Study On Regulation Mechanism Of Hoxc13 On The Keratin Family In Goat

Cashmere,characterized by periodic growth,undergoes annually cyclic transformation from the resting phase(telogen,April to June)to the growth phase(anagen,July to next February).The previous RNA-seq of skin sample during telogen and anagen,identified that the expression of Hoxc13 and keratin genes such as KRT38 and KRT84 during anagen of cashmere undercoat growth were upregulated when compared with telogen,while some other keratin genes such as KRT1 and KRT2 were downregulated in anagen.What is noteworthy is that the binding sites of transcription factor Hoxc13 were found in all these promoters of keratin gene.This is inconsistent with the report that Hoxc13 involving in the regulation of downstream keratin and keratin-associated protein.To understand the regulatory mechanism of keratin family genes by Hoxc13 during hair follicle development and cycling,firstly we constructed the expression vector of Shaanbei cashmere goat Hoxc13 gene by SSA repair pathway.Subsequently,we cloned the promoter of Shaanbei cashmere goat KRT1,KRT2,KRT38 and KRT84 gene and ovexpressed these vectors in dermal papillae of Shaanbei cashmere goat with Hoxc13 expression vector.Meanwhile,we verified the interaction between Hoxc13 and Foxn1 protein of Shaanbei cashmere goat by yeast two-hybrid assay to enrich the regulatory mechanism of keratin family genes by Hoxc13.The result obtained from above study was:1.We constructed the expression vector of Shaanbei cashmere goat Hoxc13 gene in the way that linearized expression vector with 42 bp,101 bp and 333 bp of direct repeats were repaired via SSA pathway in E.coli.Similarly,we constructed the Hoxc13 expression vector with mutation(c.812A>G)in the same way using 25 bp direct repeats.2.The promoters' transcriptional activity of KRT38 and KRT84 gene were intensive significantly(P<0.05)when ovexpressed Hoxc13 gene in dermal papillae cells of Shaanbei cashmere goat(P<0.05),while that of KRT1 was reduced notably.But there was no significant difference observed for the promoters' transcriptional activity of KRT2(P>0.05).3.When both Hoxc13 and Foxn1 gene were overexpressed in dermal papillae,the transcriptional activity of KRT84 gene promoters were higher than the overexpression group of Hoxc13 or Foxn1.As for yeast two-hybrid assay,blue substrate was observed in pBD-Hoxc13/pAD-Foxn1 group when no blue substrate was also observed in the pBD-Hoxc13/pAD group,which demonstrated that Hoxc13 may interact with Foxn1.Our results further uncovered the molecular mechanism that Hoxc13 was involved in the transcriptional regulation of keratin gene and its role of in Shaanbei cashmere goat skin during hair follicle development and cycling,which provided the reference for the study on hair follicle cyclic transformation of Shaanbei cashmere goat.