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Cell Stress and Chaperones

, Volume 23, Issue 4, pp 617–628 | Cite as

DNA methylation rather than single nucleotide polymorphisms regulates the production of an aberrant splice variant of IL6R in mastitic cows

  • Yan Zhang
  • Xiuge Wang
  • Qiang Jiang
  • Haisheng Hao
  • Zhihua Ju
  • Chunhong Yang
  • Yan Sun
  • Changfa Wang
  • Jifeng Zhong
  • Jinming Huang
  • Huabin Zhu
Original Paper

Abstract

Interleukin-6 receptor-alpha (IL6R) interacts with IL6 and forms a ligand–receptor complex, which can stimulate various cellular responses, such as cell proliferation, cell differentiation, and activation of inflammatory processes. Both genetic mutation and epigenetic modification regulate gene transcription. We identified a novel splice variant of bovine IL6R, designated as IL6R-TV, which is characterized by the skipping of exon 2 of the NCBI-referenced IL6R gene (IL6R-reference). The expression levels of IL6R-TV and IL6R-reference transcripts were lower in normal mammary gland tissues. These transcripts play a potential role during inflammatory infection. We also detected two putative functional SNPs (g.19711 T > C and g.19731 G > C) located within the upstream 100 bp of exon 2. These SNPs formed two haplotypes (T-G and C-C). Two mutant pSPL3 exon-trapping plasmids (pSPL3-T-G and pSPL3-C-C) were transferred into the bovine mammary epithelial cells (MAC-T) and human embryonic kidney 293 T cells (HEK293T) to investigate the relationship between the two SNPs and the aberrant splicing of IL6R. DNA methylation levels of the alternatively spliced exon in normal and mastitis-infected mammary gland tissues were quantified through nested bisulfate sequencing PCR (BSP) and cloning sequencing. We found that DNA methylation regulated IL6R transcription. The DNA methylation level was high in mastitis-infected mammary gland tissues and stimulated IL6R expression, thereby promoting the inclusion of the alternatively spliced exon. The upregulated expression of the two transcripts was due to DNA methylation modification rather than genetic mutations.

Keywords

IL6R Splice variant Single nucleotide polymorphism Cattle Mastitis DNA methylation 

Abbreviations

IL6R

Interleukin-6 receptor-alpha

SNPs

Single nucleotide polymorphisms

MAC-T

Bovine mammary epithelial cells

HEK293T

Human embryonic kidney 293 T cells

BSP

Bisulfate sequencing PCR

E. coli

Escherichia coli

S. aureus

Staphylococcus aureus

ASEs

Alternatively spliced exons

TPI

Total performance index

SCS

Somatic cell score

Q-PCR

Quantitative real-time PCR

DMEM-F12

Dulbecco’s modified Eagle medium/Ham’s F-12 medium

DMEM

Dulbecco’s modified Eagle medium

TFBS

Transcriptional factor binding sites

ESE

Exonic splicing enhancer

DMRs

Differentially methylated regions

ETS family

E26 transformation-specific family

mIL6R

Membrane-bound interleukin-6 receptor-alpha

sIL6R

Soluble interleukin-6 receptor-alpha

SR family

Serine/arginine (SR) family

Notes

Acknowledgements

We gratefully acknowledge Professor Kerong Shi from Shandong Agricultural University for kindly providing the MAC-T cell line. We thank Dr. Guili Song of the Institute of Hydrobiology, Chinese Academy of Sciences, for providing the pSPL3 vector.

Funding information

This work was supported by grants from the National Natural Science Foundation of China (31401049, 31671286 and 31672397), the Agricultural Science and Technology Innovation Program (ASTIP-IA S06), and the Key Research and Development Program of Shandong (2015GNC110002).

Compliance with ethical standards

All experiments were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals published by the Ministry of Science and Technology, China, in 2004. The procedures were approved by the Animal Care and Use Committee of the Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Shandong, People’s Republic of China.

Supplementary material

12192_2017_871_MOESM1_ESM.pdf (575 kb)
ESM 1 (PDF 575 kb)

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Copyright information

© Cell Stress Society International 2018

Authors and Affiliations

  1. 1.Institute of Animal Sciences (IAS)Chinese Academy of Agricultural Sciences (CAAS)BeijingPeople’s Republic of China
  2. 2.Dairy Cattle Research CenterShandong Academy of Agricultural SciencesJinanPeople’s Republic of China
  3. 3.College of Life SciencesShandong Normal UniversityJinanPeople’s Republic of China

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