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 HuangEmail author
  • Huabin ZhuEmail author
Original Paper


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.


IL6R Splice variant Single nucleotide polymorphism Cattle Mastitis DNA methylation 



Interleukin-6 receptor-alpha


Single nucleotide polymorphisms


Bovine mammary epithelial cells


Human embryonic kidney 293 T cells


Bisulfate sequencing PCR

E. coli

Escherichia coli

S. aureus

Staphylococcus aureus


Alternatively spliced exons


Total performance index


Somatic cell score


Quantitative real-time PCR


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


Dulbecco’s modified Eagle medium


Transcriptional factor binding sites


Exonic splicing enhancer


Differentially methylated regions

ETS family

E26 transformation-specific family


Membrane-bound interleukin-6 receptor-alpha


Soluble interleukin-6 receptor-alpha

SR family

Serine/arginine (SR) family



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)


  1. Aitken SL, Corl CM, Sordillo LM (2011) Immunopathology of mastitis: insights into disease recognition and resolution. J Mammary Gland Biol Neoplasia 16(4):291–304. CrossRefPubMedGoogle Scholar
  2. Benakanakere M, Adbolhosseini M, Hosur K, Finoti LS, Kinane DF (2015) TLR2 promoter hypermethylation creates innate immune dysbiosis. J Dent Res 94(1):183–191. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bradley T, Cook ME, Blanchette M (2015) SR proteins control a complex network of RNA-processing events. RNA 21(1):75–92. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Breiling A, Lyko F (2015) Epigenetic regulatory functions of DNA modifications: 5-methylcytosine and beyond. Epigenetics Chromatin 8(24).
  5. Brett D, Pospisil H, Valcárcel J, Reich J, Bork P (2001) Alternative splicing and genome complexity. Nat Genet 30:29–30CrossRefPubMedGoogle Scholar
  6. Buitenhuis B, Røntved CM, Edwards SM, Ingvartsen KL, Sørensen P (2011) In depth analysis of genes and pathways of the mammary gland involved in the pathogenesis of bovine Escherichia coli-mastitis. BMC Genomics 12(130).
  7. Chang G, Petzl W, Vanselow J, Günther J, Shen X, Seyfert HM (2015) Epigenetic mechanisms contribute to enhanced expression of immune response genes in the liver of cows after experimentally induced Escherichia coli mastitis. Vet J 203(3):339–341. CrossRefPubMedGoogle Scholar
  8. ElSharawy A, Manaster C, Teuber M, Rosenstiel P, Kwiatkowski R, Huse K, Platzer M, Becker A, Nürnberg P, Schreiber S, Hampe J (2006) SNPSplicer: systematic analysis of SNP-dependent splicing in genotyped cDNAs. Hum Mutat 27(11):1129–1134. CrossRefPubMedGoogle Scholar
  9. Fontes JA, Rose NR, Čiháková D (2015) The varying faces of IL-6: from cardiac protection to cardiac failure. Cytokine 74(1):62–68. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Gao Q, Ju Z, Zhang Y, Huang J, Zhang X, Qi C, Li J, Zhong J, Li G, Wang C (2014) Association of TNP2 gene polymorphisms of the bta-miR-154 target site with the semen quality traits of Chinese Holstein bulls. PLoS One 9(1):e84355. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Garbers C, Aparicio-Siegmund S, Rose-John S (2015) The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition. Curr Opin Immunol 34:75–82. CrossRefPubMedGoogle Scholar
  12. Garcia-Blanco MA, Baraniak AP, Lasda EL (2004) Alternative splicing in disease and therapy. Nat Biotechnol 22(5):535–546. CrossRefPubMedGoogle Scholar
  13. García-Ruiz A, Cole JB, VanRaden PM, Wiggans GR, Ruiz-López FJ, Van Tassell CP (2016) Changes in genetic selection differentials and generation intervals in US Holstein dairy cattle as a result of genomic selection. Proc Natl Acad Sci U S A 113(28):E3995–E4004. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Gomes F, Henriques M (2016) Control of bovine mastitis: old and recent therapeutic approaches. Curr Microbiol 72(4):377–382. CrossRefPubMedGoogle Scholar
  15. Günther J, Esch K, Poschadel N, Petzl W, Zerbe H, Mitterhuemer (2011) Comparative kinetics of Escherichia coli- and Staphylococcus aureus-specific activation of key immune pathways in mammary epithelial cells demonstrates that S. aureus elicits a delayed response dominated by interleukin-6 (IL-6) but not by IL-1A or tumor necrosis factor alpha. Infect Immun 79(2):695–707. CrossRefPubMedGoogle Scholar
  16. Günther J, Koy M, Berthold A, Schuberth HJ, Seyfert HM (2016) Comparison of the pathogen species-specific immune response in udder derived cell types and their models. Vet Res 47(1, 22)Google Scholar
  17. Heyn H, Moran S, Hernando-Herraez I, Sayols S, Gomez A, Sandoval J, Monk D, Hata K, Marques-Bonet T, Wang L, Esteller M (2013) DNA methylation contributes to natural human variation. Genome Res 23(9):1363–1372. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Hinchs D, Bennewitz J, Stamer E, Junge W, Kalm E, Thaller G (2011) Genetic analysis of mastitis data with different models. J Dairy Sci 94(1):471–478. CrossRefGoogle Scholar
  19. Hou Q, Huang J, Ju Z, Li Q, Li L, Wang L et al (2012) Identification of splice variants, targeted microRNAs and functional SNPs of the BOLA-DQA2 gene in dairy cattle. DNA Cell Biol 31(5):739–744. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Huang CS, Shen CY, Wang HW, Wu PE, Cheng CW (2007) Increased expression of SRp40 affecting CD44 splicing is associated with the clinical outcome of lymph node metastasis in human breast cancer. Clin Chim Acta 384(1–2):69–74. CrossRefPubMedGoogle Scholar
  21. Huang J, Luo G, Zhang Z, Wang X, Ju Z, Qi C, Zhang Y, Wang C, Li R, Li J, Yin W, Xu Y, Moisá SJ, Loor JJ, Zhong J (2014) iTRAQ—proteomics and bioinformatics analyses of mammary tissue from cows with clinical mastitis due to natural infection with Staphylococci aureus. BMC Genomics 15(1):839. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hunter CA, Jones SA (2015a) IL-6 as a keystone cytokine in health and disease. Nat Immunol 16(5):448–457. CrossRefPubMedGoogle Scholar
  23. Hunter CA, Jones SA (2015b) IL-6 as a keystone cytokine in health and disease. Nat Immunol 16(5):448–457. CrossRefPubMedGoogle Scholar
  24. Iannone C, Valcárcel J (2013) Chromatin’s thread to alternative splicing regulation. Chromosoma 122(6):465–474. CrossRefPubMedGoogle Scholar
  25. Jones SA, Scheller J, Rose-John S (2011) Therapeutic strategies for the clinical blockade of IL-6/gp130 signaling. J Clin Invest 121(9):3375–3383. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Ju Z, Wang C, Wang X, Yang C, Sun Y, Jiang Q, Wang F, Li M, Zhong J, Huang J (2015) Role of an SNP in alternative splicing of bovine NCF4 and mastitis susceptibility. PLoS One 10(11):e0143705. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kumar H, Raj U, Gupta S, Tripathi R, Varadwaj PK (2015) Systemic review on chronic myeloid leukemia: therapeutic targets, pathways and inhibitors. J Nucl Med Radiat Ther 6(6)Google Scholar
  28. Larriba E, del Mazo J (2016) Role of non-coding RNAs in the transgenerational epigenetic transmission of the effects of reprotoxicants. Int J Mol Sci 17(4):452. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Lev Maor G, Yearim A, Ast G (2015) The alternative role of DNA methylation in splicing regulation. Trends Genet 31(5):274–280CrossRefPubMedGoogle Scholar
  30. Lewandowska-Sabat AM, Boman GM, Downing A, Talbot R, Storset AK, Olsaker I (2013) The early phase transcriptome of bovine monocyte-derived macrophages infected with Staphylococcus aureus in vitro. BMC Genomics 14(1):891. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Li L, Huang J, Ju Z, Li Q, Wang C, Qi C, Zhang Y, Hou Q, Hang S, Zhong J (2013) Multiple promoters and targeted microRNAs direct the expressions of HMGB3 gene transcripts in dairy cattle. Anim Genet 44(3):241–250. CrossRefPubMedGoogle Scholar
  32. Li H, Rokavec M, Hermeking H (2015) Soluble IL6R represents a miR-34a target: potential implications for the recently identified IL-6R/STAT3/miR-34afeed-back loop. Oncotarget 6(16):14026–14032. PubMedPubMedCentralCrossRefGoogle Scholar
  33. Lopez-Lasanta M, Julià A, Maymó J, Fernández-Gutierrez B, Ureña-Garnica I, Blanco FJ, Cañete JD, Alperi-López M, Olivè A, Corominas H, Tornero J, Erra A, Almirall M, Palau N, Ortiz A, Avila G, Rodriguez-Rodriguez L, Alonso A, Tortosa R, Gonzalez-Alvaro I, Marsal S (2015) Variation at interleukin-6 receptor gene is associated to joint damage in rheumatoid arthritis. Arthritis Res Ther 17(242).
  34. Lund T, Miglior F, Dekkers JCM, Burnside EB (1994) Genetic-relationships between clinical mastitis, somatic-cell count, and udder conformation in Danish Holsteins. Livest Prod Sci 39(3):243–251. CrossRefGoogle Scholar
  35. Maunakea AK, Chepelev I, Cui K, Zhao K (2013) Intragenic DNA methylation modulates alternative splicing by recruiting MeCP2 to promote exon recognition. Cell Res 23(11):1256–1269. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Messier TL, Gordon JA, Boyd JR, Tye CE, Browne G, Stein JL et al (2016) Histoen H3 lysine 4 acetylation and methylation dynamics define breast cancer subtypes. Oncotarget 7(5):5094–5109. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Niwa T, Tsukamoto T, Toyoda T, Mori A, Tanaka H, Maekita T et al (2010) Inflammatory processes triggered by Helicobacter pylori infection cause aberrant DNA methylation in gastric epithelial cells. Cancer Res 70(4):11430–11440CrossRefGoogle Scholar
  38. Oviedo-Boyso J, Valdez-Alarcón JJ, Cajero-Juárez M, Ochoa-Zarzosa A, López-Meza JE, Bravo-Patiño A et al (2007) Innate immune response of bovine mammary gland to pathogenic bacteria responsible for mastitis. J Inf Secur 54(4):399–409Google Scholar
  39. Rainard P, Cunha P, Gilbert FB (2016) Innate and adaptive immunity synergize to trigger inflammation in the mammary gland. PLoS One 11(4):e0154172. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Rawstron AC, Fenton JA, Ashcroft J, English A, Jones RA, Richards SJ et al (2000) The interleukin-6 receptor alpha-chain (CD126) is expressed by neoplastic but not normal plasma cells. Blood 96(12):3880–3886PubMedGoogle Scholar
  41. Resch A, Xing Y, Alekseyenko A, Modrek B, Lee C (2004) Evidence for a subpopulation of conserved alternative splicing events under selection pressure for protein reading frame preservation. Nucleic Acids Res 32(4):1261–1269. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Runyon RS, Cachola LM, Rajeshuni N, Hunter T, Garcia M, Ahn R, Lurmann F, Krasnow R, Jack LM, Miller RL, Swan GE, Kohli A, Jacobson AC, Nadeau KC (2012) Asthma discordance in twins is linked to epigenetic modifications of T cells. PLoS One 7(11):e48796. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Song M, He Y, Zhou H, Zhang Y, Li X, Yu Y (2016) Combined analysis of DNA methylome and transcriptome reveal novel candidate genes with susceptibility to bovine Staphylococcus aureus subclinical mastitis. Sci Rep 6(29390)Google Scholar
  44. Tryndyak VP, Han T, Fuscoe JC, Ross SA, Beland FA, Pogribny IP (2016) Status of hepatic DNA methylome predetermines and modulates the severity of non-alcoholic fatty liver injury in mice. BMC Genomics 17(298).
  45. Vanselow J, Yang W, Herrmann J, Zerbe H, Schuberth HJ, Petzl W, Tomek W, Seyfert HM (2006) DNA-remethylation around a STAT5-binding enhancer in the alphaS1-casein promoter is associated with abrupt shutdown of alphaS1-casein synthesis during acute mastitis. J Mol Endocrinol 37(3):463–477. CrossRefPubMedGoogle Scholar
  46. Wang XX, Wu Z, Huang HF, Han C, Zou W, Liu J (2013a) Caveolin-1, through its ability to negatively regulate TLR4, is a crucial determinant of MAPK activation in LPS-challenged mammary epithelial cells. Asian Pac J Cancer Prev 14(4):2295–2299. CrossRefPubMedGoogle Scholar
  47. Wang XS, Zhang Y, He YH, Ma PP, Fan LJ, Wang YC, Zhang YI, Sun DX, Zhang SL, Wang CD, Song JZ, Yu Y (2013b) Aberrant promoter methylation of the CD4 gene in peripheral blood cells of mastitic dairy cows. Genet Mol Res 12(4):6228–6239. CrossRefPubMedGoogle Scholar
  48. Wang X, Zhong J, Gao Y, Ju Z, Huang J (2014) A SNP in intron 8 of CD46 causes a novel transcript associated with mastitis in Holsteins. BMC Genomics 15(1):630. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Wiggans GR, Cole JB, Hubbard SM, Sonstegard TS 2016 Genomic selection in dairy cattle: the USDA experience. Annu Rev Anim BiosciGoogle Scholar
  50. Yang X, Han H, De Carvelho DD, Lay FD, Jones PA, Liang G (2014) Gene body methylation can alter gene expression and is a therapeutic target in cancer. Cancer Cell 26(4):577–590. CrossRefPubMedPubMedCentralGoogle Scholar
  51. Yearim A, Gelfman S, Shayevitch R, Melcer S, Glaich O, Mallm JP, Nissim-Rafinia M, Cohen AHS, Rippe K, Meshorer E, Ast G (2015) HP1 is involved in regulating the global impact of DNA methylation on alternative splicing. Cell Rep 10(7):1122–1134. CrossRefPubMedGoogle Scholar
  52. Zhang G, Tsang CM, Deng W, Yip YL, Lui VW, Wong SC, al e (2013) Enhanced IL-6/IL6R signaling promotes growth and malignant properties in EBV-infected premalignant and cancerous nasopharyngeal epithelial cells. PLoS One 8(5):e62284. CrossRefPubMedPubMedCentralGoogle Scholar
  53. Zhang Z, Wang X, Li R, Ju Z, Qi C, Zhang Y et al (2015) Genetic mutations potentially cause two novel NCF1 splice variants up-regulated in the mammary gland, blood and neutrophil of cows infected by Escherichia coli. Microbiol Res 174:24–32CrossRefPubMedGoogle Scholar

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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