MyoD targets chromatin remodeling complexes to the myogenin locus prior to forming a stable DNA-bound complex

The activation of muscle-specific gene expression requires the coordinated action of muscle regulatory proteins and chromatin-remodeling enzymes. Microarray analysis performed in the presence or absence of a dominant-negative BRG1 ATPase demonstrated that approximately one-third of MyoD-induced genes were highly dependent on SWI/SNF enzymes. To understand the mechanism of activation, we performed chromatin immunoprecipitations analyzing the myogenin promoter. We found that H4 hyperacetylation preceded Brg1 binding in a MyoD-dependent manner but that MyoD binding occurred subsequent to H4 modification and Brg1 interaction. In the absence of functional SWI/SNF enzymes, muscle regulatory proteins did not bind to the myogenin promoter, thereby providing evidence for SWI/SNF-dependent activator binding. We observed that the homeodomain factor Pbx1, which cooperates with MyoD to stimulate myogenin expression, is constitutively bound to the myogenin promoter in a SWI/SNF-independent manner, suggesting a two-step mechanism in which MyoD initially interacts indirectly with the myogenin promoter and attracts chromatin-remodeling enzymes, which then facilitate direct binding by MyoD and other regulatory proteins.     

Mice with a targeted disruption of the Fgfrl1 gene die at birth due to alterations in the diaphragm

FGFRL1 is a recently discovered member of the fibroblast growth factor receptor family that is lacking the intracellular tyrosine kinase domain. To elucidate the function of the novel receptor, we created mice with a targeted disruption of the Fgfrl1 gene. These mice develop normally until term, but die within a few minutes after birth due to respiratory failure. The respiratory problems are explained by a significant reduction in the size of the diaphragm muscle, which is not sufficient to inflate the lungs after birth. The remaining portion of the diaphragm muscle appears to be well developed and innervated. It consists of differentiated myofibers with nuclei at the periphery. Fast and slow muscle fibers occur in normal proportions. The myogenic regulatory factors MyoD, Myf5, myogenin and Mrf4 and the myocyte enhancer factors Mef2A, Mef2B, Mef2C and Mef2D are expressed at normal levels. Experiments with a cell culture model involving C2C12 myoblasts show that Fgfrl1 is expressed during the late stages of myotube formation. Other skeletal muscles do not appear to be affected in the Fgfrl1 deficient mice. Thus, Fgfrl1 plays a critical role in the development of the diaphragm.     

MyoD cannot compensate for the absence of myogenin during skeletal muscle differentiation in murine embryonic stem cells

myogenin (-/-) mice display severe skeletal muscle defects despite expressing normal levels of MyoD. The failure of MyoD to compensate for myogenin could be explained by distinctions in protein function or by differences in patterns of gene expression. To distinguish between these two possibilities, we compared the abilities of constitutively expressed myogenin and MyoD to support muscle differentiation in embryoid bodies made from myogenin (-/-) ES cells. Differentiated embryoid bodies from wild-type embryonic stem (ES) cells made extensive skeletal muscle, but embryoid bodies from myogenin (-/-) ES cells had greatly attenuated muscle-forming capacity. The inability of myogenin (-/-) ES cells to generate muscle was independent of endogenous MyoD expression. Skeletal muscle was restored in myogenin (-/-) ES cells by constitutive expression of myogenin. In contrast, constitutive expression of MyoD resulted in only marginal enhancement of skeletal muscle, although myocyte numbers greatly increased. The results indicated that constitutive expression of MyoD led to enhanced myogenic commitment of myogenin (-/-) cells but also indicated that committed cells were impaired in their ability to form muscle sheets without myogenin. Thus, despite their relatedness, myogenin's role in muscle formation is distinct from that of MyoD, and the distinction cannot be explained merely by differences in their expression properties.     

Transcriptional analysis of the titin cap gene

Mutations in titin cap (Tcap), also known as telethonin, cause limb-girdle muscular dystrophy type 2G (LGMD2G). Tcap is one of the titin interacting Z-disc proteins involved in the regulation and development of normal sarcomeric structure. Given the essential role of Tcap in establishing and maintaining normal skeletal muscle architecture, we were interested in determining the regulatory elements required for expression of this gene in myoblasts. We have defined a highly conserved 421 bp promoter proximal promoter fragment that contains two E boxes and multiple putative Mef2 binding sequences. This promoter can be activated by MyoD and myogenin in NIH3T3 fibroblast cells, and maintains the differentiated cell-specific expression pattern of the endogenous Tcap in C2C12 cells. We find that while both E boxes are required for full activation by MyoD or myogenin in NIH3T3 cells, the promoter proximal E box has a greater contribution to activation of this promoter in C2C12 cells and to activation by MyoD in NIH3T3 cells. Together, the data suggest an important role for MyoD in activating Tcap expression through the promoter proximal E box. We also show that myogenin is required for normal expression in vivo and physically binds to the Tcap promoter during embryogenesis.     

Cyclin-mediated inhibition of muscle gene expression via a mechanism that is independent of pRB hyperphosphorylation.

It was recently demonstrated that ectopic expression of cyclin D1 inhibits skeletal muscle differentiation and, conversely, that expression of cyclin-dependent kinase (cdk) inhibitors facilitates activation of this differentiation program (S. S. Rao, C. Chu, and D. S. Kohtz, Mol. Cell. Biol. 14:5259-5267, 1994; S. S. Rao and D. S. Kohtz, J. Biol. Chem. 270:4093-4100, 1995; S. X. Skapek, J. Rhee, D. B. Spicer, and A. B. Lassar, Science 267:1022-1024, 1995). Here we demonstrate that cyclin D1 inhibits muscle gene expression without affecting MyoD DNA binding activity. Ectopic expression of cyclin D1 inhibits muscle gene activation by both MyoD and myogenin, including a mutated form of myogenin in which two potential inhibitory cdk phosphorylation sites are absent. Because the retinoblastoma gene product, pRB, is a known target for cyclin D1-cdk phosphorylation, we determined whether cyclin D1-mediated inhibition of myogenesis was due to hyperphosphorylation of pRB. In pRB-deficient fibroblasts, the ability of MyoD to activate the expression of muscle-specific genes requires coexpression of ectopic pRB (B. G. Novitch, G. J. Mulligan, T. Jacks, and A. B. Lassar, J. Cell Biol., 135:441-456, 1996). In these cells, the expression of cyclins A and E can lead to pRB hyperphosphorylation and can inhibit muscle gene expression. The negative effects of cyclins A or E on muscle gene expression are, however, reversed by the presence of a mutated form of pRB which cannot be hyperphosphorylated. In contrast, cyclin D1 can inhibit muscle gene expression in the presence of the nonhyperphosphorylatable form of pRB. On the basis of these results we propose that G1 cyclin-cdk activity blocks the initiation of skeletal muscle differentiation by two distinct mechanisms: one that is dependent on pRB hyperphosphorylation and one that is independent of pRB hyperphosphorylation.