We have characterized the molecular mechanism by which the muscle-specific transcription factor MyoD activates transcription from the RB gene promoter. Our data reveal a novel mechanism of MyoD-dependent transactivation of target genes, linking the activity of MyoD to that of the ubiquitous transcription factor CREB.
Skeletal muscle differentiation is regulated by the MyoD family of transcription factors, which activate transcription by heterodimerizing with ubiquitously expressed E-proteins, to bind a consensus DNA motif (E-box) found in the regulatory region of many muscle genes.
In proliferating myoblasts, differentiation stimuli trigger the MyoD activation required to both promote cell-cycle arrest and initiate the transcriptional cascade leading to muscle-specific gene expression. These two MyoD functions, though tightly coordinated, are temporally separated and controlled by distinct mechanisms. MyoD-mediated growth arrest relies upon the ability to induce the expression of at least three critical cell-cycle regulators: the retinoblastoma (RB) growth suppressor, the CDK inhibitor p21, and cyclin D3. These MyoD-activated genes share the properties of being non-muscle-specific genes already expressed in proliferating myoblasts, and of having their expression levels raised by MyoD, at the onset of differentiation.
We have previously demonstrated that MyoD stimulates RB gene promoter activity by an E-box-independent mechanism. The present study identifies a cAMP-responsive element (CRE) as the target of RB-promoter activation by MyoD. We have determined that CREB is the main transcription factor recognizing the RB promoter CRE in differentiating myoblasts, and that the DNA binding activity of CREB is required by MyoD to enhance transcription from such promoter. We show that the expression levels of CREB and its phosphorylation are induced at the onset of differentiation, and remain at high levels in differentiated myocytes. We provide functional and biochemical evidence that in such cells MyoD becomes associated with CREB, and targeted to the RB-promoter CRE in a complex containing also the p300 and P/CAF coactivators. Our results strongly suggest that MyoD stimulates RB transcription by facilitating the recruitment of p300 and P/CAF on the promoter-bound phospho-CREB.
High levels of pRb are needed for both the growth arrest and differentiation of muscle cells, and for their survival. We found that the ectopic expression in myoblasts of a dominant-negative inhibitor of CREB (A-CREB) induced apoptosis in the early phase of differentiation. The A-CREB ability to induce apoptosis in differentiating myoblasts is consistent with previously described CREB roles in the survival of other cell types, and with the CREB regulatory links with survival-associated factors and genes. We suggest that RB is one of the CREB target genes participating in the maintenance of cell survival.
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