Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

TALEN-mediated enhancer knockout influences TNFAIP3 gene expression and mimics a molecular phenotype associated with systemic lupus erythematosus

Genes & Immunity volume 17, pages 165–170 (2016)Cite this article

Subjects

Abstract

Linkage disequilibrium poses a major challenge to the functional characterization of specific disease-associated susceptibility variants. Precision genome-editing technologies have provided new opportunities to address this challenge. As proof of concept, we employed TALEN (transcription activation-like effector nuclease)-mediated genome editing to specifically disrupt the TT>A enhancer region to mimic candidate causal variants identified in the systemic lupus erythematosus-associated susceptibility gene, tumor necrosis factor-α-induced protein 3 (TNFAIP3), in an isogenic HEK293T cell line devoid of other linkage disequilibrium-associated variants. Targeted disruption of the TT>A enhancer impaired its interaction with the TNFAIP3 promoter by long-range DNA looping, thereby reducing TNFAIP3 gene expression. Loss of TNFAIP3 mRNA and its encoded protein, A20, impaired tumor necrosis factor-α-induced receptor-mediated downregulation of nuclear factor-κB signaling, a hallmark of autoimmunity. Results demonstrate that the TT>A enhancer variants contribute to causality and function independently of other variants to disrupt TNFAIP3 expression. Furthermore, we believe this approach can be implemented to independently examine other candidate casual variants in the future.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Cui Y, Sheng Y, Zhang X . Genetic susceptibility to SLE: recent progress from GWAS. J Autoimmun 2013; 41: 25–33.

    Article  CAS  Google Scholar 

  2. Relle M, Weinmann-Menke J, Scorletti E, Cavagna L, Schwarting A . Genetics and novel aspects of therapies in systemic lupus erythematosus. Autoimmun Rev 2015; 14: 1005–1018.

    Article  CAS  Google Scholar 

  3. Adrianto I, Wang S, Wiley GB, Lessard CJ, Kelly JA, Adler AJ et al. Association of two independent functional risk haplotypes in TNIP1 with systemic lupus erythematosus. Arthritis Rheum 2012; 64: 3695–3705.

    Article  CAS  Google Scholar 

  4. Wang S, Adrianto I, Wiley GB, Lessard CJ, Kelly JA, Adler AJ et al. A functional haplotype of UBE2L3 confers risk for systemic lupus erythematosus. Genes Immun 2012; 13: 380–387.

    Article  CAS  Google Scholar 

  5. Graham RR, Cotsapas C, Davies L, Hackett R, Lessard CJ, Leon JM et al. Genetic variants near TNFAIP3 on 6q23 are associated with systemic lupus erythematosus. Nat Genet 2008; 40: 1059–1061.

    Article  CAS  Google Scholar 

  6. Bentham J, Morris DL, Cunninghame Graham DS, Pinder CL, Tombleson P, Behrens TW et al. Genetic association analyses implicate aberrant regulation of innate and adaptive immunity genes in the pathogenesis of systemic lupus erythematosus. Nat Genet 2015; 47: 1457–1464.

    Article  CAS  Google Scholar 

  7. Ma A, Malynn BA . A20: linking a complex regulator of ubiquitylation to immunity and human disease. Nat Rev Immunol 2012; 12: 774–785.

    Article  CAS  Google Scholar 

  8. Lee EG, Boone DL, Chai S, Libby SL, Chien M, Lodolce JP et al. Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice. Science 2000; 289: 2350–2354.

    Article  CAS  Google Scholar 

  9. Tavares RM, Turer EE, Liu CL, Advincula R, Scapini P, Rhee L et al. The ubiquitin modifying enzyme A20 restricts B cell survival and prevents autoimmunity. Immunity 2010; 33: 181–191.

    Article  CAS  Google Scholar 

  10. Chu Y, Vahl JC, Kumar D, Heger K, Bertossi A, Wojtowicz E et al. B cells lacking the tumor suppressor TNFAIP3/A20 display impaired differentiation and hyperactivation and cause inflammation and autoimmunity in aged mice. Blood 2011; 117: 2227–2236.

    Article  CAS  Google Scholar 

  11. Hovelmeyer N, Reissig S, Xuan NT, Adams-Quack P, Lukas D, Nikolaev A et al. A20 deficiency in B cells enhances B-cell proliferation and results in the development of autoantibodies. Eur J Immunol 2011; 41: 595–601.

    Article  Google Scholar 

  12. Yang W, Tang H, Zhang Y, Tang X, Zhang J, Sun L et al. Meta-analysis followed by replication identifies loci in or near CDKN1B, TET3, CD80, DRAM1, and ARID5B as associated with systemic lupus erythematosus in Asians. Am J Hum Genet 2013; 92: 41–51.

    Article  CAS  Google Scholar 

  13. Jostins L, Ripke S, Weersma RK, Duerr RH, McGovern DP, Hui KY et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 2012; 491: 119–124.

    Article  CAS  Google Scholar 

  14. Genetic Analysis of Psoriasis Consortium & the Wellcome Trust Case Control Consortium 2, Strange A, Capon F, Spencer CC, Knight J et al. A genome-wide association study identifies new psoriasis susceptibility loci and an interaction between HLA-C and ERAP1. Nat Genet 2010; 42: 985–990.

    Article  Google Scholar 

  15. Kochi Y, Okada Y, Suzuki A, Ikari K, Terao C, Takahashi A et al. A regulatory variant in CCR6 is associated with rheumatoid arthritis susceptibility. Nat Genet 2010; 42: 515–519.

    Article  CAS  Google Scholar 

  16. Stahl EA, Raychaudhuri S, Remmers EF, Xie G, Eyre S, Thomson BP et al. Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci. Nat Genet 2010; 42: 508–514.

    Article  CAS  Google Scholar 

  17. Dubois PC, Trynka G, Franke L, Hunt KA, Romanos J, Curtotti A et al. Multiple common variants for celiac disease influencing immune gene expression. Nat Genet 2010; 42: 295–302.

    Article  CAS  Google Scholar 

  18. Han JW, Zheng HF, Cui Y, Sun LD, Ye DQ, Hu Z et al. Genome-wide association study in a Chinese Han population identifies nine new susceptibility loci for systemic lupus erythematosus. Nat Genet 2009; 41: 1234–1237.

    Article  CAS  Google Scholar 

  19. Musone SL, Taylor KE, Lu TT, Nititham J, Ferreira RC, Ortmann W et al. Multiple polymorphisms in the TNFAIP3 region are independently associated with systemic lupus erythematosus. Nat Genet 2008; 40: 1062–1064.

    Article  CAS  Google Scholar 

  20. Plenge RM, Cotsapas C, Davies L, Price AL, de Bakker PI, Maller J et al. Two independent alleles at 6q23 associated with risk of rheumatoid arthritis. Nat Genet 2007; 39: 1477–1482.

    Article  CAS  Google Scholar 

  21. Wang S, Wen F, Wiley GB, Kinter MT, Gaffney PM . An enhancer element harboring variants associated with systemic lupus erythematosus engages the TNFAIP3 promoter to influence A20 expression. PLoS Genet 2013; 9: e1003750.

    Article  CAS  Google Scholar 

  22. Adrianto I, Wen F, Templeton A, Wiley G, King JB, Lessard CJ et al. Association of a functional variant downstream of TNFAIP3 with systemic lupus erythematosus. Nat Genet 2011; 43: 253–258.

    Article  CAS  Google Scholar 

  23. Wright DA, Li T, Yang B, Spalding MH . TALEN-mediated genome editing: prospects and perspectives. Biochem J 2014; 462: 15–24.

    Article  CAS  Google Scholar 

  24. Kim Y, Kweon J, Kim JS . TALENs and ZFNs are associated with different mutation signatures. Nat Methods 2013; 10: 185.

    Article  Google Scholar 

  25. Gaj T, Gersbach CA, Barbas CF 3rd . ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 2013; 31: 397–405.

    Article  CAS  Google Scholar 

  26. Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, Cathomen T . A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res 2011; 39: 9283–9293.

    Article  CAS  Google Scholar 

  27. Doyle EL, Booher NJ, Standage DS, Voytas DF, Brendel VP, Vandyk JK et al. TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res 2012; 40 (Web Server issue): W117–W122.

    Article  CAS  Google Scholar 

  28. Dahlem TJ, Hoshijima K, Jurynec MJ, Gunther D, Starker CG, Locke AS et al. Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome. PLoS Genet 2012; 8: e1002861.

    Article  CAS  Google Scholar 

  29. Siggers T, Chang AB, Teixeira A, Wong D, Williams KJ, Ahmed B et al. Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-kappaB family DNA binding. Nat Immunol 2012; 13: 95–102.

    Article  CAS  Google Scholar 

  30. Gilmore TD . Introduction to NF-kappaB: players, pathways, perspectives. Oncogene 2006; 25: 6680–6684.

    Article  CAS  Google Scholar 

  31. Sanger F, Coulson AR . A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol 1975; 94: 441–448.

    Article  CAS  Google Scholar 

  32. Sanger F, Nicklen S, Coulson AR . DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74: 5463–5467.

    Article  CAS  Google Scholar 

  33. Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke-Kaiser H . Quantitative real-time RT-PCR data analysis: current concepts and the novel "gene expression's CT difference" formula. J Mol Med (Berl) 2006; 84: 901–910.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National Institutes of Health 5 R01 AR056360-05, 5 R01 AR063124-04 and P30 GM110766.

Author information

Authors and Affiliations

  1. Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA

    S Wang, F Wen, K L Tessneer & P M Gaffney

  2. Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, China

    S Wang

  3. The Bethune Institute of Epigenetic Medicine, First Hospital of Jilin University, Changchun, China

    S Wang

  4. Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    F Wen

Authors
  1. S Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. F Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K L Tessneer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P M Gaffney
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to P M Gaffney.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on Genes and Immunity website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, S., Wen, F., Tessneer, K. et al. TALEN-mediated enhancer knockout influences TNFAIP3 gene expression and mimics a molecular phenotype associated with systemic lupus erythematosus. Genes Immun 17, 165–170 (2016). https://doi.org/10.1038/gene.2016.4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gene.2016.4

Search

Advanced search

Quick links