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

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.

Figure 1. Design of TALENs for targeting the TT>A enhancer

A. Location of 5’ and 3’ TALEN nuclease binding sites. B. Western blotting of transfected HA-tagged 5’ TALEN arm and FLAG-tagged 3’ TALEN arm in HEK293T cells. C. Location of TALEN-mediated mutations of the TT>A enhancer in two HEK293T cell lines. Mutant 1 has a 26 base pair deletion of the TT>A enhancer including the NF-κB and SATB1 binding sites. Mutant 2 has a 9 base pair deletion and 4 base pair insertion (indel) of the TT>A enhancer that disrupts the NF-κB binding site. Red letters in A and C indicate the SLE risk TT>A polymorphisms.

Figure 2. Mutating the TT>A enhancer region by TALEN engineering impairs TNFAIP3 expression

A. Quantitative RT-PCR analysis of TNFAIP3 mRNA expression normalized to control HMBS mRNA in control and clonal mutant HEK293T cell lines. B. Western blotting analysis of A20 protein normalized to β-actin in parental (control) and clonal mutant HEK293T cell lines. Statistical significance was calculated using paired student t-test. For all experiments: n = 3, *p < 0.05.

Figure 3. TALEN mutagenesis of the TT>A enhancer reduces interaction with the TNFAIP3 promoter

A. Schematic representation of the allele-specific 3C and clonal assay analyses of the engineered heterozygous clonal mutant HEK293T cell lines. Wild type (WT) allele experiences higher frequencies of long-range DNA looping, thus higher proportions of 3C events are observed relative to Mutant 1 or Mutant 2 alleles. B. Genomic DNA isolated from 72 clonal expansions from Mutant 1 or Mutant 2 HEK293T cells without 3C was analyzed for wild type and mutant allele frequencies. The expected clonal frequency of approximately 50% wild type and 50% mutant allele was observed. C. Analysis of 72 mutant 1 or 2 clonal expansions from the 3C capture were analyzed for relative crosslinking frequencies between wild-type or mutated TT>A enhancer and TNFAIP3 promoter. P values were calculated using the Fisher’s exact test and are as indicated.

Figure 4. TALEN mutagenesis of the TT>A enhancer region delayed TNFα-inducible inactivation of NF-κB signaling

A. Western blotting of phospho-IκBα at designated time points following TNFα stimulation of parental (wild type) and clonal mutant HEK293T cells. B. Densitometry analyses of Western blotting data represented in (A) normalized to β-actin levels; n = 3, P value calculated by two-way ANOVA and are as indicated.

Figure 5. SLE-associated TT>A enhancer risk allele is causal for SLE-associated A20 protein reduction and elevated NF-κB signaling

Schematic depiction of the TT>A enhancer function in non-risk and SLE-associated risk alleles. SLE-associated polymorphisms in the TT>A enhancer impairs long-range DNA looping and TT>A enhancer interaction with, and delivery of transcription factors including NF-κB, to the TNFAIP3 gene promoter. Subsequent reductions in TNFAIP3 mRNA transcription and A20 protein translation impair A20-mediated ubiquitination and degradation of NF-κB, thus preventing attenuation of pro-inflammatory NF-κB signaling.