Lupus Susceptibility Region Containing CDKN1B rs34330 Mechanistically Influences Expression and Function of Multiple Target Genes, Also Linked to Proliferation and Apoptosis

Abstract

Objective In a recent genome‐wide association study, a significant genetic association between rs34330 of CDKN1B and risk of systemic lupus erythematosus (SLE) in Han Chinese was identified. This study was undertaken to validate the reported association and elucidate the biochemical mechanisms underlying the effect of the variant. Methods We performed an allelic association analysis in patients with SLE, followed by a meta‐analysis assessing genome‐wide association data across 11 independent cohorts (n = 28,872). In silico bioinformatics analysis and experimental validation in SLE‐relevant cell lines were applied to determine the functional consequences of rs34330. Results We replicated a genetic association between SLE and rs34330 (meta‐analysis P = 5.29 × 10−22, odds ratio 0.84 [95% confidence interval 0.81–0.87]). Follow‐up bioinformatics and expression quantitative trait locus analysis suggested that rs34330 is located in active chromatin and potentially regulates several target genes. Using luciferase and chromatin immunoprecipitation–real‐time quantitative polymerase chain reaction, we demonstrated substantial allele‐specific promoter and enhancer activity, and allele‐specific binding of 3 histone marks (H3K27ac, H3K4me3, and H3K4me1), RNA polymerase II (Pol II), CCCTC‐binding factor, and a critical immune transcription factor (interferon regulatory factor 1 [IRF‐1]). Chromosome conformation capture revealed long‐range chromatin interactions between rs34330 and the promoters of neighboring genes APOLD1 and DDX47, and effects on CDKN1B and the other target genes were directly validated by clustered regularly interspaced short palindromic repeat (CRISPR)–based genome editing. Finally, CRISPR/dead CRISPR‐associated protein 9–based epigenetic activation/silencing confirmed these results. Gene‐edited cell lines also showed higher levels of proliferation and apoptosis. Conclusion Collectively, these findings suggest a mechanism whereby the rs34330 risk allele (C) influences the presence of histone marks, RNA Pol II, and IRF‐1 transcription factor to regulate expression of several target genes linked to proliferation and apoptosis. This process could potentially underlie the association of rs34330 with SLE.

A multilayered post-GWAS analysis pipeline defines functional variants and target genes for systemic lupus erythematosus (SLE)

“…To assess the impact of the SNP on target gene expression, we used qRT-PCR on WT, CRISPRa, and CRISPRi cells, as described elsewhere(32). RNA was isolated from WT and CRISPRa/i cells using an RNA Mini kit (Zymo Research) and reverse transcribed using iScript Reverse Transcription Supermix cDNA synthesis kit (Bio-Rad).…”

“…qRT-PCR was performed on WT, CRISPRa, and CRISPRi cells, as described elsewhere 53 . RNA was isolated from WT and CRISPRa/i cells using an RNA Mini kit (Zymo Research).…”

A Non-Coding Variant inSLC15A4Modulates Enhancer Activity and Lysosomal Deacidification Linked to Lupus Susceptibility

“…To investigate whether the rs35907548 region has enhancer activity, we utilized the well-established Dual-Luciferase Reporter Assay System. A detailed description of the method is provided elsewhere [27, 28]. Briefly, we cloned the 300 bp region, rs35907548 at the middle (chr12:129,282,013-129,282,312, hg 19) locus into pGL4.26 (Promega) and co-transfected with pGL4.74 (internal control) in HEK293, LCL, Jurkat, and HL-60 cells.…”

“…492024, Thermo-Fisher, Waltham, MA, USA), following manufacturer’s guidelines. A detailed description of the method is provided elsewhere [27, 28]. Briefly, 1.5-2 × 10 6 homozygous rs35907548 risk-“TT” (GM18603) and non-risk-“CC” (GM18624) genotype B-lymphoblastoid LCLs (Coriell) were cross-linked with 1% paraformaldehyde, washed, and sonicated.…”

“…To investigate the chromatin interactions between the promoters of the target genes and the rs35907548 region in an ex vivo context, we utilized 3C-qPCR in LCL and Jurkat cells. Detailed protocols are provided elsewhere [27, 28]. Briefly, cells were suspended in complete media with 10% FBS (1 × 10 6 /mL media) at 70-80% confluency and cross-linked with 1% paraformaldehyde at room temperature for 10 minutes.…”