CAUSEL: an epigenome- and genome-editing pipeline for establishing function of noncoding GWAS variants

Sándor Spisák, Kate Lawrenson, Yanfang Fu, István Csabai, Rebecca T Cottman, Ji-Heui Seo, Christopher Haiman, Ying Han, Romina Lenci, Qiyuan Li, Viktória Tisza, Zoltán Szállási, Zachery T Herbert, Matthew Chabot, Mark Pomerantz, Norbert Solymosi, The GAME-ON/ELLIPSE Consortium, Simon A Gayther, J Keith Joung, Matthew L Freedman
Source: Nat Med
Publication Date: (2015)
Issue: 21(11): 1357-63
Research Area:
Cancer Research/Cell Biology
Gene Expression
Basic Research
Molecular Biology
Cells used in publication:
Species: human
Tissue Origin: prostate
Species: human
Tissue Origin: prostate
4D-Nucleofector® X-Unit
Transfection. 22Rv1 or LNCaP cells were plated the day before transfection, to reach 70–80% confluence at the time of transfection. 1 × 106 and 0.4 × 106 cells per transfection were collected for each cell line, respectively. Cells were transfected with 1 mg of TAL nuclease or TALE effector or control empty vector plasmid DNA by Nucleofection with SF Cell Line 4D-Nucleofector X Kit (Lonza) using 20-ml Nucleocuvette Strips, as described by the manufacturer (Program EN120 and EN150). Cells were immediately resuspended in 100 ml of culturing medium and plated into 1.5 ml of pre-warmed culturing medium in 24-well tissue culture plates. The T7E1 assay, gene expression assays or single-cell cloning were performed 72 h after Nucleofection.
The vast majority of disease-associated single-nucleotide polymorphisms (SNPs) mapped by genome-wide association studies (GWASs) are located in the non-protein-coding genome, but establishing the functional and mechanistic roles of these sequence variants has proven challenging. Here we describe a general pipeline in which candidate functional SNPs are first evaluated by fine mapping, epigenomic profiling, and epigenome editing, and then interrogated for causal function by using genome editing to create isogenic cell lines followed by phenotypic characterization. To validate this approach, we analyzed the 6q22.1 prostate cancer risk locus and identified rs339331 as the top-scoring SNP. Epigenome editing confirmed that the rs339331 region possessed regulatory potential. By using transcription activator-like effector nuclease (TALEN)-mediated genome editing, we created a panel of isogenic 22Rv1 prostate cancer cell lines representing all three genotypes (TT, TC, CC) at rs339331. Introduction of the 'T' risk allele increased transcription of the regulatory factor 6 (RFX6) gene, increased homeobox B13 (HOXB13) binding at the rs339331 region, and increased deposition of the enhancer-associated H3K4me2 histone mark at the rs339331 region compared to lines homozygous for the 'C' protective allele. The cell lines also differed in cellular morphology and adhesion, and pathway analysis of differentially expressed genes suggested an influence of androgens. In summary, we have developed and validated a widely accessible approach that can be used to establish functional causality for noncoding sequence variants identified by GWASs.