Integrated Analyses Of Zhongwei Goat Hair Follicle Development Based On Genome-wide DNA Methylome And Transcriptomic Profile

As a rare and precious fur goat breed,Chinese zhongwei goat has a reputation for its pelts worldwide,which are obtained at about 35 days of age.Nevertheless,these natural and exquisite patterns of fleeces are disappearing within two months,causing decreasing economic value with the curly wool straightening.Significantly,the exact reasons of this dynamic transformation are still elusive.Epigenetic mechanisms play essential roles in regulating epidermis development,regeneration,aging,periodic growth of hair follicles and other biological processes.In this study we sampled shoulder skin tissues from the same Chinese zhonwei goats in their 45 days and 108 days after birth,to get the DNA methylation landscape and transcriptional expression by conducting whole genome bisulfite sequencing(WGBS)and RNA sequencing(RNA-seq).Integrated analysis(WGBS and RNA-seq)was performed using differentially methylated regions(DMRs)and differentially expressed m RNAs,and candidate gene PDGFC was selected to performe the following biological function validation on human hair follicle inner root sheath(HHIRS)cells.The main results we acquired are summarized as follows.1.Morphological analysis of hair follicles using HE staining of goat scapular skin detected the immature structure of hair follicle in 45 days comparing with hair follicle of108 days(p<0.01),which can not support and encompass the hair shaft completely.2.Based on high throughput sequencing analysis and a series of quality control,the unique mapped reads were annotated and got 25641 genes,among which there were 326 differentially expressed genes(DEGs),including 186 up-regulated and 140 down-regulated genes.Functional enrichment analysis was conducted on DEGs,showing that skin development,epidermal cell differentiation,epithelium differentiation,keratinization and cytoskeleton were related GO terms.These DEGs were also enriched in signaling pathways that associated nucleotide metabolism,neuron development and intercellular communication,indicating the existence of differentially transcriptional regulations in dynamic curl transformations.3.Different alternative splicing events analysis was performed and 1945 events was found between samples in two stages,with skipped exon(SE)had the most number of events(1403 events),and the alternative 3’ splice site ranked the second(223 events).4.Whole genome bisulfite sequencing(WGBS)was introduced and every sample got more than 90 Gb of clean data.1250 uniquely and differentially methylated genes(DMGs)out of 3379 DMRs were annotated using these unique mapped reads.Further analysis of DMGs was exerted to functional enrichment,which were mainly correlative with system development,plasma membrane regions,ions channel regulation,synapse development.Glutamate synapse,calcium channel,adherens junction were mainly signaling pathway within DMGs,highlighting the importance of cellular interaction during the development of epidermis.5.14 overlapped genes were obtained after we conducted integrated analysis using DEGs and DMGs.The result derived from protein-protein interaction network showed that NOD1,DICER1,SMAD3,PDGFC had a relative strong interacting effects as post-translational level,which were enriched in TGF-β,Hippo,and Fox O signaling pathways that involved in stem cells proliferation and differentiation.6.Overexpression and inhibition of PDGFC in HHIRS cells were performed in this study.The results showed that higher expression of PDGFC improved cellular migration and increased the proliferation rate.Also,PDGFC had direct or indirect effects on hair follicle development-related genes GJA1 and JAK1,hypothesizing the epigenetic patterns of PDGFC could alter the expression tendency of downstream genes that in the same regulatory pathways,to control the growth and development of postnatal hair follicles.In conclusion,the dynamic expression profile driving by DNA methylation mechanism may have potential role in modulating hair follicle development of Zhongwei lamb,and candidate gene PDGFC can regulate the function of hair follicle inner root sheath cells in vitro.