Study Of Myogenesis Mechanism Regulated By Histone Modifications Of Porcine Satellite Cells

Porcine skeletal muscle satellite cells(PSCs)play an important role in muscle development and regeneration in swine.During porcine myogenesis,skeletal muscle satellite cells were regarded as muscle stem cells,which would activate and differentiate into myotubes to participate in muscle formation and repairation.Previous studies have shown that a series of factors regulated proliferation and differentiation of muscle satellite cells,such as signaling pathways,transcription factors and epigenetic modifications.Epigenetic modifications would active the expression of muscle-specific genes,though regulating the covalent modifications of histone and transcription factors and chromatin remodeling,to controll the cell fate of muscle stem cells and the precise transition between differential cell phases.However,little was known about the epigenetic mechanisms during PSCs differentiation.Therefore,in this study,we isolated and in-vitro cultured primary PSCs from newborn piglets.RNA-Seq and ChIP-Seq were performed in proliferation cells and terminal myotubes to interrogate the transcriptome profiles and the distributions of three histone markers,H3K4me3,H3K27me3 and H3K27 ac,together with transcription factor RNA polymerase II(RNAPII),in order to explain the mechanism of histone modifications in regulating the differential expression of transcriptome profiles during PSCs differentiation.The main results are as follows:(1)Establish and optimize the isolation and culture methods of porcine satellite cells.In this study,two PSCs isolation methods were compared.It was found that tissue piece enzyme digestion method was more efficient,which would obtain large mount of satellite cells.The PSCs were proliferated and renewed using this method and cultured with strong myogenic characteristics,which were able to fuse into myotubes after differentiated in medium for two days.Adding leukocyte inhibitory factor(LIF),GSK3β and MEK1/2 inhibitors would maintain the characteristics of muscle stem cells,which up-regulated the hallmark gene Pax7.(2)The purity,passage ability and transfection efficiency of primary PSCs were identified.PSCs would proliferate and differentiated into mytubes after passage.The transfection efficiency was calculated,which revealed 46% GFP+ satellite cells after transfection.In addition,PAX7+ satellite cells were detected 93% using immunofluorescent staining.Taken together PSCs were able to use as a tool for later studies.(3)The expression pattern of myogenic marker genes was identified during PSCs differentiation.PAX7 and MYOD1 were highly expressed in proliferation cells,while MYOG and MYOSIN would only detect in differentiated myotubes.(4)The transcriptome profiles were deeply excavated in proliferation and differentiation satellite cells and the network of differential expressed genes were also reaveled.Transcriptional analysis detected 917 differential expressed genes(DEGs)during PSCs differentiation,which composed of 406 and 511 down-and up-regulated genes,mainly enriched in muscle development and cell cycle-related biological processes.Even some histone deacetylases and chromatin remodeling related genes were found differential expressed during cell differentiation.While the qPCR validation of five up-regulated and nine down-regulated genes indicated consistent results with sequencing data analysis.(5)The genome-wide distribution of epigenetic marks was displayed,and the degree of three histone modifications and the transcription factor RNAPII were globally shrunken during cell differentiation.Among them,H3K27me3 modification showed remarkable depletion by 50% from 22,060 to 11,573.And for RNAPII,3,958 peaks were decreased at transcription start site(TSS)and genebody regions,regulating gene expression pattern and further promoting myogenic differentiation of PSCs.(6)Integrative analysis of histone modification and gene characterization in myogenesis of PSCs were illustrated.The transcription start site of the activated genes was deposited with H3K4me3 and H3K27 ac modifications as well as transcription factor RNAPII.While H3K27me3 modification enriched in the inhibitory chromatin conformation,which regulated the transcription of 179 genes.(7)The regulation mechanism between histone modifications and differential expressed genes was interrogated.During cell differentiation,the H3K27me3 modification decreased,accompanying with the elevating of H3K4me3 and H3K27 ac together with RNAPII,which was able to up-regulated specific genes.However,H3K27me3 reinforcement participated in transcription obstruction of the cell-related genes,and its depletion up-regulated 139 differential expressed genes,promoting PSCs differentiation.In summary,during PSCs differentiation,the H3K27me3 modification depleted at the transcriptional start site of specific myogenic transcription factor to up-regulate the expression of myogenic genes,including MYOG and MEF2 C,which activated the downstream genes,promoting PSCs differentiation and myotube formation.Our findings in this study provide new insights of epigenomic mechanism in myogenic differentiation and shed light on explaining chromatin states and dynamics underlying the process of porcine myogenesis.