Study under Thrap3 role in skeletal muscle differentiation

Abstract

Skeletal muscle differentiation is controlled by transcriptional networks that integrate extracellular and metabolic cues, however the mechanism of transcriptional cofactors to early myogenesis remains largely unknown. Here, we identify Thrap3 as a previously uncharacterized regulator of myogenic differentiation and FoxO3a-dependent transcription. By integrating multiple independent myogenic transcriptome datasets from GEO, we defined a core set of differentially expressed genes that are consistently regulated during myogenic induction and annotated transcriptional cofactors within this set using AnimalTFDB4 and TcoF-DB. Thrap3 emerged as one of a few shared cofactors, and its mRNA levels were consistently decreased after myogenic induction. Consistent with these data, Thrap3 protein expression was reduced at early time points after induction of differentiation in C2C12 cells. Functional loss of Thrap3 revealed a requirement for this cofactor in myogenic differentiation. Stable Thrap3 knockdown in C2C12 cells markedly attenuated the induction of the myogenic markers MyoD, MyoG, and MyHC and resulted in reduced myotube formation compared with control cells. To explore a mechanistic link, we focused on FoxO3a, a forkhead transcription factor that regulates myogenic gene expression and the E3 ubiquitin ligase atrogin-1 (Fbxo32). Co-immunoprecipitation experiments in HEK293T cells showed that Thrap3 physically interacts with FoxO3a. In proliferating C2C12 myoblasts, Thrap3 knockdown decreased FoxO3a Thr32 phosphorylation and MyoD protein abundance, while Myod1 mRNA remained unchanged, and concurrently increased atrogin-1 at both mRNA and protein levels. Together with previous reports that atrogin-1 targets MyoD for ubiquitin- dependent degradation, these findings support a model in which Thrap3 constrains a FoxO3a–atrogin- 1 axis that otherwise promotes MyoD protein loss. Thus, our data identify Thrap3 as a myogenesis-associated transcriptional cofactor that is downregulated but functionally required during early differentiation and suggest that Thrap3 modulates a FoxO3a–atrogin-1 pathway controlling MyoD protein stability and early myogenic commitment. Future in vivo and genome-wide studies will be needed to define how the Thrap3–FoxO3a–atrogin-1 axis is integrated into broader myogenic signaling networks.