Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (12): 2395-2406.doi: 10.3864/j.issn.0578-1752.2023.12.013

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

Effect of Heat Stress on DNA Methylation of GNAS Promoter Region in Dairy Cows

CHEN YuMei1(), ZHANG CongCong1, HU LiRong2, FANG Hao1, DOU JinHuan3, GUO Gang4, WANG Yan4, LIU QiaoXiang5, WANG YaChun2, XU Qing1()   

  1. 1 Institute of Life Science and Bioengineering, Beijing Jiaotong University, Beijing 100044
    2 College of Animal Science and Technology, China Agricultural University, Beijing 100193
    3 Beijing University of Agriculture, Beijing 102206
    4 Beijing Sunlon Livestock Development Company Limited, Beijing 100029
    5 Beijing Innovation Consortium of Bio-breeding, Beijing 100125
  • Received:2022-02-09 Accepted:2022-07-28 Online:2023-06-16 Published:2023-06-27

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Abstract:

【Objective】Heat stress has seriously impaired the production and health of dairy cows, causing the subsequent limitation in sustainable development of dairy industry. DNA methylation is an important epigenetic regulatory mechanism involved in an animal’s heat stress response, but the potential functions and molecular mechanisms of which are not clear. The current study was conducted to detect the DNA methylation related to heat stress in dairy cows and to identify target genes related to DNA methylation, so as to provide a better insight into the epigenetics mechanism of heat stress in dairy cows.【Method】In the study, 24 Chinese Holstein lactation cows (same lactation stage and same parity) in Sanyuan dairy farm were used for the blood samples collection in heat stress period (July in the summer of 2017) and non-heat stress period (April in spring 2017), respectively, followed by DNA extraction. To explore the DNA methylation differences in dairy cows from different heat stress period, 15 of 24 animals were randomly assigned to 3 groups (N=5 animals/group), 5 DNA samples in one group were mixed together to get a single pooled DNA sample, thus 6 pooled DNA samples including 3 from spring and 3 from summer were used for the DNA methylation detection by the whole-genome bisulfite sequencing (WGBS), then the differential methylation region (DMR; 1000 bp windows, 500 bp overlap, P<0.05) and key gene were identified. PROMO and Methprimer software were used to predict transcription factor binding sites and CpG islands, respectively. Then, the bovine mammary gland epithelial cells (Mac-T) were treated at 39 ℃ for 24 h, 48 h, and 72 h, and the cell viability were detected by MTT method. Finally, using the bisulfite sequencing PCR (BSP), the methylation levels of target gene promoter in 24 dairy cows in spring and summer and Mac-T cells treated in 39 ℃ were examined, respectively. 【Result】Based on the DNA methylation analysis of WGBS data, 49 861 differential methylation regions (DMRs) associated with heat stress were identified. One of DMRs was attributed to the promoter area of GNAS complex locus (GNAS), whose methylation level significantly increased in heat-stressed animals (P<0.001). Also, there was a 352 bp CpG island in the promoter of GNAS containing potential binding sites for Sp1, C/EBP and other important transcription factors. Further the methylation status of the GNAS gene promoter region in heat stressed dairy cows were verified by BSP, and the average methylation level in all cytosine of 31 CpG sites was higher in heat stress cows than that in control groups (P<0.05), which corresponding to the above WGBS results. Moreover, the 21 (-113 bp, Chr13:57532733) and 27 (-63 bp, Chr13:57532683) CpG sites showed significant differences between the spring and summer groups (P<0.05). In Mac-T cells, after 48 h and 72 h heat treatment, the cell viability decreased significantly (P<0.01), but the overall CG methylation level of 31 CpG sites in the GNAS gene promoter region increased significantly (P<0.05), and also the similar significant methylation changes appeared in the site 21 and 27 CpG in cell. 【Conclusion】 Heat stress increased the methylation levels of the promoter region of the GNAS in dairy cows as well as in cells, which indicated that GNAS was a potential target gene regulated DNA methylation in heat stress response of dairy cows.

Key words: heat stress, dairy cows, WGBS, DNA methylation, GNAS

Fig. 1

The analytical workflow performed in this study"

Table 1

Primer for GNAS gene in nested PCR"

引物名称
Primer name		 引物序列
Primer sequence		 产物长度
The length of product (bp)
外侧引物
Outer primer		 F：5′-GTTTGAGATTATTTTTTTTTAATTATAAT-3′ 676
R：5′-AAAAACACCTACCTTCCTAACCACTCAACT-3′
内侧引物
Inner primer		 F：5′-AGTTTTTGGAAAATGTTTTGGATTGGAT-3′ 598
R：5′-CCTCTACTTACCACTCTAAACAT-3′

Fig. 2

Methylation level in GNAS promoter and bioinformatics analysis A: The number of DMR in each region obtained by WGBS; B: The methylation level of GNAS promoter detected by WGBS, DMR1 refers to the average methylation level of all C sites, GNAS1-CG refers to the average methylation level of C in CG sites, * represents P<0.05, *** represents P<0.001; C: Prediction map of CpG island in GNAS promoter; D: Nucleotide sequence of GNAS1, the +1 position represents the transcription start site, 1 to 36 is the cytosine number of CG site, single underline represents the predicted transcription factor binding site, and italic bold is the primer binding region of nested PCR"

Fig. 3

Changes of GNAS1 methylation level after heat stress in dairy cows A: Peak diagram of BSP sequencing, color represents base type, red, blue, black, and green distribution represents T, C, G, and A bases, and heavy peak indicated by arrow refers to partial methylation sites (C/T); B: Mean methylation levels of all CG sites in GNAS1; C: Methylation level of each CG site in GNAS1. * represents P<0.05"

Fig. 4

Morphology of Mac-T cells A: Cell morphology at low density (100×); B: Cell morphology at high density (100×)"

Fig. 5

Immunofluorescence identification of Mac-T cells"

Fig. 6

Cell viability of Mac-T cells after heat treatment ** represents P<0.01"

Fig. 7

Changes of GNAS1 methylation level in Mac-T cells after heat treatment A: GNAS1 methylation level in Mac-T cells treated at 39 ℃ for different times; B: Methylation levels of 21 and 27 CG sites in Mac-T cells treated at 39 ℃ for different times. * represents P<0.05; ** represents P<0.01"

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