Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose: Emerging technologies for precise, targeted genome editing provide new opportunities for the functional elucidation of causal genetic variants and genomic elements without the confounding effects of correlated variants. Transcription Activation-Like Effector Nucleases (TALENs) represents an efficient tool to mediate site-specific genome modification in human cell lines. Previously, we described the influence of systemic lupus erythematosus (SLE)-associated functional variants (rs148314165, rs200820567; collectively referred to as TT>A) on TNFAIP3 gene expression. The TT>A variants, which lie 42-kbs downstream of the TNFAIP3 promoter, are located in a putative enhancer and were recently proposed as causal variants responsible for genetic association of SLE in the region of TNFAIP3. In this study, we generated a TT>A enhancer knockout in human cell lines to further characterize the role of the enhancer in regulating TNFAIP3 gene expression.
Methods: The genomic DNA sequence surrounding the TT>A enhancer was scanned for potential TALEN target sites using TALEN Targeter 2.0. A modified TALEN Golden Gate assembly system was then used to generate RVD repeat arrays, which were then subcloned into the expression vectors pCS2TAL3DD and pCS2TAL3RR. TALENs were expressed in HEK293T cells by transient transfection, and single-cell clones were isolated. Targeted cell clones were selected based on high-resolution melt analysis of PCR amplicons from each cell clone. Sanger sequencing was performed to determine the sequence of the targeted allele. Protein expression of A20 and mRNA expression of TNFAIP3 were determined by Western blot and RT-qPCR assay, respectively. Chromatin conformation capture (3C) assay was performed to determine the interaction frequency between the TT>A enhancer and the TNFAIP3 promoter.
Results: We assembled RVD repeat arrays targeting the TT>A enhancer, generated TALEN constructs, and expressed TALENs in HEK293T cells. Using high-resolution melt analysis, we identified an engineered HEK293T cell that carries a 26-bp deletion at the TT>A enhancer, which leads to alteration of NF-κB binding sites predicted using the UniProbe database. Assessing the effect of the mutant TT>A enhancer on the expression of TNFAIP3 at the protein and transcript level, we demonstrated low-level expression of TNFAIP3 mRNA and A20 protein in the mutant cell line. Follow up allele-specific 3C assay with the engineered cells demonstrated significantly fewer interactions with the TNFAIP3 promoter as a result of the mutant allele. This is consistent with the naturally occurring SLE-associated TT>A polymorphism, in which the risk allele reduces gene expression of TNFAIP3.
Conclusion: The SLE-associated TT>A enhancer plays an important role in regulating gene expression of TNFAIP3. By generating an engineered TT>A enhancer mutant HEK293T cell line, we have characterized the influences of the mutated enhancer on TNFAIP3 gene expression. These results suggest that TALEN-mediated genome editing has potential to be used for the identification and characterization of complex disease-associated causal variants.
Disclosure:
S. Wang,
None;
F. Wen,
None;
B. He,
None;
P. M. Gaffney,
None.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/transcription-activation-like-effector-nuclease-mediated-enhancer-knockout-influences-tnfaip3-gene-expression-and-mimics-a-functional-phenotype-associated-with-systemic-lupus-erythematosus/