藏鸡低氧适应HIF-1关键靶基因的鉴定及其表观遗传调控机制研究

The transcription factor HIF-1 has been recognized as the central regulator that mediates the hypoxic response. Epigenetics plays a crucial role in regulation of genes expression. The both work hand in hand with the hypoxic adaptation responses and the maintenance of oxygen homeostasis in animals. Base on previously research about physiology of hypoxic adaptation and genomic transcription profiles in Tibet chicken, the eggs from 4 chicken populations, Tibet chicken raised in Tibet, Tibet chicken raised in Beijing, lowland chicken, and lowland chicken raised in Tibet, are collected, and incubated under normoxia and hypoxia conditions. The heart and CAM tissues of embryo at day 8 and 16 are the subjects for this study. By utilizing ChIP-Seq technology, the different enrichment binding sites of HIF-1? are selected in vital organs of hypoxic embryo. Combining with the measurement of genes expression in mRNA and protein levels, the key HIF1-target genes for hypoxic adaptation function are identified. The SNPs, methylated-DNA and methyl-histone in promoter regions of the key gene are measured, and the global epigenetic changes and the expression of epigenetic related enzymes are determined, to analyze the role and regulation of the key gene for hypoxic adaptation in Tibet chicken embryo, thus to discover the genetic and epigenetic mechanisms of hypoxic adaptation.

低氧诱导因子1（HIF-1）是调节低氧反应的核心转录因子，而表观遗传修饰是调控基因表达的重要途径，两者共同参与调控基因的低氧适应性反应，平衡氧稳态。本研究在前期对藏鸡低氧适应生理基础和基因组表达差异研究的基础上，收集西藏藏鸡、北京饲养的藏鸡、低地对照鸡和西藏饲养的低地鸡4个群体的种蛋，进行常氧和低氧孵化，以孵化第8和16天的胚胎心脏和尿囊绒毛膜（CAM）组织作为研究材料。采用ChIP-Seq技术筛选低氧孵化胚胎重要器官中HIF-1α差异富集靶基因，结合基因mRNA和蛋白质低氧表达变化，确定低氧适应关键靶基因；通过关键基因的启动子区域SNP位点、DNA甲基化和组蛋白修饰测定以及基因组整体表观修饰水平分析，结合表观遗传修饰相关酶基因的表达，分析关键基因对藏鸡胚胎低氧适应的功能及其调控机制，从而揭示低氧适应的遗传和表观遗传机理。

低氧诱导因子1（HIF-1）是调节低氧反应的核心转录因子，而表观遗传修饰是基因表达的重要途径，两者共同参与调控基因的低氧适应性反应，平衡氧稳态。本项目收集了藏鸡和对照低地鸡群体种蛋进行常氧和低氧孵化，测定了低氧相关的胚胎发育、胚胎成活、器官发育、血液生理、心肺组织显微结构等表型指标，鉴定了藏鸡胚胎低氧适应性的生理机制。采用ChIP-Seq技术分析胚胎低氧适应性相关的HIF-1与DNA结合位点，结合基因mRNA和蛋白质低氧表达变化，鉴定藏鸡低氧条件特异转录调控的靶基因。通过基因组整体水平的DNA甲基化水平和表观遗传相关酶基因的表达分析，结合关键基因的启动子区域SNP位点、DNA甲基化修饰，分析关键基因对藏鸡胚胎低氧适应的功能及其调控机制。采用RT-PCR、Western blot等技术测定了EGLN1、HIF-1α、EPAS1以及下游靶基因 eNOS、ACE 、HO-1、HSPA8、BMP4、CTGF、HSP90AA1、PPAR等基因的表达，分析了这些重要基因表达对藏鸡胚胎低氧适应作用的表达调控机制。