(Received for publication, September 8, 1994; and in revised form, November 30, 1994)
From the
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.
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) ()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
p45MafK 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 p45
MafK
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.
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).
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 (p45
Nd) 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 pEFp45
Nd.
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 -LCR
in front of a rabbit
-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 -LCR HS-2. A, sequence of the mouse
-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.
Mouse bone marrow cells (1
10
) 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
10
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
10
cells were collected and analyzed.
After analysis, 1
10
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.
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, ()it remains possible that these
differences are due to alleic variations.
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 -globin gene enhancer
(C
E2 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. CE2 was from the NF-E2 site of chicken
-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 C
E 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 p45MafK 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
-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 C
E2 probe was more prominent than that of the
MafK homodimer (see below).
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
p45Nd on MafK-mediated transcription repression. Various amounts
of pEFp45 or pEFp45
Nd 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 pEFp45
Nd. The reporter activity in the absence of
pEFmMafK was set as 100%. The results are the mean of four (pEFp45, closedtriangle) or two (pEFp45
Nd, 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.
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 pEFp45Nd (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 C
E 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 C
E 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 p45Nd
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
p45
Nd.
These results described above verified heterodimer
complex formation between MafK and NF-E2 p45 or p45Nd 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
MafK
p45 heterodimer. The p45
MafK 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, p45Nd 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.
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.
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.
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.
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 -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 Maf
Fos
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 (p45Nd). 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 p45
Nd
MafK 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 p45
Nd 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 p45
MafK 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). 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D42124[GenBank].