Developmental Cell
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Switch NFix Developmental Myogenesis Daniela Palacios1 and Pier Lorenzo Puri1,2,* 1Dulbecco
Telethon Institute (DTI), IRCCS Fondazione Santa Lucia and European Brain Research Institute, 00161 Roma, Italy Medical Research Institute, La Jolla, CA 92037, USA *Correspondence:
[email protected] or
[email protected] (P.L.P.) DOI 10.1016/j.devcel.2010.03.005 2Sanford-Burnham
During development, skeletal muscles adapt to stage-specific functional and metabolic challenges by switching the expression of specific subset of genes. The mechanism that governs these changes is still enigmatic. In a recent issue of Cell, Messina and coworkers shed light on this issue through the identification of a transcription factor—NFix—that coordinates the switch in gene expression at the transition from embryonic to fetal myoblasts. Every transition in life requires that the preexisting status be erased prior to stepping into a new stage. For instance, it is notoriously difficult to move into a new relationship if the previous one has not been resolved. An analogous situation applies to embryo development, during the transition from one stage to another. Skeletal myogenesis occurs in successive steps, each of them involving distinct progenitor cell types and specific patterns of gene expression (BrysonRichardson and Currie, 2008). The first muscle fibers in the embryo appear by day e11 from the fusion of embryonic myoblasts. By day 16 a second wave of myogenesis is driven by fetal myoblasts, which give rise to most of the adult musculature. In postnatal life, muscle growth and regeneration occurs at the expense of a heterogeneous population of adult muscle progenitors—the satellite cells. Embryonic, fetal, and adult muscle progenitors show different patterns of gene expression that comply with stagespecific activities (Gunning and Hardeman, 1991). Switching on or off specific subsets of genes at each transition is therefore a critical challenge faced by developmental myogenesis. Despite the knowledge of the molecular networks
that specify the myogenic lineage and activate skeletal myogenesis by the cooperative activity of different transcription factors (Guasconi and Puri, 2009), the identities of the cellular factors that coordinate gene repression and activation at each transition remains elusive. In a recent Cell paper, Messina et al. (2010) demonstrate that a single transcription factor— nuclear factor I-x (Nfix)—is necessary and sufficient to mediate the transcriptional switch between embryonic and fetal myogenesis. Nfix belongs to a class of transcription factors consisting of four closely related genes—Nfia, Nffb, Nfic, and Nfix—that are involved in the control of gene expression in a variety of cell types and tissues (Gronostajski, 2000). Nfi-binding sites have been implicated both in gene activation and repression, but the mechanism by which they modulate transcription is still obscure. Nfi proteins bind to DNA either as homodimers or heterodimers with other family members through an N-terminal region; the C-terminal region is highly variable, as the result of extensive alternative splicing, and contains domains responsible for transcriptional activation or repression (Gronostajski, 2000). Mouse models in which the
340 Developmental Cell 18, March 16, 2010 ª2010 Elsevier Inc.
expression of the different family members has been ablated have revealed the role of Nfia and Nfib in brain development (with Nfib being also essential for lung maturation) and of Nfic in correct tooth formation. Nfix-deficient mice die soon after birth with defects in brain, intestine, and skeleton (Pekarik and Belmonte, 2008). Thus, the discovery by Messina et al. (2010) that Nfix has a crucial role in skeletal myogenesis is unanticipated. A genome-wide screen in fetal versus embryonic myoblasts, previously performed by the same group, showed an abundant and preferential expression of Nfix in fetal myoblasts (Biressi et al., 2007). Messina et al. (2010) now use a combination of assays in established and primary (embryonic or fetal) mouse muscle cells to demonstrate the role of Nfix in the activation of gene expression typical of fetal myoblasts. In vivo experiments show that conditional ablation of Nfix in MyoD-expressing cells prevents the initiation of fetal-specific transcription. Consistently, premature expression of Nfix in embryonic myoblasts leads to an anticipated activation of fetal genes and suppression of embryonic genes. This evidence led the authors to conclude that Nfix coordinates gene expression
Developmental Cell
Previews during the embryonic-to-fetal transition by promoting fetal gene expression and repressing embryonic genes. Interestingly, the mechanism of Nfix-mediated regulation of gene expression appears extremely multifaceted and gene-specific. Nfix-mediated activation of the fetal muscle-specific enzyme MCK occurs through an interaction with MEF2A and PKC-theta, leading to PKC-mediated phosphorylation of MEF2A that enables the latter to activate transcription. Curiously, Nfix and MEF2A occupy the enhancer/promoter regions of MCK in the absence of PCK-theta, suggesting two spatially separated activities of Nfix—one to allow PKC-mediated phosphorylation of MEF2A, presumably away from the chromatin, and another to promote MEF2A-directed transcription of MCK, through chromatin interactions. By contrast, activation of beta enolase in fetal muscle does not require the formation of a PKC-containing complex. Repression of embryonic genes, such as slow embryonic myosin heavy chain (MyHC), is achieved by an indirect mechanism consisting of Nfix-mediated repression of NFATc4, which is the physiological activator of embryonic MyHC transcription. Thus, further studies will be needed to elucidate the molecular mechanism involved in Nfix-mediated regulation of target genes. Another interesting issue raised by this work relates to the putative role of Nfix during adult muscle regeneration. In fetal myoblasts, Nfix is activated by the transcription factor Pax7, which is typically expressed in satellite cells. Two pieces of evidence from previous work (discussed in Messina et al., 2010) support the idea of a more general role for Nfix during myogenesis, beyond the embryonic-to-fetal transition. Nfi-binding sites are present on the myogenin promoter, and Nfi can form a complex with this transcription factor, increasing its affinity for some target genes. Future work will determine if Nfix mediates Pax7 downstream functions in satellite cells.
Overall, the work of Messina et al. (2010) points to a general role for Nfi proteins in the control of cell fate and lineage switching during development. This concept is supported by a recent study showing that Nfia expression dictates the differentiation of hematopoietic progenitors along the erythroid lineage while suppressing granulocyte differentiation (Starnes et al., 2009). In analogy with the myogenic program, this effect is mediated, at least in part, by a dual and opposite effect in the activation and repression of erythroid and granulocytic genes. The idea that a single factor can both activate and repress different subsets of genes within the same cell is highly provocative and opens a number of interesting avenues for future work. The ability of Nfix to do so implies that it is simultaneously recruited to the chromatin of different target genes as part of distinct complexes endowed with either activating or repressive activities. It is likely that intracellular signaling cascades, triggered by extrinsic signals in the embryo, dictate the chromatin distribution of Nfix together with other muscle transcription factors during successive waves of myogenesis. Since Nfix binds to palindromic consensus sequences, it is likely that sequences adjacent to Nfix binding sites determine the recruitment of coactivators or corepressors that ultimately endow Nfix with the ability to either promote or repress gene transcription. For instance, when Nfix is recruited to enhancer/promoters regions of muscle genes in the proximity of MEF2-binding sites, it might synergize with MEF2-associated activating complexes. Given the presence of MEF2-binding sites and Ebox sequences (bound by muscle bHLH proteins) on enhancer/promoter elements of muscle genes, an open question relates to the possibility that Nfix can promote the functional cooperation between MEF2 and muscle bHLH proteins (Molkentin and Olson, 1996) to activate fetal muscle
genes. Since MEF2 factors can associate either with transcriptional activators or repressors in response to specific signaling cascades (Potthoff and Olson, 2007), it is also possible that MEF2-associated Nfix obeys the same signal-dependent regulation. Alternatively, it is possible that the presence of other regulatory sequences result in a repressive function for Nfix. Future work, including conditional knock-out strategies, should extend our knowledge of the function of Nfix during skeletal myogenesis. Moreover, the analysis of mice whose adult muscles have developed in the absence of Nfix will reveal whether—like relationships that falter when former attachments still linger—failure to coordinate gene expression during embryonicto-fetal gene switch has catastrophic consequences.
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