(Blum et al., 2012, Asp et al., 2011, Acosta-Alvear et al., 2007; Blais et al., 2005)
We have also used ChIP-on-chip to begin deciphering the regulatory networks that control myogenic differentiation. Myogenesis is orchestrated through a series of transcriptional controls governed by myogenic regulatory factors (MRFs). MyoD, a basic helix-loop-helix (bHLH) transcription factor that binds sequence elements termed E-boxes, is the founding member of the MRF family which includes the closely related Myf5, myogenin, and MRF4 proteins. These proteins cooperate with a second family of transcription factors, called Myocyte Enhancer Factor 2 (MEF2). MRFs are known to activate the expression of genes that specify muscle. The first steps in the regulatory cascade involve expression of MyoD and Myf5, which subsequently leads to expression of myogenin and MEF2, promoting conversion of myoblasts to myotubes. MyoD collaborates with myogenin to regulate the expression of genes necessary for terminal differentiation. Myogenic differentiation proceeds through irreversible cell cycle arrest of precursor cells (myoblasts), followed by a gradual increase in expression of muscle function genes, leading to fusion of myoblasts into multinucleate myofibers in the animal. This process can be recapitulated in vitro, wherein myoblasts can be converted to myotubes with high efficiency in well-established models. In adult skeletal muscle, a pool of self-renewing stem cells (satellite cells) proliferate and differentiate in response to specific stimuli, such as injury or exercise.
However, relatively few physiological targets of MRFs and MEF2 had been identified, and the degree of functional redundancy within the MRF family and the functional impact of binding by individual family members to their targets remained largely undefined. Thus, identification of direct transcriptional targets of MRFs and MEF2 and elucidation of the transcriptional regulatory networks that operate in muscle cells are essential to comprehensively understand not only how muscle differentiates but also how it responds to stress and damage, thereby allowing regeneration.
Given the similarities between regeneration and the muscle differentiation program and the observation that deficiency of certain muscle regulatory factors (MRFs) leads to defects in muscle regeneration, we sought to identify the critical downstream targets of two MRFs, MyoD and the related MRF, myogenin, which is known to play a critical role in the later stages of muscle development. We used our genome-scale approach and C2C12 mouse myoblasts, which represent a well-established in vitro model for mouse muscle differentiation. Proliferating C2C12 myoblasts can be induced to differentiate into multinucleate myotubes by growing them to confluence and switching them to reduced serum (differentiation medium, DM). Under these conditions, most cells fuse to form myotubes after four days. ChIP-on-chip analysis identified a large number of previously undisclosed targets of the muscle regulatory factors MyoD, myogenin, and MEF2C. We found that transcription factors represent the most prominent functional group of MRF and MEF2 targets. This has important implications for myogenesis, as the involvement of multiple transcription factors promotes the propagation and amplification of signals initiated by the MRFs, and the regulation of genes by multiple transcription factors in feed-forward loops lends robustness to the network. Another striking and novel outcome of our location analysis was the identification of a cohort of 40 genes involved in the stress and unfolded protein response (UPR)(Image 3). Notably, sixteen transcriptional regulators (including XBP1) cluster within this group, suggesting that the transcriptional regulatory program initiated by MRFs extends to pathways involved in response to, or protection from, stressful cues. These target genes take part in responding to diverse types of stress, including hypoxia, the UPR, heat hock, and oxidative damage. Moreover, another set of MRF targets (CARP/Ankrd1, Ankrd2, Csrp3/MLP, and calcineurin subunits) appears to be involved in responding to muscle-specific stresses, such as stretch, denervation, alpha-adrenergic stimulation, and hypertrophy, through which increased load/exercise results in enhanced muscle mass. The identification of a large number of targets (in particular transcription factor genes) with a potential role in stress response underscores how this response may be entrained by MRFs. Interestingly, our recent studies with one target of MRFs, the transcription factor XBP1, has indicated how activation of the UPR is linked mechanistically to myogenic differentiation (Acosta et al., 2007).
More recent, extensive ChIP-on-chip studies to identify downstream links in the MRF network has yielded additional targets in unappreciated functional categories that we are currently exploring. We hope that these studies will shed important new light on the myogenic transcriptional network and reveal novel insights into muscle function and disease.
Image 3: Defining transcriptional regulatory networks that underlie myogenic differentiation