©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Mouse MRF4 Promoter Is trans-Activated Directly and Indirectly by Muscle-specific Transcription Factors (*)

(Received for publication, November 18, 1994; and in revised form, December 13, 1994)

Brian L. Black (§) James F. Martin (¶) Eric N. Olson (**)

From the Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

MRF4 is a member of the basic helix-loop-helix (bHLH) family of muscle-specific transcription factors, which also includes MyoD, myogenin, and myf5. The myocyte enhancer binding factor 2 (MEF2) proteins also serve as important muscle-specific transcription factors. In addition to activating the expression of many muscle-specific structural genes, various members of these two classes of proteins activate their own expression and the expression of each other in a complex transcriptional network that results in the establishment and maintenance of the muscle phenotype. To begin to determine how the expression of MRF4 is regulated by other muscle-specific transcription factors, we have isolated a region of the MRF4 gene that confers muscle-specific expression and have analyzed this promoter region for cis-acting elements involved in trans-activation by the myogenic bHLH and MEF2 transcription factors. Here, we show that in 10T1/2 fibroblasts the MRF4 promoter is trans-activated by myogenin, MyoD, myf5, and by the MEF2 factors, but that MRF4 does not activate expression of its own promoter. Myogenin activated the MRF4 promoter directly by an E box-dependent mechanism, while MEF2 factors activated the promoter through an indirect pathway. The E box-dependent regulation of the MRF4 promoter is in contrast to the regulation of the myogenin and MyoD promoters and may represent a mechanism for the differential expression of these factors during myogenesis.


INTRODUCTION

During skeletal muscle development, a wide array of muscle-specific genes are expressed in an ordered pattern, which results in the myogenic phenotype. One set of transcription factors that is involved in the regulation of the muscle transcriptional network is the myogenic basic helix-loop-helix (bHLH) (^1)family. This family of transcription factors includes MyoD(1) , myogenin(2, 3) , myf5(4) , and MRF4(5, 6, 7) . These factors induce transcription by binding as heterodimers with ubiquitously expressed E-proteins to the E box consensus sequence (CANNTG), which is found in the control regions of numerous muscle-specific genes(8) . When expressed in a variety of non-muscle cells, each of these four factors is capable of inducing myogenic conversion and differentiation(1, 2, 3, 5, 7) . In addition to activating the expression of muscle-specific structural genes, in many cell types, these myogenic bHLH factors have been shown to activate their own and each other's expression(9, 10, 11) .

The MEF2 family of MADS box transcription factors has also been shown to play a role in the activation of muscle-specific gene transcription (12) . MEF2 factors are the products of four separate genes, mef2a, mef2b, mef2c, and mef2d(13, 14, 15, 16, 17, 18, 19, 20) , and they activate transcription by binding to the consensus MEF2 site sequence, CTA(A/T)(4)TA(G/A), as homo- and heterodimers(13, 20, 21, 22) . Recently, the MEF2 binding site has been shown to be required for expression of several of the myogenic bHLH factors(18, 23, 24, 25, 26) ; likewise, members of the myogenic bHLH family can activate the expression of MEF2 factors(19, 22, 27) . Thus, the MEF2 factors and the myogenic bHLH proteins appear to function in a complex network by auto- and cross-activating their own and each others expression to establish and maintain the expression of muscle genes that give rise to the myogenic phenotype.

During mouse development, each of the myogenic bHLH factors is expressed in a precise temporal and spatial pattern to give rise to muscle(28) . In the developing myotome, for example, myf5 is the first of the bHLH factors to be expressed, followed shortly thereafter by the expression of myogenin(28) . MRF4 is expressed in a biphasic pattern in the somite and is the last of the myogenic bHLH factors to be expressed in the developing muscle of the limb(29, 30) . It is the most highly expressed of the bHLH factors at birth and is the only myogenic bHLH factor to be expressed at high levels in adult muscle(5, 7, 29, 30) . Based on these observations, it has been proposed that MRF4 primarily functions downstream of the other myogenic bHLH factors(7, 11, 29, 31, 42) . In this regard, MRF4 has been postulated to be involved in myofiber formation (11, 29, 31) and in the maintenance of the muscle phenotype(7, 29) .

Based on these hypotheses, which suggest that MRF4 plays a downstream role in myogenesis, we wanted to determine if MRF4 expression is directly activated by other muscle-specific transcription factors. To begin to define the mechanisms involved in the activation of MRF4 expression, we have isolated a region of the MRF4 promoter that confers muscle-specific expression and have analyzed this promoter region for muscle-specific sequence elements involved in activation by the myogenic bHLH and MEF2 transcription factors. Here, we show that the MRF4 promoter is trans-activated directly by the myogenic bHLH factor myogenin via an E box-dependent mechanism and is activated by MEF2 proteins via an indirect pathway.


MATERIALS AND METHODS

Isolation of MRF4 Upstream Region

To isolate MRF4 genomic clones, a mouse genomic library was screened by hybridization to the full-length rat MRF4 cDNA(5) . Hybridizations and purification of phage clones were performed using standard techniques(32) . PCR was then used to isolate the 390-bp upstream fragment used in these studies using 5` primer 5`-CTACTACTACTAAAGCTTGTGTTAATCCCCAGT TGT-3` and 3` primer 5`-CATCATCATCATTCTAGACTCCTCCTTGGCCTCTGA-3`.

Plasmids, Mutagenesis, and Sequence Analysis

The 390-bp mouse MRF4 promoter fragment was isolated using the primers described above. This fragment was then cloned into the polylinker of plasmid pCAT-BASIC (Promega, Inc.) to create plasmid pMRF4.CAT. The E2 mutant MRF4 promoter was cloned by the same strategy except that the following 5` primer was used to remove the E2 E box from the 5` end of the fragment: 5`-AAAACTGCAGTGAAGTTGCCTGGTTAGCAGG-3`. All other mutants were made by utilizing the PCR mutagenesis technique of gene splicing by overlap extension (``gene SOEing'') (33) creating the following mutant sequences in the wild-type promoter context: E1, 5`-AATTAAATGATATCTGGGTGGCTCC-3`; and MEF2, 5`-TAGCTAGTATATAAGCAGCTGGGTCGA-3`. The MEF2 mutagenic primers mutate the outer sequences of the MEF2 consensus, while leaving the TATA motif contained within the larger MEF2 sequence intact(26, 34) . Each of the MRF4 promoter-CAT constructs was sequenced from both ends to confirm that the mutations were in place and that no unintentional mutations were introduced by the PCR. MEF2 expression plasmids were made by cloning the coding regions for MEF2A(14) , MEF2C(19) , and MEF2D(20) , into the polylinker of plasmid pCDNAI/amp (Invitrogen, Inc.). The expression plasmids encoding myogenin(2) , MyoD(1) , myf5(4) , and MRF4 (5) all contain the complete coding region cDNAs cloned into plasmid pEMSVscribe(1) . Plasmid pCDNAIII.CAT (Invitrogen, Inc.) encodes the CAT gene under control of the cytomegalovirus immediate-early promoter and was used as a positive control for expression.

Cell Culture and Transfections

C3H10T1/2 (10T1/2) and C2C12 (C2) cells were maintained essentially as described previously(2) . Chick primary myoblasts were isolated and grown overnight as described previously(35) . Differentiation medium (DM) is Dulbecco's modified Eagle's medium supplemented with 5 mML-glutamine, 1 mM sodium pyruvate, 100 IU of penicillin, 100 µg/ml streptomycin, and 2% horse serum. Growth medium (GM) is Dulbecco's modified Eagle's medium supplemented with 5 mML-glutamine, 1 mM sodium pyruvate, and either 10% fetal calf serum (10T1/2) or 15% fetal calf serum (C2C12, chick myoblasts).

All transfections were performed by calcium phosphate precipitation for 12 h as described previously(36) . In each transfection, 20 µg of plasmid DNA was transfected. Chick primary myoblasts and late C2C12 myotubes were transfected in DM. 10T1/2 cells and early C2C12 myotubes were transfected in GM. Following transfection, early C2C12 myotubes and 10T1/2 cells were grown for 12 h in GM followed by 48 h in DM prior to harvesting, and chick primary myoblasts were maintained in DM supplemented with chick embryo extract (35) and without antibiotics for 48 h prior to harvesting. Late C2C12 myotubes were allowed to differentiate for 5 days in DM, transfected in DM for 12 h, then maintained for 48 h in DM prior to harvesting.

CAT Assays

Transfected cells were harvested, and extracts were prepared by three freeze-thaw cycles and heat inactivation as described previously(37) . Cell lysates were then quantitated for total protein(38) , and an equivalent amount of cell lysate (normalized for total protein) from each transfection was assayed for CAT activity as described previously(37) . 100 µg (10T1/2 and C2C12 transfected cells) or 20 µg (chick primary myoblasts) of total cellular protein was assayed for each reaction. Reactions were conducted for 5 h at 37 °C. Conversion to acetylated forms was analyzed by thin-layer chromatography and quantitated by phosphorimager analysis (Molecular Dynamics, Inc.).


RESULTS

The Upstream Region of the Mouse MRF4 Gene Contains Putative Muscle-specific cis-Acting Regulatory Elements

To begin to define the muscle-specific regulation of MRF4 gene transcription, we isolated a 390 bp fragment extending from -300 to +90 relative to the primary transcription start site at +1. Location of the transcription start site is based on complete sequence homology to the published transcription start site of the rat MRF4 gene (39) . The sequence of this region of the mouse gene is shown in Fig. 1. We chose to focus on this region of sequence surrounding the MRF4 transcription start site because it contains several potential muscle-specific cis-acting elements. Furthermore, sequence analysis of this 390-bp fragment showed that it had a strong overall resemblance to the proximal region of the myogenin gene promoter(23) . Two E boxes are present in this region, one at +22 to +27 and the other at -287 to -282, designated as E1 and E2, respectively. There is also an A+T-rich element in this region at -26 to -17, which serves as the primary transcription start site(39) , as well as meeting the consensus sequence requirements for MEF2 transcription factor binding(13, 22) . An additional A+T-rich region, which resembles a TATA motif, is present at +11 to +19.


Figure 1: Nucleotide sequence of the upstream region of the mouse MRF4 gene. Figure shows nucleotide sequence of the 390-bp fragment of the MRF4 gene used in this study. The E boxes (E1 and E2) and MEF2/TATA element are underlined. Numbers are relative to the transcriptional start site at +1(39) .



The Upstream Region of the MRF4 Gene Mediates Muscle-specific Expression

Based on its similarity to the myogenin promoter, we predicted that this region of the mouse MRF4 gene would serve as a muscle-specific promoter. To test this hypothesis and determine if this region could provide muscle-specific expression, we subcloned this 390 bp of 5`-flanking sequence upstream of the CAT reporter gene into plasmid pCATBASIC to create plasmid pMRF4.CAT and we transfected this plasmid into both muscle and non-muscle cell types (Fig. 2). The results showed that this MRF4 upstream fragment is incapable of directing expression of CAT in 10T1/2 fibroblast cells but mediates significant expression in the C2 muscle cell line and in primary chick muscle cells. These results indicate that this 390 bp of MRF4 5`-flanking sequence contains promoter and enhancer elements sufficient to mediate muscle-specific activation, and that this promoter element is transcriptionally silent in 10T1/2 fibroblasts. While the activity of the MRF4 promoter was relatively weak in C2 myotubes, it was clearly muscle-specific. The relatively weak activity of the promoter most likely reflects the fact that MRF4 is up-regulated late in the differentiation program and is not expressed at high levels in C2 myotubes(5, 6, 7, 29, 30) . It is also possible that while the promoter sequence used here contains the elements necessary for muscle-specificity, it may not contain all of the enhancer elements required for high level expression in these cell types.


Figure 2: Muscle-specific activity of the 390 bp MRF4 fragment. 10T1/2 cells, early and late C2C12 myotubes, and day 12 chicken primary myoblasts were transfected with equal amounts of plasmid pMRF4.CAT (MRF4), pCATBASIC (BASIC), or pCDNAIII.CAT (CMV) or were untransfected (UNTR). Transfection of each cell type was performed as described under ``Materials and Methods.'' Reactions were analyzed by thin-layer chromatography, an autoradiograph of which is shown. The MRF4 promoter-CAT construct exhibited 1.0-, 2.9-, 3.8-, and 5.2-fold activation over the activity of the pCATBASIC vector in 10T1/2, early C2C12, late C2C12, and chick myoblasts, respectively. CAT activity of cell extracts was determined by the percent conversion of [^14C]chloramphenicol (Cm) to acetylated forms (AcCm). Percent conversion of each reaction is shown at the top of the figure. All four cell types were similarly transfected as the activation of the positive control plasmid pCDNAIII.CAT was approximately equivalent in each. Quantitation was by phosphorimager analysis (Molecular Dynamics, Inc.). Comparable results were obtained in three separate sets of experiments.



Muscle-specific Transcription Factors trans-Activate the MRF4 Promoter

Since the MRF4 promoter contains potential myogenic bHLH and MEF2 binding sites (Fig. 1) and was capable of directing muscle-specific transcription (Fig. 2), we wanted to determine if these two classes of transcription factors were involved in activation of the MRF4 promoter. We cotransfected pMRF4.CAT into 10T1/2 fibroblasts along with activator plasmids encoding one of the four myogenic bHLH factors or MEF2A, MEF2C, or MEF2D. As shown in Fig. 3, myogenin, myf5, and MyoD all mediate a strong trans-activation (about 20-fold) of the MRF4 promoter. In contrast, MRF4 was unable to activate its own promoter in this assay. All of the MEF2 factors tested were able to reproducibly activate the MRF4 promoter from 4-10-fold. These trans-activations were specific for the MRF4 promoter and were not the result of a general transcriptional activation, since neither the bHLH nor the MEF2 activators affected the level of CAT expression when CAT was under the control of a constitutively active viral promoter (data not shown). These results indicate that the MRF4 promoter can be trans-activated by muscle-specific transcription factors and suggest that the putative E boxes and MEF2 sites in the promoter may be involved in this activation.


Figure 3: trans-Activation of the MRF4 promoter by muscle-specific transcription factors. Expression plasmids encoding myogenin, myf5, MyoD, MRF4, MEF2A, MEF2C, or MEF2D were cotransfected into 10T1/2 cells along with the MRF4 promoter reporter plasmid, pMRF4.CAT. In each case 10 µg of reporter and 10 µg of activator were cotransfected. The data are expressed as the -fold activation of the activator cotransfection over a control cotransfection in which the activator plasmid encoded the neo gene rather than one of the muscle-specific transcription factors. CAT activity of cell extracts was determined by thin-layer chromatography and was quantitated by phosphorimager analysis (Molecular Dynamics, Inc.). The data are the average of three independent experiments. Error bars indicate the standard error of the mean for the three experiments.



Myogenin trans-Activation of the MRF4 Promoter is E Box-dependent

In order to determine whether the two E boxes and the single MEF2 site in the MRF4 promoter were involved in trans-activation of the promoter by myogenic bHLH and MEF2 proteins, we mutated each of these elements singly and in combination to produce each possible double and triple mutant. The wild-type 390-bp MRF4 promoter and each of the seven mutant promoter-CAT constructs were then cotransfected into 10T1/2 cells along with a myogenin expression plasmid or with a control plasmid expressing the neomycin resistance (neo) gene. Following transfection, the cells were harvested and assayed for CAT activity. The results in Fig. 4A show that at least one E box is required for trans-activation of the MRF4 promoter by myogenin. Mutation of either one of the E boxes (E1(-) or E2(-)) had no effect on promoter activity, suggesting that only one E box is necessary and sufficient for full activation of the promoter by myogenin. These data also show that the MEF2 site in the MRF4 promoter is not required for indirect activation by myogenic bHLH proteins, since the MEF2 mutations had no effect on trans-activation by myogenin. The activity of the promoter was greatly reduced only when both E boxes were mutated. These results suggest that in 10T1/2 cells, trans-activation of the MRF4 promoter by myogenin, MyoD, and myf5 is direct and requires at least one intact E box motif. The direct nature of this interaction is further supported by gel shift analyses, which showed that both E boxes specifically bound myogenin-E12 heterodimers (data not shown).


Figure 4: Effect of MRF4 promoter mutations on trans-activation by myogenin and MEF2A. Myogenin (A) or MEF2A (B) expression plasmid was cotransfected into 10T1/2 cells with either the wild-type (wt) or any of seven mutants of the MRF4 promoter-CAT plasmid pMRF4.CAT. The data are expressed as fold activation of the myogenin (A) or MEF2A (B) cotransfection over a neo-only activator cotransfection using the same MRF4 reporter construct. In each case 10 µg of reporter and 10 µg of activator were cotransfected. CAT activity of cell extracts was determined by thin-layer chromatography and was quantitated by phosphorimager analysis (Molecular Dynamics, Inc.). The results presented are from a representative cotransfection analysis. The same results were obtained from two independent transfections and analyses. Similar results to those in A were also obtained with myf5 and MyoD activators. Similar results to those in B were also obtained with MEF2C and MEF2D activators. MRF4 promoter mutants: wt, wild-type; E1(-), mutant E1 E box; E2(-), deleted E2 E box; E1/E2(-), mutant E1 E box and deleted E2 E box; M2(-), mutant MEF2 site (TATA site remains intact); M2/E1(-), mutant MEF2 site and mutant E1 E box; M2/E2(-), mutant MEF2 site and deleted E2 E box; M2/E1/E2(-), mutant MEF2 site, mutant E1 E box, and deleted E2 E box.



We also tested the MRF4 promoter mutants for the ability to be trans-activated by MEF2 factors. Whereas the MEF2 factors were able to activate the MRF4 promoter in 10T1/2 cells (Fig. 3), this activation was through an indirect pathway since all of the mutant reporters were as active as the wild-type construct (Fig. 4B). This result is also supported by gel shift data, which showed that this consensus MEF2 binding sequence in the promoter is bound only very weakly by in vitro translated MEF2 proteins (data not shown). The indirect nature of the MEF2 trans-activation seen in Fig. 4B suggests that in vivo the MEF2 consensus sequence in the MRF4 promoter serves as the TATA box as has been previously demonstrated (39) but that it does not function as a MEF2 site.


DISCUSSION

The data presented in this study provide evidence for direct and indirect trans-activation of the MRF4 promoter by muscle-specific transcription factors. We have shown that a small region of the MRF4 gene surrounding the transcription start site directs muscle-specific expression. Myogenin, MyoD, and myf5 strongly trans-activate the MRF4 promoter while MRF4 is incapable of efficiently activating the expression of its own promoter. This activation by other members of the bHLH family is direct and requires at least one intact E box motif. Likewise, members of the MEF2 family of transcription factors are able to trans-activate the MRF4 promoter; however, this activation is weak and appears to function via an indirect mechanism that does not require an intact MEF2 site or E box sequence elements in the promoter. The indirect nature of the MEF2 trans-activation of the MRF4 promoter probably occurs through protein-protein interactions with the basal transcription machinery. Such an interaction would imply that the sequence of the TATA element in the MRF4 promoter imparts a degree of muscle specificity since MEF2 activates the expression of the MRF4 promoter but not the CMV promoter in these cotransfection analyses. This type of functional heterogeneity of TATA elements resulting in muscle-specific activation has been proposed previously(40) . The low level of activation seen using the E1/E2(-) mutants in the myogenin cotransfection (Fig. 4A) may be a result of myogenin activation of endogenous MEF2 factors, which could then indirectly regulate the MRF4 promoter, as in Fig. 4B. Depending on which muscle-specific transcription factors are present, both the direct and indirect pathways described in this study may function in muscle cells in vivo. It is also possible that the regulatory pathways that govern MRF4 expression in muscle cells may vary from these as a result of a more complex set of muscle-specific trans-acting factors present at times during muscle development. It is also likely that additional complexity in MRF4 regulation exists in additional enhancer sequences since 6.5 kb of MRF4 upstream sequence linked to lacZ in transgenic mice recapitulated only part of the pattern of endogenous MRF4 expression(41) .

In spite of the similarity of the MRF4 promoter to the proximal myogenin promoter, the two promoters are regulated quite differently. The myogenin promoter is trans-activated by myogenic bHLH factors via an indirect pathway dependent upon the MEF2 site in the promoter(23) , while the MRF4 promoter is regulated directly by the other bHLH factors. The regulation of the MRF4 promoter is also in contrast to the regulation of the Xenopus and chicken MyoD promoters, which are regulated independently of their E box motifs(26, 40) . Thus, while all of the myogenic bHLH factors are able to mediate myogenesis, it is becoming clear that they are regulated by different mechanisms. It is these different mechanisms that are likely to account for proper expression of each of these bHLH factors during myogenesis.


FOOTNOTES

*
This work was supported in part by grants from the National Institutes of Health, the Muscular Dystrophy Association, and the Robert A. Welch Foundation (to E. N. O.). 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) U18131[GenBank].

§
American Cancer Society postdoctoral fellow.

Supported by a National Institutes of Health training grant.

**
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Box 117, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-3648; Fax: 713-791-9478.

(^1)
The abbreviations used are: bHLH, basic helix-loop-helix; MEF2, myocyte enhancer-binding factor 2; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; DM, differentiation medium; GM, growth medium; bp, base pair(s).


ACKNOWLEDGEMENTS

We appreciate the sequence analysis provided by Mike Chase and the help with chicken cell culture provided by Janet Mar and Mei Zhang. We also thank Janet Mar for critical review of the manuscript.


REFERENCES

  1. Davis, R. L., Weintraub, H., and Lassar, A. B. (1987) Cell 51, 987-1000 [Medline] [Order article via Infotrieve]
  2. Edmondson, D. G., and Olson, E. N. (1989) Genes & Dev. 3, 628-640
  3. Wright, W. E., Sassoon, D. A., and Lin, V. K. (1989) Cell 56, 607-617 [Medline] [Order article via Infotrieve]
  4. Braun, T., Buschhausen-Denker, G., Bober, E., Tannich, E., and Arnold, H. H. (1989) EMBO J. 8, 701-709 [Abstract]
  5. Rhodes, S. J., and Konieczny, S. F. (1989) Genes & Dev. 3, 2050-2061
  6. Braun, T., Bober, E., Winter, B., Rosenthal, N., and Arnold, H. H. (1990) EMBO J. 9, 821-831 [Abstract]
  7. Miner, J. H., and Wold, B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1089-1093 [Abstract]
  8. Edmondson, D. G., and Olson, E. N. (1993) J. Biol. Chem. 268, 755-758 [Free Full Text]
  9. Braun, T., Bober, E., Buschhausen-Denker, G., Kotz, S., Grzeschlik, K.-H., and Arnold, H. H. (1989) EMBO J. 8, 3617-3625 [Abstract]
  10. Weintraub, H. (1993) Cell 75, 1241-1244 [Medline] [Order article via Infotrieve]
  11. Olson, E. N., and Klein, W. H. (1994) Genes & Dev. 8, 1-8
  12. Olson, E. N. (1992) Dev. Biol. 154, 261-272 [Medline] [Order article via Infotrieve]
  13. Pollock, R., and Treisman, R. (1991) Genes & Dev. 5, 2327-2341
  14. Yu, Y.-T., Breitbart, R. E., Smoot, L. B., Lee, Y., Mahdavi, V., and Nadal-Ginard, B. (1992) Genes & Dev. 6, 1783-1798
  15. Chambers, A. E., Kotecha, S., Towers, N., and Mohun, T. J. (1992) EMBO J. 11, 4981-4991 [Abstract]
  16. Leifer, D., Krainc, D., Yu, Y.-T., McDermott, J., Breitbart, R. E., Heng, J., Neve, R. L., Kosofsky, B., Nadal-Ginard, B., and Lipton, S. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1546-1550 [Abstract]
  17. McDermott, J., Cardosa, M. C., Yu, Y.-T., Andres, V., Leifer, D., Krainc, D., Lipton, S. A., and Nadal-Ginard, B. (1993) Mol. Cell. Biol. 13, 2564-2577 [Abstract]
  18. Breitbart, R. E., Liang, C., Smoot, L. B., Laheru, D. A., Mahdavi, V., and Nadal-Ginard, B. (1993) Development 118, 1095-1106 [Abstract/Free Full Text]
  19. Martin, J. F., Schwarz, J. J., and Olson, E. N. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5282-5286 [Abstract]
  20. Martin, J. F., Miano, J. M., Hustad, C. M., Copeland, N. G., Jenkins, N. A., and Olson, E. N. (1994) Mol. Cell. Biol. 14, 1647-1656 [Abstract]
  21. Gossett, L. A., Kelvin, D. J., Sternberg, E. A., and Olson, E. N. (1989) Mol. Cell. Biol. 9, 5022-5033 [Medline] [Order article via Infotrieve]
  22. Cserjesi, P., and Olson, E. N. (1991) Mol. Cell. Biol. 11, 4854-4862 [Medline] [Order article via Infotrieve]
  23. Edmondson, D. G., Cheng, T.-C., Cserjesi, P., Chakraborty, T., and Olson, E. N. (1992) Mol. Cell. Biol. 12, 3665-3677 [Abstract]
  24. Cheng, T.-C., Wallace, M. C., Merlie, J. P., and Olson, E. N. (1993) Science 261, 215-218 [Medline] [Order article via Infotrieve]
  25. Yee, S.-P., and Rigby, P. W. J. (1993) Genes & Dev. 7, 1277-1289
  26. Leibham, D., Wong, M.-W., Cheng, T. C., Schroeder, S., Weil, P. A., Olson, E. N., and Perry, M. (1994) Mol. Cell. Biol. 14, 686-699 [Abstract]
  27. Lassar, A. B., Davis, R. L., Wright, W. E., Kadesch, T., Murre, C., Voronova, A., Baltimore, D., and Weintraub, H. (1991) Cell 66, 305-315 [Medline] [Order article via Infotrieve]
  28. Sassoon, D. A. (1993) Dev. Biol. 156, 11-23 [CrossRef][Medline] [Order article via Infotrieve]
  29. Hinterberger, T. J., Sassoon, D. A., Rhodes, S. J., and Konieczny, S. F. (1991) Dev. Biol. 147, 144-156 [Medline] [Order article via Infotrieve]
  30. Bober, E., Lyons, G. E., Braun, T., Cossu, G., Buckingham, M., and Arnold, H. H. (1991) J. Cell Biol. 113, 1255-1265 [Abstract]
  31. Rudnicki, M. A., Schnegelsberg, P. N. J., Stead, R. H., Braun, T., Arnold, H. H., and Jaenisch, R. (1993) Cell 75, 1351-1359 [Medline] [Order article via Infotrieve]
  32. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., pp. 2.60-2.125, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  33. Horton, R. M. (1993) in PCR Protocols: Current Methods and Applications (White, B. A., ed) Vol. 15, pp. 251-261, Humana Press, Inc., Totowa, NJ
  34. Wobbe, C. R., and Struhl, K. (1990) Mol. Cell. Biol. 10, 3859-3867 [Medline] [Order article via Infotrieve]
  35. Mar, J. H., Antin, P. B., Cooper, T. A., and Ordahl, C. P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6404-6408 [Abstract]
  36. Sternberg, E. A., Spizz, G., Perry, W. M., Vizard, D., Weil, T., and Olson, E. N. (1988) Mol. Cell. Biol. 8, 2896-2909 [Medline] [Order article via Infotrieve]
  37. Black, B. L., and Lyles, D. S. (1992) J. Virol. 66, 4058-4064 [Abstract]
  38. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  39. Hinterberger, T. J., Mays, J. L., and Konieczny, S. F. (1992) Gene (Amst.) 117, 201-207 [CrossRef][Medline] [Order article via Infotrieve]
  40. Dechesne, C. A., Wei, Q., Eldridge, J., Gannoun-Zaki, L., Millasseau, P., Bouguelere, L, Caterina, D., and Paterson, B. M. (1994) Mol. Cell. Biol. 14, 5474-5486 [Abstract]
  41. Patapoutian, A., Miner, J. H., Lyons, G. E., and Wold, B. (1993) Development 118, 61-69 [Abstract/Free Full Text]
  42. Hasty, P., Bradley, A., Morris, J. H., Edmondson, D. G., Venuti, J. M., Olson, E. N., and Klein, W. H. (1993) Nature 364, 501-506 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.