(Received for publication, June 26, 1995)
From the
The muscle creatine kinase gene enhancer contains two regulatory elements (MCK-R and MCK-L) with the consensus E-box sequence (CAnnTG). A myocyte specific protein complex, MEF1, binds the MCK-R site. MEF1 contains several basic H-L-H myogenic determination factors (MDFs), each dimerized with ubiquitous members of the bH-L-H family (e.g. E12/E47). We now demonstrate that the ubiquitous bH-L-H factor E2-2 is a major component of the endogenous MCK-R site specific complex.
Previous studies described the MCK-L site as a similar but low affinity MDF/bH-L-H heterodimer binding site. However, we find that the MCK-L site exhibits preferential binding of an unknown ubiquitous factor which contains neither E12/E47 nor E2-2, and that it exhibits differential transcriptional activity with muscle and non-muscle cells. The differential behavior of the MCK-L and MCK-R sites may be a general trait of E-box elements since one among several E-boxes in the MLC 1/3 enhancer also binds preferentially to the MCK-L factor. From our studies we now propose separate consensus sequences for MCK-R and MCK-L E-box types: AACAc/gc/gTGCa/t and GGa/cCANGTGGc/gNa/g. Our results suggest that while many muscle gene E-boxes are capable of binding the previously characterized spectrum of MDF/bH-L-H heterodimers in vitro, MCK-L type E-boxes probably bind qualitatively different factors in vivo.
Skeletal muscle differentiation involves the coordinate
activation and regulation of many hundreds of muscle specific genes.
Muscle gene expression is thought to occur via the interaction of
muscle specific and ubiquitous transcription factors with a common
subset of cis regulatory elements(1) . Among the most widely
found muscle gene control elements are sequences containing the
canonical E-box motif CANNTG(2) . The study reported below
focuses on the analysis of two E-box elements within the enhancer
region of the muscle creatine kinase (MCK) ()gene, and
proposes that muscle genes contain at least two classes of E-boxes
with qualitatively different functions.
The two E-box sequences in the MCK enhancer are designated the MCK-L site and the MCK-R or MEF1 site(2, 3) . Similar E-box sequences are found in the enhancers of the human, rat, and rabbit MCK genes(4, 5, 6) . Mutation of either site in the mouse MCK enhancer causes a dramatic decrease in the transcriptional activity of reporter genes when tested in skeletal muscle cells(2, 3) , and significantly smaller decreases when tested in cardiac muscle cells(7) . In gel shift assays the MCK-R site binds a myocyte specific complex called MEF1 (2) that contains the myogenic determination factors (MDFs), MyoD, and/or myogenin(2, 8, 9) . All four myogenic factors (MRF4, myf5, MyoD, and myogenin) can bind the MCK-R site as homodimers in gel shift assays but they exhibit much greater binding affinity as heterodimers with the ubiquitous H-L-H factor products of the E2A gene, E12 and E47(10, 11, 12, 13, 14) . Studies using antibodies suggest that E12 and E47 are naturally occurring participants in the MEF1 complex(15) . Based on gel mobility shift studies using in vitro translated and bacterially synthesized E-proteins such as E2-2 and HEB, it is known that other ubiquitous E-proteins can also form heterodimers with the MDFs and that these exhibit binding to E-boxes(16, 17) . However, it was not known whether any of the latter heterodimers were naturally occurring in muscle nuclei. The participation of E2-2 as part of the MEF1 complex is demonstrated in this study.
Much less is known about the MCK-L site. Studies with MyoD-glutathione S-transferase fusion protein and in vitro translated MyoD- or myogenin-E12 heterodimers have been interpreted as indicating that the MCK-L site is a low affinity MEF1 binding site(8, 9) . The finding that high level constitutive expression of MyoD in non-muscle cells can activate a thymidine kinase promoter reporter gene construct with four inserted MCK-L sites has also been interpreted as indicating interaction between MyoD and the MCK-L site(18, 19) . The possibility that the MCK-L site might bind a ubiquitous factor was suggested by analysis of the rat MCK enhancer(20) . However, the ubiquitous factor(s) in that study bound a region containing the MCK-L site as well as the adjacent A/T-rich site, which is known to bind both ubiquitous (21) and more restricted transcription factors(22) , thus the existence of a distinct MCK-L site binding factor was unclear.
The CAnnTG E-box core is important for the regulation of both muscle-specific genes, and non-muscle genes, such as the immunoglobulins. Therefore, the mechanism of tissue specific gene regulation via E-box sequences must also involve sequences flanking the core CAnnTG and/or the internal undefined bases. A consensus sequence for generic muscle E-box binding sites based on sequence comparisons of the MCK-L and MCK-R sites as well as the E-boxes found in the regulatory regions of other muscle genes was proposed several years ago (2) . Subsequent experimental evidence from polymerase chain reaction based MDF/E2A protein binding site selection protocols suggested very similar consensus sequences and confirmed the importance of the flanking sequences for determining the unique binding sites for MyoD/E2A and myogenin/E2A heterodimers(23, 24) . Functional evidence for the effects of flanking sequence differences on E-box activity was obtained by studies of the troponin I enhancer in which the endogenous E-box and flanking sequences were replaced with E-box and flanking sequences from both muscle and non-muscle genes(25) . These studies are all consistent with the concept that sequences flanking the core E-box in muscle-specific genes play an important role in regulating gene expression.
To better understand the complexities of muscle-specific gene regulation, we have studied differences between the MCK E-box sites with respect to their transcriptional activities and their binding factor interactions. We have also examined transcriptional differences between the MCK-L and MCK-R sites in the absence of other muscle regulatory sequences. To determine if the two E-box elements are simply low and high affinity sites for the same nuclear factors we analyzed qualitative differences in the factors which preferentially bind the two sites. Then by examining the binding preferences of E-box sequences found in the myosin light chain 1/3 enhancer we investigated whether factor binding differences between the MCK E-boxes are specific to the MCK gene or represent a more general mechanism of muscle gene regulation. The results suggest that factor interaction at muscle E-box regulatory sites is more complex than can be explained by the hypothesis of high and low affinity MDF binding sites. We propose that E-box elements of the MCK-L type interact with a ubiquitous binding complex that contains none of the common MDF/E-protein heterodimers that bind E-boxes of the MCK-R type.
NIH3T3 cells were grown and transfected as above except that the cells were switched into conditioned media after glycerol shock. Conditioned medium was F-10C, 1.5% horse serum that had been incubated on confluent NIH3T3 cells until there was no apparent cell division (typically 48 h after medium addition). Statistical analysis of transfection results was done by ANOVA using the SAS computer program.
Figure 1: Transcriptional differences between the MCK-L and MCK-R sites. Transcriptional differences between the MCK E-box sites were assessed by transfecting three cell types with CAT reporter constructs driven by the tk promoter with and without four MCK E-box sequences. A, mitogen-starved NIH 3T3 cultures; B, proliferating MM14 myoblast cultures; C, differentiated mitogen-starved MM14 myocyte cultures. All cultures were harvested 26 h after transfection. 95% of the cells in differentiated MM14 myocyte cultures were myosin-positive as assessed by immunostaining for myosin heavy chain at the time of harvest, whereas less than 5% of the cells in proliferating myoblast cultures were myosin-positive. Since the background of differentiated cells in the proliferating myoblast cultures was not 0%, some or all the CAT activity detected in 4RtkCAT-transfected myoblasts could be due to expression of 4RtkCAT by the small number of differentiated muscle cells in these cultures. Relative CAT activity was calculated as follows: the CAT activity (minus background cpm) was divided by the placental alkaline phosphatase activity to normalize for transfection efficiency between plates. In all cases CAT activity was at least 2 times background. In each experiment CAT/placental alkaline phosphatase values were normalized to those of the basal construct (tkCAT) which was set at 1.0. n equals the total number of transfected plates. Panels A, B, and C represent data from at least two different plasmid preparations and three experiments. Error bars are standard deviations. p value as determined by ANOVA was equal to p < 0.0001 for 4RtkCAT and 4LtkCAT compared to tkCAT in all cell types tested.
The absolute activity levels of tkCAT are similar in 3T3 cells myoblasts, and myocytes, but activity differences are observed when MCK-L or MCK-R sites are combined with the tk promoter. In 3T3 cells, the activity of 4LtkCAT is 2-fold higher than that of the tk promoter alone while the 4RtkCAT construct is 4-fold lower than that of the tk promoter alone (Fig. 1A). This indicates that the MCK-L site is not muscle-specific when removed from the context of the MCK enhancer. In addition, the decreased activity of 4RtkCAT suggests that the MCK-R site may bind factors in non-muscle cells that repress expression from the ubiquitously active tk promoter (see ``Discussion'').
Results from the myoblast transfection study are similar to those with 3T3 cells: the activity of 4LtkCAT is 5-fold higher than that of tkCAT while that of 4RtkCAT is 2-fold lower (Fig. 1B). Since it is impossible to grow myoblast cultures without a low background of differentiated myocytes (see Fig. 1legend), the true relative repression of 4RtkCAT in myoblasts is probably severalfold greater than the 2-fold repression level observed. In contrast to their behavior in fibroblasts and myoblasts, both minimal E-box promoters exhibit significantly elevated expression in differentiated muscle cells (15-20-fold) over that of tkCAT alone (Fig. 1C). However, although both constructs have similar absolute expression levels in myocytes, 4RtkCAT exhibits a 50-100-fold induction in activity during the transition from myoblasts to myocytes, while the induction of 4LtkCAT during differentiation is only 3-fold, since this construct is also expressed at relatively high levels in myoblasts. Based on these results we conclude that the MCK-L and MCK-R sites exhibit quantitatively distinct transcriptional enhancements when tested in the absence of other muscle regulatory elements. In addition, the MCK-R site appears to bind factors that repress gene expression in non-muscle cells and in replicating myoblasts.
Figure 2:
Detection of MCK-L and MCK-R site binding
complexes by gel mobility shift assays of an NIH3T3 nuclear extract. 2
µg of an NIH3T3 nuclear extract was incubated with labeled MCK-L or
MCK-R site oligomers and with unlabeled competitors (MCK enhancers with
mutations in either the MCK-L or MCK-R sites). A, partial
sequence of the wild type MCK enhancer and the mutations used in
competition assays. B, gel mobility shift assays demonstrated
that MCK-L (band 1) and MCK-R (band 2) specific
binding complexes exist in the fibroblast extract. The enhancer was in
35 molar excess compared to probe. Lanes 1 and 2, 2 µg of NIH3T3 nuclear extract was incubated with the
MCK-L probe and the indicated enhancer fragments. Lanes 3 and 4, 2 µg of the extract was incubated with the MCK-R probe
and indicated enhancer fragment.
Figure 3:
Partial purification of MCK-L and MCK-R
factors from a myocyte nuclear extract. A, 1 mg of MM14
myocyte nuclear extract protein was run over a heparin-agarose column
and eluted with a step gradient of increasing KCl concentrations (see
``Experimental Procedures''). 125-ml fractions were collected
and an aliquot was assayed for protein. The x axis represents
the fraction number, the y axis is an arbitrary value based on
the protein assay. Fractions were pooled as indicated by bars above peaks. B, column pools were analyzed for MCK-L and
MCK-R binding activities by gel mobility shift assays. 0.5 µg of
protein from each column pool plus 180 ng of poly(dI-C)(dI-C) as
a nonspecific competitor was mixed with either end-labeled MCK-L
oligomer (lanes 1-4) or end-labeled MCK-R oligomer (lanes 5-8). FP is free oligomer
probe.
To determine where potential MCK-L and MCK-R factors elute from the heparin-agarose column, the pooled fractions were analyzed by gel mobility shift assays using MCK-L or MCK-R site oligomers as probes (Fig. 3B). With the MCK-R probe, a binding activity which is characteristic of the previously reported MEF1 complex(2) , is detected in fractions C and D as a broad region containing at least two shifted complexes (Fig. 3B, lanes 7 and 8). Studies from our laboratory and by other investigators suggest that the upper MEF1 band contains MyoD and the lower band contains myogenin(16, 31) . The MCK-R probe also exhibits binding to a broad band (complex 3), but this binding is nonspecific (see Fig. 4B, lanes 4 and 5). A similar nonspecific complex is observed when the heparin column fractions are assayed with an MCK-L site probe. However, the MCK-L probe also exhibits specific binding (complex 1). The complex 1 factor is found predominately in fraction D, with smaller amounts in fraction C (Fig. 3B, lanes 3 and 4). Complex 1 appears to be MCK-L site-specific because it is not seen in fraction D when examined with the MCK-R probe (compare Fig. 3B, lanes 4 and 8). Furthermore, under these conditions, MEF1 does not bind the MCK-L probe (Fig. 3B, lanes 3 and 7).
Figure 4:
MCK-L
and MCK-R site binding specificity of heparin-agarose fractions C and
D. A, the specificity of the binding activities separated by
heparin-agarose column chromatography was determined with unlabeled
enhancer BamHI/HindIII fragments from pUC-E
containing mutations 1 or 2 in the MCK-L site or a mutation in the
MCK-R site. All three mutations decrease enhancer activity in myocyte
transient transfection assays. B, 0.5 µg of column
fraction protein was incubated with end-labeled oligonucleotide
corresponding to the MCK-L site (lanes 1-3) or the MCK-R
site (lanes 4 and 5). 35 molar excess of
unlabeled fragments were used as competitors; lanes 1 and 4, the competitor was the 206-bp MCK enhancer containing the
mutated MCK-R site and the wild type MCK-L site; lanes 2 and 5, the competitor was the same enhancer fragment containing
mutation 1 of the MCK-L site and the wild type MCK-R site; lane
3, the competitor was the same enhancer fragment containing
mutation 2 of the MCK-L site and the wild type MCK-R
site.
The specificity of DNA binding activity detected in the heparin-agarose column fractions was determined by competition gel mobility shift assays using various unlabeled MCK enhancer fragments. The DNA fragments used as competitors were the BamHI/HindIII fragments from pUC-E that contain mutations in either the MCK-R or MCK-L site (Fig. 4A). These mutations are known to decrease enhancer function in muscle cell culture(2, 7, 8) . Competition with an enhancer fragment containing a mutated MCK-L site (mt1) but a wild type MCK-R site decreases binding of the MEF1 complex to the MCK-R probe (Fig. 4B, lane 5). However, an enhancer fragment with a mutant MCK-R site but wild type MCK-L site does not compete for MEF1 binding to the MCK-R probe (Fig. 4B, compare lanes 4 and 5). This demonstrates that only wild type MCK-R sites compete well for the MCK-R site binding activity (MEF1) in fraction C. In contrast, an enhancer fragment containing a wild type MCK-L site, but not enhancer fragments containing either a 5-bp MCK-L mutation (mt1) or a 2-bp MCK-L mutation (mt2), competes for binding of the MCK-L site complex-1 to the MCK-L site probe (Fig. 4B, compare lanes 1, 2, and 3). These results indicate that complex-1 contains a MCK-L site-specific binding factor. Significantly, it is not necessary to fractionate the nuclear extract to detect MCK-L site binding, since high levels of this activity are also detected in unfractionated myocyte nuclear extract (data not shown).
Figure 5: Methylation interference indicates the core E-box CATGTG region is at the center of the MCK-L factor binding site. A, the methylation interference pattern of the MCK-L site with MCK-L factor complex-1 determined with labeled bottom strand (lanes 1-3) or top strand (lanes 4-6) is indicated by the lines. B, the corresponding sites are marked in the partial sequence of the enhancer by the open arrow. The circle represents an under-methylated G residue in the total probe.
Figure 6: Tissue specificity of the MCK-L binding site activity. Nuclear extracts from several mouse tissues were incubated with end-labeled MCK-L oligomer. The competitor enhancer fragments contained either MCK-L mutation 1 and a wild type MCK-R site, or the wild type MCK-L site and a mutation in the MCK-R site. The competitor was used at 35-fold molar excess. LIV, liver; SK, skeletal muscle; BR, brain; KID, kidney; HRT, heart; MB, myoblast cell culture; and FR-D, myocyte nuclear extract heparin column fraction D.
Figure 7: The ubiquitous H-L-H proteins E12/E47 and E2-2 are not present in the MCK-L complex but are present in the MCK-R complex, MEF1. A, myocyte fractions C and D from the heparin-agarose column containing MEF1 and the MCK-L factor were incubated with 1 µl of E12/E47 antiserum, lanes 1 and 3; or control sera, lanes 2 and 4. B, fractions C (lanes 1-4) and D (lanes 5 and 6) were incubated with purified preimmune (C) or immune (I) IgGs raised against mE2-2. The specificity of the supershifted complex (ss) in lane 1 was determined with enhancer fragments that contained a mutant or wild type MCK-R site. The nonspecific binding (ns) in the control lanes 3 and 4 was not competed with the enhancer fragments.
To examine the presence of E2-2 in the MCK-L site binding complex, we cloned a 1.4-kilobase cDNA from a myocyte expression library that encodes roughly 75% of the murine homolog of the human H-L-H factor, E2-2 (ITF2) (data not shown). The predicted amino acid sequence includes the bH-L-H domains which are very similar with those in E12. The predicted murine sequence of mE2-2 is greater than 95% identical to the predicted sequence of the human homolog(32) . A synthetic peptide (DAANHGQMM) representing the C terminus of mE2-2 was then used to produce polyclonal mE2-2 antiserum. The C terminus peptide has only a 33% similarity to the corresponding E12/E47 peptide (EAHNPAGHM). Purified IgGs from either the mE2-2 preimmune serum or E2-2 antiserum do not affect binding of the MCK-L factors to the MCK-L probe (Fig. 7B, lanes 5 and 6). However, incubation of the MCK-R fraction with E2-2 IgGs causes a supershift (ss) of the MCK-R factors (Fig. 7B, lane 1), and binding of the supershift complex to the MCK-R site probe is competed by an enhancer containing a wild type MCK-R site but not by an enhancer containing the mutated MCK-R site (Fig. 7B, compare lanes 1 and 2). When control preimmune IgG was used, a band (NS) with slightly faster mobility than the supershift is observed (Fig. 7B, lanes 3 and 4); however, the NS band represents nonspecific binding of the IgG fraction since the NS complex is not competed by the enhancer fragment. The mE2-2 IgGs did not cross-react with pure E12 under gel mobility shift assay conditions (data not shown). These results suggest that mE2-2 is not a participant in the MCK-L complex, but that it is a substantial component of the MCK-R complex, MEF1.
Figure 8:
E-box sites in the myosin light chain 1/3
enhancer preferentially bind the MCK-L and MCK-R binding complexes.
Double stranded oligo probes corresponding to the MCK-R (R),
MCK-L (L), MCL-A (A), MLC-B (B), and MLC-C (C) sites (see ``Experimental Procedures'' for
sequences) were labeled to the same specific activity and 5
10
cpm of each probe was incubated with equal amounts of
myocyte fraction C (lanes 1-5) or fraction D (lanes
6-10).
Previous studies have shown that 10T1/2 cells transfected with either of the minimal E-box ``muscle'' gene constructs (4RtkCAT or 4LtkCAT) and co-transfected with constitutively active MyoD exhibit elevated expression of both reporter genes(18, 19) . However, these results do not prove that elevated expression from either test gene is directly attributable to increased MyoD or MyoD/E-protein interaction with the L or R E-boxes.
In this study we have expanded the concept of muscle gene regulation via E-box sequences by demonstrating that E-box sites in two muscle regulatory regions, the MCK and MLC 1/3 enhancers, exhibit preferential binding for different transcription factors. The MEF1 type E-box binds heterodimers containing MDFs dimerized with E12/E47, and as shown in this report, E2-2. The MDFs participating in these heterodimers are muscle-specific, whereas E12/E47 and E2-2 are relatively ubiquitous. In contrast, the MCK-L type E-box exhibits preferential binding for a different ubiquitous complex that contains neither MDFs nor E12/E47 or E2-2. Based on a comparison of the highly conserved MCK-L sites in the mouse, rat, human, and rabbit MCK enhancers and the myosin light chain 1/3 enhancer MLC-B site, which also appears to bind the MCK-L factor (Fig. 8), we propose a ``L'' site E-box consensus sequence, GGa/cCANGTGGc/gNa/g. We have also refined the consensus MCK-R sequence by eliminating sequence comparisons to the MCK-L and MLC 1/3-B sites and by including comparisons to the sequences identified as optimal MyoD and myogenin/E-protein binding sites(23, 24) . The newly proposed muscle-specific E-box consensus sequence is AACAc/gc/gTGCa/t. Further analysis of binding preferences of the E-box sites found in other muscle gene regulatory regions will be necessary to refine these consensus sequences: but the proposed MCK-R and MCK-L site consensus sequences should serve as useful interim models for predicting qualitative differences in muscle gene E-box/transcription factor interactions.
Our binding studies show that the MCK-L site binds an apparently ubiquitous factor. We have carried out extensive screening of both skeletal and cardiac myocyte expression libraries with the MCK-L site and have yet to identify a definitive MCK-L factor. Based on the methylation interference pattern, the E-box (CATGTG) is at the core of the MCK-L binding site. This suggests that the MCK-L factor may be a ubiquitous H-L-H protein. However, neither E12, E47, nor E2-2 appear to be present in the MCK-L complex. Possible candidates that may bind the MCK-L site include members of the basic helix-loop-helix-leucine zipper protein family (bH-L-H-ZIP), such as TFE3(35, 36) , AP4(37) , USF(38) , and TFEB(39) . The core of the TFE3 binding site, µE3 (CATGTGGC), in the immunoglobulin heavy chain enhancer, is identical to the MCK-L site core. Furthermore, the MCK-L site competes for TFE3 binding to the µE3 site while the MCK-R site does not(36) . Interestingly, the H-L-H differentiation inhibiting protein Id is not capable of forming heterodimers with the bH-L-H-ZIP family of transcription factors(40) . Thus if the ubiquitous MCK-L factor were a member of this family its activity would not be inhibited by the Id present in myoblasts or fibroblasts.
The two MCK enhancer E-box sites have different transcriptional activities(2, 7, 8) . The MCK-L site increases the activity of a tk promoter in fibroblasts and replicating myoblasts, while the MCK-R site represses expression from the tk promoter in these cells (Fig. 1). The different transcriptional activities of the MCK-L and MCK-R sites are consistent with the findings of Yutzey et al.(1992) that E-box and flanking sequences (referred to as the muscle E-box consensus sequence) are not functionally equivalent. However, while the previous study proposed that these E-box transcriptional differences could be accounted for by low and high affinity MDF binding, we now provide evidence that the differences are due to the preferential binding of distinct factors.
One intriguing aspect of the MCK-L site's transcriptional activity is that it appears to be regulated during the developmental transition from proliferating skeletal muscle myoblasts to differentiated myocytes. The activity of the MCK-L site in fibroblasts and myoblasts is consistent with the ubiquitous distribution of the MCK-L binding activity. However, after differentiation of myoblasts to myocytes the total activity of the MCK-L site increases, suggesting a qualitative and/or quantitative change in the MCK-L factor (Fig. 1). Several regulatory mechanisms could account for the increase in MCK-L site activity, including increased levels of MCK-L factor in myocytes versus myoblasts, changes in accessory factors, or post-translational modification of the MCK-L factor (see below).
An alternative for E-box transcriptional regulation could
involve the competitive binding of repressor factors to certain E-box
motifs. This model is supported by our transcriptional studies of the
MCK-R site in 3T3 cells and myoblasts, in which the MCK-R site
represses the activity of the thymidine kinase promoter (Fig. 1, A and B). The possibility that repressors may
regulate the MEF1-type (MCK-R site) E-box activity is consistent with
two recent reports. First, mutations of a MEF1 type E-box in the
subunit of acetylcholine receptor (
AChR) promoter lead to higher
expression in fibroblasts and myoblasts compared to the wild type
promoter(41) . In addition, an E-box binding activity that may
be responsible for repression via the
AChR E-box was detected in
myoblast nuclear extracts(41) . The MCK-R site-specific binding
activity we detect in fibroblast nuclear extracts could represent the
same or similar repressor complex (Fig. 2). Second, mutation of
the µE5 site in the IgH enhancer renders the enhancer, which is
normally unresponsive to MyoD, sensitive to MyoD
transactivation(42) . The sequences responsible for the
repression of MyoD activation are four bases flanking the core E-box.
Interestingly, three of these four are present in the MCK-R site at the
same positions.
Previous studies had shown that E12 forms heterodimers with the MDFs and that E12 is part of the MEF1 complex(15) . It had also been shown that E2-2 forms heterodimers with all MDFs in vitro and that the complexes can bind muscle E-box sites when tested via gel mobility shift assays(43) , However, it was not known whether E2-2 was a component of the naturally occurring MCK-R site binding complex, MEF1. The involvement of E2-2 has now been demonstrated by the antibody studies shown in Fig. 7B. Participation of E2-2 in MCK-R site binding complexes may help explain why disruption of the E2A gene in embryonic stem cells has no effect on the ability of the cells to form muscle colonies(44) , because under these circumstances E2-2 may function in place of the missing E2A products, E12, E47, and ITF1.
Although our studies indicate the existence of a ubiquitous nuclear factor that exhibits preferential binding to the MCK-L site, this does not prove that the MCK-L factor is the exclusive occupant of muscle control elements of the MCK-L site consensus type in vivo. For example, since high levels of bacterially produced H-L-H proteins exhibit MCK-L site binding activity(3) , it is possible that one or more of the H-L-H heterodimers does interact with the MCK-L consensus sequences in living cells. However, if this were so, a regulatory mechanism which would enable the H-L-H heterodimers to out-compete the MCK-L site factor for occupancy of these control elements would also be necessary. For reasons of simplicity we thus favor a model in which E-box control elements resembling the MCK-L site and MLC 1/3 enhancer B site interact with a ubiquitous nuclear factor that is qualitatively different from factors which bind the MCK-R and MLC 1/3 enhancer A sites.