The Role And Mechanism Of HRP2-DPF3a-BAF Epigenetic Regulatory Complex In Myogenesis And Injury-induced Muscle Regeneration

Histone modification and ATP-dependent chromatin remodeling are two important aspects of epigenetics.The crosstalk between histone modification and ATP-dependent chromatin remodeling plays critical roles in epigenetic regulation,however the underlying molecular mechanisms and biological function remain elusive.Epigenetic regulation plays a major role in the determination of cell fate.Myogenesis,the process from myoblasts to myotubes,is a classical model for studying the role of epigenetic regulation in cell fate decisions.Therefore,we hope to find new epigenetic regulatory molecules and further explore the molecular mechanisms and functions of the regulator in myogenesis.To identify epigenetic factors that regulate myogenesis,we first generated C2C12-E3MCK-luc M21 cell line transfected with E3MCK-luc which is a muscle-specific reporter vector.Myogenesis can be monitored by detection of luciferase activity.Using a customized siRNA library that targets 220 epigenetic modifiers,we identified hepatoma-derived growth factor-related protein 2(HRP2)as a key epigenetic regulatory factor of myogenesis in the engineered C2C12-E3MCK-luc M21 cells.Next,to confirm an essential role for HRP2 in myogenesis,we performed a knockdown assay in C2C12.We found that knockdown of HRP2 greatly reduces the expression of two differentiation markers(Myog and MHC).To further investigate the functions of HRP2 in vivo,we generated Hrp2-knockout mice.To examine the role of HRP2 in muscle regeneration,the muscles were degenerated by cardiotoxin(CTX)treatment.Importantly,we observed that Hrp2-knockout mice exhibit impaired muscle regeneration after injury,further emphasizing the importance of HRP2 in myogenesis.To study the molecular mechanisms,we carried out a proteomics analysis to identify HRP2 binding proteins.We identified HRP2 associates with the BRG1/BRM-associated factor(BAF)chromatin remodeling complex by mass spectrometry analysis.Moreover,through literature search,sequence alignment,and biochemical experiments,we demonstrated that HRP2 associates with BAF chromatin remodeling complex by interacting directly with the DPF3 a subunit through its HIV integrase binding domain(IBD).Furthermore,we observed that depletion of DPF3 a dramatically diminishes the expression of two differentiation markers(Myog and MHC)in C2C12.The IBD domain of HRP2,mediating the interaction with DPF3 a,is important.So we further analyzed the structure of IBD domain and found that there is low-sequence complexity regions(LCRs)in this region.Recent studies have shown that proteins with LCRs are likely to have the ability to form phase-separated droplets,so we explored whether the IBD domain of HRP2 can form phase-separated droplets.In vitro,we purified GFP-HRP2-IBD and IBD mutant(R527A / R528A)proteins.Then we proved that HRP2-IBD has the ability to form phase-separated droplets through a series of experiments including changing protein concentration,ionic strength of the buffer,addition of precipitators and fluorescence recovery after photobleaching(FRAP)analysis.At the same time,we purified Cherry-DPF3 a and co-incubated it with HRP2-IBD.We observed that DPF3 a is incorporated into HRP2-IBD liquid droplets.These results suggest that HRP2 and DPF3 a may exist in the same phase-separated droplets to regulate myogenesis.In vivo,we also demonstrated that HRP2 forms phase-separated droplets through a series of experiments including immunofluorescence staining and 1,6-hexanediol treatment.Finally,in order to find their target genes and the regulatory mechanisms,we performed RNA-seq,Ch IP-seq and ATAC-seq.Combined analyses revealed that HRP2 colocalizes with DPF3 a across the genome and that the recruitment of HRP2/DPF3 a to chromatin is dependent on H3K36me2.HRP2 and DPF3 a activated myogenic genes by increasing chromatin accessibility through recruitment of BAF complex to regulate myogenesis.In this study,we find that HRP2 is indispensable for myogenesis and that its deficiency impairs post-injury muscle regeneration in mice.HRP2 preferentially binds to H3K36me2 and recruits the BAF chromatin remodeling complex,by interacting directly with its DPF3 a subunit,to myogenic gene promoters.Thus,HRP2 activates muscle-specific gene expression by increasing chromatin accessibility to regulate myogenesis.And the result also suggests that HRP2 can regulate muscle-specific gene expression through the formation of phase-separated droplets to regulate myogenesis.