Boyarchuk Ekaterina, Robin Philippe, Fritsch Lauriane, Joliot Véronique, Ait-Si-Ali Slimane
Epigenetics and Cell Fate, UMR 7216 CNRS, Centre National de la Recherche Scientifique CNRS - Université Paris Diderot, Sorbonne Paris Cité
Epigenetics and Cell Fate, UMR 7216 CNRS, Centre National de la Recherche Scientifique CNRS - Université Paris Diderot, Sorbonne Paris Cité;
J Vis Exp. 2016 May 17(111):53924. doi: 10.3791/53924.
Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.
骨骼肌终末分化始于多能中胚层前体细胞向成肌细胞的定向分化。这些细胞仍具有增殖能力，或者它们可以分化并融合形成多核肌管，多核肌管进一步成熟形成肌纤维。骨骼肌终末分化是由多种转录因子协同作用调控的，特别是肌肉调节因子（MRFs）家族成员（MyoD、肌细胞生成素、Myf5和MRF4），也被称为生肌bHLH转录因子家族。这些因子在精细的转录调控网络中与染色质重塑复合物协同作用，以实现骨骼肌生成。在此过程中，MyoD被认为是触发肌肉终末分化的主要生肌转录因子。MyoD能够将非肌肉细胞转化为骨骼肌细胞，这一能力进一步强化了这一观点。在这里，我们描述了一种用于全面鉴定MyoD蛋白伴侣的方法，以阐明参与骨骼肌终末分化的不同因子。长期目标是了解骨骼肌基因（即MyoD靶基因）调控中涉及的表观遗传机制。通过串联亲和纯化（TAP标签）从与质谱（MS）表征相结合的异源系统中鉴定MyoD伴侣，随后验证相关伴侣在骨骼肌终末分化过程中的作用。生肌因子的异常形式或其异常调控与多种肌肉疾病相关：先天性肌无力、强直性肌营养不良、横纹肌肉瘤以及肌肉再生缺陷。因此，无论是在导致疾病本身的机制方面，还是在可改善疾病治疗的再生机制方面，生肌因子都为肌肉疾病提供了一系列潜在的治疗靶点。因此，详细了解分子间相互作用以及生肌因子控制的遗传程序对于合理设计有效的治疗方法至关重要。
