Effect of acute heat stress on gene expression and DNA methylation in the hypothalamus of male L2 strain Taiwan country chickens

• Main Authors: Huang-Chun Hsueh, 薛凰均
• Other Authors: 黃三元
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/9b4fc7

碩士 === 國立中興大學 === 動物科學系所 === 107 === Heat stress causes poor growth performance, reproductive performance, and high mortality in farm animals. The hypothalamus acts as a crucial center to regulate body temperature and detect environmental temperature changes. Epigenetics is the study of genomic structural modifications that affects gene expression without altering the underlying nucleotide sequence. DNA methylation modifications on the CpG sites of promoter are generally thought to be related to gene silencing, and heat stress may alter epigenetic modifications to allow animals withstand or adapt to the environment stress. The purpose of this study was to investigate the effect of acute heat stress on gene expression and to explore whether the modification of DNA methylation affects gene expression in layer-type L2 strain Taiwan country chickens (TCCs). A total of 197 30-week-old roosters of L2 strain TCCs were used in this study. One hundred and nighty-two roosters were acute heat-stressed at 38°C and 55% RH for 4 h. After acute heat stress, the roosters were grouped according to the body temperature change (highest body temperature during heat stress minus pre-heat stress body temperature, ΔT), including sensitive group (Sen.) with ΔT≧6.5 and resistant group (Res.) with ΔT≦2.5. The other five roosters were kept at 25°C and 55% RH as a control group (Ctl.). The hypothalamus of five roosters in each group were collected for RNA sequencing (RNA-seq) and DNA methylation analysis by pyrosequencing (Pyro-seq) and methylation specific PCR (MS-PCR). There were 1,690 genes upregulated and 819 genes downregulated among the differentially expressed genes (DEGs) after acute heat stress in the hypothalamus of L2 strain TCCs. Gene ontology annotation indicated that most of the DEGs were involved in biological processes of cellular process, metabolic process and biological regulation. The expressions of genes in HSP family (HSPB1, HSPB8, HSPB9 and DNAJA4), antioxidant (GPX1), hypothalamus function (FKBP5), and DNA methylation and transcription (DNMT3A and CREBBP) were significantly changed and were selected for further DNA methylation analysis. Result of MS-PCR revealed that HSPB1 was partially methylated and HSPB8 was highly methylated in the three groups of roosters; HSPB9 was partially methylated in CTL group and SEN group and hypo- to partially methylated in RES group; GPX1 was partially methylated in CTL group and SEN group and hypomethylated to partially methylated in RES group. Pyro-seq revealed that the methylation level of specific CpG site in HSPB8、GPX1 and DNMT3A were higher in SEN group than RES group. In summary, acute heat stress changes the expression of genes related to the response to stimuli, antioxidant, and hypothalamus function of hypothalamus in TCCs. Moreover, some of the Pyro-seq results were partially conflict with the RNA-seq results. The possible reason may be that the sequences analyzed are not the transcription factor binding sites, thus the increase of methylation level did not reduce the level of gene expression. Therefore, whether the sequences are transcription factor binding sites require further exploration.