Abstract
The transcriptional mechanisms driving lineage specification during development are still largely unknown, as the interplay of multiple transcription factors makes it difficult to dissect these molecular events. Using a cell-based differentiation platform to probe transcription function, we investigated the role of the key paraxial mesoderm and skeletal myogenic commitment factors—mesogenin 1 (Msgn1), T-box 6 (Tbx6), forkhead box C1 (Foxc1), paired box 3 (Pax3), Paraxis, mesenchyme homeobox 1 (Meox1), sine oculis-related homeobox 1 (Six1), and myogenic factor 5 (Myf5)—in paraxial mesoderm and skeletal myogenesis. From this study, we define a genetic hierarchy, with Pax3 emerging as the gatekeeper between the presomitic mesoderm and the myogenic lineage. By assaying chromatin accessibility, genomic binding and transcription profiling in mesodermal cells from mouse and human Pax3-induced embryonic stem cells and Pax3-null embryonic day (E)9.5 mouse embryos, we identified conserved Pax3 functions in the activation of the skeletal myogenic lineage through modulation of Hedgehog, Notch, and bone morphogenetic protein (BMP) signaling pathways. In addition, we demonstrate that Pax3 molecular function involves chromatin remodeling of its bound elements through an increase in chromatin accessibility and cooperation with sine oculis-related homeobox 4 (Six4) and TEA domain family member 2 (Tead2) factors. To our knowledge, these data provide the first integrated analysis of Pax3 function, demonstrating its ability to remodel chromatin in mesodermal cells from developing embryos and proving a mechanistic footing for the transcriptional hierarchy driving myogenesis.
Highlights
Embryonic development is characterized by a highly regulated cascade of cell fate choices, involving the concerted action of signaling pathways and transcriptional responses, which results in the specification of progenitors for a specific organ or tissue
We identify that cooperation with sine oculis-related homeobox 4 (Six4) and TEA domain family member 2 (Tead2) is instrumental for the robust paired box 3 (Pax3)-mediated myogenic commitment, indicating that combination of transcription factor (TF) and chromatin remodeling are both required for lineage commitment
To study the role of TFs involved in the specification of the presomitic mesoderm toward the myogenic lineage (Fig 1A), we used an inducible cassette exchange system [23], which allowed for the generation of dox-inducible mouse embryonic stem (ES) cell lines expressing mesogenin 1 (Msgn1), T-box 6 (Tbx6), FoxC1, Pax3, Paraxis, mesenchyme homeobox 1 (Meox1), sine oculis-related homeobox 1 (Six1), or myogenic factor 5 (Myf5) (S1A Fig)
Summary
Embryonic development is characterized by a highly regulated cascade of cell fate choices, involving the concerted action of signaling pathways and transcriptional responses, which results in the specification of progenitors for a specific organ or tissue. Transcription factors (TFs) modulate the expression of lineage-specific genes by binding genomic elements, located either in proximity to transcription start sites (TSSs) or at distal intra- and intergenic regions, referred to as enhancers [1]. The enhancer landscape is instrumental for lineage-specific transcription, and chromatin remodeling at these sites is associated with transcriptional changes [2]. The nucleosome can represent a physical barrier for TF binding to the DNA, a specific class of TFs known as “pioneers” can overcome this inhibition by engaging their targets on nucleosomal DNA [3]. Since lineage specification often involves multiple classes of TFs, the interplay between pioneering activity and formation of macromolecular complexes that initiate transcription is critical for the successful activation of the differentiation program
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