Study On The Role Of DNA Methylation And Chromatin Accessibility During Yak Preadipocyte Differentiation

The yak(Bos grunniens)is a large herbivorous ruminant mainly living in the Qinghai-Tibet Plateau.Adipose tissue not only serves as an important barrier to prevent body heat dissipation in the cold environment,but also provides the energy requirements to maintain normal body metabolism.Therefore,studies on adipogenesis in yaks can help further understand the importance of adipose tissue for the homeostasis of the body’s internal environment under high altitude environmental conditions.Adipogenesis involves the process of preadipocytes differentiation into adipocytes,and epigenetic modification plays an important role in cell differentiation.In this study,m RNA-seq,WGBS and ATAC-seq were used to detect the changes of gene expression and epigenetic modification before and after the differentiation of yak preadipocytes.The characteristics of DNA methylation modification and chromatin accessibility during yak adipocyte differentiation were described.The DNA methylation and chromatin accessibility at the DNA level were integrated and analyzed,and the role of transcription factor RUNX1 in adipocyte differentiation was studied.The major results of the study were as follows:1.A total of 15,045 and 7683 ATAC-seq peaks were detected in yak preadipocytes and adipocytes,respectively.The distribution of peaks in the genome of the two cells was similar,mainly located in the intergenic region,followed by introns,promoters,5’UTR,exons,gene downstream and 3’UTR.Differential chromatin accessibility analysis found 1293 differential peaks,including 1265 down-regulated peaks and 28up-regulated peaks.Differential peaks related genes were significantly enriched in PPAR,Wnt,regulation of actin cytoskeleton and ECM-receptor interaction signaling pathways.Combined analysis of chromatin accessibility and gene expression during adipocyte differentiation revealed that high levels of gene expression were associated with high levels of chromatin accessibility,especially in the 1 kb upstream and downstream of the TSS.The regulatory network diagram of the transcription factors enriched by differential peaks was constructed,and it was found that the transcription factors with the highest connectivity were FOS,JUNB and JUND.2.There are three methylation contexts CG,CHG and CHH(H = A/T/C)in yak preadipocytes and adipocytes.CG is the main methylation context in the whole genome,and the methylation level is the highest.The three methylation contexts showed similar methylation level distribution in preadipocytes and adipocytes.The CG methylation level in different regions of the genome was higher than that of CHG and CHH,and the CG methylation level in exons and introns was higher than that in promoters.A total of862 CG differentially methylated regions(DMRs)were identified,of which 472 were hypermethylated and 390 were hypomethylated.CG DMRs related genes were significantly enriched in c AMP,PI3K-Akt,c GMP-PKG,Rap1 and Hedgehog signaling pathways.The combined analysis of WGBS and m RNA-Seq showed that the genes with high expression had the lowest CG methylation level in their promoter regions.The Spearman correlation coefficient between CG methylation level and gene expression level further illustrated the negative correlation between them.3.There are 46 genes shared between CG DMRs and differential chromatin accessibility,which are significantly enriched in the Hedgehog and PI3K-Akt signaling pathways related to adipogenesis and metabolism.ATAC-seq peaks located in the promoter(≤ 2 kb from TSS)and distal(> 2 kb from TSS)regions have high chromatin opening and low methylation levels.The interaction network diagram of transcription factors enriched by differential peaks and shared genes between CG DMRs and differential chromatin accessibility was constructed,and core transcription factors such as FOS,JUN,JUNB,JUND,SP1,CREB1 and PPARγ were found.4.The expression of RUNX1 showed first a rising and then a falling trend during yak adipocyte differentiation,and the expression level was the highest on the 8th day.Interference with RUNX1 significantly increased the expression of fatty acid transport gene CD36 and adipocyte differentiation marker genes C/EBPα,PPARγ and FABP4(P< 0.05),and significantly decreased the expression of fatty acid synthesis genes ACC,FASN and SCD1(P < 0.05).On the contrary,overexpression of RUNX1 significantly reduced the expression of CD36,C/EBPα,PPARγ and FABP4(P < 0.05),and significantly increased the expression of FASN(P < 0.05).RUNX1 is affected by DNA methylation modification.In summary,two epigenetic modifications at the DNA level,DNA methylation and chromatin accessibility,regulated the differentiation process of yak adipocytes.Differential chromatin accessibility and CG DMRs-related genes are enriched in signaling pathways related to adipogenesis and metabolism.RUNX1 has a negative regulatory effect on the differentiation of yak adipocytes.FOS,JUN,JUNB,JUND,SP1,CREB1 and PPARγ may be the core transcription factors regulating adipocytes differentiation.These results will provide research materials for exploring the mechanism of different epigenetic modifications and their interactions in adipocyte differentiation.