total RNAs were isolated from mouse livers of 12, 14, or 16 days after gestation or livers of 0, 7, or 14 days after birth. Hybridizations were with the mafK- (above) and p45- (below) specific probes.



Taking advantage of the fact that the mouse fetal liver is a hematopoietic organ(39) , we examined the expression of both mafK and p45 mRNAs in mouse liver at various stages of development (Fig. 6B). Consistent with the fact that the mafK cDNA clones reported here were isolated from a mouse fetal liver cDNA library, mafK RNA was detected in that tissue. The levels of mafK and p45 mRNAs peaked around 14 days of gestation and declined thereafter to a still detectable level in the adult liver. This observation suggests that the majority of mafK mRNA in mouse fetal liver is derived from hematopoietic lineage cells. The expression profiles of mafK and p45 mRNAs also suggest that there may be a coordinated induction of mafK and p45 mRNA expression in hematopoietic cells.

Expression of mafK mRNA in Isolated Populations of Bone Marrow Cells from the Adult Mouse

To determine the expression profile of mafK and p45 mRNAs within hematopoietic lineage cells of adult mice, we separated primary bone marrow cells into four fractions by cell sorting using antibodies that recognize a variety of hematopoietic lineage markers (c-Kit, Sca-1, CD4, CD8, Mac-1, B-220, Gr-1, and TER119). These four fractions were 1) Kit/Sca-1/Lin (hematopoietic stem cell compartment), 2) Kit/Sca-1/Lin (committed progenitor cells), 3) Kit/TER119/Gr-1/B220 (differentiated non-erythroid cells), and 4) Kit/TER119/Gr-1/B220 (differentiated erythroid lineage cells), respectively (fractions1-4 in Fig. 7A). RNAs were isolated from these sorted cell fractions and subjected to semi-quantitative RT-PCR assay (see ``Materials and Methods''). Whereas both mafK and p45 mRNAs were found to be expressed in all four fractions, the levels of expression for both were higher in Kit/TER119 cells (Fig. 7B). These results are in clear contrast to the expression profile determined for GATA-1, another erythroid transcription factor, in these same fractions (Fig. 7B). GATA-1 mRNA was expressed abundantly in both the committed progenitor fraction (Kit/Sca-1/Lin, fraction2) and erythroid lineage cells (Kit/TER119, fraction4), but only marginally in fractions representing stem cells (Kit/Sca-1/Lin, fraction1) or differentiated, nonerythroid cells (Kit/TER119, fraction3). Thus, the expression of both mafK and p45 mRNAs are not restricted in erythroid lineage cells.


Figure 7: Expression of mafK mRNA in hematopoietic cells. A, FACS analysis of mouse primary bone marrow cells. Mouse bone marrow cells were sorted from 1) Sca/Kit/Lin, 2) Sca/Kit/Lin, 3) Kit/TER119/B-220/Gr-1, and 4) Kit/TER119/B-220/Gr-1 fractions, which represent hematopoietic stem cells, committed progenitor cells, differentiated non-erythroid cells, and erythroid lineage cells (fractions1-4), respectively. B, semi-quantitative RT-PCR assays for mafK and p45 RNA expression. Polyadenylated RNAs were prepared from these sorted mouse bone marrow cell fractions and used as templates for semi-quantitative RT-PCR analysis. Products were separated on agarose gels, transferred onto nylon membranes, and hybridized with mafK-, p45-, or GATA-1-specific DNA probes.




DISCUSSION

Combinatorial usage of transcription factors may be a fundamental strategy for organisms to achieve refined patterns of gene expression during development and differentiation. Formation of homodimers and heterodimers of transcription factors with a leucine zipper structure is a typical example of such strategies(40, 41, 42, 43, 44, 45) . Here, we report the molecular cloning of murine mafK cDNA and participation of its protein product in the regulation of NF-E2 activity. The results have important implications for understanding of the erythroid-specific as well as global gene regulation in higher eukaryotic cells in several respects. First, MafK, an unusually small b-zip protein, is expressed in a wide range of tissues and can act as a transcriptional repressor. This may be one of the fundamental mechanisms generating differential regulation of various genes with T-MARE-like elements by members of the Maf and AP-1 families. Second, the cell lineage-restricted transcription factor p45 NF-E2 can convert the function of MafK from the repressor to the activator through heterodimer formation. Since both MafK and p45 mRNAs are expressed from early stages of hematopoiesis, the finding points to mafK gene expression as an important component of the gene regulation in hematopoietic cells. Third, the presence of transcriptional regulatory mechanisms similar to that of NF-E2 may operate in various non-erythroid tissues by using tissue-restricted p45 homologues, since mafK mRNA is expressed in a wide range of tissues other than erythroid cells(5, 46) .

One of the striking features of the chicken MafK, MafG, and MafF structures is their small size. These proteins consist essentially of only a b-zip domain. The deduced primary structure encoded by the mouse cDNA clone shows the highest similarity to chicken MafK among the small Maf family members (see Fig. 1C). We therefore assigned this clone as mouse mafK homologue. In addition, where the two cDNAs overlap, the sequence of mafK cDNA reported here is virtually identical to that of the mouse p18 NF-E2 cDNA reported by Andrews et al.(10) , indicating that the p18 subunit is encoded by mafK. We additionally found that out of the 156 amino acid residues encoding mouse MafK, 151 residues are identical to those of chicken MafK. The basic domain, which is important for recognition of DNA, is perfectly conserved. It should also be noted that the amino-terminal and carboxyl-terminal small regions of mouse and chicken MafK are also highly conserved, whereas these regions are less conserved when compared with other chicken small Maf proteins. These regions are likely to endow MafK with some significant function other than dimer formation/DNA binding, which may be unique to MafK among the small Maf family proteins. The high degree of conservation of the primary structures of MafK in chicken and mouse suggests that this small b-zip protein carries out an important role in gene regulation.

Some of the known TRE- or cAMP-responsive element-like elements are likely to be regulated by Maf family members(2, 47) . The NF-E2 site was the obvious candidate for such elements because the consensus sequence of NF-E2 binding shares extensive similarity with that of T-MARE(2) , and transient cotransfection experiments in fibroblast cell lines described here and in a previous study (11) indicate that at least some of the NF-E2 sites are regulated by both AP-1-like factors and the small Maf family proteins. The small Maf family proteins powerfully antagonize transcriptional activation by endogenous factors through the NF-E2 sites (Fig. 3). Since a homodimer of MafK expressed in E. coli can bind to the chicken beta-globin enhancer NF-E2 site and since MafK lacks a canonical activation domain, one explanation for MafK function could be that MafK competed for the sites on the reporter plasmid with endogenous factors and, as a result, repressed transcription. Alternatively, MafK may heterodimerize with endogenous factors and thus inactivate them. The latter hypothesis is supported by the recent finding that chicken small Maf family proteins are able to form heterodimers with Fos(56) . The small MafbulletFos heterodimers bind to an NF-E2 site but cannot activate transcription. We think that both of these kinds of mechanisms are involved in MafK repressor function, depending on the sequences of target cis-regulatory elements. The present study suggests that antagonism of MafK and other small Maf family proteins to AP-1-like activity is an important constituent of transcription regulation through TRE-like elements in various tissues.

The expression of p45 converts MafK into a transcriptional activator effecting NF-E2 sites. To determine structural requirements for p45 to convert MafK function, we expressed a mutant p45 that lacks the amino-terminal half of the polypeptide but contains all of the b-zip domain (p45DeltaNd). Even though this mutant efficiently formed a heterodimer with MafK within transfected cells (Fig. 4), it failed to antagonize the transcriptional repression caused by MafK (Fig. 3). The failure to antagonize the repression by MafK is not due to its inability to translocate into nucleus, since p45DeltaNdbulletMafK heterodimer was detected in nuclear extracts (data not shown). In accord with this result, a mutant p45 molecule with a more extended amino-terminal truncation mutation than described here was also shown to accumulate in nuclear fraction in transfected cells(7) . The amino-terminal region of p45 that is deleted in p45DeltaNd is rich in proline(7, 9, 48) , which is one of the hallmarks of transcription activation domains. These observations, taken together, strongly suggest that the p45bulletMafK heterodimer activates transcription through binding to the NF-E2 sites and that the amino-terminal proline-rich domain of p45 mediates the transcription activation. Viewed from the other side, the small Maf family proteins are indispensable for the transcriptional activation by p45, as they are required for DNA binding of p45. Thus, both positive as well as negative regulation can be achieved from the same cis-regulatory element, depending on the composition of the transcription factor protein that binds to that element.

In vivo footprinting analyses showed that the NF-E2 sites of the LCR HS-2 are bound by proteins not only in erythroid cells but also in non-erythroid cells(49, 50, 51) . It was suggested that the binding activities present in non-erythroid cells repress the enhancer activities of the HS-2 region in non-erythroid cells(49) . Since we showed that MafK represses the enhancer activity of HS-2 (Fig. 5B) and since MafK is expressed in a wide range of tissues (Fig. 6A), it is tempting to speculate that small Maf family proteins are responsible for the occupancy of the NF-E2 sites and thus cause the repression of HS-2 enhancer activity in non-erythroid cells.

To gain insight into functional roles that MafK and p45 play in gene regulation during hematopoiesis, we analyzed the expression of mRNAs encoding MafK and p45 in FACS-purified mouse primary bone marrow cells. Consistent with previous reports examining human bone marrow cells (52) and chicken hematopoietic progenitor cells(53) , GATA-1 mRNA was expressed abundantly in both committed progenitor cells and in differentiated cells with erythroid markers. In contrast, both mafK and p45 mRNAs are expressed in all cell fractions. This observation suggests that both MafK and p45 are intimately involved in the regulation of cell differentiation within various hematopoietic cell lineages. Even though currently known NF-E2 sites are associated with erythroid-specific genes (e.g. globin genes), regulatory regions for some other hematopoietic lineage cell genes may also contain cis-regulatory elements similar to NF-E2 sites, and these genes may be regulated by MafK and/or p45.

In chicken, all of the mafF, mafG, and mafK mRNAs are expressed in erythroid and lymphoid cells (11) . In contrast, even though we screened a cDNA library derived from fetal mouse liver with a mixture of three chicken small maf probes, all four clones isolated thus far were found to encode mafK. Therefore, an obvious question is whether mafK is the only small maf family gene expressed in hematopoietic lineage cells in the mouse. The amino acid sequences of fragments generated from the 18-kDa polypeptide that was copurified in a stoichiometric ratio with p45 from MEL cell extract were found to contain only those homologous to chicken MafK but not to MafF or MafG (10) . This observation suggests that MafK is the major small Maf family protein expressed in MEL cells. However, p45 mRNA is expressed more abundantly than mafK mRNA in fetal liver (Fig. 6).^3 This observation suggests that MafK may not be the predominant small Maf family protein in the fetal mouse liver. Thus, as there may be mafF and mafG homologues in mouse, the precise roles of mafK during erythropoiesis should be interpreted in the context of regulatory networks achieved by the small maf family genes.

Recently, it was shown that there are several NF-E2 p45-related transcription factors in cells of various lineages(54) . It seems highly likely that these p45 homologues also interact with the small Maf family proteins in certain tissues, generating tissue-specific transcriptional regulators. Furthermore, there is a possibility that recognition sequence of the small Maf family proteins can be modulated as a result of heterodimeric association with partner proteins. These processes potentially generate a vast network of gene regulation in various tissues.


FOOTNOTES

*
This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture, the Uehara Memorial Foundation, the Naito Foundation, and the Gonryo Foundation for Promotion of Medical Sciences (to K. I. and M. Y.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D42124[GenBank].

(^1)
The abbreviations used are: b-zip, basic region leucine zipper; TRE, 12-O-tetradecanoylphorbol-13-acetate-responsive element; T-MARE and C-MARE, the TRE-type and cAMP-responsive element-type Maf recognition element, respectively; MEL, murine erythroleukemia; MBP, maltose binding protein; LCR, locus control region; FACS, fluorescence-activated cell sorter; kb, kilobase(s); bp, base pair(s); RT-PCR, reverse transcriptase-polymerase chain reaction; ORF, open reading frame; PBGD, porphobilinogen deaminase; EGMSA, electrophoretic gel mobility shift analysis.

(^2)
H. Ohtsu, unpublished observation.

(^3)
K. Igarashi, H. Motohashi, and M. Yamamoto, unpublished observations.

(^4)
K. Kataoka and M. Nishizawa, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. S.-I. Nishikawa and M. Ogawa for providing the fetal mouse liver cDNA library and anti-c-Kit antibody and T. Kina for the TER119 antibody. We also thank Drs. T. Sasaki, T. Nagai, K. Kataoka, H. Ohtani, S. Sassa, H. Fujita, Y. Muraosa, H. Ohtsu, and Y. Nakamura for discussion.


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