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. 2016 Sep 14;12(9):e1006292.
doi: 10.1371/journal.pgen.1006292. eCollection 2016 Sep.

The Rheumatoid Arthritis Risk Variant CCR6DNP Regulates CCR6 via PARP-1

Gang Li  1 , Pierre Cunin  1 , Di Wu  1   2   3 , Dorothée Diogo  1   4   5 , Yu Yang  1 , Yukinori Okada  6   7   8 , Robert M Plenge  1 , Peter A Nigrovic  1   9
Affiliations

The Rheumatoid Arthritis Risk Variant CCR6DNP Regulates CCR6 via PARP-1

Gang Li et al. PLoS Genet. .

Abstract

Understanding the implications of genome-wide association studies (GWAS) for disease biology requires both identification of causal variants and definition of how these variants alter gene function. The non-coding triallelic dinucleotide polymorphism CCR6DNP is associated with risk for rheumatoid arthritis, and is considered likely causal because allelic variation correlates with expression of the chemokine receptor CCR6. Using transcription activator-like effector nuclease (TALEN) gene editing, we confirmed that CCR6DNP regulates CCR6. To identify the associated transcription factor, we applied a novel assay, Flanking Restriction Enhanced Pulldown (FREP), to identify specific association of poly (ADP-ribose) polymerase 1 (PARP-1) with CCR6DNP consistent with the established allelic risk hierarchy. Correspondingly, manipulation of PARP-1 expression or activity impaired CCR6 expression in several lineages. These findings show that CCR6DNP is a causal variant through which PARP-1 regulates CCR6, and introduce a highly efficient approach to interrogate non-coding genetic polymorphisms associated with human disease.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Characterization of mutated HCT116 clones generated by TALENs targeting the CCR6DNP.
(a) Partial genomic arrangement of CCR6 showing alternative transcripts CCR6-a and CCR6-b and the location of CCR6DNP. Only CCR-b could be detected in HCT116 and Jurkat T cells by real time PCR (see S3 Fig). (b) Sequence of the three alleles of CCR6DNP in 7 representative targeted HCT116 clones. The CCR6DNP is underlined. (c) and (d) Expression of CCR6 in the 7 targeted clones by qPCR and Western blot. For qPCR, data are shown as mean±s.d. (n = 3). Sequence, sequence trace and expression data for all 17 mutated HCT116 clones are in S1 and S2 Figs. Statistical significance in 1c reflects comparison to WT.
Fig 2
Fig 2. Flanking Restriction Enhanced Pulldown (FREP).
(a) An 82 bp biotinylated DNA fragment is conjugated to streptavidin-coated magnetic Dynabeads (Invitrogen). This fragment is engineered to include a 31bp gene specific (“bait”) sequence (black box), flanked by restriction enzyme cleavage sites for BamH I proximally (blue box) and EcoR I distally (red box), as well as 20bp DNA fragments to allow PCR amplification of the whole unit. (b) DNA-beads are mixed with nuclear extract. A free 82 bp non-biotinylated DNA fragment can be included in the control reaction at this stage as a specific competitor. (c) Magnetic separation and wash to remove non-DNA binding proteins. (d) EcoR I digestion to release 3’ DNA end-binding proteins, yielding the EcoR I fraction. (e) BamH I digestion to separate the sequence specific DNA binding proteins, BamH I fraction, from proteins that bind 5’ DNA and Dynabeads. (f) Mass spectrometry (MS) to identify proteins remaining within the BamH I fraction.
Fig 3
Fig 3. FREP to identify the binding of PARP-1 with CCR6DNP.
(a) Silver stain and (b) densitometry to show proteins pulled down from CCR6DNP/TG-beads mixed with nuclear extract from THP-1 cells. Lane 1, BamH I fraction; Lane 2, BamH I fraction where pulldown was performed in the presence of a 40x excess of competitor (free CCR6DNP/TG). Arrow indicates the specific band sent for mass spectrometry analysis.
Fig 4
Fig 4. The association of PARP-1 with CCR6DNP is sequence dependent and allele specific.
(a) FREP+Western blot for PARP-1 with BamH I fractions. Lane 1: nuclear extract (NE) input, lane 2: NE with CCR6DNP/TG-beads, lane 3: NE with CCR6DNP/TG-beads with cold competitor (Free TG), and lane 4: NE with a non-specific DNA sequence (NS-beads). (b) To detect allele specificity, FREP was modified to cut directly with BamH I only, yielding a combined BamH I+EcoR I fraction, and SDS-PAGE was probed with anti-PARP-1. Anti-Ku86 antibody was used as the internal loading control to ensure comparable amounts of competitor DNA. CCR6DNP/TG-beads were incubated with NE and competed with equal amount of free competitors. Lane 1: no competitor; lane 2: free CCR6DNP/TG; lane 3: CCR6DNP/CG; lane 4: free CCR6DNP/CA; and lane 5: free non-specific DNA. Upper: Western blot; Lower: relative density from the Western blot. (c) ChIP assay with an anti-PARP-1 antibody on WT HCT116 cells and 5 mutants as indicated in Fig 1 showing impaired binding of PARP-1 to the CCR6DNP. (d) ChIP assay with an anti-PARP-1 antibody on WT Jurkat T cells showing significant enrichment of the CCR6DNP fragments comparing to anti-IgG antibody. (e) Sequencing of the CCR6DNP fragments from the ChIP with WT HCT116 cells showing the significant enrichment of the T allele over the C allele by an anti-PARP-1 antibody. Results from (a), (b), (c), and (d) representative of 3 experiments. Data are shown as mean±s.d. (n = 3).
Fig 5
Fig 5. CCR6 expression in PARP-1 knockdown human cells.
HCT116 cells (a) and Jurkat T cells (b) by Western blot (left), and qPCR analysis on PARP-1 (middle) and on CCR6 (right). For Western blot, whole cell extract was isolated and separated on SDS-PAGE gel. Western blot was detected with mouse anti-human PARP-1, CCR6 and α-tubulin antibodies simultaneously. Lane 1: negative control for shRNA. Lane 2: shRNA treatment for HCT116 cells or siRNA treatment for Jurkat T cells. For qPCR, data are shown as mean±s.d. (n = 3).
Fig 6
Fig 6. CCR6 expression in human cells treated with 3-aminobenzamide.
(a) qPCR and (b) Western blot to show expression of CCR6 and PARP-1 in Jurkat T cells, HCT116 and Hela cells. Cells were treated with 0 mM (lane 1), 5 mM (lane 2) and 10 mM (lane 3) 3-AB for 72 hrs. For qPCR, data are shown as mean±s.d. (n = 3).
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