Identification Of DNA-methylated Gene Related To Muscle Growth And Development And Transcription Regulation Of IGF2and ZBED6Genes

With the high speed development of molecular biology techniques, as well as thecompletion of genome map of each species, the Post-genome era is coming. The cattle breedresources are rich in China, and has a lot of good genes, but has not been fully exert and use.Marker-assisted selection (MAS) and whole genome wide selection breeding way areattracting more and more attention, and to control the function of the (economic) growth traitsgenetic screening and in-depth study has become a hot spot. This research is the basis ofgenetic and epigenetic, using the methylation of DNA immunization co-precipitationsequencing (MeDIP-Seq), bisulfite sequencing method (BSP), bisulfite treatment and enzymedigestion analysis (COBRA), quantitative real time PCR (QPCR), DNA pool sequencing,Forced-PCR-RFLP, gene cloning, cell culture, adenovirus vector packaging, and eukaryoticexpression of the adenovirus modern biological technology, this research have two aspects:first, study the genome-wide DNA methylation modify map for back muscle tissue inQinchuan fetal and adult bovine, select methylation significant differences genes and theirrole in signaling pathways; Second, analysis the genetic variants and associated with growthtraits in ZBED6and its target genes IGF2, analyzed the expression profile and promoteractivity, methylation and transcriptional regulation function at the cellular level.The main research contents and results are as follows:1. High throughput sequencing analysis of Genome-Wide Landscapes of DNAMethylomes in Bovine Muscle Tissue(1) MeDIP-seq high-throughput sequencing analysis of bovine muscle tissuegenome-wide methylation pattern: it is the first time to map genome-wide landscapes of DNAmethylomes in bovine muscle tissue, we found a large number of DNA methylationmodification differences between the two sample groups. They have a high level ofconcentration reads at each end of a chromosome than other regions, and the cattle genemethylation patterns is similar the other species.(2) DNA methylation distribution trend of different gene expression level in all the gene:the expression level is higher, the degree of DNA methylation modification is lower, and vice versa. Especially in gene transcription start site (TSS). DNA methylation level higher in adultbovine than that is in fetal bovine, DNA methylation level gradually lowered starting from thegene upstream2kb TSS, then the DNA methylation level has a sharp rise behind TSS, therewas high levels of methylation in the intragenic, DNA methylation achieve to the highest intranscription termination site (TES), the DNA methylation level gradually lowered after TES.(3) The number of differentially methylation genes in the differentially expression level:in the whole genome,1885genes are up-regulation,4889genes are down-regulation ofexpression level;235genes are up-regulation methylation level in the promoter,143genes aredown-regulation methylation level in the promoter;3504genes are up-regulation methylationlevel in the genebody,2313genes are down-regulation methylation level in the genebody.(4) GO function analysis of the differences methylation genes: in the upstream2k, thereare143genes are down-regulation methylation level; In the molecular function category,there are one significant enrichment item, its function is associated with methyl transferaseactivity. In CDS region,1076genes are up-regulation, in the molecular function category,1item of this biology function category, there are9significant enrichment items, most of thesefunctions are combined with nucleotides and phosphatase activity related adjustment. In CDSregion,792genes are down-regulation, in the molecular function category, there are8significant enrichment items, most of these functions are combined with nucleotides andtransportation related activity; In the process of biological classification, most functions areassociated with methyl transferase activity, there is one significant enrichment is locomotion.(5) Cellular pathways analysis of the differences methylation genes: in the upstream2kregion,93pathways are down-regulation,143pathways are up-regulation; in the CDS,195pathways are down-regulation,209pathways are up-regulation; In the CDS,1significantlyenriched pathways is focal adhesion, there are34gene significantly enriched to this pathway.(6) GO function analysis of the negative correlation genes: in the promoter region, thereare77negative correlation genes between methylation and expression level; in the genebody,there are1054negative correlation genes between methylation and expression level. Therewere no significant enrichment GO item in these regions of the negative correlation genes.(7) Cellular pathways analysis of the negative correlation genes: in the promoter region,77negative correlation genes involved in the81pathway, there are2significantly enrichedsignaling pathways, they are citric acid cycle (Citrate cycle) and Axon guidance (Axonguidance). in the gene body region,1054negative correlation genes involved in the204pathway, there are3significantly enriched signaling pathways, they are closely connected(Tight junction), regulating actin cytoskeleton (Regulation of actin cytoskeleton) anddigestion and absorption of vitamins (Vitamin digestion and absorption).(8) Identification and validation of the negative correlation gene of methylation and expression: For validation and identification the expression levels changes and gain insightinto the possible roles of the negative correlation genes of methylation and expression atdifferent developmental stages or in different tissues/organs in Qinchuan cattle, the QPCRanalysis of expression was performed in fetal bovine (FB, day90bovine embryos), newbornbovine (NB,3-day-old) and adult bovine (AB,2-year-old) skeletal muscle, heart, liver, spleen,lungs, kidney, stomach, small intestine, and fat. In the present study, we randomly picked outseveral high-read genes, including a total of37negative correlation genes of methylation upand expression down and11negative correlation genes of methylation down and expressionup. The expression patterns and level of48genes in all tested bovine tissues suggests thatthese genes may be more relevant to the highly conserved biological process in mammalians.2. DNA methylation status and expression level in different developmental stages inbovine ZBED6and IGF2genesQPCR, BSP and COBRA analysis results indicate that ZBED6and IGF2genesexpression levels were negatively associated with the methylation status of the different DMRduring the two developmental stages. ZBED6gene expression in the liver, lung and spleenvolume increased, and there was decreased in the methylation level; IGF2gene expressed inmuscle, heart, lung and spleen were decreased, and there was increased in the methylationleve.3. Assessment of association between variants and haplotypes of the ZBED6and IGF2genes in beef cattleFour single nucleotide polymorphisms (SNPs:1-4) were identified in the bovine IGF2bysequencing pooled DNA samples. Compared with previously reported sequence from NCBI,four SNPs were identified in these animals, and these SNPs are the non-coding mutations,respectively named-826-G> A,680C> G and1043A>G. The statistical analyses indicatedthat the4SNPs and18combined genotypes or haplotypes are associated with the bodyweight at18and24months in Jiaxian cattle population (P<0.05or P<0.01).There are one mutation (SNP1) in the promoter region and two missense mutations(SNP2and3) in the coding region (single exon) within the bovine ZBED6gene, respectivelynamed IVS8-17G>A,-220C>T,-221A>G and-1393A>G. The statistical analyses indicatedthat SNPs, combined genotypes (NY:26; JX:23) and haplotypes were significantly associatedwith growth traits in NY or JX cattle population (P<0.05or P<0.01) at five different ages.4. Polymorphisms modulate promoter activity and gene expression of the growthsuppressor gene ZBED6in cattleThe promoter and coding region are the key regions for gene function; polymorphisms inthese regions can alter gene expression. Quantitative real-time PCR (qPCR) analysis showedthat ZBED6has a broad tissue distribution in cattle and is highly expressed in skeletal muscle. Eleven promoter-detection vectors have been constructed which enable the cloning andanalysis of its transcriptional activity by luciferase reporter gene assays. The core region ofthe basal promoter of bovine ZBED6is located within the region-866to-556, the activity ofWT-826G-pGL3in driving reporter gene transcription is significantly higher than theM-826A-pGL3construct (P <0.01). Analysis of gene expression patterns of homozygousindividuals from full-siblings of Qinchuan cattle, the mutant-type Hap-AGG exhibited a lowermRNA level than the wild-type Hap-GCA (P <0.05) in longissimus dorsi muscle (LDM);Moreover, ZBED6mRNA expression was low in mutant-type ZBED6-overexpression(pcDNA3.1+-Hap-GG) C2C12cell (P <0.01). Our results suggest that the polymorphisms inthe promoter and coding regions may modulate the promoter activity and gene expression ofbovine ZBED6in the skeletal muscles of these cattle breeds.5. Construction of recombinant ZBED6adenoviral shRNA vector and its expression invitroAdenovirus shRNA vector of bovine ZBED6gene was constructed, after packaging andamplification in293cell lines, high-titer of pENTR/CMV-GFP/U6-shRNA-906was infectedwith bovine primary muscle cells. ZBED6expression is down-regulation, IGF2geneexpression is up-regulation; According to the expression of the IGF2gene we found thatshRNA of ZBED6gene can promote the generation of muscle cell growth differentiation.6. Construction of recombinant ZBED6adenoviral over-expression vector and itsexpression in vitroAdenovirus vector of bovine ZBED6gene was constructed, after packaging andamplification in293cell lines, high-titer of Ad-ZBED6was infected with bovine primarymuscle cells. ZBED6expression quantity increases, IGF2gene expression quantity obviouslylowered; According to the expression of the IGF2gene we found that over-expression ofZBED6gene can inhibit the growth of cattle primary muscle cells differentiation.The above results show that there are large amounts of DNA methylation modifydifference genes over the two key growth period in Qinchuan cattle muscle tissue. Thisresearch is the basis of genetic and epigenetic, we found a large number of excellent geneticresources in the local cattle breeds in China. The results of this study also provide valuableepigenetic markers to improve the production performance in beef cattle, and providescientific data for the development of modern high-tech beef cattle industry, and these resultsas the theoretical basis for Chinese beef cattle breeding program.