Study On The Function Of Dsg4 In Dermal Papilla Cells Of Cashmere Goats And Its Regulation By Transcription Factors

The hair follicle development process of cashmere goats is spatio-temporal specific,and it is regulated by various factors at different stages of development.Based on our previous RNA-seq sequencing data of skin tissues of cashmere goats in different periods,it was found that Dsg4 gene differentially expressed in different periods,and the existing studies indicated that the key transcription factors related to hair follicle development,HOXC13 and FOXN1 could regulate the expression of Dsg4 gene.But the function and regulation mechanism of this gene have not been reported.Therefore,based on skin tissue sequencing data at different stages of cashmere goat hair follicle development,combined with MTT,Ed U and quantitative PCR(RT-q PCR)techniques to explore the effect of Dsg4 gene on the proliferation of cashmere dermal papilla cells.And dual-luciferase reporter assay and chromatin immunoprecipitation(Ch IP)assay were used to explore the regulatory effects of HOXC13 and FOXN1 on Dsg4.The results obtained were as follows:1.RT-q PCR technology was used to detect the expression of Dsg4 gene in the skin tissues from different stages of cashmere goat.The results showed that the expression of Dsg4 gene in anagen was significantly higher than that in telogen(P<0.05).The expression level at 120 days was significantly higher than that at 66 days during embryonic development(P<0.01).2.The Dsg4 gene overexpression vector was contracted and packaged with adenovirus,then was transfected into the primary dermal papilla cells.MTT assay showed that the activity of hair papilla cells was significantly reduced after the overexpression of Dsg4 gene compared with the control(P<0.01).The Ed U result further found that the proportion of positive proliferating cells was significantly reduced with the overexpression of Dsg4 gene,indicating that Dsg4 gene had the ability to inhibit the proliferation of dermal papilla cells.3.The yeast two-hybrid results showed that there was an interaction between HOXC13 and FOXN1,and the dual-luciferase reporter assay found that the promoter viability of Dsg4 gene was significantly promoted under the interaction of HOXC13 and FOXN1(P<0.01),indicating that the two transcription factors could form co-activator to positively regulate target genes.In addition,two binding sites of HOXC13 were predicted at-1078 bp and-349 bp upstream at the promoter region of Dsg4.Ch IP assay enriched the DNA sequence bound by HOXC13,and the-349 bp fragment could be amplified by PCR method,indicating that HOXC13 could bind to the promoter region of Dsg4 as a basic transcription factor.In this study,the expression characteristics,function and regulatory mechanism of cashmere goat Dsg4 gene in different hair follicle development stages were explored,which would provide a theoretical basis for elaborating the molecular regulation mechanism of hair follicle development and molecular breeding of cashmere goats.