Promoter Methylation Changes in KRT17: A Novel Epigenetic Marker for Wool Production in Angora Rabbit

Abstract

Wool production is an important economic trait of Angora rabbits. Exploring molecular markers related to wool production is one of the essentials of Angora rabbits’ breeding. KRT17 (Keratin 17) is an important gene of hair follicle development, which must be explored for genetic/epigenetic variation to assess its effect on wool production. Based on the effective wool production data of 217 Angora rabbits, the high and low yield groups were screened with 1.5 standard deviations of the population mean. The full-length sequence of KRT17 was obtained by rapid amplification of cDNA ends technology, and the polymorphism was analyzed in the promoter, exon, and intron regions by direct sequencing. KRT17, SP1 over-expression plasmids, and siRNA were constructed and transfected into dermal papilla cells. The mRNA expressions of relevant genes were analyzed by RT-qPCR. The methylation level of the KRT17 promoter was determined by Bisulfite Sequencing PCR. Dual-luciferase system, site-directed mutagenesis, and electrophoretic mobility shift assays were used to analyze the binding relationship between SP1 and the promoter of KRT17. The structure map of KRT17 was drawn, and no SNPs were found in the promoter, exon, and intron, indicating a relatively conserved structure of KRT17. Expression of KRT17 was significantly higher in cutaneous tissues than in other tissues and was significantly upregulated in the high-yield group compared to the low-yield group (p < 0.05). Furthermore, the overall high methylation levels of KRT17 CpG I and CpG III showed significant association with low wool yield; the methylation levels of 5 CpG locus (CpG I site 4 and CpG III site 2–5) were significantly different between the high and low yield groups (p < 0.05). The methylation levels of 3 CpG locus (CpG I site 4 and CpG III site 4, 14) showed a significant correlation with KRT17 expression (p < 0.05). Overall, CpG III site 4 significantly affects wool production and KRT17 expressions (p < 0.05). This site promotes SP1 binding to the KRT17 promoter region (CGCTACGCCC) to positively regulate the KRT17 expression. KRT17 CpG III site 4 can be used as candidate epigenetic markers for the breeding of high wool-producing Angora rabbits.