The Caenorhabditis elegans protein, CeMyoD, is related to the vertebrate myogenic regulatory factors MyoD, myogenin, MRF-4 and Myf-5. Like its vertebrate counterparts, CeMyoD accumulates in the nucleus of striated muscle cells prior to the onset of terminal differentiation. CeMyoD also shares functional similarities with the vertebrate myogenic regulatory factors. Viral LTR driven expression of CeMyoD in mouse 10T1/2 cells can convert this cell line into myoblasts as well as efficiently Zrans-activate mouse muscle-specific promoters. Furthermore, mouse MyoD expression can activate a CeMyoD-P-galactosidase reporter construct in a 10T1/2 co-transfection assay.

The myogenic regulatory genes encode a subgroup of the helix-loop-helix (HLH) family of transcription factors (Murre et al., 1989b). Originally described in vertebrates, the myogenic subgroup consists of the gene products MyoD (Davis et al., 1987), myogenin (Wright et al., 1989), MRF-4/Herculin/Myf-6 (Rhodes and Konieczny, 1989; Braun et al., 1990a; Miner and Wold, 1990), and Myf-5 (Braun et al., 1989). All share a high degree of sequence conservation across a 60 amino acid stretch that includes the HLH region and an adjacent basic (B) domain (Benezra et al., 1990). The basic region has been shown to constitute the DNA-binding motif of these proteins whereas the HLH region is a dimerization motif that allows multimerization with other HLH proteins (Murre et al., 1989a,b; Davis et al., 1990).

The expression of each of these myogenic factors by itself is sufficient to convert many different cultured cells into myoblasts that will, in turn, differentiate into striated muscle when placed in the appropriate growth conditions (Weintraub et al., 1989; Choi et al., 1990). The myogenic activity of these factors is due to their direct action as transcription factors; they can positively auto-activate themselves, cross-activate each other, and bind to and activate enhancer elements regulating muscle-specific promoters (Braun et al., 1989; Thayer et al" 1989; Edmondson and Olson, 1989; Lassar et al., 1989; Murre et al., 1989a; Braun et al., 1990b; Davis et al., 1990). This ability to convert non-myogenic cells to myoblasts, coupled with an early onset of expression during development, has led to the notion that these myogenic factors play a key role in regulating myogenesis in vertebrates (Weintraub et al., 1991).

CeMyoD and developmental expression

The nematode Caenorhabditis elegans has a gene, hlh-1 (Aelix-/oop-Aelix), that encodes a protein, CeMyoD, that is related to the myogenic regulatory factors of vertebrates (Krause et al., 1990). To date, no other related genes have been identified in the nematode. With in the B-HLH region, 80% of the amino acid residues of CeMyoD are identical to the vertebrate myogenic factors (Table 1), and CeMyoD preferably binds to the E-box (CANNTG) motif (K. Black-well, M. K. and H. W” unpublished data). Outside of the B-HLH region, there is no obvious similarity between CeMyoD and any other myogenic regulatory factor yet described.

Table 1.

Comparison of the basic-helix-loop-helix region of the myogenic factors

Comparison of the basic-helix-loop-helix region of the myogenic factors
Comparison of the basic-helix-loop-helix region of the myogenic factors

As in the vertebrates, CeMyoD is expressed in striated muscle cells and their precursors and begins to accumulate prior to any overt signs of muscle differentiation. CeMyoD expression occurs in two waves during C. elegans development. The first wave is transient, beginning at the 28-cell stage of development and lasting only for two cell divisions. During this stage, low levels of CeMyoD are detected in only one founder cell lineage, the MS (Krause et al., 1990; Chen et al., 1992). Although some descendants of these CeMyoD positive blastomeres will become striated muscle cells, others will become pharyngeal cells, neurons and non-muscle mesodermal cell types. Thus, low level expression of CeMyoD is not sufficient, at this stage of development, to ensure that these cells become committed to the striated muscle cell fate.

A second wave of CeMyoD expression begins at about the 90-cell stage of embryogenesis; expression at this stage is stable, that is, once a blastomere becomes CeMyoD positive, it and its descendants remain CeMyoD positive throughout development (Krause et al., 1990). With one exception, all stable CeMyoD positive blastomeres will become striated muscle cells and only striated muscle cells. Moreover, stable CeMyoD expression precedes terminal differentiation (expression of muscle structural proteins) by up to four cell divisions in some lineages. This early onset of expression is analogous to that seen for the myogenic factors during mammalian development (Bober et al., 1991).

There is one exception to the rule that all stable CeMyoD positive cells become striated muscle. Late in embryogenesis a set of six cells (glial-like cells) near the nerve ring become CeMyoD positive; these cells are not muscle cells and do not stain with antibodies raised against muscle structural proteins. Interestingly, these six cells form gap junctions with some of the striated muscle cells in the head of the animal. It is possible that intercellular signaling through these gap junctions results in CeMyoD accumulation in these six non-muscle cells. Accumulation of CeMyoD in these six cells appears to occur only after they have been born and begun differentiating as glial-like cells. CeMyoD expression, therefore, appears to be unable to alter the fate of a previously differentiated cell type.

CeMyoD can convert murine 10T1/2 cells to muscle

The similarities between the nematode and vertebrate myogenic factors suggested they might also be functionally related. A transient transfection assay of the murine fibroblast cell line C3H10T1/2 (10T1/2) (Taylor and Jones, 1979) has been used to address the question of functional conservation.

A viral LTR expression vector, EMSVscribe (Davis et al., 1987), was engineered to transcribe a full length cDNA encoding CeMyoD in either the sense or anti-sense orientation. These constructs, along with the vector alone or an EMSV mouse MyoD sense construct, were transfected independently into the murine fibroblast cell line 10T1/2. After two days, cells were placed in differentiation media (DMEM supplemented with insulin and transferin) for an additional two days and assayed for myogenesis as previously described (Davis et al., 1987; Davis et al., 1990).

Transfection of the EMSV mouse MyoD construct resulted in a high frequency of myogenic conversion as expected (2%-5% of the cell population). In contrast, both the EMSV vector alone and the EMSV CeMyoD anti-sense construct resulted in no myogenesis as assayed by visual inspection and staining for myosin heavy chain gene expression. However, transfection of the EMSV CeMyoD sense construct resulted in low level myogenesis with approximately 0.1% of the cells forming bipolar cells expressing myosin heavy chain gene. Although weak, this myogenic activity of the EMSV CeMyoD sense construct is significant as both negative controls give no myogenic conversion.

The level of myogenesis observed following transfection of 10T1/2 cells with EMSV CeMyoD sense could be attributable to activation of the mouse myogenic regulatory genes. Steady state mRNA levels of endogenous mouse myogenic gene products were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of total cellular RNA isolated from cells maintained in either proliferative or differentiation media. Specifically, levels of mRNA for transfected CeMyoD and the endogenous genes encoding mouse MyoD, myogenin and muscle creatine kinase (MCK) were compared (Fig. 1).

Fig. 1.

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of gene expression in transfected 10T1/2 cells. (A) 10T 1/2 cell cultures were transfected with either EMSV-CeMyoD (lanes 1 and 3) or EMSV-mouse MyoD (lanes 2 and 4) and grown in differentiation (lanes 1 and 2) or proliferation (lanes 3 and 4) conditions; note similar results are obtained regardless of growth conditions. Total RNA isolated from each culture was used to prime a cDNA reaction with (+) or without (-) the addition of reverse transcriptase. One fifth of the cDNA reaction was subsequently used in a standard PCR reaction to detect the presence or absence of cDNA products corresponding to the following mRNAs: CeMyoD, the transfected nematode MyoD gene; MmMyoD. the endogenous mouse MyoD gene; Myogenin. the endogenous mouse myogenin gene; MCK, the endogenous mouse muscle creatine kinase gene. Transfected mouse MyoD gene products were not assayed and are not detected by the primer pair used to assay the endogenous mouse MyoD gene products. (B) Same as in A except only the endogenous mouse MyoD and myogenin gene products were assayed. CeMyoD expression elicits a weak, but detectable, level of endogenous MyoD and myogenin expression (lanes 1 and 3). For comparison, notice the high levels of endogenous mouse gene expression activated by transfection of the EMSV-mouse MyoD construct (lanes 2 and 4). also binds the E-box (CANNTG) motif, completely eliminates myogenic activity (Davis et al., 1990).

Fig. 1.

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of gene expression in transfected 10T1/2 cells. (A) 10T 1/2 cell cultures were transfected with either EMSV-CeMyoD (lanes 1 and 3) or EMSV-mouse MyoD (lanes 2 and 4) and grown in differentiation (lanes 1 and 2) or proliferation (lanes 3 and 4) conditions; note similar results are obtained regardless of growth conditions. Total RNA isolated from each culture was used to prime a cDNA reaction with (+) or without (-) the addition of reverse transcriptase. One fifth of the cDNA reaction was subsequently used in a standard PCR reaction to detect the presence or absence of cDNA products corresponding to the following mRNAs: CeMyoD, the transfected nematode MyoD gene; MmMyoD. the endogenous mouse MyoD gene; Myogenin. the endogenous mouse myogenin gene; MCK, the endogenous mouse muscle creatine kinase gene. Transfected mouse MyoD gene products were not assayed and are not detected by the primer pair used to assay the endogenous mouse MyoD gene products. (B) Same as in A except only the endogenous mouse MyoD and myogenin gene products were assayed. CeMyoD expression elicits a weak, but detectable, level of endogenous MyoD and myogenin expression (lanes 1 and 3). For comparison, notice the high levels of endogenous mouse gene expression activated by transfection of the EMSV-mouse MyoD construct (lanes 2 and 4). also binds the E-box (CANNTG) motif, completely eliminates myogenic activity (Davis et al., 1990).

Although EMSV mouse MyoD transfection led to high level expression from the three endogenous genes monitored, EMSV CeMyoD sense resulted in only low levels of expression (Fig. 1A,B). For both transfected expression constructs, the level of endogenous myogenic regulatory gene expression correlates with their respective strengths as myogenic activators; high for EMSV mouse MyoD and low for EMSV CeMyoD sense. Immunological staining of EMSV CeMyoD transfected cells shows that mouse MyoD is detected within the nuclei of the myotubes that arise. Taken together, these results suggest that the CeMyoD induced myogenic conversion events are mediated by activation of endogenous myogenic regulatory genes.

It is important to note that regardless of whether CeMyoD directly, or indirectly, activates the myogenic program in 10T1/2 cells, it clearly can function as a myogenic factor within these mouse cells. This myogenic ability is not a general feature of B-HLH proteins but rather is unique to the myogenic subgroup of B-HLH proteins. In fact, replacement of the mouse MyoD basic residues with the corresponding basic region from the HLH protein El2, that

CeMyoD activates mouse muscle-specific promoters

The EMSV CeMyoD constructs have also been used in a co-transfection assay of 10T1/2 cells with two mouse muscle-specific reporter gene constructs linked to bacterial chloramphenicol acteyltransferase (CAT): MCK-CAT (Jaynes et al., 1988) and desmin-CAT (Pieper et al., 1987). CAT activity was quantitated biochemically using a Relabelled acetyl CoA incorporation assay (Sleigh, 1986).

Table 2 shows the results of CAT activity from this type of transient co-transfection experiment. Both MCK-CAT and desmin-CAT are activated to comparable levels (200-to 300-fold above background) by either EMSV mouse MyoD or EMSV CeMyoD in the sense orientation. In contrast. EMSV CeMyoD anti-sense gives only background levels of activation of both CAT reporters.

Table 2.

Trans-activation of CAT reporter gene constructs

Trans-activation of CAT reporter gene constructs
Trans-activation of CAT reporter gene constructs

As discussed above, EMSV CeMyoD activates very low levels of endogenous mouse MyoD and myogenin. To eliminate the possibility that these endogenous mouse myogenic gene products were responsible for the high levels of MCK-CAT and desmin-CAT activity in these experiments, a CAT reporter construct driven by a tandemly repeated mouse MyoD binding site derived from the MCK enhancer was tested. This reporter construct, called 4R-CAT, is efficiently irans-activated by mouse MyoD and myogenin and serves as an indicator of either exogenous or endogenous mouse myogenic B-HLH protein activity (Weintraub et al., 1990). If, for example, CeMyoD-induced low level expression of the endogenous mouse MyoD was activating high levels of MCK-CAT and desmin-CAT, then it should similarly activate high levels of 4R-CAT. However, when EMSV CeMyoD constructs are co-transfected into 10TI/2 cells with 4R-CAT in a transient assay, the 4R-CAT reporter is not activated. Although it is formally possible that EMSV CeMyoD is specifically inhibiting 4R-CAT expression in these co-transfections, a more likely explanation is that CeMyoD positively activates the high level expression of MCK-CAT and desmin-CAT independently of the endogenous B-HLH myogenic regulatory genes. It is not known if CeMyoD activates the MCK and desmin promoters directly or works by interacting with, or activating, other endogenous mouse transcription factors.

Mouse myoD trans-activates CeMyoD

One characteristic of the vertebrate myogenic regulatory factors is that they auto-activate their own expression and can trans-activate expression of at least some of the other myogenic genes. For example, transfection of EMSV mouse MyoD into 10T1/2 cells will activate the endogenous MyoD and myogenin genes (Braun et al., 1989a; Thayer et al., 1989). To determine if mouse MyoD could positively act on sequences of the nematode gene hlh-1 that encodes CeMyoD, EMSV mouse MyoD was co-transfected with three hlh-1 P-galactosidase reporter constructs into 10T1/2 cells. All three hlh-1 reporter constructs were activated by EMSV mouse MyoD but not by the EMSVscribe vector alone (Fig. 2). A p-galactosidase reporter gene construct lacking hlh-1 sequences was not trans-activated suggesting that one or more sites within the hlh-1 region is responsible for the positive effect by mouse MyoD.

Fig. 2.

C. elegans hlh-1 gene structure and p-galactosidase reporter gene fusions. The intron-exon structure of the C. elegans gene hlh-1, that encodes CeMyoD, is shown at top with the exons numbered 1 to 6. The 5 ′ end of the hlh-1 transcript is generated by a trans-splicing reaction indicated by the offset, and unnumbered, exon box labeled SL-1. A potential autoregulatory enhancer site is indicated by a circle within the large intron between exons 1 and 2. Distance (in base pairs) and representative restriction enzyme sites are depicted along a line beneath the gene structure. Three WA-7-β-galactosidase reporter gene constructs are also diagrammed and labeled 37.48, 39.47 and 38.82. The relative efficiency of trans-activation of these reporter genes in murine 10T1/2 cells following co-transfection of EMSV-mouse MyoD is indicated by plus (+) signs.

Fig. 2.

C. elegans hlh-1 gene structure and p-galactosidase reporter gene fusions. The intron-exon structure of the C. elegans gene hlh-1, that encodes CeMyoD, is shown at top with the exons numbered 1 to 6. The 5 ′ end of the hlh-1 transcript is generated by a trans-splicing reaction indicated by the offset, and unnumbered, exon box labeled SL-1. A potential autoregulatory enhancer site is indicated by a circle within the large intron between exons 1 and 2. Distance (in base pairs) and representative restriction enzyme sites are depicted along a line beneath the gene structure. Three WA-7-β-galactosidase reporter gene constructs are also diagrammed and labeled 37.48, 39.47 and 38.82. The relative efficiency of trans-activation of these reporter genes in murine 10T1/2 cells following co-transfection of EMSV-mouse MyoD is indicated by plus (+) signs.

Studies of the nematode hlh-1 promoter elements has shown that the 3 kb upstream of the CeMyoD coding region is sufficient, in transgenic nematode strains, to give the correct temporal and spatial pattern of CeMyoD expression during development (Krause et al., 1990). It was surprising then that the hlh-1 reporter construct that lacks much of these upstream promoter sequences (38.82) responded as well, if not better, than those constructs with an intact promoter region (37.48 and 39.47) (Fig. 2). An inspection of the hlh-1 intron sequences that are retained in construct 38.82 revealed a cluster of seven E-box (CANNTG) elements spanning a 100 bp region; this region could serve as a site for mouse MyoD binding and trans-activation. Preliminary evidence (gel mobility shift and inter-species hlh-1 gene sequence comparisons) supports this notion and suggests that this 100 bp region is a site for CeMyoD auto-activation (T. Gerber, M. K. and H. W., unpublished data).

A comparison of the vertebrate myogenic regulatory factors with the related nematode protein CeMyoD reveals a remarkable degree of evolutionary conservation. In addition to the sequence similarity across the B-HLH domain of these proteins, they share similar temporal and spatial patterns of expression during development. Vertebrate and nematode myogenic proteins also share functional similarities; CeMyoD is able to convert the murine cell line 10T1/2 to myoblasts and efficiently.trans-acüvate mouse muscle-specific promoters in transient transfection assays. Furthermore, mouse MyoD appears to recognize, and activate, a CeMyoD autoregulatory site within the nematode hlh-1 gene.

This is not the first reported instance of a non-mammalian myogenic factor acting in the 10T1/2 cell line. Venuti et al. (1991) isolated a sea urchin analogue of the vertebrate myogenic factors called SUM-1. Transfection of EMSV SUM-1 can convert 10T1/2 cells, and trans-activate muscle-specific reporter gene expression, as efficiently as EMSV mouse MyoD. More recently, the Xenopus laevis MyoDb gene has been used to convert this same cell line to muscle (R. Rupp and H. W., unpublished data).

The ability of these non-mammalian myogenic factors to convert 10T1/2 cells to myoblasts should, however, be kept in perspective; 10T1/2 cells are a cultured line that grow outside the developmental context in which these factors normally operate. One should be cautious, therefore, in ascribing developmental functions to these myogenic factors based solely on this assay.

Related to this point, recent results in C. elegans demonstrate that myogenesis can occur in the absence of zygotic CeMyoD (Chen et al., 1992). This was shown by generating chromosomal deficiencies spanning the hlh-1 (and flanking) genes. Mutant embryos homozygous for these deficiencies arrest at the two-fold stage of embryogenesis but still make striated muscle. Although it is still possible that maternal hlh-1 products or a second myogenic gene (as yet undiscovered) is functioning in these homozygous deficiency animals, these unanticipated results suggest that we do not yet understand the role, if any, of CeMyoD in determining muscle cell fate in the nematode.

Given the similarities between the nematode and vertebrate myogenic factors, one might also reconsider the role of the myogenic regulatory factors in vertebrate myogenesis. Is the developmental process of myogenesis absolutely conserved through evolution or have these distantly related organisms diverged to the point that their respective myogenic factors function in fundamentally different programs? Presently one can make circumstantial arguments in support of either side of this issue. For example, based on the sequence, DNA binding, 10T1/2 cell conversion assay, and temporal and spatial pattern of expression one would conclude that the vertebrate and nematode myogenic proteins are very similar. However, no other B-HLH protein has been uncovered in C. elegans, leading one to wonder if the CeMyoD dimerization partners (E2a and Id gene products) exist in the nematode. Furthermore, no muscle structural gene promoter studied to date in C. elegans has been shown to require an E-box (CANNTG) site for expression.

Circumstantial arguments do not answer the question of whether developmentally relevant functional similarities exist between the vertebrate and nematode myogenic regulatory factors. Fortunately, these are questions that can be answered experimentally through a combination of nematode genetics and vertebrate gene disruptions.

Benezra
,
R.
,
Davis
,
R. L.
,
Lockshon
,
D.
,
Turner
,
D. L.
and
Weintraub
,
H.
(
1990
).
The protein Id: a negative regulator of helix-loop-helix DNA binding proteins
.
Cell
61
,
49
59
.
Bober
,
E.
,
Lyons
,
G. E.
,
Braun
,
T.
,
Cossu
,
G.
,
Buckingham
,
M.
and
Arnold
,
H. H.
(
1991
).
The muscle regulatory gene, Myf-6, has a biphasic pattern of expression during early mouse development
.
J. Cell Biol
.
113
,
1255
1265
.
Braun
,
T.
,
Bober
,
E.
,
Buschhausen-Denker
,
G.
,
Kotz
,
S.
,
Grzeschik
,
K H.
and
Arnold
,
H. H.
(
1989
).
Differential expression of myogenic determination genes in muscle cells: possible autoactivation by Myf gene products
.
EMBOJ
.
8
,
3617
3625
.
Braun
,
T.
,
Bober
,
E.
,
Winter
,
B.
,
Rosenthal
,
N.
and
Arnold
,
H. H.
(
1990a
).
Myf-6, a new member of the human gene family of myogenic determination factors: evidence for a gene cluster on chromosome 12
.
EMBOJ
.
9
,
821
831
.
Braun
,
T.
,
Winter
,
B.
,
Bober
,
E.
and
Arnold
,
H. H.
(
1990b
).
Transcriptional activation domain of the muscle-specific gene-regulatory protein myf5
.
Nature
346
,
663
665
.
Chen
,
L.
,
Krause
,
M.
,
Draper
,
B.
,
Weintraub
,
H.
and
Fire
,
A.
(
1992
).
Body-wall muscle formation in Caenorhabditis elegans embryos that lack the MyoD homolog hlh-1
.
Science
256
,
240
243
.
Choi
,
J.
,
Costa
,
M. L.
,
Mermelstein
,
C. S.
,
Chagas
,
C.
,
Holtzer
,
S.
and
Holtzer
,
H.
(
1990
).
MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle and retinal pigment epithelial cells into striated, mononucleated myoblasts and multinuceated myotubes
.
Proc. Nat. Acad. Sci. USA
87
,
7988
7992
.
Davis
,
R. L.
,
Weintraub
,
H.
and
Lassar
,
A. B.
(
1987
).
Expression of a single transfected cDNA converts fibroblasts to myoblasts
.
Cell
51
,
9871000
.
Davis
,
R. L.
,
Chen
,
P. F.
,
Lassar
,
A. B.
and
Weintraub
,
H.
(
1990
).
The MyoD DNA binding domain contains a recognition code for musclespecific gene activation
.
Cell
60
,
733
746
.
Edmondson
,
D. G.
and
Olson
,
E. N.
(
1989
).
A gene with homology to the myc similarity region of MyoDl is expressed during myogenesis and is sufficient to activate the muscle differentiation program
.
Genes Dev
.
3
,
628
640
.
Jaynes
,
J. B.
,
Johnson
,
J. E.
,
Buskin
,
J. N.
,
Gartside
,
C. L.
and
Hauschka
,
S.
(
1988
).
The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer
.
Mol. Cell Biol
.
8
,
62
70
.
Krause
,
M.
,
Fire
,
A.
,
White-Harrison
,
S.
,
Priess
,
J.
and
Weintraub
,
H.
(
1990
).
CeMyoD accumulation defines the body wall muscle cell fate during C. elegans embryogenesis
.
Cell
63
,
907
919
.
Lassar
,
A. B.
,
Buskin
,
J. N.
,
Lockshon
,
D.
,
Davis
,
R. L.
,
Apone
,
S.
,
Hauschka
,
S. D.
and
Weintraub
,
H.
(
1989
).
MyoD is a sequencespecific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer
.
Cell
58
,
823
831
.
Miner
,
J. H.
and
Wold
,
B.
(
1990
).
Herculin, a fourth member of the MyoD family of myogenic regulatory genes
.
Proc. Nat. Acad. Sci. USA
87
,
1089
1093
.
Murre
,
C.
,
McCaw
,
P. S.
and
Baltimore
,
D.
(
1989a
).
The new DNA binding and dimerization motif in immunoglobulin enhancer binding daughterless, MyoD, and myc proteins
.
Cell
56
,
777
783
.
Murre
,
C.
,
McCaw
,
P. S.
,
Vassin
,
H.
,
Caudy
,
M.
,
Jan
,
L. Y.
,
Jan
,
Y. N.
,
Cabrera
,
C. V.
,
Buskin
,
J. N.
,
Hauschka
,
S. D.
,
Lassar
,
A. B.
,
Weintraub
,
H.
and
Baltimore
,
D.
(
1989b
).
Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence
.
Cell
58
,
537
544
.
Pieper
,
F. R.
,
Slobbe
,
R. L.
,
Ramaekers
,
F. C. S.
,
Cuypers
,
H. T.
and
Bloemendal
,
H.
(
1987
).
Upstream sequences of the hamster demin and vimentin genes regulate expression during in vitro myogenesis
.
EMBO J
.
6
,
3611
3618
.
Rhodes
,
S. J.
and
Konieczny
,
S. F.
(
1989
).
Identification of MRF4: a new member of the muscle regulatory factor gene family
.
Genes Dev
3
,
20502061
.
Sleigh
,
M. J.
(
1986
).
A non-chromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells
.
Ann. Biochem
.
156
,
254
256
.
Taylor
,
S. M.
and
Jones
,
P. A.
(
1979
).
Multiple new phenotypes induced in 10T1/2 cells treated with 5-azacytidine
.
Cell
17
,
771
779
.
Thayer
,
M. J.
,
Tapscott
,
S. J.
,
Davis
,
R. L.
,
Wright
,
W. E.
,
Lassar
,
A. B.
and
Weintraub
,
H.
(
1989
).
Positive autoregulation of the myogenic determination gene MyoDl
.
Cell
58
,
241
248
.
Venuti
,
J. M.
,
Goldberg
,
L.
,
Chakraborty
,
T.
,
Olson
,
E. N.
and
Klein
,
W. H.
(
1991
).
A myogenic factor from sea urchin embryos cabable of programming muscle differentiation in mammalian cells
.
Proc. Nat. Acad. Sci. USA
88
,
6219
6223
.
Weintraub
,
H.
,
Tapscott
,
S. J.
,
Davis
,
R. L.
,
Thayer
,
M. J.
,
Adam
,
M. A.
,
Lassar
,
A. B.
and
Miller
,
A. D.
(
1989
).
Activation of muscle-specific genes in pigment, nerve, fat, liver and fibroblast cell lines by forced expression of MyoD
.
Proc. Nat. Acad. Sci. USA
86
,
5434
5438
.
Weintraub
,
H.
,
Davis
,
R.
,
Lockshon
,
D.
and
Lassar
,
A.
(
1990
).
MyoD binds cooperatively to two sites in a target enhancer sequence: Occupancy of two sites is required for activation
.
Proc. Nat. Acad. Sci. USA
87
,
56235627
.
Weintraub
,
H.
,
Davis
,
R.
,
Tapscott
,
S. J.
,
Thayer
,
M.
,
Krause
,
M.
,
Benezra
,
R.
,
Blackwell
,
T. K.
,
Turner
,
D.
,
Rupp
,
R.
,
Hollenberg
,
S.
,
Shuang
,
Y.
and
Lassar
,
A.
(
1991
).
The MyoD gene family: Nodal point during specification of the muscle cell lineage
.
Science
251
,
761
766
.
Wright
,
W. E.
,
Sassoon
,
D. A.
and
Lin
,
V. K.
(
1989
).
Myogenin, a factor regulating myogenesis, has a domain homologous to MyoDl
.
Cell
56
,
607
617
.