A novel regulatory element upstream of TNFAIP3 promoter may be influenced by SLE risk variants

Almost all of Genome Wide Association Studies (GWAS) hits come from non-coding DNA elements. Data from chromatin interaction analyses suggest a long-range interaction with a putative enhancer upstream of TNFAIP3. Disrupting the enhancer may impair TNFAIP3 expression and enhance SLE risk. Two variants, rs10499197 and rs58905141 carried on the SLE risk haplotype are situated near this enhancer and could affect its function. Cloning the regulatory region surrounding rs10499197 into an expression plasmid containing a CRISPR-Cas9 backbone, and then performing a genome editing assay, we found that the variant is located near an enhancer. And any changes to the SNP region might impair enhancer and its ability to regulate TNFAIP3 expression.


Introduction
Completion of the human genome sequence has enabled unparalleled exploration of the human genome. Availability of dense SNP panels with high-throughput, cost-effective genotyping and sequencing platforms, aid in performing unbiased high-resolution genome-wide surveys needed to identify DNA segments that harbor complex disease causal variants.

Understanding SLE, Why it matters. Systemic Lupus Erythematosus (SLE) is an autoimmune
disease characterized by dysregulated interferon response and loss of self-tolerance to cellular antigens. Autoantibody production leads to immune complex formation resulting in wide scale inflammation and multiple organ failure. SLE afflicts women at a rate nine times that of men (Chen et al., 2017;Deng and Tsao, 2017). The precise etiology of SLE is complex and poorly defined, requiring the interplay between inherited common risk alleles, genetic predisposition and environmental triggers.

TNFAIP3, a key SLE-risk locus.
Currently, there are more than 90 robust SLE susceptibility loci (Mohan and Putterman, 2015;Chen et al., 2017;Deng and Tsao, 2017). Testing association of 311,238 genotyped SNPs in 431 unrelated SLE cases and 2155 controls, three regions met a specified threshold for genome-wide significance: the previously defined HLA, IRF5 regions and a novel SLE locus at chromosome 6q23 within the TNFAIP3 gene (Dieude P. et al, 2010).
Tumor necrosis factor alpha-inducible protein 3 (TNFAIP3) is a prominent risk locus identified in many autoimmune diseases including SLE. TNFAIP3 encodes the ubiquitin-editing enzyme, A20 that negatively regulates Nuclear Factor kappa- (NF-κB) signaling to attenuate proinflammatory immune responses and protect against the damaging effects of chronic inflammation (Han et al., 2009;Cai et al., 2010).
Human GWAS of autoimmune diseases have identified single nucleotide polymorphisms (SNPs) on the TNFAIP3 locus that encodes A20. However, due to strong linkage disequilibrium, GWAS cannot distinguish causal from benign SNPs. Our group previously identified several potential causal SNPs within the TNFAIP3 gene locus associated with SLE.
In this study, we aim to briefly evaluate the function of a novel regulatory element located upstream of the TNFAIP3 promoter.

Antibodies and cell lines
Jurkat, Human embryonic kidney cells 293 (HEK293T) and THP-1 were purchased from ATCC. Epstein Barr Virus (EBV)-transformed B cell lines were obtained from the Lupus

Western Blotting
Two million EBV-transformed B-cells were pelleted and washed in cold PBS and lysed with RIPA lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, pH 8, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate) containing proteases (EMD Millipore, Burlington, MA) and Halt phosphatase inhibitors (Pierce, Rockford, IL). Cells were lysed for 15 minutes on ice followed by syringe lysis with a 27g needle then cleared by centrifugation at max speed for 20 minutes. Protein concentrations were determined by Qubit assay (Life Technologies, Carlsbad, CA). Proteins were denatured in 2X SDS loading buffer by heating to 100 °C for 5-10 min. Proteins were eluted by SDS-PAGE and analyzed by immunoblotting as indicated.

HiChIP Assay
A modified version of a previously published HiChIP method was used (Mumbach et al., 2016). Briefly, EBV-transformed B cells were crosslinked with 1% formaldehyde for 10 min at room temperature, then intact nuclei were isolated and DNA digested using MboI restriction enzyme (New England BioLabs, Inc., Ipswich, MA) for 4 hours at 37 °C. The ends of the DNA fragments were filled and labeled with dCTP, dGTP, dTTP, and biotin-dATP. Proximity ligation was performed at room temperature overnight, and then the ligated chromatin was fragmented using a Covaris S2 sonicator (Woburn, MA) and previously described parameters (Mumbach et al., 2016 was performed on a Roche LightCycler 480 system using primers specific to the HiSeq flowcell. Using the template size determined from the Qubit, the pools were diluted to 2 nM and re-quantified using qPCR to confirm the starting concentration. The pooled samples were denatured for 5 min with 0.1N NaOH, and further diluted to a loading concentration of 1.8 pM. Libraries were sequenced on the Illumina NextSeq 500 sequencer on a 150-cycle paired-end Mid flowcell. Analysis pipeline: HiChIP raw reads (fastq files) were aligned to the hg19 human reference genome using HiC-Pro [Servant, et al., 2015]. Aligned data were processed and analyzed through hichipper pipeline [hichipper MS]. We used MAC2 for anchor calling in the pipeline based on ChIP-enriched regions. Loops were derived from the linked paired-end reads that overlap with anchors. A good quality sample has a minimum of 15% intrachromosomal interactions.

CRISPR-Cas9 Genome Editing
A CRISPR-Cas9 system from reagent design, construction, validation and cell line expansion Completed and sequence-verified pSpCas9 (gRNA) plasmids and optional repair templates for facilitating HDR were then transfected into HEK293T cells and assayed for their ability to mediate targeted cleavage. Finally, transfected cells were clonally expanded to derive isogenic cell lines with defined mutations. Chimeric DNA generated was extracted and cleaned using a QIAGEN DNA purification kit (QIAGEN Inc., Valencia, CA). DNA was then amplified by PCR and subjected to Sanger sequencing to decode the clones.

Epigenomic analyses revealed a putative enhancer upstream of the TNFAIP3 promoter.
Dynamic organization of chromatin in three-dimensional space, specifically enhancer-gene promotor looping, plays a crucial role in regulating gene transcription. To investigate the threedimensional organization of the TNFAIP3 locus, we performed highly integrative chromatin immunoprecipitation (HiChIP) analysis on chromatin obtained from Epstein Barr virustransformed B cells (EBV) using antibodies against the epigenetic mark of an active enhancer, acetylation of Histone 3 lysine 27 (H3K27ac). We used hichipper and DNAlandscaper (Lareau and Aryee, 2018) to visualize, in two dimensions, several long-range enhancer-promotor interactions in and around the TNFAIP3 locus (Figure 1). Notably, our H3K27ac HiChIP data revealed a prominent long-range looping event between the TNFAIP3 promoter and a region ~55 kb upstream that is enriched with epigenetic marks of an enhancer (H3K4me1, H3K27ac) and clusters of transcription factor binding sites defined by ENCODE ChIP-seq data. The putative enhancer also overlapped SLE risk variants rs10499197 and rs58905141 located on the most 5' end of the SLE-associated ~109 kb risk haplotype (Figure 1). Collectively these results suggest that the putative enhancer located ~55 kb upstream of TNFAIP3 may play an important role in modulating TNFAIP3 expression during SLE pathogenesis.

TNFAIP3 expression is regulated by upstream DNA element.
To further analyze the functional potential of the putative enhancer ~ 55kb upstream of TNFAIP3, we generated CRISPR-Cas9 constructs to modify the region using the rs10499197 variant as a landmark. We used two sets of small RNAs (guide (g)-RNA) to enable Cas9 mediated genome editing at the target site in HEK293T cells: gRNA#1 was located 31 bp upstream of rs10499197, and gRNA#10 was located 4 bp downstream of rs10499197 (Supplementary Figure). Several isogenic clones were Sanger sequenced (Supplementary Table), but only three cell clones exhibited varying patterns of CRISPR editing near the target site. Clone#10-14 (from gRNA#10) had a 22-nucleotide deletion around rs10499197 on one of the alleles and 62-nucleotide deletion around rs10499197 on the other (Figure 2a). Clone#10-9 had a 11-nucleotide deletion around rs10499197 on one allele and a 9-nucleotide deletion downstream of the target site on the other allele (Figure 2a). Clone#1-6 (from gRNA#1) had a homozygous 5-nucleotide deletion upstream of rs10499197 (Figure 2a). Western blotting for A20 protein in whole-cell lysates from the CRISPR clones revealed that the homozygous 5-nucleotide deletion upstream of the rs10499197 significantly reduced TNFAIP3 expression (Figure 2b). Our CRISPR technology provided an orthologus approach to support the ENCODE data by demonstrating that the ~ 55 kb region upstream of the TNFAIP3 promoter functions as a likely enhancer necessary to sustain TNFAIP3 expression. The data also suggests that the rs10499197 may be a causal variant that disrupts TNFAIP3 gene expression by disrupting the enhancer.

Discussion
In this study, we describe the functional characterization of the rs10499197 variant that is techniques. The end result, as a consequence, is a list of variants that methodically characterized to understand the underlying biological function in relation to complex diseases.
The current work offers a functional explanation for the genetic association between SLE and the alleles of the variant rs10499197. Thereby making rs10499197 a potential SLE risk haplotype. The TNFAIP3 locus exhibits complex genetic architecture. A study looking at the genetic associations of SLE and TNFAIP3 identified three key risk haplotypes around the TNFAIP3 gene body (Plenge R.M. et al., 2007): 185kb haplotype upstream of TNFAIP3, 109kb haplotype that spans the TNFAIP3 coding region and a 249kb haplotype downstream of TNFAIP3. Our work focuses on a non-coding DNA element 55kb upstream of the TNFAIP3 promoter. This upstream DNA element harbors SLE variants believed to affect TNFAIP3 and in turn SLE pathophysiology.

Conclusions
Our future work would include, Chromatin conformation capture assays (3C) that would determine how the upstream DNA element physically interacts with the TNFAIP3 promoter.
An understanding of which will help us establish a novel mechanism for TNFAIP3 transcriptional regulation.