猪低氧适应相关DNA甲基化位点筛选及表观修饰酶表达分析
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摘要
因为高原地区的低气压与低氧分压严重地影响着人类与动物的生存与发展,所以低氧反应和低氧适应一直都是国内外生物学和医学研究的重要热点之一。为了应对低氧,机体除了激活和稳定转录调控因子外,还会启动一些基因的DNA甲基化等表观调控机制。藏猪生活在青藏高原,能够适应恶劣的高寒气候和低劣的饲养管理条件,是研究低氧适应的宝贵的物种资源。而低氧适应是一个复杂的性状,至今藏猪适应低氧的分子遗传学机制以及表观调控机制尚未完全清楚。本研究选取生活在高原环境的藏猪(TH)和移居高原环境的大约克猪(YH),以及移居在低地饲养的藏猪(TL)和低地大约克猪(YL)4组猪群体作为研究对象,利用甲基化DNA免疫共沉淀(MeDIP),并结合二代测序技术来筛选影响低氧适应过程中DNA甲基化差异的关键基因,联合转录组测序结果分析,挑选基因进行DNA甲基化和表达量验证,旨在阐明藏猪低氧适应的表观遗传学机制,并筛选重要的低氧适应候选基因。本研究对5个重要的表观修饰酶进行了mRNA水平的半定量和定量分析,以揭示不同猪种的表观修饰酶的mRNA表达差异。
MeDIP-Seq共得到486M reads,每个个体得到约60000个DNA甲基化peak。用MEDIPS软件包计算不同组别两两之间的差异甲基化区域(DMR),并利用UCSC数据库进行DMR的注释。对MeDIP-Seq得到的DMR进行聚类分析,结果显示环境对个体甲基化水平影响远小于品种的影响。对4种组间比较得到的差异甲基化基因,进行GO及KEGG Pathway富集分析后,发现低氧时,两品种差异甲基化基因与低氧适应相关通路(PPAR)及脂肪合成代谢相关；常氧变为低氧时,藏猪差异甲基化基因与低氧适应有关(VEGF、PPAR等通路)。
从既差异甲基化又差异表达的基因中筛选到5个可能与低氧适应相关的基因：EPHX2、RXRG、 BCKDHB、UBD和GOT2。对于这5个基因通过克隆测序和荧光定量PCR方法进行验证基因的差异甲基化程度和差异表达水平。结果表明,RXRG基因的差异甲基化水平和差异表达水平与高通量测序结果一致,即它们同时在4组中差异甲基化和差异表达。而BCKDHB和EPHX2,虽然差异甲基化,但mRNA表达水平并不存在显著性差异,GOT2和UBD基因不存在甲基化差异位点,表达上也无显著性差异。但所有基因的DNA甲基化和mRNA表达在不同组间的趋势与高通量测序相同。
5个表观修饰酶基因的半定量结果表明,它们在高原藏猪(TH)的表达具有组织特异性。Dnmt1和Uhrfl表达量最高的组织是心脏、肺脏、垂体,而其他三个基因表达量最高的组织均为肺脏、肾脏、垂体。肺脏中所有表观修饰酶在TH组的表达量均高于大约克猪；肾脏中,常氧变为低氧时,藏猪表观修饰酶表达量下调,而大约克猪表达上调。
本研究利用高通量测序法对4组猪的心脏组织进行DNA甲基化测序,筛选并验证得到了RXRG基因在不同组间差异DNA甲基化和差异表达。对Dnmtl,Dnmt3a,Kdm3a,Hdac1, Uhrf15个表观修饰酶进行了表达分析,发现肺脏和肾脏可能是低氧适应中表观修饰相关功能器官,且肺脏中低氧时,可能藏猪大部分基因相对于大约克猪下调,肾脏中,常氧变为低氧时,低氧反应/适应可能导致大约克猪很多基因表达受到抑制。本研究对于完善藏猪低氧适应的表观遗传学调控提供了依据,也为进一步研究低氧适应相关基因提供了方向和目标。
Low pressure and low partial pressure of oxygen of plateau affects survival and development of human and animals, so hypoxia response and hypoxia adaptation are always focus of biological and medicine. To deal with hypoxia, the organism will activate and stable HIFs, also needs some epigenetic regulation, DNA methylation is one of the most important epigenetic machnisms. Tibet pigs live at Qinghai-Tibet Plateau, their adaptation to hypoxia makes them precious species resource and object for hypoxia adaptation machnism. We selected four groups of pig as the study object, which were Tibet pig in highland (TH), Yorkshire in highland (YH), Tibet pig in lowland (TL), Yorkshire in lowland (YL). Using methylated DNA immunoprecipitation and subsequent high throughput sequencing (MeDIP-Seq), and combining RNA-Seq results, we filtered key genes related to hypoxia adaptation that were differentially methylated and differentially expressed simultaneously. We conducted clone sequencing and real time PCR to validate these high throughput results. mRNA semi-quantitative and quantitative assay were conducted for five epigenetic modifying enzymes to reveal their expression differences.486M reads was obtained from MeDIP-Seq, and there were about60K peaks for each individual. Then we used MEDIPS package to obtain differentially methylated regions which were annotated in UCSC. Cluster analysis of differentially methylated genes (DMGs) from MeDIP-Seq assay showed that breed affect DNA methylation level much more than altitude. GO and KEGG pathway analysis results showed that, in condition of hypoxia, DMGs between two breeds were related to PPAR pathway and Fatty acid elongation GO term. From normoxia to hypoxia, Tibet pigs changed DNA methylation of hypoxia adaptation related pathway(VEGF and PPAR).
We selected five genes that may play important roles in hypoxia adaptation from DMRs that were both differentially methylated and expressed:EPHX2, RXRG, BCKDHB, UBD and GOT2. Clone sequencing and real time quantitative PCR were implemented for these five genes, and validation results showed that RXRG was both differentially methylated and expressed between different groups; although BCKDHB and EPHX2was differentially methylated, there were no significantly difference between four groups; and there were neither significant difference of methylation nor significant difference of expression between different groups. The methylation difference and expression difference of these five genes had the same trend with that resulted from MeDIP-Seq and RNA-Seq.
These five epigenetic modifying genes presented tissue specific expression patterns. Dnmtl and Uhrfl expressed highly in heart, lung and pituitary gland, and the other three genes expressed highly in lung, kidney and pituitary gland. In lung, all these genes expressed higher in TH than that in Yorkshire. In kidney, from nomoxia to hypoxia, genes expressed lower in Tibet pigs, but higher in Yorkshire.
This research obtained DNA methylation profile of heart using four groups of pigs, then selected and validated five genes whih may play roles in hypoxia adaptation, validation turned out that RXRG was both differentially methylated and expressed between different groups. We also conducted semi-quantitative expression analysis and quantitative expression analysis of Dnmtl, Dnmt3a, Kdm3a, Hdacl and Uhrfl, results suggested that lung and kidney may play important roles of epigenetic regulation to adapt to hypoxia. In lung, in condition of hypoxia, most genes in Tibet pigs maybe were down regulated compared to Yorkshire. In kidney, from nomoxia to hypoxia, hypoxic response or adaptation may induce many genes' decreased expression. All these results will be useful to reveal the epigentic regulation of hypoxia adaptation and useful to provide suggestions to do research towards hypoxia adaptation related genes.
MeDIP-Seq共得到486M reads,每个个体得到约60000个DNA甲基化peak。用MEDIPS软件包计算不同组别两两之间的差异甲基化区域(DMR),并利用UCSC数据库进行DMR的注释。对MeDIP-Seq得到的DMR进行聚类分析,结果显示环境对个体甲基化水平影响远小于品种的影响。对4种组间比较得到的差异甲基化基因,进行GO及KEGG Pathway富集分析后,发现低氧时,两品种差异甲基化基因与低氧适应相关通路(PPAR)及脂肪合成代谢相关；常氧变为低氧时,藏猪差异甲基化基因与低氧适应有关(VEGF、PPAR等通路)。
从既差异甲基化又差异表达的基因中筛选到5个可能与低氧适应相关的基因：EPHX2、RXRG、 BCKDHB、UBD和GOT2。对于这5个基因通过克隆测序和荧光定量PCR方法进行验证基因的差异甲基化程度和差异表达水平。结果表明,RXRG基因的差异甲基化水平和差异表达水平与高通量测序结果一致,即它们同时在4组中差异甲基化和差异表达。而BCKDHB和EPHX2,虽然差异甲基化,但mRNA表达水平并不存在显著性差异,GOT2和UBD基因不存在甲基化差异位点,表达上也无显著性差异。但所有基因的DNA甲基化和mRNA表达在不同组间的趋势与高通量测序相同。
5个表观修饰酶基因的半定量结果表明,它们在高原藏猪(TH)的表达具有组织特异性。Dnmt1和Uhrfl表达量最高的组织是心脏、肺脏、垂体,而其他三个基因表达量最高的组织均为肺脏、肾脏、垂体。肺脏中所有表观修饰酶在TH组的表达量均高于大约克猪；肾脏中,常氧变为低氧时,藏猪表观修饰酶表达量下调,而大约克猪表达上调。
本研究利用高通量测序法对4组猪的心脏组织进行DNA甲基化测序,筛选并验证得到了RXRG基因在不同组间差异DNA甲基化和差异表达。对Dnmtl,Dnmt3a,Kdm3a,Hdac1, Uhrf15个表观修饰酶进行了表达分析,发现肺脏和肾脏可能是低氧适应中表观修饰相关功能器官,且肺脏中低氧时,可能藏猪大部分基因相对于大约克猪下调,肾脏中,常氧变为低氧时,低氧反应/适应可能导致大约克猪很多基因表达受到抑制。本研究对于完善藏猪低氧适应的表观遗传学调控提供了依据,也为进一步研究低氧适应相关基因提供了方向和目标。
Low pressure and low partial pressure of oxygen of plateau affects survival and development of human and animals, so hypoxia response and hypoxia adaptation are always focus of biological and medicine. To deal with hypoxia, the organism will activate and stable HIFs, also needs some epigenetic regulation, DNA methylation is one of the most important epigenetic machnisms. Tibet pigs live at Qinghai-Tibet Plateau, their adaptation to hypoxia makes them precious species resource and object for hypoxia adaptation machnism. We selected four groups of pig as the study object, which were Tibet pig in highland (TH), Yorkshire in highland (YH), Tibet pig in lowland (TL), Yorkshire in lowland (YL). Using methylated DNA immunoprecipitation and subsequent high throughput sequencing (MeDIP-Seq), and combining RNA-Seq results, we filtered key genes related to hypoxia adaptation that were differentially methylated and differentially expressed simultaneously. We conducted clone sequencing and real time PCR to validate these high throughput results. mRNA semi-quantitative and quantitative assay were conducted for five epigenetic modifying enzymes to reveal their expression differences.486M reads was obtained from MeDIP-Seq, and there were about60K peaks for each individual. Then we used MEDIPS package to obtain differentially methylated regions which were annotated in UCSC. Cluster analysis of differentially methylated genes (DMGs) from MeDIP-Seq assay showed that breed affect DNA methylation level much more than altitude. GO and KEGG pathway analysis results showed that, in condition of hypoxia, DMGs between two breeds were related to PPAR pathway and Fatty acid elongation GO term. From normoxia to hypoxia, Tibet pigs changed DNA methylation of hypoxia adaptation related pathway(VEGF and PPAR).
We selected five genes that may play important roles in hypoxia adaptation from DMRs that were both differentially methylated and expressed:EPHX2, RXRG, BCKDHB, UBD and GOT2. Clone sequencing and real time quantitative PCR were implemented for these five genes, and validation results showed that RXRG was both differentially methylated and expressed between different groups; although BCKDHB and EPHX2was differentially methylated, there were no significantly difference between four groups; and there were neither significant difference of methylation nor significant difference of expression between different groups. The methylation difference and expression difference of these five genes had the same trend with that resulted from MeDIP-Seq and RNA-Seq.
These five epigenetic modifying genes presented tissue specific expression patterns. Dnmtl and Uhrfl expressed highly in heart, lung and pituitary gland, and the other three genes expressed highly in lung, kidney and pituitary gland. In lung, all these genes expressed higher in TH than that in Yorkshire. In kidney, from nomoxia to hypoxia, genes expressed lower in Tibet pigs, but higher in Yorkshire.
This research obtained DNA methylation profile of heart using four groups of pigs, then selected and validated five genes whih may play roles in hypoxia adaptation, validation turned out that RXRG was both differentially methylated and expressed between different groups. We also conducted semi-quantitative expression analysis and quantitative expression analysis of Dnmtl, Dnmt3a, Kdm3a, Hdacl and Uhrfl, results suggested that lung and kidney may play important roles of epigenetic regulation to adapt to hypoxia. In lung, in condition of hypoxia, most genes in Tibet pigs maybe were down regulated compared to Yorkshire. In kidney, from nomoxia to hypoxia, hypoxic response or adaptation may induce many genes' decreased expression. All these results will be useful to reveal the epigentic regulation of hypoxia adaptation and useful to provide suggestions to do research towards hypoxia adaptation related genes.
引文
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