Differential DNA Methylation Levels Of Growth Regulating Genes In Antler Tips Among Male And Female Reindeer (Rangifer Tarandus) And Sika Deer(Cervus Nippon)

Anlter is the only mammalian appendage that can be completely regenerated.It can rapidly proliferate without cancer.It is a unique model for the research of trauma repair,organ regeneration and cancer.Antler is usually a male trait of deer,and reindeer is the only deer species in both sexes produce antlers.There are many apparent differences in antlers among the reindeer and other deers,such as branch size,number,complexity,regeneration and abscission time.Exploring the intrinsic molecular mechanism of the apparent differences is a challenging scientific problem.Genes involved in regulating antler growth are being gradually discovered,and regulatory functions are also being revealed.It lays the foundation for further studies on the regulation difference of genes in antler and other tissue of same deer,antlers of different deers and antlers of male and female reindeer.As an important epigenetic mechanism,DNA methylation plays an important regulatory role in the life process by regulating the transcription and expression of target genes.Therefore,it has great scientific value and significance to study the DNA methylation differences of genes in antlers of the female,male reindeer and sika deer.In this study,we choosed the same age and healthy reindeer and sika deer as research object,taking antler tip mesenchymal and blood of male,female reindeer and sika deer as experimental materials.We selected 10 genes(COL1A1,COL6A3,DKK1,IGF1,KGF,NGF,OPN,RUNX1,S100A4,STAT1)that be with antler growth regulation and lead directly to the apparent relative genetic difference,then cloned gene promoters and carried out bioinformatics analysis.Bisulfite sequencing PCR was used to analysis methylation patterns of 10 genes in antler mesenchymal and blood of the female,male reindeer and sika deer.Then we studied the difference of methylation level in antler mesenchymal and blood tissue,and both of them in the same deer.The results are as follows:1.Promoters of 10 antler related genes were successfully cloned from reindeer and sika deer.The homologies were above 98.3%with white tailed deer,and above 85.8%with the other artiodactylous animals(cattle,sheep).The core promoter elements were distributed,such as CpG island region and core promoter region.2.There were different methylation patterns in 10 genes of the same tissue and the same gene of different tissues,methylation rate was from(0.00±0.00)%to(91.70±1.39)%.COL6A3,NGF,OPN and S100A4 were methylation in different tissues,methylation rate of COL6A3 in blood was the highest,from(73.50±0.75)%to(83.53±.99)%.While COL1A1,DKK1,IGF1,KGF,RUNX1 and STAT1 were not methylation in all tissues.Methylation rate of KGF in male reindeer and sika deer were from(45.53±3.87)%to(42.20±1.91)%,there was no methylation in female reindeer.RUNX1 had a low level of methylation in the female reindeer,which was(3.33±1.44)%.3.Methylation patterns of different gene promoters were affected by the methylation rate of CpG dinucleotide loci in their promoter sequences.Methylation patterns of different tissues in same gene were influenced by the methylation differences of CpG dinucleotide loci.4.The differences of methylation were analyzed in antler tip mesenchymal and blood of the male and female reindeer,sika deer:Antler tip mesenchymal of female and male reindeer:(1)Methylation rate of the female was significantly higher than that of the male in COL6A3,NGF,OPN,RUNX1 and S100A4(P<0.05),methylation rate of them were as follows,(16.83±1.10)%and(6.63±1.31)%,(33.90±3.57)%and(17.20±3.57)%,(36.83±5.86)%and(18.10±0.87)%,(3.33±1.44)%and(0.00±0.00)%,(27.80±1.28)%and(21.67±2.17)%,respectively.(2)Methylation rate of the female was significantly lower than the male in IGF1 and KGF(P<0.05),methylation rate of them were as follows,(6.40±0.52)%and(7.20±1.28)%,(0.00±0.00)%and(45.5±3.87)%,respectively.(3)They were both not methylation in COL1A1.There were no significantly differences in other two genes.Antler tip mesenchymal of reindeer and sika deer:(1)Methylation rate of the female reindeer was significantly higher than that of sika deer in COL6A3,NGF,RUNX1 and S100A4,and significantly lower in KGF(P<0.05).Methylation rate of them were as follows,(16.83±1.10)%and(7.10±1.93)%,(33.90±3.57)%and(21.57±1.21)%,(3.33±1.44)%and(0.00±0.00)%,(27.80±1.28)%and(5.03±1.44)%,(0.00±0.00)%and(42.20±1.91)%,respectively.(2)Methylation rate of the male reindeer was significantly higher than that of sika deer in IGF1,KGF and S100A4,and significantly lower in COL1A1,NGF and OPN(P<0.05).Methylation rate of them were as follows,(2.23±0.92)%and(0.00±0.00)%,(91.70±1.39)%and(90.30±0.52)%,(21.67±2.17)%and(5.03±1.44)%,(0.00±0.00)%and(2.40±0.00)%,(17.20±3.57)%and(21.57±1.21)%,(18.10±0.87)%and(35.70±1.40)%,respectively.They were both not methylation in RUNX1.Blood of female and male reindeer:(1)Methylation rate of the female was significantly higher than that of the male in COL6A3 and OPN,and significantly lower in COL1A1 and KGF(P<0.05).Methylation rate of them were as follows,(83.53±2.99)%and(77.10±4.03)%,(80.00±1.40)%and(52.40±0.87)%,(3.47±0.61)%and(6.40±0·00)%,(86.70±0.00)%and(91.70±1.39)%,(6.70±0.90)%and(10.03±2.25)%,respectively.(2)There was no significant difference in methylation rate of other 5 genes(P>0.05).Blood of reindeer and sika deer:(1)Methylation rate of female reindeer was significantly higher than that of sika deer in COL6A3,significantly lower in COL1A1 and KGF(P<0.05).Methylation rate of them were as follows,(83.53±2.99)%and(73.50±0.75)%,(3.47±0.61)%and(5.87±0.46)%,(86.70±0.00)%and(90.30±0.52)%,respectively.(2)Methylation rate of male reindeer was significantly lower than that of sika deer in OPN(P<0.05).methylation rate were(52.40±0.87)%and(78.07±2.97)%.(3)There was no significant difference in blood tissue in 5,7,9 genes of female and male reindeer,female reindeer and sika deer,and male reindeer and sika deer,respectively(P>0.05).Antler tip mesenchymal and blood of female reindeer:Methylation rate of antler tip mesenchymal was significantly lower than that of blood in COL1A1,COL6A3,IGF1,KGF,OPN and STAT1(P<0.05).Methylation rate were as follows,(0.00±0.00)%and(3.47±0.61)%,(16.83± 1.10)%and(83.53±2.99)%,(0.00±0.00)%and(6.40±0.52)%,(0.00±0.00)%and(86.70±0.00)%,(36.83±5.86)%and(80.00±1.40)%,(0.27±0.12)%and(1.53±0.29)%,(33.90±3.57)%and(27.33± 1.19)%,(3.33±1.44)%and(0.00±0.00)%,(27.80±1.28)%and(6.70±0.90)%,respectively.Antler tip mesenchymal and blood of male reindeer:(1)Methylation rate of antler tip mesenchymal was significantly lower than that of blood in COL1A1,COL6A3,IGF1,KGF,NGF,OPN and STAT1,and significantly higher in S100A4(P<0.05).Methylation rate were as follows,(0.002±0.00)%and(6.40±0.00)%,(6.63±1.31)%and(77.10±4.03)%,(2.23±0.92)%and(7.20±1.28)%,(45.53±3.87)%and(91.70±1.39)%,(17.20±3.57)%and(29.23±1.37)%,(18.10±0.87)%and(52.40±0.87)%,(0.20±0.20)%and(1.70±0.20)%,(21.67±2.17)%and(10.03±2.25)%,respectively.(2)There was significant difference in 9,8 genes in female and male reindeer,respectively(P<0.05).Antler tip mesenchymal and blood of sika deer:Methylation rate of antler mesenchymal was significantly lower than that of blood in COL1A1,COL6A3,IGF1,KGF,NGF,OPN,S100A4 and STAT1(P<0.05).Methylation rate were as follows,(2.40±0.00)%and(5.87±0.46)%,(7.10±1.93)%and(73.50±0.75)%,(0.00±0.00)%and(5.83±1.44)%,(42.20±1.91)%and(90.30±0.52)%,(21.57±1.21)%and(28.57±1.40)%,(35.70±1.40)%and(78.07±2.97)%,(5.03±1.44)%and(8.90±0.52)%,(0.00±0.00)%and(1.40±0.36)%,respectively.The difference of gene methylation was closely related to the difference in the regulatory function of the gene.The DNA methylation differences of the genes related to the antler growth were obtained in the study.It would provide theoretical support for further revealing antler regulation mechanism and the differences between male and female reindeer antlers,and be conducive to the study of a series of human medical problems,such as tissue and organ regeneration,damage repair and cancer.