Il fattore di rimodellamento della cromatina BRM ha un ruolo specifico nel corso del differenziamento muscolare
Author(s)
Albini, Sonia
Date Issued
April 4, 2006
Type
Doctoral Thesis
Abstract
Myogenic differentiation is achieved by myogenic bHLH regulatory factors, whose prototype is MyoD, which act in cooperation with the MEF2 family of transcription factors. Upon differentiation signals, MyoD is activated and induces the transcription of muscle-specific genes as well as that of genes involved in cell cycle arrest. Recent studies revealed the requirement of SWI/SNF chromatin-remodelling complexes in MyoD-induced myogenesis. Within the SWI/SNF complexes, Brm and BRG1 are mutually exclusive subunits and harbour the ATPase activity, indicating that two versions of the SWI/SNF complex, associated with either the BRG1 or the BRM ATP-ase subunit, coexist in mammalian cells.
To start investigating the specific role of SWI/SNF complexes (BRG1- or BRM-containing) in muscle differentiation, we analysed the expression of BRM and BRG1 in growing versus differentiating C2 myoblasts. Our data indicate that the levels of Brm mRNA and protein are considerably induced, whereas those of BRG1 remain relatively constant, suggesting that BRM might play a specific role during muscle differentiation. According to this hypothesis is our observation that the expression of dominant negative versions of Brm, carrying mutations in crucial regions of the protein, severely inhibit the ability of MyoD to activate muscle-specific genes. These data while clearly indicating that functional SWI/SNF complexes are required for muscle gene transcription, do not allow distinguishing the specific functions of BRG1 and BRM. In fact, because both BRG1- and BRM-based SWI/SNF complexes share many of the same subunits, introduction of BRM (wt or mutant) may deleteriously affect also the ability of BRG1 to form functional complexes.
To investigate the specific requirement of BRM, we examined MyoD-mediated induction of muscle differentiation in primary embryo fibroblasts derived from BRM -/- mice as compared to their WT counterpart. The results of such analysis indicate that MyoD induces greatly reduced levels of several differentiation markers in cells lacking BRM. By contrast, the absence of BRM does not appear to affect the ability of MyoD to induce cell cycle arrest.
To ascertain that the differentiation defects observed in BRM-deficient cells were truly due to the absence of BRM, and not to additional mutations necessary for proliferation in culture, we re-expressed BRM in these cells by means of retroviral infection. Both a pooled population and independent clones expressing ectopic BRM were then infected with a MyoD-encoding retrovirus, and analyzed for their ability to differentiate. Our results indicate that the re-introduction of BRM in BRM-deficient cells is sufficient to restore normal expression levels of muscle differentiation markers. To establish whether Brm is recruited on muscle gene promoters, we performed experiments of chromatin immunoprecipitation in stable clones expressing Brm as compared to the control cells lacking Brm, after induction of muscle differentiation by ectopic MyoD. Our results indicate that Brm is recruited on muscle promoters together with MyoD, MEF2C, p300 and P/CAF in the BRM +/+ clone, while these factors do not appear to gain access on muscle loci in BRM -/- cells. These data suggest that Brm is required to build a MyoD-transcriptionally active complex on muscle regulatory regions and rise the question of how Brm is targeted on muscle-specific promoters. To address this issue we analysed the ability of Brm and BRG1 to interact with myogenic bHLH and MEF2 transcription factors. Our results highlighted a specific and selective interaction between Brm and MEF2C, while in our experimental conditions we did not find any interaction between Brm and Myod or myogenin. Taken together these data suggest that MEF2C may be the transcription factor implied in the recruitment of Brm on muscle-specific promoters.
The defective differentiation phenotype observed in BRM -/- overlaps that observed previously in Rb -/- cells, suggesting that Brm and Rb could be part of the same pathway during the late phase of muscle differentiation. This hypothesis was supported by recent evidences according to which Brm expression is negatively affected by ras and ras expression is negatively regulated by pRb, suggesting an indirect mechanism by which Rb could affect the expression of Brm. Our data indicate that in Rb -/- myogenic cells in which we reintroduced Rb expression, the levels of Brm mRNA, but not BRG1, are greatly enhanced together with those of late markers of differentiation. These results reveal that the Rb-mediated induction of Brm may be a novel mechanism by which Rb promotes terminal differentiation.
Additional information
Dottorato di ricerca in Genetica e biologia cellulare
Subjects
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