![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Molecular and Cellular Biology, September 2002, p. 6199-6208, Vol. 22, No. 17
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.17.6199-6208.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392
Received 17 January 2002/ Returned for modification 26 March 2002/ Accepted 4 June 2002
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The MRFs typically form heterodimers with widely expressed bHLH factors such as E12, E47, HEB, and ITF-1 (25, 30, 37, 49). The MRF-E-protein heterodimer binds to the DNA consensus sequence CANNTG, which is found in the enhancer and promoter regions of most muscle-specific genes. Binding of the MRF-E-protein heterodimer to target genes leads to their transcriptional activation and the subsequent differentiation of myoblasts into functional myotubes. Specificity in DNA binding is mediated by the unique combinatorial interactions of the bHLH regions of individual MRF and E-protein dimers (11, 26, 33, 53), enabling subtle alterations in the targets, as well as the levels of gene transactivation. In addition, cooperative interactions with other DNA binding transcription factors, such as the MEF2 family of transcription factors, is involved in enhancement of the transcription of muscle-specific genes by the MRFs (3, 36, 39). To date, most of the DNA binding and transactivation studies reported for MRFs have focused on terminal differentiation events. Very few studies have examined the transcriptional activity of the MRFs in undifferentiated myoblasts. Most models suggest that the MRFs exhibit no specific function in myoblasts but exist to prime cells for differentiation when the environmental conditions become permissive (i.e., cessation of cellular proliferation signals). The activity of MRFs toward muscle-specific genes is negatively regulated when conditions remain restrictive for differentiation (reviewed in references 32 and 41). Mechanisms mediating this negative regulation include posttranslational modifications leading to reduced heterodimer formation and DNA binding capabilities (16, 29), increased protein turnover (6, 18, 48), and induction of dominant-negative HLH proteins of the Id protein family (25, 33).
One of the remaining challenges in understanding the molecular basis of muscle development is the identification of genes involved in the specification of myogenic stem cells (24). Fundamental differences must exist between the predetermined cells of the epithelial somite and the specified myogenic precursors of the dermomyotome. MyoD and Myf-5 are expressed in myogenic stem cells and thus have long been suspected of specifying these changes. However, no direct target genes have been identified for these MRFs in committed myogenic stem cells and the most attention has been focused on the genes activated by MyoD during the initial stages of differentiation (1, 2, 15, 19, 28, 53).
To identify MyoD transcriptional targets in undifferentiated myoblasts, we utilized C3H10T1/2 (10T1/2) mouse fibroblasts expressing a MyoD protein fused to the estrogen receptor (ER) (19). 10T1/2-MyoD-ER cells are maintained in an undifferentiated state by providing cells with normal growth medium (GM) containing serum. In the presence of ß-estradiol, the MyoD-ER protein undergoes a conformational change and becomes available for dimerization with E-proteins. In GM plus ß-estradiol, the cells remain undifferentiated myoblasts. When cells are switched to differentiation medium (DM) in the absence of ß-estradiol, they remain undifferentiated. However, when ß-estradiol is added to DM, the cells rapidly differentiate and activate the expression of an entire set of muscle-specific gene products (19). By establishing tight control over the timing of ß-estradiol addition to the 10T1/2 MyoD-ER cell line, it is possible to examine the initial events associated with MyoD activity in proliferating cells.
We utilized suppression subtractive hybridization to compare changes in gene expression associated with the induction of MyoD in undifferentiated cells. Our studies have identified two genes, Id3 and NP1 (neuronal pentraxin 1), that are induced in 10T1/2-MyoD-ER cells when the cells are provided ß-estradiol but are not expressed in ß-estradiol-treated 10T1/2 fibroblasts or in 10T1/2-MyoD-ER cells grown in the absence of ß-estradiol. Id3 and NP1 gene expression is detected in undifferentiated myoblasts from the C2C12, 23A2, and L6A1 myogenic cell lines and can be induced in control 10T1/2 cells following transfection with expression plasmids encoding MyoD or myogenin. Interestingly, MyoD-induced activation of Id3 and NP1 gene expression is not subject to the negative regulation imposed on terminal differentiation events by an activated H-Ras G12V protein. Our results reveal the novel finding that MRFs are transcriptionally active in myogenic stem cells, prior to overt terminal differentiation, and have identified two genetic targets for MRF activity that may be essential to the establishment and maintenance of the myogenic stem cell phenotype.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The 10T1/2-MyoD-ER inducible myogenic cell line (9-5) was a generous gift of Stephen J. Tapscott (Fred Hutchinson Cancer Research Center, Seattle, Wash.) and has been described previously (19). 10T1/2-MyoD-ER cells were maintained in high-glucose DMEM supplemented with 10% FBS, 1% penicillin, and streptomycin plus 250 µg of G418 (Mediatech) per ml. Myogenic differentiation was induced by incubation in low-glucose DMEM supplemented with 1% ITS, 1% penicillin and streptomycin, and 36.7 nM 17-ß-estradiol (Sigma) diluted in ethanol.
Immunohistochemistry. Immunohistochemistry was performed on the 10T1/2-MyoD-ER cell line as described previously (35). Briefly, cells were fixed in 4% paraformaldehyde for 10 min and then permeabilized in phosphate-buffered saline containing 0.1% Triton X-100 (PBS-T; Sigma) for 30 min. Cells were treated with 10% calf serum in PBS-T for 30 min and washed three times for 5 min each time with PBS-T, and then primary antibodies supplemented with 0.1% bovine serum albumin were incubated with the cells for 1 h at room temperature. Cells were washed three times for 5 min each time with PBS-T, fluorescently labeled secondary antibodies were added, and the mixture was incubated for 1 h. Primary antibodies used in these studies were obtained from the University of Iowa Developmental Hybridoma Bank and included the antibodies against embryonic myosin heavy chain (MyHC) (MF-20), troponin T (TnT) (CT3), and tropomyosin (Tm) (CH1).
Western blot analysis. Whole-cell protein extracts were harvested from cells by aspirating the medium and adding 25 µl of 4x sodium dodecyl sulfate-Laemmli loading buffer per 60-mm-diameter dish. The crude lysate was sonicated for 15 s and then incubated at 95°C for 5 min. Approximately 20 µg of whole-cell lysate was electrophoresed through a sodium dodecyl sulfate-polyacrylamide gel, and the separated proteins were transferred onto Immobilon-P polyvinylidene difluoride membranes (Bio-Rad) with a semidry transfer apparatus. Membranes were blocked for 30 min with 5% BLOTTO (nonfat dry milk in TBS-T), incubated in primary antibodies for 1 h, rinsed, and incubated for 1 h with secondary antibodies. The blot was then developed with an ECL kit (Pierce) and subsequently exposed to film.
PCR-Select suppression subtractive hybridization. Total RNA was harvested from cell cultures with Trizol (Gibco BRL). Poly(A) mRNA was purified from total RNA with the PolyATract mRNA isolation system (Promega). The poly(A) RNA was used as the starting material for the PCR-Select suppression subtractive hybridization kit (Clontech, Inc.). All reactions were performed exactly as described in the manufacturer's instructions. Briefly, 2 µg of poly(A) RNA from each experimental group (10T1/2-MyoD-ER cells maintained in GM with and without ß-estradiol) was used to generate first- and second-strand cDNA synthesis. The generated cDNA was digested with RsaI, and two different adapters were ligated to separate tester cDNA pools (plus ß-estradiol; groups A and B). The tester and driver (minus ß-estradiol; 100-fold excess) cDNAs were hybridized overnight, and the remaining single-stranded tester A and B cDNAs were hybridized to each other. Double-stranded complexes were amplified by PCR with primers that anneal to the appropriate adapters. Amplified selected cDNAs were cloned into the pT-Adv plasmid (Clontech, Inc.) for further characterization. Inserts were amplified by PCR directly from individual colonies, separated on agarose gels, transferred to nylon membranes, and hybridized with 32P-labeled tester and driver cDNA reaction mixtures that were used in the original subtraction procedure. Potential clones that hybridized specifically to tester cDNAs were characterized further as described in Results.
Expression analysis.
RNA blot hybridizations were performed by separating 20 µg of total RNA through a 1% agarose-6.5% formaldehyde gel. The samples were then transferred to Hybond-N+ (Amersham) membranes and hybridized with [
-32P]dCTP-labeled cDNA probes at 42°C in 50% formamide by following standard procedures. RNA expression also was detected with reverse transcriptase (RT)-coupled PCR. Briefly, 2 µg of total RNA was used for each RT reaction and an aliquot of this material was amplified by PCR with gene-specific primers. All primers crossed intron junctions, and all products were verified by DNA sequencing, nucleic acid hybridization, and restriction enzyme digestion. The DNA primers used in the PCR amplifications are described in Table 1.
|
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
PCR-Select subtractive hybridization. Precise control of MyoD-ER activity in the 10T1/2 MyoD-ER cell line provides a system for investigation of the initial stages of myogenesis. Previous groups have used this system to identify immediate targets of MyoD during the first few hours of differentiation (2, 14, 19). However, little information is available regarding the genetic targets of MyoD activity in cells maintained in GM. To identify potential MyoD gene targets in proliferating myoblasts, we compared gene expression profiles of 10T1/2-MyoD-ER cells cultured for 18 h in GM plus ethanol (driver RNA) or cultured in GM containing ß-estradiol (tester RNA). PCR-generated products that survived the subtractive hybridization procedure were cloned. The initial procedure yielded 5,352 individual colonies containing inserts, of which 557 were chosen for further analysis. Inserts were amplified by PCR, and the DNA fragments were resolved on four duplicate agarose gels, transferred to Hybond N+ nylon membranes, and hybridized with radiolabeled probes representing each subtraction. Inserts that displayed specific hybridization to the forward subtraction, but not to the reverse subtraction, were chosen as representing transcripts whose expression was up-regulated as a result of MyoD-ER transactivation. Of the 30 clones identified as being up-regulated in the MyoD-ER cells exposed to ß-estradiol, several contained overlapping fragments corresponding to the mouse Id3 and NP1 genes. Id3 encodes an HLH nuclear protein that is known to serve as a negative regulator of bHLH factors (7, 31, 34), while NP1 encodes a secreted glycoprotein that previously has been thought to be expressed primarily in neuronal cells (12, 47).
Characterization of Id3 and NP1 gene expression. In order to examine and confirm the regulated expression of Id3 and NP1 in 10T1/2-MyoD-ER cells, total RNA isolated from cells maintained in GM for 18 h in the presence or absence of ß-estradiol was analyzed by RT-PCR. The results demonstrate that Id3 and NP1 transcripts are not detected in 10T1/2-MyoD-ER cells maintained in GM, even after 33 amplification cycles, while transcripts from Id3 and NP1 are detected in cells treated with GM plus ß-estradiol (Fig. 4A). Semiquantitative PCR shows that the expression of these genes is fairly equivalent since the transcripts are not detected after 18 PCR cycles but appear by 23 PCR cycles. A similar profile of expression is observed for the control ß-actin-encoding gene. Id3 and NP1 gene expression is specific for cells containing MyoD and is not an artifact of ß-estradiol treatment since control 10T1/2 cells do not produce Id3 and NP1 transcripts (Fig. 4B).
|
|
|
|
15 min) (2), these results suggest that MyoD activation of the Id3 and NP1 genes represents a stable, heritable change in gene expression patterns that no longer rely on continued MyoD activity.
|
|
The identity of committed myoblasts versus terminally differentiated muscle cells is an important distinction when defining stem cell populations. Previous studies in our laboratory have shown that expression of a constitutively active H-Ras G-protein can block MyoD-induced terminal differentiation (23, 35), although it was unclear whether H-Ras solely blocks terminal differentiation events or represses the myoblast commitment stage. In order to examine the effect of H-Ras G12V expression on Id3 and NP1 gene activity, 10T1/2 fibroblasts were transfected with expression vectors encoding MyoD, or MyoD plus H-Ras G12V, and then maintained in GM or DM. As shown in Fig. 10, MyoD cells in GM express Id3 and NP1. Similarly, MyoD cells in DM activate expression of the Id3 and NP1 genes, as well as the differentiation-specific genes myogenin and myosin. Interestingly, cells expressing H-Ras G12V continue to express Id3 and NP1 even though H-Ras G12V clearly represses expression of differentiation markers (myogenin and myosin) (Fig. 10), as well as formation of myotubes (data not shown). These results show, for the first time, that H-Ras G12V blocks terminal differentiation events but does not alter the molecular identity of myoblast stem cells.
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The main targets for the MRFs are the genes involved in the terminal differentiation process itself, and numerous studies have revealed that activation of these genes occurs only when myoblasts are provided the proper environmental conditions, namely, reduced growth. Myogenic stem cells maintained in a high concentration of serum remain undifferentiated, whereas cells switched to a low concentration of serum generate transcriptionally active MRFs and initiate a complex pattern of gene expression required for sarcomere formation and muscle contraction (1, 15). The tight control of the MRFs is known to occur through a variety of intracellular signal transduction pathways, including regulation of the phosphorylation (16, 29), acetylation (43, 46), and ubiquitination (6, 18, 48) status of the proteins. Posttranslational regulation of the MRFs ensures that myogenic cell differentiation occurs at the correct developmental time and embryological location. Unregulated muscle differentiation can lead to severe abnormalities within the developing embryo.
Given the need to properly control the activity of the MRFs, it remains unclear why MyoD and Myf-5 are expressed in undifferentiated myoblasts. Current models contend that these MRFs are present to allow myoblasts to differentiate rapidly under appropriate environmental conditions. However, it is possible that MyoD (for example) exhibits transcriptional activity in myogenic stem cells and that the correct gene targets for MyoD have not been identified. Indeed, Thayer et al. (50) provided the first evidence that MyoD is transcriptionally active in myoblasts when they reported that exogenously expressed MyoD is capable of activating the endogenous MyoD gene in undifferentiated myoblasts, even though the obvious MyoD target genes (myosin, troponin, and M-creatine kinase) remain transcriptionally silent. The unique activity of MyoD has also been studied by Gerber et al. (14), who showed that MyoD is able to mediate transcriptional activation of muscle-specific genes in repressive chromatin with an efficiency 10-fold greater than that of myogenin. These results demonstrate that the MRFs exhibit distinct molecular activities, as well as unique embryonic expression patterns.
In this study, we have taken advantage of the inducible 10T1/2-MyoD-ER cell line to examine if MyoD is transcriptionally active in myogenic stem cells. Our data demonstrate that the Id3 and NP1 genes are activated by MyoD in undifferentiated myoblasts, prior to overt terminal differentiation. Both genes are transcriptionally silent in control 10T1/2 fibroblasts, in 10T1/2-MyoD-ER cells maintained in the absence of ß-estradiol in GM, and in 10T1/2-MyoD-ER cells maintained in the absence of ß-estradiol in DM. However, once ß-estradiol is provided, effectively activating the constitutively expressed MyoD-ER fusion protein, Id3 and NP1 gene transcription is induced. Expression of NP1 and Id3 is also obtained by transient expression of MyoD or myogenin in 10T1/2 fibroblasts, confirming that the expression of these genes in the 10T1/2-MyoD-ER cell line is not restricted to this experimental system. Indeed, analysis of undifferentiated myoblasts from established myogenic cell lines confirms that the Id3 and NP1 genes are expressed in these myogenic stem cells. Although MyoD is responsible for the initial activation of the Id3 and NP1 genes, continued expression does not require further MyoD activity. These results suggest that activation of the Id3 and NP1 genes represents a stable, heritable event that is associated with myoblast stem cells.
One of the most interesting results of this study is the observation that H-Ras G12V does not alter Id3 and NP1 gene expression although the signaling pathways through which H-Ras functions are potent inhibitors of terminal differentiation events (23, 35). This observation implies that the molecular events by which MyoD activates gene expression programs in myogenic stem cells versus during terminal differentiation are subject to distinct regulatory mechanisms. Indeed, it seems likely that MyoD activity in myoblasts is unaffected by the negative influences of serum growth factors that block terminal differentiation. Understanding these distinct intracellular pathways through which MyoD functions will be an important next step in the characterization of the complete phenotype of myogenic stem cells.
The isolation of the NP1 gene in this study was surprising since NP1 has been shown to be a neuron- and brain-specific gene (12, 47). At the same time, recent investigation of the Myf-5 gene has demonstrated that, in addition to muscle expression, Myf-5 is also expressed in the brain (10). With this in mind, it remains possible that MyoD is expressed in these regions, stimulating NP1 gene expression in the brain, as well as in myogenic stem cells. Since the exact function of NP1 is not fully understood (12, 22, 47), it seems likely that NP1 also serves an important role in myogenic stem cell development. Future studies examining the activity of NP1 should include analyses of skeletal muscle and neuronal tissues.
The identification of Id3 as a MyoD-regulated gene in undifferentiated myoblasts is an intriguing finding. Id3 is expressed in myoblasts and has been reported to function to block MyoD-induced myogenic differentiation of cultured cells (7, 31). However, there is no direct evidence that Id3 blocks the activities of MyoD in proliferating myoblasts. Indeed, members of the Id family of HLH factors are known to function as repressors of terminal differentiation events by forming DNA binding inactive complexes with the normal MyoD bHLH partner proteins E12, E47, ITF-1, and HEB (8, 20, 33). These Id HLH proteins are also known to enhance cellular proliferation, possibly ensuring that cells remain undifferentiated (31, 42). The ability of MyoD to simultaneously activate expression of the Id3 and NP1 genes suggests that Id3 does not inhibit MyoD transcriptional activity in undifferentiated myoblasts and that the functional complex(es) for MyoD in undifferentiated myoblasts and in differentiated myotubes may be quite different. For instance, the preferred complex within differentiated myotubes is a MyoD-E-protein heterodimer (20, 33), and thus, if Id3 functions to sequester the E-protein partners from MyoD, differentiation events are blocked. In contrast, functional MyoD complexes in undifferentiated myoblasts may be significantly different, possibly existing as MyoD homodimers (20) or as heterodimers with unknown protein partners. These complexes may be resistant to the repressor activity attributed to Id3 in differentiated myotubes. Each of these models suggests a mechanism by which MyoD is unaffected by the signaling events (i.e., H-Ras) that block the ability of MyoD to transcriptionally activate differentiation-specific genes. These models also provide a possible mechanism(s) by which MyoD can specify the myoblast lineage through activation of myoblast-specific gene products and, at the same time, ensure that the proper molecular mechanisms are in place to prevent untimely terminal differentiation events. Further studies are needed to identify the mechanism by which MyoD activates expression of the Id3 gene and to understand the developmental consequences of Id3 gene expression in the context of myogenic stem cells.
The revelation that MyoD initiates the expression of genes in proliferating myoblasts is a fundamental step toward understanding the complete myogenic program. Further analyses must be undertaken to identify additional genes that may help to shape the identity of a proliferating myoblast. More importantly, the true regulation of Id3 and NP1 gene expression needs to be investigated and the molecular interactions, cellular localizations, and functions of these proteins should be defined to clarify their roles in myoblast determination. Characterization of the myoblast phenotype in its earliest stages could yield a wealth of information relevant to other types of cellular determination and differentiation events in which lineage-specific bHLH proteins are involved.
|
ACKNOWLEDGMENTS |
|---|
This work was supported by National Institutes of Health grant AR41115 to S.F.K.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Bergstrom, D. A., B. H. Penn, A. Strand, R. L. S. Perry, M. A. Rudnicki, and S. J. Tapscott. 2002. Promoter-specific regulation of MyoD binding and signal transduction cooperate to pattern gene expression. Mol. Cell 9:587-600.[CrossRef][Medline]
3. Black, B., and E. N. Olson. 1998. Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu. Rev. Cell. Dev. Biol. 14:167-196.[CrossRef][Medline]
4. Brand-Saberi, B., and B. Christ. 2000. Evolution and development of distinct cell lineages derived from somites. Curr. Top. Dev. Biol. 48:1-42.[Medline]
5. Braun, T., M. A. Rudnicki, H. H. Arnold, and R. Jaenisch. 1992. Targeted inactivation of the muscle regulatory factor gene Myf-5 results in abnormal rib development and perinatal death. Cell 71:369-382.[CrossRef][Medline]
6. Breitschopf, K., E. Bengal, T. Ziv, A. Admon, and A. Ciechanover. 1998. A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein. EMBO J. 17:5964-5973.[CrossRef][Medline]
7. Chen, B., B. H. Han, X.-H. Sun, and R. W. Lim. 1997. Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function. Nucleic Acids Res. 25:423-430.
8. Chen, B., and R. W. Lim. 1997. Physical and functional interactions between the transcriptional inhibitors Id3 and ITF-2b. J. Biol. Chem. 272:2459-2463.
9. Dale, K., and O. Pourquie. 2000. A clock-work somite. Bioessays 22:72-83.[CrossRef][Medline]
10. Daubas, P., S. Tajbakhsh, J. Hadchouel, M. Primig, and M. Buckingham. 2000. Myf5 is a novel early axonal marker in the mouse brain and is subjected to post-transcriptional regulation in neurons. Development 127:319-331.[Abstract]
11. Davis, R. L., P.-F. Cheng, A. B. Lassar, and H. Weintraub. 1990. The MyoD binding domain contains a recognition code for muscle-specific gene activation. Cell 60:733-746.[CrossRef][Medline]
12. DeGregorio-Rocasolano, N., T. Gasull, and R. Trullas. 2001. Overexpression of neuronal pentraxin 1 is involved in neuronal death evoked by low K+ in cerebellar granule cells. J. Biol. Chem. 276:796-803.
13. Dockter, J. 2000. Sclerotome induction and differentiation. Curr. Top. Dev. Biol. 48:77-127.[Medline]
14. Gerber, A. N., T. R. Klesert, D. A. Bergstrom, and S. J. Tapscott. 1997. Two domains of MyoD mediate transcriptional activation of genes in repressive chromatin: a mechanism for lineage determination in myogenesis. Genes Dev. 11:436-450.
15. Halevy, O., B. G. Novitch, D. B. Spicer, S. X. Skapek, J. Rhee, G. J. Hannon, D. Beach, and A. B. Lassar. 1995. Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD. Science 267:1018-1021.
16. Hardy, S., Y. Kong, and S. F. Konieczny. 1993. Fibroblast growth factor inhibits MRF4 activity independently of the phosphorylation status of a conserved threonine residue within the DNA-binding domain. Mol. Cell. Biol. 13:5943-5956.
17. Hasty, P., A. Bradley, J. H. Morris, D. G. Edmondson, J. M. Venuti, E. N. Olson, and W. H. Klein. 1993. Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature 364:501-532.[CrossRef][Medline]
18. Hatoum, O. A., S. Gross-Mesilaty, K. Breitschopf, A. Hoffman, H. Gonen, A. Ciechanover, and E. Bengal. 1998. Degradation of myogenic transcription factor MyoD by the ubiquitin pathway in vivo and in vitro: regulation by specific DNA binding. Mol. Cell. Biol. 18:5670-5677.
19. Hollenberg, S. M., P. F. Cheng, and H. Weintraub. 1993. Use of a conditional MyoD transcription factor in studies of MyoD trans-activation and muscle determination. Proc. Natl. Acad. Sci. USA 90:8028-8032.
20. Kadesch, T. 1993. Consequences of heteromeric interactions among helix-loop-helix proteins. Cell Growth Differ. 4:49-55.[Medline]
21. Kaul, A., M. Koster, H. Neulhaus, and T. Braun. 2000. Myf-5 revisited: loss of early myotome formation does not lead to a rib phenotype in homozygous Myf-5 mutant mice. Cell 102:17-19.[CrossRef][Medline]
22. Kirkpatrick, L. L., M. M. Matsuki, D. C. Dodds, and M. S. Perin. 2000. Biochemical interactions of the neuronal pentraxins. J. Biol. Chem. 275:17786-17792.
23. Kong, Y., S. E. Johnson, E. J. Taparowsky, and S. F. Konieczny. 1995. Ras p21val inhibits myogenesis without altering the DNA binding or transcriptional activities of the muscle regulatory factors. Mol. Cell. Biol. 15:5205-5213.[Abstract]
24. Konieczny, S. F., and C. P. Emerson. 1984. 5-Azacytidine induction of stable mesodermal stem cell lineages from 10T1/2 cells: evidence for regulatory genes controlling determination. Cell 38:791-800.[CrossRef][Medline]
25. Langlands, K., X. Yin, G. Anandi, and E. V. Prochownik. 1997. Differential interactions of Id proteins with basic helix loop helix transcription factors. J. Biol. Chem. 272:19785-19793.
26. Lassar, A., J. N. Buskin, D. Lockshon, R. L. Davis, S. Apone, S. D. Hauschka, and H. Weintraub. 1989. MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer. Cell 58:823-831.[CrossRef][Medline]
27. Lemercier, C., R. Q. To, R. A. Carrasco, and S. F. Konieczny. 1998. The basic helix-loop-helix transcription factor Mist1 functions as a transcriptional repressor of MyoD. EMBO J. 17:1412-1422.[CrossRef][Medline]
28. Li, H., and Y. Capetanaki. 1992. Regulation of the mouse desmin gene: transactivation by MyoD, myogenin, MRF4, and Myf5. Nucleic Acids Res. 21:335-343.
29. Li, L., J. Zhou, G. James, R. Heller-Harrison, M. P. Czech, and E. N. Olson. 1992. FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domains. Cell 71:1181-1194.[CrossRef][Medline]
30. Lin, H., and S. F. Konieczny. 1992. Identification of MRF4, myogenin, and E12 oligomer complexes by chemical crosslinking and two-dimensional gel electrophoresis. J. Biol. Chem. 267:4773-4780.
31. Loveys, D. A., M. B. Streiff, and G. J. Kato. 1996. E2A basic-helix-loop-helix transcription factors are negatively regulated by serum growth factors and by the Id3 protein. Nucleic Acids Res. 24:2813-2820.
32. Ludolph, D. C., and S. F. Konieczny. 1995. Transcription factor families: muscling in on the myogenic program. FASEB J. 9:1595-1604.[Abstract]
33. Massari, M. E., and C. Murre. 2000. Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms. Mol. Cell. Biol. 20:429-440.
34. Meinikova, I. N., and B. A. Christy. 1996. Muscle cell differentiation is inhibited by the helix-loop-helix protein Id3. Cell Growth Differ. 7:1067-1079.[Abstract]
35. Mitin, N., A. J. Kudla, S. F. Konieczny, and E. J. Taparowsky. 2001. Signaling through NF
B is not required for Ras-induced inhibition of skeletal muscle differentiation. Oncogene 20:1276-1286.[CrossRef][Medline]
36. Molkentin, J. D., B. L. Black, J. F. Martin, and E. N. Olson. 1995. Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins. Cell 83:1125-1136.[CrossRef][Medline]
37. Murre, C., P. S. McCaw, and D. Baltimore. 1989. A new binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell 56:777-783.[CrossRef][Medline]
38. Nabeshima, Y., K. Hanaoka, M. Hayasaka, E. Esumi, S. Li, I. Nonaka, and Y. Nabeshima. 1993. Myogenin gene disruption results in perinatal lethality because of severe muscle defect. Nature 364:532-535.[CrossRef][Medline]
39. Naya, F., and E. N. Olson. 1999. Mef2: a transcriptional target for signaling pathways controlling skeletal muscle growth and differentiation. Curr. Opin. Cell Biol. 11:683-688.[CrossRef][Medline]
40. Olson, E., H. H. Arnold, P. W. J. Rigby, and B. J. Wold. 1996. Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell 85:1-4.[CrossRef][Medline]
41. Perry, R. L. S., and M. A. Rudnicki. 2000. Molecular mechanisms regulating myogenic determination and differentiation. Front. Biosci. 5:750-767.
42. Peverali, F. A., T. Ramqvist, R. Saffrich, R. Pepperkok, R. V. Barone, and L. Philipson. 1994. Regulation of G1 progression by E2A and Id helix-loop-helix proteins. EMBO J. 13:4291-4301.[Medline]
43. Polesskaya, A., I. Naguibneva, L. Fritsch, A. Duquet, S. A.-S. Ali, P. Robin, A. Vervisch, L. L. Pritcahrd, P. Cole, and A. Harel-Bellan. 2001. CBP/p300 and muscle differentiation: no HaT, no muscle. EMBO J. 20:6816-6825.[CrossRef][Medline]
44. Rudnicki, M., T. Braun, S. Hinuma, and R. Jaenisch. 1992. Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Cell 71:383-390.[CrossRef][Medline]
45. Rudnicki, M., P. N. J. Schnegelsberg, R. H. Stead, T. Braun, H. H. Arnold, and R. Jaenisch. 1993. MyoD or Myf-5 is required for the formation of skeletal muscle. Cell 75:1351-1359.[CrossRef][Medline]
46. Sartorelli, V., P.L Puri, Y. Hamamori, V. Ogryzko, G. Chung, Y. Nakatani, J. Y. Wang, and L. Kedes. 1999. Acetylation of MyoD directed by PCAF is necessary for the execution of the muscle program. Mol. Cell 4:725-734.[CrossRef][Medline]
47. Schlimgen, A. K., J. A. Heims, H. Vogel, and M. S. Perin. 1995. Neuronal pentraxin, a secreted protein with homology to acute phase proteins of the immune system. Neuron 14:519-526.[CrossRef][Medline]
48. Song, A., Q. Wang, M. G. Goebl, and M. A. Harrington. 1998. Phosphorylation of nuclear MyoD is required for its rapid degradation. Mol. Cell. Biol. 18:4994-4999.
49. Sun, X. H., and D. Baltimore. 1991. An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but not in E12 heterodimers. Cell 64:459-470.[CrossRef][Medline]
50. Thayer, M. J., S. J. Tapscott, R. L. Davis, W. E. Wright, A. B. Lassar, and H. Weintraub. 1969. Positive autoregulation of the myogenic determination gene MyoD1. Cell 58:241-248.
51. Vaidya, T. B., S. J. Rhodes, E. J. Taparowsky, and S. F. Konieczny. 1989. Fibroblast growth factor and transforming growth factor ß repress expression of the myogenic regulatory gene MyoD1. Mol. Cell. Biol. 9:3576-3579.
52. Venuti, J., J. H. Morris, J. L. Vivian, E. N. Olson, and W. H. Klein. 1995. Myogenin is required for late but not early aspects of myogenesis during development. J. Cell Biol. 128:563-576.
53. Yutzey, K. E., S. J. Rhodes, and S. F. Konieczny. 1990. Differential trans activation associated with the muscle regulatory factors MyoD1, myogenin, and MRF4. Mol. Cell. Biol. 10:3934-3944.
54. Zhang, W., R. R. Behringer, and E. N. Olson. 1995. Inactivation of the myogenic bHLH gene MRF4 results in up-regulation of myogenin and rib abnormalities. Genes Dev. 9:1388-1399.
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. |
|---|
