elifeeLifeeLife2050-084XeLife Sciences Publications, Ltd7283710.7554/eLife.72837Research ArticleComputational and Systems BiologyGenetics and GenomicsSTAT3-mediated allelic imbalance of novel genetic variant Rs1047643 and B-cell-specific super-enhancer in association with systemic lupus erythematosusZhangYanfenghttps://orcid.org/0000-0002-3859-3839yanfengzhang1984@outlook.com1DayKenneth2AbsherDevin Mdabsher@hudsonalpha.org1https://ror.org/04nz0wq19HudsonAlpha Institute for BiotechnologyHuntsvilleUnited Stateshttps://ror.org/05av6m066Zymo Research CorpIrvineUnited StatesGuoXingyiReviewing Editorhttps://ror.org/02vm5rt34Division of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center, and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of MedicineUnited StatesDiamondBettySenior Editorhttps://ror.org/05dnene97The Feinstein Institute for Medical ResearchUnited States

Department of Population and Data Sciences, University of Texas Southwestern Medical Center, Dallas, United States

21022022202211e728370608202118022022This manuscript was published as a preprint at .20092021© 2022, Zhang et al2022Zhang et alhttp://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Mapping of allelic imbalance (AI) at heterozygous loci has the potential to establish links between genetic risk for disease and biological function. Leveraging multi-omics data for AI analysis and functional annotation, we discovered a novel functional risk variant rs1047643 at 8p23 in association with systemic lupus erythematosus (SLE). This variant displays dynamic AI of chromatin accessibility and allelic expression on FDFT1 gene in B cells with SLE. We further found a B-cell restricted super-enhancer (SE) that physically contacts with this SNP-residing locus, an interaction that also appears specifically in B cells. Quantitative analysis of chromatin accessibility and DNA methylation profiles further demonstrated that the SE exhibits aberrant activity in B cell development with SLE. Functional studies identified that STAT3, a master factor associated with autoimmune diseases, directly regulates both the AI of risk variant and the activity of SE in cultured B cells. Our study reveals that STAT3-mediated SE activity and cis-regulatory effects of SNP rs1047643 at 8p23 locus are associated with B cell deregulation in SLE.

allelic imbalancesuper-enhancerSTAT3systemic lupus erythematosusb-lymphocytechromatin accessibilityResearch organismHumanHudsonAlpha Institute for biotechnology fundsZhangYanfengAbsherDevin MThe funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Author impact statementGenetic analyses reveal biological insight of a novel risk locus into systemic lupus erythematosus susceptibility.
Introduction

Super-enhancers (SEs) are recently discovered large domains of clustered enhancers (Parker et al., 2013; Whyte et al., 2013). The extraordinary feature of SEs is the extremely high and broad enrichment of enhancer-related transcription factors (TFs), H3K4me1 and H3K27ac modifications, resulting in high capability to enhance gene expression programs (Whyte et al., 2013). A large quantity of SEs show cell/tissue specificity (Vahedi et al., 2015), thereby they have become principal determinants of cell identity (Hnisz et al., 2013). Nonetheless, disease-associated SEs, in particular those exhibiting aberrant activity in autoimmune diseases, are less characterized.

Signal transducer and activator of transcription 3 (STAT3), as one of seven STAT family members, is activated by phosphorylation at tyrosine 705 (Y705) and/or at serine 727 (S727) (Decker and Kovarik, 2000). After import to the nucleus, the phospho-STAT3 (pSTAT3) modulates gene transcription by binding its target sequence (Levy and Darnell, 2002). STAT3 has gained broad attention because it plays a key role in a variety of pathophysiological immune responses related to lymphocyte development and differentiation, and in other cellular processes of normal and tumor cells (Yu et al., 2009).

Systemic lupus erythematosus (SLE) is an autoimmune disease that is known to be associated with an array of abnormal immune cell signaling. B-cell hyperactivity in auto-antigen recognition and interaction with T-cells, which ultimately results in multi-organ damage, is central to the pathogenesis of SLE (Rahman and Isenberg, 2008). Genetic factors conferring a predisposition to the development of SLE have been widely characterized. Over 100 loci have been implicated in SLE by genome-wide association studies (GWAS) (Catalina et al., 2020; Yin et al., 2021). Among them, several genes and/or loci are potent as putative drivers of the disease. For example, genetic risk variants at the promoter of BLK at 8p23 locus alter BLK transcription activity and thus contribute to autoreactive B-cell responses (Guthridge et al., 2014). Nonetheless, the GWAS-identified genetic variants together explained approximately 30% of the heritability of SLE (Sun et al., 2016; Morris et al., 2016), suggesting a requirement of further efforts to explain the missing heritability of SLE. Meanwhile, there is growing evidence that genetic risk factors behave in a context-dependent or cell-specific manner (Guthridge et al., 2014; Gallagher and Chen-Plotkin, 2018). Thus, for SLE and other autoimmune diseases, there is a need to identify the regulatory programs in which these genetic factors impact the immune cell developmental processes.

One approach for tying genetic risk to function in the post-GWAS era (Gallagher and Chen-Plotkin, 2018), is a measurement of allelic imbalance (AI) on two alleles at a given heterozygous locus, typically at single-nucleotide polymorphism (SNP). The genes and/or loci with SNPs exhibiting AI could provide a strong foundation for implicating the genetic or epigenetic mechanisms linked to complex traits or diseases (Pastinen and Hudson, 2004; Yan et al., 2002). As a readout of AI, analyses of allele-specific chromatin accessibility and allele-specific RNA expression have accumulated a wealth of interesting findings, including functional cis-regulation (Li et al., 2013; Zhang et al., 2020b), genomic imprinting (Pollard et al., 2008), X-chromosome inactivation or escape (Zhang et al., 2020a). Therefore, tracking AI difference in a comparison between diseases and controls may enable to uncover novel functional variants associated with complex diseases. In this study, we describe one such strategy through integrative multi-omics analysis to discover known or novel functional variants associated with SLE, and report on the identification of a novel risk variant rs1047643 and B-cell-specific SE in B cells with SLE. We further demonstrate that the resultant allelic imbalanced variant and SE activity are directly controlled by STAT3, a master TF that plays a critical role in B cell development and highly associates with autoimmune diseases.

ResultsMulti-omics data summary

Functional genomics sequencing data sets comprising 279 samples from eleven studies were collected (Supplementary file 1). Of eleven studies, seven are SLE case-control studies with data across three immune cell types including B cells, T cells, and Neutrophils (Supplementary file 2). Also included in the present study were SNP microarray data from a SLE GWAS study (n = 2279).

Identification of SLE-associated variant showing AI at both chromatin and RNA levels

We next designed a two-stage study (Figure 1) to identify putative SLE-associated functional variants. In stage I, also named as the discovery stage, two chromatin accessibility (ATAC-seq) data sets (Accession ID: GSE118253 and GSE71338, Supplementary file 1) comprised 49 samples were analyzed. We focused on those variants displaying difference in AI of chromatin accessibility at heterozygous SNP sites in a comparison between SLE and controls (see Materials and methods in detail). In total, 18,456 (the GSE118253 dataset) and 4319 (the GSE71338 dataset) SNPs were tested. We collected the resulting SNPs with nominal (unadjusted) p < 0.05. From the reciprocal validation between two data sets, three SNPs (rs1047643, rs246367, and rs72642993) were identified to show the significant AI (combined p < 0.01) in B cells from patients with SLE, relative to controls (Figure 2A and Figure 2—figure supplement 1). Based on annotations from the GWAS central (Beck et al., 2020) and HaploReg (Ward and Kellis, 2016) databases, we then focused on the rs1047643 because it is the most promising variant. Meanwhile, in B cells at different stages, the allelic preference of chromatin accessibility for SNP rs1047643 is alterable. For example, the T allele exhibits more preferential chromatin accessibility in activated B cells from patients, relative to the C allele. However, the direction is reversed in SLE naive B cells.

Schematic of the study design.

On the basis of the functional genomic data feature, a two-stage study was designed. Summary of data sets is available in Supplementary files 1-2.

Change of allelic chromatin accessibility and expression in B cell subtypes from SLE patients and controls.

(A) Forest plot showing AI of allelic chromatin state of SNP rs1047643 in both resting naive (rN) and activated (Non-rN) B cells in patients of SLE compared with healthy controls. The p-value per study and combined p-value (summary) are calculated based on the linear regression model and Fisher’s method, respectively. The plot in the right panel displays the 95% of confidence interval of beta-value. (B–C) Boxplots showing allelic expression of SNP rs1047643 in both rN and activated B cells in patients with SLE as compared with healthy individuals. All raw data are available in Figure 2—source data 1.

Source files for presenting results in <xref ref-type="fig" rid="fig2">Figure 2</xref>.

This zip archive contains all source data used for the quantitative analyses shown in Figure 2.

Change of allelic chromatin accessibility in B cell subtypes from SLE patients and controls.

Forest plots showing AI of allelic chromatin state of SNP rs246367 (A) and rs72642993 (B) in both rN and activated (Non-rN) B cells in patients of SLE compared with healthy controls. The plots in the right panel display the 95% of confidence interval of beta-value.

Expression pattern of FDFT1 and BLK across B cell subtypes in patients with SLE and healthy controls.

The data showing expression profiles for FDFT1 (A) and BLK (B) in B cell subtypes were from a case-control study (Accession ID: GSE118254).

Expression pattern of FDFT1 and BLK across B cell subtypes in patients with SLE and healthy controls.

The data showing expression profiles for FDFT1 (A) and BLK (B) in B cell subtypes were from a case-control study (Accession ID: GSE92387).

Because the rs1047643 is located in the first exon of FDFT1 gene (Figure 3E), it enables us to test the functionality of this variant at the transcriptional level. Analyzing RNA-seq data (Accession ID: GSE118254), we determined the AI of RNA transcripts for the rs1047643 by comparing the AR values (see Materials and methods in detail) between SLE and controls. In line with results shown above, we observed the dynamic AI pattern on the transcriptional level for the rs1047643 (Figure 2B). Meanwhile, this dynamic allelic expression pattern is specific during B cell development with SLE (Figure 2C).

Association analysis and functional prediction of SNP rs1047643.

(A) Association results for the SNP rs1047643 with SLE risk in single marker analyses. MAF, minor allele frequency; OR, odds ratio; CI, confidence interval. Adjusted p-trend: after adjustment for 12 GWAS index SNPs (shown in E) in a logistic regression model. (B) Haplotype analyses of the two SNPs (SNP1: GWAS indexed SNP rs17807624; SNP2: rs1047643) in relation to SLE risk. Baseline (the reference haplotype) represents the alleles associated with a reduced risk in two SNPs. (C) Barplot showing the genomic length of chromHMM-annotated enhancer state on the super-enhancer region (blue highlighted in 3 C) in 43 epigenomes. (D) Plot shows the eQTL result of SNP rs1047643 in whole blood or B cells from three databases (shown in y-axis). (E) Genomic annotations of the SNP rs1047643. The three tracks show locations of 13 GWAS index SNP, gene annotation and 15-state chromatin segments in CD19+ B cells at 8p23 locus, respectively. Vertical blue and purple lines, represents the location of super-enhancer and SNP rs1047643, respectively. (F) Long-range interaction between a super-enhancer and SNP rs1047643. The two tracks show chromatin interactions from two independent studies using whole-genome Hi-C and capture Hi-C technologies, respectively. Orange curves show the interactions between the super-enhancer and the SNP rs1047643. (G) Heatmaps showing the 3D DNA interactions at 8p23.1 locus in eight cell lines. The rectangle represents interactions between the super-enhancer and the SNP rs1047643. All raw data are available in Figure 3—source data 1.

Source files for presenting results in <xref ref-type="fig" rid="fig3">Figure 3</xref>.

This zip archive contains all source data used for the quantitative analyses shown in Figure 3.

Chromatin interactions with <italic>FDFT1</italic> promoter region (marked in green arrow) on 8p23 locus from CHi-C data with duplicates in two types of normal T cells.

Orange arrow represents the location of super-enhancer identified in this study.

Heatmaps of Long-range chromatin interactions from Hi-C data in 8p23 locus at 10 kb (or 20 kb) resolution in a panel of human tissues (n = 9) from the 3D Genome Browser.

The circles shown on heatmaps are the interaction density between SNP rs1047643 and SE region.

Association with SLE risk in American Hispanic and European populations

Because SNP rs1047643 has not been reported to be associated with the susceptibility of SLE and other autoimmune diseases, we next tested the association using a dataset from an SLE GWAS case-control study. Employing the univariate analysis for SNP rs1047643 in samples from Hispanic populations, we identified an association of the rs1047643 with SLE risk (OR per C effect allele = 0.77, 95% CI 0.54–0.99, p = 0.023 after adjusting for covariates, Figure 3A), albeit not reaching the significance after adjustment for 12 GWAS index SNPs (the top track in Figure 3E, where one SNP rs2736336 is excluded due to its multivariate alleles). We also examined an association in European population (n = 19,468) from Bentham et al., 2015 study. A similar result was observed for the rs1047643 (OR per C effect allele = 0.91, 95% CI 0.84–0.98, p = 0.02, Supplementary file 3).

An analysis of linkage disequilibrium (LD) with each of 12 GWAS tag SNPs showed that there was no strong LD (r2 <0.1) between SNP rs1047643 and GWAS SNPs in European population (Supplementary file 4). This result indicates that SNP rs1047643 is a potentially SLE GWAS independent functional variant. Of the 12 index SNPs, indeed, one index SNP rs17807624 with the statistical significance with p < 1.5 × 10–3 using the univariate analysis, is the top signal to which the SNP rs1047643 is conditional. Thus, we performed haplotype analyses on these two SNPs (index SNP rs17807624 and rs1047643, Figure 3B). Compared with the reference haplotype, which carries the alleles associated with a reduced risk in two SNPs, haplotype 2, which carries the risk-associated alleles, showed a significant association (p = 0.03).

Functional annotation

An analysis of eQTL data derived from three independent cohorts indicated both proximal ( < 200 kb) and distal ( > 200 kb) regulatory potential for the SNP rs1047643 in normal B or blood cells (Figure 3D). Interestingly, besides correlated with three adjacent genes (FDFT1, CTSB and NEIL2), the rs1047643 is also an eQTL linked with an upstream BLK gene in a distance of ~300 kb, a result that is detected in two independent data sets. An analysis of RNA-seq data from two independent studies (Accession ID: GSE118254 and GSE92387, Supplementary file 1) consistently showed that expression patterns for two representative genes (BLK and FDFT1) are gradually increased in a developmental process from naive to memory B cells, in particular, the double negative memory B cell subset in patients with SLE, the pattern that is not observed in controls (Figure 2—figure supplements 2 and 3).

By searching for enhancers and other regulatory elements across 8p23 locus from a dataset of the 127 epigenomes from Roadmap, we identified a SE with a length of 7 kb in the upstream of BLK gene in CD19+ B cells (Epigenome ID: E032, Figure 3E). An analysis of enhancer elements across the 127 epigenomes showed 43 (33.9%) epigenomes had enhancers at this SE region. Comparative analysis of the enhancer length at this SE region on the 43 epigenomes further showed that this SE is specific in CD19+ B cells (Epigenome ID: E032, Figure 3C).

Analyzing Hi-C data sets from two independent studies in GM12878 cells, we observed a DNA looping between the SNP rs1047643 within FDFT1 and the SE region (Figure 3F). More importantly, in GM12878 B-lymphoblastic cells, this SE region has a wealth of long-range interactions with adjacent genes (e.g., BLK) and functional elements. In contrast, in another seven cells (Figure 3G), as well as in normal T cells (Figure 3—figure supplement 1) and nine selected tissues (Figure 3—figure supplement 2), these interactions are either much weaker or completely absent. These results indicate that the physical interaction between SNP rs1047643 and SE region, and many interactions with this SE, are specific to B-lymphocytes.

Specificity in B cells

We then hypothesized that the SE region may show aberrant activity in B cells from SLE patients. To test this hypothesis, we conducted quantitative analysis on the same ATAT-seq data (Accession ID: GSE118253 and GSE71338) used in stage I (see Materials and methods in detail). Comparison of SE activity in a quantitative manner between SLE patients and controls indicated that the SE activity is gradually increased through B cell development in SLE patients (Figure 4A–B), with a hyper-activity being observed in double negative (DN) B cells in patients, relative to controls (Figure 4B–C). It should be noted that such a pattern is not observed in the background sampling (Figure 4—figure supplement 1). Similarly, the rs1047643-containing promoter activity also shows up-regulation toward B cell development in SLE patients (Figure 4D–E). In a comparison of B cell development on activities of SE and FDFT1 promoter regions in two individuals, the chromatin accessibility on both regions in an individual with SLE is increased during B cell development, but remains relatively unchanged in the healthy individual (Figure 4F–H).

Aberration of super-enhancer and <italic>FDFT1</italic> promoter region in B cell subtypes from SLE patients.

(A) Empirical cumulative distribution of TPM values per 50 bp window across the 7 kb SE region in B cell subsets for disease and control groups. (B) Plots showing the TPM values at the third quartile (Q3) across B cell subtypes as a comparison between SLE and controls. (C) Empirical cumulative distribution of TPM values on the SE region (same as shown in A) in a comparison between two groups across four B cell subtypes. (D) Boxplots showing the TPM values per 50 bp window at the FDFT1 promoter region in B cell subtypes for SLE and controls. The black lines and grey areas represent the linear regression results towards the B cell development from T3 to DN stages, and 95% of CI. (E) Plots showing the correlation between super-enhancer and FDFT1 promoter regions based on mean TPM values with respect to B cell subtypes in SLE and controls. (F) Wiggle plot showing the enrichment of open chromatin states at 8p23.1 locus in B cell subtypes for two individuals (a healthy individual at upper panel, and a patient with SLE at lower panel). Purple and green vertical lines represent the locations for super-enhancer and FDFT1 promoter, respectively. Quantitative comparison of chromatin accessibility states in SE (G) and FDFT1 promoter regions (H) with respect to B cell subtypes. All raw data are available in Figure 4—source data 1.

This txt file contains source data used for the quantitative analyses shown in <xref ref-type="fig" rid="fig4">Figure 4</xref>.

Genome-wide background analysis of ATAC-seq data.

Left panel: empirical cumulative distribution of TPM values per 50 bp window across randomly selected regions (n = 2,000) in B cell subsets for disease and control groups. Right panel: plots showing the TPM values at the third quartile (Q3) across B cell subtypes as a comparison between SLE and controls.

Aberration of super-enhancer in resting naive B cell subtypes from SLE patients in relation to healthy controls.

(A) Wiggle plot showing the enrichment of open chromatin states at 8p23.1 locus in resting native B cells from eight individuals. Blue and purple vertical lines represent the locations of SE and FDFT1 promoter, respectively. (B–C) Quantitative comparison of chromatin accessibility states in the SE and FDFT1 promoter regions in naive B cells in a comparison between SLE and controls.

Super-enhancer activity in T and neutrophils from SLE patients and controls.

(A–B) Empirical cumulative distribution of TPM values per 50 bp window and enrichment of ATAC-seq reads (TPM value) across the SE region in neutrophil cell subsets from SLE patients and controls. (C–D) Empirical cumulative distribution of TPM values per 50 bp window and enrichment of ATAC-seq reads (TPM value) across the SE region in two T cell subsets from SLE patients. (E) Wiggle plot showing the enrichment of open chromatin states at 8p23.1 locus in neutrophils and T cells. Blue and purple vertical lines represent the locations of SE and FDFT1 promoter, respectively.

We also quantitatively compared open chromatin states of SE and FDFT1 promoter regions in resting naive B cells (Accession ID: GSE71338). Concordant with the results from active B cell subsets, the open chromatin states on both regions are low in non-active B cells from SLE patients, relative to healthy controls (Figure 4—figure supplement 2).

We further conducted quantitative analyses on ATAC-seq data from another two independent studies in two immune cell types, T cells, and neutrophils (Accession ID: GSE139359 and GSE110017, Supplementary file 1). The results showed that there was no marked enrichment of ATAC-seq reads on both the SE and FDFT1 promoter regions in these two immune cell types for both SLE and controls (Figure 4—figure supplement 3). Collectively, these results suggest a B cell specific, rs1047643-interacting SE whose activity is aberrant in SLE B cell development.

Hypomethylation in SLE B cells

We further analyzed DNA methylation in the SE region using RRBS data in B cell development in a comparison between SLE and controls (Accession ID: GSE118255, Supplementary file 1). Our results show that DNA methylation levels on the SE region are gradually decreased in the developmental process from resting native (rN) to memory B cells in patients with SLE (Figure 5A). In contrast, there is no such obvious change of DNA methylation pattern in the control group. In addition, an analysis of DNA methylation levels on the background sampling doesn’t show such a difference between SLE and controls (Figure 5—figure supplement 1), further suggesting that epigenetic change on the SE region is biologically meaningful in B cell development in SLE.

Hypomethylation in super-enhancer region in B cell subtypes from SLE patients.

(A) Boxplots showing the CpG methylation levels per 50 bp window in 7 kb SE region in B cell subtypes for SLE and control groups. The black and red lines represent the linear regression results towards the B cell development from rN to DN stages for SLE and controls, respectively. (B) Plots showing the correlation between TPM values (y-axis) and DNA methylation levels (x-axis) averaged over each B cell type in SLE and controls. All raw data are available in Figure 5—source data 1.

This txt file contains source data used for the quantitative analyses shown in <xref ref-type="fig" rid="fig5">Figure 5</xref>.

DNA methylation comparison across randomly selected regions in B cell subtypes between patients with SLE and controls.

Boxplots showing the CpG methylation levels per 50 bp window in 2000 randomly selected regions in B cell subtypes for SLE and control groups. The black and red lines represent the linear regression results towards the B cell development from rN to DN stages for SLE and controls, respectively.

A correlation analysis also showed a marked negative correlation between open chromatin states (TPM values, also presented in Figure 4E) and DNA methylation levels at the SE region in the SLE group, relative to the healthy controls (Figure 5B). Together, these results reinforce the aberrant activity of SE in developmental process of B-lymphocytes in patients with SLE.

STAT3 binding on both super-enhancer and rs1047643-residing regions

TF-motif enrichment and binding analysis using the ENCODE TF ChIP-seq dataset (v3) predicted that STAT3 may bind to both the SNP rs1047643-containing promoter and SE regions (data not shown). To validate the finding, we designed two pairs of primers (SE5 and SE3, Figure 6A) to determine the STAT3 binding on SE region and its contribution to the SE activity using STAT3, H3K4me1 and H3K27ac ChIP-qPCR assays in GM11997 cells. Under normal culture conditions, we validated that pSTAT3, H3K4me1 and H3K27ac modifications are remarkably enriched on the SE region (Figure 6A, B and D), but not on the negative control region (Figure 6—figure supplement 1) in B-lymphoblastic cells, relative to IgG mock controls. We then conducted both the inhibition and activation of STAT3 DNA-binding activity using two small molecules. In B-lymphoblastic cells challenged with S3I-201, a STAT3 DNA binding inhibitor, both the DNA binding of STAT3 on SE region and the SE activity are significantly reduced (Figure 6A and B), relative to control. In GM11997 cells treated with ML115, a selective activator of STAT3 (41), both the STAT3 DNA-binding capability on SE region and the SE activity are significantly increased (Figure 6D and E), relative to controls. These results together demonstrate that STAT3 directly modulates the SE activity.

Contribution of STAT3 modulates the enhancer activity and SNP-residing locus in cultured GM11997 cells.

(A) ChIP-qPCR for H3K27ac (left lower panel), H3K4me1 (right lower panel) and pSTAT3 (B) at 8p23 super-enhancer region following 40 μM S3I-201 treatment for 24 hr. Upper panel: UCSC genome browser showing the location of two pairs of qPCR primers (SE5 and SE3) on the SE region (yellow). Two tracks shown below are the enrichment of H3K27ac and H3K4me1 across the SE region. (C) Allelic ChIP-qPCR for pSTAT3 binding on rs1047643 (T vs C alleles) following S3I-201 treatment for 24 hr. (D–E) ChIP-qPCR for H3K27ac (D), and pSTAT3 (E) at 8p23 super-enhancer region following 100 nM ML115 treatment for 6 hr. (F) Allelic ChIP-qPCR for pSTAT3 binding on rs1047643 in cells that have been challenged with ML115 for 6 hr as indicated. Note: the fold changes for the rs1047643-associated BLK and FDFT1 genes in response to small molecules compared to vehicle (0.1% DMSO) as control, which was set as one in all cases, are presented. NS, not significance; *, p < 0.05; **, p < 0.01; ***, p < 0.005.

Quality control of ChIP experiments in GM11997 cells.

Plots showing ChIP-qPCR results for H3K27ac (A and D), H3K4me1 (B and E) and pSTAT3 (C and F) at a negative control (NC) region with the treatment of S3I-201 and ML115, respectively.

Genotyping of SNP rs1047643 in GM11997 genomic DNA using allelic qPCR analysis.

Amplification plots are presented for two alleles.

Validation of STAT3-mediated allelic binding in GM11997 cells.

Plots showing ChIP-qPCR results for pSTAT3 at rs1047643 following 1 μM Cucurbitacin I for 24 hr (A) and 50 ng/ml IL-6 treatment for 1 h (B), respectively. NS, not significance; *, p < 0.05.

We next tested whether the STAT3 might also regulate the rs1047643-residing regions. Using allelic qPCR assay, we confirmed that genomic DNA in the GM11997 cells carries a heterozygous variant for the SNP rs1047643 (Figure 6—figure supplement 2), enabling the AI analysis in this cell model. In GM11997 cells treated with the STAT3 inhibitor S3I-201, STAT3 binding on the risk allele T is significantly reduced, relative to the rs1049643-C allele (Figure 6C). Conversely, we observed an increase of STAT3 DNA binding at the rs1049643-T allele in cells stimulated with the STAT3 activator ML115 (Figure 6F). We further confirmed the findings in cells treated with Cucurbitacin I or IL-6 that acts an inhibitor and stimulator (Figure 6—figure supplement 3) of the Janus Kinase (JAK)/STAT3 signaling pathway (Blaskovich et al., 2003; Hunter and Jones, 2015), respectively.

Consistent with findings in the STAT3 DNA-binding study, we observed a significant change of the rs1049643-T allele at the transcriptional level after treatment with S3I-201 and ML115 at different concentrations and duration times, relative to the C allele (Figure 7A and C and Figure 7—figure supplement 1). These results suggest that the risk rs1049643-T allele is preferentially bound and regulated by STAT3 in B cells.

Expression of two alleles on SNP rs1047643 and its linked genes in cultured cells.

Left panel: allelic RT-qPCR on SNP rs1047643 (T vs C alleles) following S3I-201 (A) and ML115 (C) treatment for 24 hr, respectively. Right panel: RT-qPCR analysis showing the fold changes for the rs1047643-associated BLK and FDFT1 genes in response to different concentrations of S3I-201 (B) and ML115 (D) compared to vehicle (0.1% DMSO) as control, which was set as one in all cases, are presented. *, p < 0.05; **, p < 0.01; ***, p < 0.005.

Expression of two alleles on SNP rs1047643 in B-lymphoblastic cells.

Allelic RT-qPCR in GM11997 cells that have been challenged with S3I-201 for 24 hr (A) and ML115 for 6 hr (B) as indicated, respectively.

Finally, we determined RNA expression of BLK and FDFT1, two representative genes that correlate with the risk rs1047643. The expression levels of both genes are decreased with the treatment of S3I-201 (Figure 7B), and upregulated with the STAT3 activator ML115 (Figure 7D). Together, these results suggest the STAT3-binding risk allele T is associated with increased expression of BLK and FDFT1.

Discussion

In the present study, by integrating a variety of functional genomic data, we performed AI analysis to uncover novel functional promising variants and their regulatory targets in association with SLE. Of note, the diversity of genomic data types from this comprehensive data collection for autoimmune diseases allowed us to develop an approach not used before for accessing the role of variants in SLE disease activity.

One of the most significant findings is the identification of a novel risk variant rs1047643. The association study shows that the rs1049643-T is a risk allele for SLE. Our AI analyses indicate that the rs1049643-T allele resides in more open chromatin state and has higher expression in SLE memory B cell subsets, relative to the C allele. Functional study further provides evidence that the rs1049643-T allele is preferentially bound by STAT3. The SNP rs1047643 is also an eQTL linked with both proximal and distal genes, including BLK, the gene that plays a critical role in B lymphocyte development (Saijo et al., 2003). These results demonstrate that this novel SLE-associated risk rs1047643 whose functionality is mediated by STAT3, may play a role in allele-specific control of adjacent genes at 8p23 locus in B cells. Despite no report for association with other autoimmune diseases, this SNP has been associated with multiple myeloma (Van Ness et al., 2008) and follicular lymphoma (Skibola et al., 2008), two malignant diseases whose pathogenesis is partially associated with the dysfunction of B cells. Specifically, hyperactive STAT3 has been reported to be associated with poor survival in both diseases (Huang et al., 2013; Jung et al., 2017). Therefore, our findings may provide a clue for genetic and mechanical studies on those B cell associated diseases.

Another intriguing finding in this study is the identification of an aberrant activity of a SE in lupus B cell subsets, particularly the hyperactivity in memory B cells. In contrast, there is no enhancer activity in other immune cells (T cells and neutrophils analyzed in this study) in patients with SLE. We also demonstrate that the aberrant activity of the SE can be mediated by STAT3. Some studies have consistently reported a critical role of STAT3 in the B cell maturation, differentiation, as well as the autoimmunity (Avery et al., 2010; Ding et al., 2016). These reports further support the significance of STAT3-mediated SE aberration in B cells with SLE.

Several studies have highlighted the 8p23 locus as a major SLE susceptibility region (Demirci et al., 2017). Our study further expands the significance at this locus. For example, our study and others together suggest that there are a few cis-eQTLs linked with transcriptional levels of BLK (Guthridge et al., 2014; Demirci et al., 2017). Thus, we speculate that the 8p23 locus may play functional roles in B cell development in both genetic and epigenetic fashions. Besides the SNP rs1047643 discovered in the present study, there are 13 SLE-associated GWAS leading SNPs reported in this locus. Of 13 SNPs, six SNPs (Figure 3E) directly sit in the SE region, suggesting these risk variants may play roles in a genetic interaction way, in spite of the unavailability of AI analysis, due to either low coverage (read depth <8) or homozygosity in most or all samples for the 13 SNPs. Epigenetically, the SLE-associated SE has physical interactions with adjacent genes, including BLK and FDFT1, and the risk rs1047643-residing region. This indicates a potentially complex role of the variant rs1047643 for broad regulation by physically contacting the SE. Thus, our data provide new insights into the molecular mechanisms by merging genetic susceptibility with epigenetic impacts on gene expression for autoimmune diseases.

The FDFT1 is a gene encoding for squalene synthase, the enzyme that catalyzes the early step in the cholesterol biosynthetic pathway (Tozawa et al., 1999). Previous studies have shown dyslipidemia, with elevations in total cholesterol, low-density lipoprotein, triglyceride levels in patients with lupus (Tisseverasinghe et al., 2006), especially in the active disease. Our multi-omics data indicate that the SNP rs1047643-linked FDFT1 may be aberrantly activated in B cell development in SLE patients, thereby providing an insight into the genetic implication of lipid metabolism for autoimmune diseases.

The limitations of this study include, due to the presence of six SLE GWAS tagging SNPs in SE region, we are unclear how they genetically influence the SE activity during B cell development. Second, it remains unclear how the AI pattern occurs in naive B cells with lupus. The C allele shows more open chromatin state in SLE naive B cells, this can’t be explained by STAT3 allelic DNA binding at the T allele. This implies that some other factors may also contribute to this dynamic AI pattern. Third, no functional studies on genetic manipulation at the rs1047643 prevent us draw the further conclusion about whether and how the rs1047643 impact the STAT3 binding in the present study.

In addition, it should be noted is the implementation of linear regression model in the initial step to identify the allelic difference signals between SLE and controls. Due to small sample size, there is no statistical power to analyze with more optimal statistical models, such as the logistic regression model. Meanwhile, the unavailability of other variables in regression analysis further restricts this study to remove the potential confounding factors. Together, our analysis may miss some potential AI signals and disable to evaluate the causation.

In conclusion, we identified a novel functional variant and B-cell-specific SE in association with the SLE pathogenesis, both mediated by STAT3, and influencing their gene targets. This insight into the mechanism by which manipulation of STAT3 affects the SE activity and its associated gene expression in B cells may have implications for future drug development in autoimmunity.

Materials and methods
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Chemical compound, drugML115Cayman ChemicalCayman Chemical: 15,178Madoux et al., 2010
Chemical compound, drugS3I-201Sigma-AldrichSigma-Aldrich: SML0330
Chemical compound, drugCucurbitacin ISigma-AldrichSigma-Aldrich: C4493
Chemical compound, drugRecombinant human IL-6Cell Guidance SystemsCell Guidance Systems: GFH10AF
AntibodyPhospho-STAT3 (Ser727)Thermo Fisher ScientificThermo Fisher Scientific Cat# PA5-17876; RRID:AB_10980044
AntibodyAnti-Histone H3 (acetyl K27)AbcamAbcam Cat# ab4729; RRID:AB_2118291
AntibodyH3K4me1 Recombinant Polyclonal AntibodyThermo Fisher ScientificThermo Fisher Scientific Cat# 710795; RRID:AB_2532764
Antibodynormal mouse IgGSanta Cruz BiotechnologySanta Cruz Biotechnology Cat# sc-2025; RRID:AB_737182
Antibodynormal rabbit IgGSanta Cruz BiotechnologySanta Cruz Biotechnology Cat# sc-2027; RRID:AB_737197
Cell line (H. sapiens)GM11997CoriellCoriell Cat# GM11997; RRID:CVCL_5C55
Sequence-based reagentChIP-qPCR primersThis paperSee Supplementary file 5
Sequence-based reagentRT-qPCR primersThis paperSee Supplementary file 5
Sequence-based reagentAllelic qPCR primersThis paperSee Supplementary file 5
Software, algorithmRR Foundationhttps://www.r-project.orgVersion 4.0.2
Software, algorithmHisat2Kim et al., 2019Version 2
Software, algorithmAllelic imbalance analysis and plotsThis paper (Zhang, 2021)The R code used for the AI analysis can be accessed via github at https://github.com/youngorchuang/Allelic-imbalance-analysis, (copy archived at swh:1:rev:f0db42af8fed130ebbfe0b46abf992300dadddd6)
Software, algorithmHiCUPWingett et al., 2015
Commercial assay or kitMycoplasma detection kitSigma-AldrichSigma-Aldrich:MP0025
Commercial assay or kitSuperScript III reverse transcriptaseThermo Fisher ScientificThermo Fisher Scientific:18080044
Commercial assay or kitLuna Universal qPCR Master MixNew England BiolabsNew England Biolabs:M3003X
Data collection

We collected a variety of functional genomics data, including ATAC-seq, RNA-seq, reduced-representation bisulfite sequencing (RRBS), Hi-C data (Supplementary file 1), from the Gene Expression Omnibus (GEO) and ArrayExpress databases. Meanwhile, we downloaded genotype and Epidemiological data from a SLE case-control study (accession: phs001025.v1) in Hispanic population (1393 cases and 886 controls) from the dbGaP database with approval (accessed 29 Sep 2020).

Analysis of RNA-Seq and ATAC-Seq data

RNA-seq data were analyzed as described previously with few modifications (Zhang et al., 2016). In brief, raw sequencing data were mapped to the human reference genome (hg19) using Hisat2 program (Kim et al., 2019) with the default setting. Aligned data were processed and converted into BAM files using SAMtools program (Li et al., 2009). To quantify gene expression levels, read counts were calculated using the featureCounts (version 2.0.2) program, then implemented in the edgeR package to calculate the count per million (CPM) values.

We used a similar method described previously with several modifications (Zhang et al., 2015) to process the ATAC-seq data. In brief, raw sequencing data were mapped to the human reference genome (hg19) using Bowtie2 program (Langmead and Salzberg, 2012) with the default setting. Tag per million (TPM) metric, a method commonly used for read counting normalization, was used to quantitatively present the enrichment of open chromatin states across regions of interest.

Identification of allelic imbalance sites

We used a similar approach described in our previous study to call variants and allelic analysis for both RNA-seq and ATAC-seq data (Zhang et al., 2020a). Briefly, the deduplicated reads in BAM format were realigned and recalibrated, and genetic variants were called in a multiple-sample joint manner implemented in the GATK toolkit (version 3.3). We next filtered out variants as follows: (Parker et al., 2013) mapping quality score <20, (Whyte et al., 2013) ≥ 3 SNPs detected within 10 bp distance, (Vahedi et al., 2015) variant confidence/quality by depth <2, (Hnisz et al., 2013) strand bias score >50, (Decker and Kovarik, 2000) genotype score <15 and (Levy and Darnell, 2002) read depth <8. Then, we extracted SNPs annotated from dbSNP (Build 150) that were called as heterozygotes for each sample. For a reasonable comparison, those heterozygous SNPs identified at least triple in both case and control samples were retained for further analysis.

For a given heterozygous SNP, we calculated allelic ratio (AR) based on read coverage onto two alleles. For RNA-seq data, the resulting AR values were used to compare the AI difference of RNA transcripts between cases and controls. For ATAC-seq data, by testing for associations between AR of each heterozygous SNP (as response variable) and SLE disease status as categorical variable (case/control comparison, control and case are coded as 0 and 1, respectively) implemented in the regression analysis (see below), we analyzed the AI difference of chromatin accessibility between cases and controls. Then, the p-value and beta coefficient were calculated to estimate the significance of the association, and the differences between cases and controls, respectively.

Genetic association analysis

For genotype data from a SLE case-control study in Hispanic population, all typed SNPs in chromosome eight were extracted for imputation using TOPMed Imputation Server (Das et al., 2016). To test SNP rs1047643 in association with SLE, we used a method described previously for univariate and haplotype analyses (Shi et al., 2016). In brief, the per-allele odds ratio (OR) and 95% confidence interval (CI) for the rs1047643 was estimated for SLE risk using a log-additive logistic model with covariates of five countries of the Hispanic population, sex and five principal components (PCs). We used the haplo.stats package in R for haplotype analyses with five countries of the Hispanic population, sex and five PCs as covariates. For SLE GWAS data in European population from Bentham et al., 2015 study, we downloaded summary statistical data (Accession ID: GCST003156) from GWAS catalog (Buniello et al., 2019) and extracted statistical results for the SNP rs1047643.

A dataset of GWAS leading SNPs was downloaded from the GWAS Catalog (Buniello et al., 2019). Then we extracted SLE-associated SNPs at 8p23. For each indexed SLE-associated SNP at 8p23, we tested the linkage disequilibrium (LD) score (r2) with query SNP rs1047643 from the data set of the Phase 3 of the 1,000 Genomes Project in European population using LDlink web tool (Machiela and Chanock, 2015).

Super-enhancer annotation

We downloaded whole-genome chromatin state segmentation data (core 15-state model) for 127 cell types from the Roadmap project. As Parker et al., 2013 defined, we consider contiguous genomic region marked by states 6–7 (enhancer states, annotated by chromHMM) with ≥3 kb as SE in a cell type. Then, we extracted and annotated super-enhancers on 8p23 locus.

Analysis of eQTL data

We collected eQTL data sets from three large-scale studies, the Genotype-Tissue Expression (GTEx, v8) (Laboratory, 2017), the Haploreg v4.1 dataset (Ward and Kellis, 2016) and the study by Westra, 2013. By searching for the SNP rsID or the coordinate, we extracted the linked genes with query SNPs and plotted the results based on the significance and studies.

Hi-C data analysis

For in situ Hi-C dataset (Accession ID: GSE63525), we downloaded the Hi-C binary file from Rao et al. study (Rao et al., 2014) and extracted the observed long-range interactions normalized with Knight-Ruiz matrix balancing (KR) method at 10 kb resolution across the 8p23.1 region (the coordinate: chr8:11260000–11740000, hg19).

For other genome-wide Hi-C (Accession ID: GSE113405) and capture Hi-C (CHi-C) datasets (Accession ID: GSE81503 and E-MTAB-6621), we used the Hi-C Pipeline (HiCUP) (Wingett et al., 2015) to truncate and align reads to the human reference genome. The deduplicated data were then processed using the Homer pipeline (Heinz et al., 2010) to call the significant chromatin interaction at 10 kb resolution with the support of ≥5 reads and p ≤ 0.001. The resulting interactions were visualized using UCSC Genome Browser or Sushi package in R environment.

DNA methylation analysis

We downloaded the processed RRBS dataset of DNA methylation profiles on each CpG site from Scharer et al., 2019 report, then extracted and compared CpG methylation levels on a region of interest between SLE and healthy controls.

Cell culture

GM11997 B lymphoblastic (purchased from Coriell Institute) cells were cultured in RPMI-1640 medium, supplemented with 10% FBS (Thermo Fisher Scientific), 2 mM L-glutamine and 1% penicillin-streptomycin at 37 °C with 5% CO2. These cells are mycoplasma-negative when tested with PCR-based mycoplasma detection kit. For perturbation of STAT3, B cells were plated in 12-well plates or 10 cm dishes one day prior to the experiment. Cells were then treated with S3I-201, ML115, Cucurbitacin I or IL-6. Cells were harvested, washed with PBS and analyzed for proper assays.

Reverse transcription qPCR

Total RNA was isolated from cells using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. Oneμg of total RNA was reverse transcribed using SuperScript III reverse transcriptase and random hexamer. One-tenth of the RT reaction was used as a template for real-time PCR using Luna Universal qPCR Master Mix (New England Biolabs) on a QuantStudi six system. Relative expression was calculated with 2−ΔΔCt using the average value of housekeeping gene GAPDH.

Chromatin immunoprecipitation

ChIP was performed as described previously (Whyte et al., 2013). Approximately 10 × 106 suspension cells were harvested and in 10 ml PBS with 1% formaldehyde for 10 min at room temperature, followed by adding 0.125 M glycine for 5 min. Cells were washed and pelleted by centrifugation and lysed with buffer (50 mM Tris-HCl, pH 7.5, 1% IGEPAL CA-630, 1 mM EDTA, 0.1% SDS, plus 1 mM PMSF) in the presence of protease inhibitors and incubated on ice for 30 min. Cell lysate was sonicated to shear DNA to a length of 200–600 bp. The lysates were centrifuged, and supernatant transferred to new tubes. For immunoprecipitation, approximately 2 × 106 cells and 2–3 μg of antibodies or isotype matched IgG as control were used per ChIP and incubated with supernatant at 4 °C on a rotating wheel overnight. Chromatin-antibody complexes were sequentially washed with low-salt buffer, high-salt buffer, LiCl buffer, and TE buffer. Cross-links were reversed by addition of 100 μl of 1% SDS plus 100 mM NaHCO3 and by heating at 65 °C overnight. Following phenol/chloroform/isoamyl alcohol extraction, immunoprecipitated DNA was precipitated with isopropyl alcohol and resuspended in nuclease-free water. For the identification of the specific regions of interest, ~ 10 ng of purified DNA was quantified to determine the percentage of each analyzed region, as well as a negative control region from Fullwood et al., 2009, against input DNA. The PCR primers are shown in Supplementary file 5.

Additional statistical analysis

Data were presented as mean ± standard deviation (SD) of three replicates unless stated otherwise. Correlation analysis was performed using Pearson’s correlation coefficient. Statistical significance was considered at two-sided P-values less than 0.05.

Additional informationCompeting interests

No competing interests declared

No competing interests declared

Author contributions

Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Writing – original draft, Writing – review and editing

Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

Data curation, Funding acquisition, Investigation, Resources, Supervision, Writing – review and editing

Additional filesSummary of data sets used in the study.

Functional genomics data sets, including ATAC-seq, RNA-seq and RRBS-seq data sets from seven SLE case-control studies (Supplementary file 2), and Hi-C data sets in multiple cell lines, and a SNP microarray data set from a lupus GWAS study.

List of data sets from seven SLE case-control studies.Association results for the SNP rs1047643 with SLE risk in European population.LD score (r<sup>2</sup>) between SNP rs1047643 and 12 GWAS tag SNPs in European population.List of primers used in this study.
Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 2-5.

The following previously published datasets were used:

ScharerCDBossJM2019Accessible chromatin profiles of B cell subsets from healthy and SLE subjectsNCBI Gene Expression OmnibusGSE118253

ScharerCDBossJ2016Effects of biobanking on chromatin accessibilityNCBI Gene Expression OmnibusGSE71338

MistryPNakaboSO'NeilLGoelRRJiangKGuptaSDell'OrsoSGutierrez-CruzGSunHWKaplanMJ2019Transcriptomic, epigenetic and functional analyses implicate neutrophil diversity in the pathogenesis of systemic lupus erythematosus [ATAC-seq]NCBI Gene Expression OmnibusGSE139359

CaielliSVeigaDFDomicBMuratEBanchereauRXuZChandraMAthaleSChungCWaltersLBaischJWrightTPunaroMUcarDUenoHZhouJBanchereauJPascualV2018A novel CD4 T cell population expanded in SLE blood provides B cell help through IL10 and succinate [ATAC-seq]NCBI Gene Expression OmnibusGSE110017

ScharerCDBossJM2019Transcriptome profiles of B cell subsets from healthy and SLE subjectsNCBI Gene Expression OmnibusGSE118254

SanzIJenksSMarigortaUM2018Gene expresison studies of lupus and healthy B cell subsets through RNA sequencingNCBI Gene Expression OmnibusGSE92387

ScharerCDBossJM2019DNA methylation profiles profiles of B cell subsets from healthy and SLE subjectsNCBI Gene Expression OmnibusGSE118255

RaoSHuntleyMLieberman AidenE2014A three-dimensional map of the human genome at kilobase resolution reveals prinicples of chromatin loopingNCBI Gene Expression OmnibusGSE63525

WalterJMankeT2018HepG2 Hi-CNCBI Gene Expression OmnibusGSE113405

CairnsJFreire-PritchettPWingettSWVárnaiCDimondAPlagnolVZerbinoDSchoenfelderSJavierreBOsborneCFraserPSpivakovM2016CHiCAGO: Robust Detection of DNA Looping Interactions in Capture Hi-C dataNCBI Gene Expression OmnibusGSE81503

WellsADChesiAManduchiEJohnsonMELeonardMERombergNDGrantSFALuS2020Promoter capture-C of primary human T Follicular Helper (TFH) cells and naive CD4-positive helper T cells from tonsils of healthy volunteersArrayExpressE-MTAB-6621

Disease-Specific (Systemic lupus erythematosus, NPU, MDS, RD)2017GWAS in an Amerindian Ancestry Population Reveals Novel Systemic Lupus Erythematosus Risk Loci and the Role of European AdmixtureNCBI dbGAPphs001025.v1

Acknowledgements

We thank Dr. Le Su for insightful suggestions, technical discussions and critical reading of the manuscript. This study was supported by the HudsonAlpha institutional funds. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Funding This study was supported by the HudsonAlpha Institute Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

ReferencesAveryDTDeenickEKMaCSSuryaniSSimpsonNChewGYChanTDPalendiraUBustamanteJBoisson-DupuisSChooSBleaselKEPeakeJKingCFrenchMAEngelhardDAl-HajjarSAl-MuhsenSMagdorfKRoeslerJArkwrightPDHissariaPRimintonDSWongMBrinkRFulcherDACasanovaJ-LCookMCTangyeSG2010B cell-intrinsic signaling through IL-21 receptor and STAT3 is required for establishing long-lived antibody responses in humansThe Journal of Experimental Medicine20715517110.1084/jem.2009170620048285BeckTShorterTBrookesAJ2020GWAS Central: a comprehensive resource for the discovery and comparison of genotype and phenotype data from genome-wide association studiesNucleic Acids Research48D933D94010.1093/nar/gkz89531612961BenthamJMorrisDLGrahamDSCPinderCLTomblesonPBehrensTWMartínJFairfaxBPKnightJCChenLReplogleJSyvänenA-CRönnblomLGrahamRRWitherJERiouxJDAlarcón-RiquelmeMEVyseTJ2015Genetic association analyses implicate aberrant regulation of innate and adaptive immunity genes in the pathogenesis of systemic lupus erythematosusNature Genetics471457146410.1038/ng.343426502338BlaskovichMASunJCantorATurksonJJoveRSebtiSM2003Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer cells in miceCancer Research631270127912649187BunielloAMacArthurJALCerezoMHarrisLWHayhurstJMalangoneCMcMahonAMoralesJMountjoyESollisESuvegesDVrousgouOWhetzelPLAmodeRGuillenJARiatHSTrevanionSJHallPJunkinsHFlicekPBurdettTHindorffLACunninghamFParkinsonH2019The NHGRI-EBI GWAS Catalog of published genome-wide association studies, targeted arrays and summary statistics 2019Nucleic Acids Research47D1005D101210.1093/nar/gky112030445434CatalinaMDOwenKALabonteACGrammerACLipskyPE2020The pathogenesis of systemic lupus erythematosus: Harnessing big data to understand the molecular basis of lupusJournal of Autoimmunity11010235910.1016/j.jaut.2019.10235931806421DasSForerLSchönherrSSidoreCLockeAEKwongAVriezeSIChewEYLevySMcGueMSchlessingerDStambolianDLohP-RIaconoWGSwaroopAScottLJCuccaFKronenbergFBoehnkeMAbecasisGRFuchsbergerC2016Next-generation genotype imputation service and methodsNature Genetics481284128710.1038/ng.365627571263DeckerTKovarikP2000Serine phosphorylation of STATsOncogene192628263710.1038/sj.onc.120348110851062DemirciFYWangXMorrisDLFeingoldEBernatskySPineauCClarkeARamsey-GoldmanRManziSVyseTJKambohMI2017Multiple signals at the extended 8p23 locus are associated with susceptibility to systemic lupus erythematosusJournal of Medical Genetics5438138910.1136/jmedgenet-2016-10424728289186DingCChenXDascaniPHuXBolliRZhangH-GMcleishKRYanJ2016STAT3 Signaling in B Cells Is Critical for Germinal Center Maintenance and Contributes to the Pathogenesis of Murine Models of LupusJournal of Immunology (Baltimore, Md1964477448610.4049/jimmunol.150204327183592FullwoodMJLiuMHPanYFLiuJXuHMohamedYBOrlovYLVelkovSHoAMeiPHChewEGYHuangPYHWelborenW-JHanYOoiHSAriyaratnePNVegaVBLuoYTanPYChoyPYWansaKDSAZhaoBLimKSLeowSCYowJSJosephRLiHDesaiKVThomsenJSLeeYKKaruturiRKMHerveTBourqueGStunnenbergHGRuanXCacheux-RataboulVSungW-KLiuETWeiC-LCheungERuanY2009An oestrogen-receptor-alpha-bound human chromatin interactomeNature462586410.1038/nature0849719890323GallagherMDChen-PlotkinAS2018The Post-GWAS Era: From Association to FunctionAmerican Journal of Human Genetics10271773010.1016/j.ajhg.2018.04.00229727686GuthridgeJMLuRSunHSunCWileyGBDominguezNMacwanaSRLessardCJKim-HowardXCobbBLKaufmanKMKellyJALangefeldCDAdlerAJHarleyITWMerrillJTGilkesonGSKamenDLNiewoldTBBrownEEEdbergJCPetriMARamsey-GoldmanRReveilleJDViláLMKimberlyRPFreedmanBIStevensAMBoackleSACriswellLAVyseTJBehrensTWJacobCOAlarcón-RiquelmeMESivilsKLChoiJJooYBBangS-YLeeH-SBaeS-CShenNQianXTsaoBPScofieldRHHarleyJBWebbCFWakelandEKJamesJANathSKGrahamRRGaffneyPM2014Two functional lupus-associated BLK promoter variants control cell-type- and developmental-stage-specific transcriptionAmerican Journal of Human Genetics9458659810.1016/j.ajhg.2014.03.00824702955HeinzSBennerCSpannNBertolinoELinYCLasloPChengJXMurreCSinghHGlassCK2010Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identitiesMolecular Cell3857658910.1016/j.molcel.2010.05.00420513432HniszDAbrahamBJLeeTILauASaint-AndréVSigovaAAHokeHAYoungRA2013Super-enhancers in the control of cell identity and diseaseCell15593494710.1016/j.cell.2013.09.05324119843HuangXMengBIqbalJDingBBPerryAMCaoWSmithLMBiCJiangCGreinerTCWeisenburgerDDRimszaLRosenwaldAOttGDelabieJCampoEBrazielRMGascoyneRDCookJRTubbsRRJaffeESArmitageJOVoseJMStaudtLMMcKeithanTWChanWCYeBHFuK2013Activation of the STAT3 signaling pathway is associated with poor survival in diffuse large B-cell lymphoma treated with R-CHOPJournal of Clinical Oncology314520452810.1200/JCO.2012.45.600424220563HunterCAJonesSA2015IL-6 as a keystone cytokine in health and diseaseNature Immunology1644845710.1038/ni.315325898198JungS-HAhnS-YChoiH-WShinM-GLeeS-SYangD-HAhnJ-SKimY-KKimH-JLeeJ-J2017STAT3 expression is associated with poor survival in non-elderly adult patients with newly diagnosed multiple myelomaBlood Research5229329910.5045/br.2017.52.4.29329333406KimDPaggiJMParkCBennettCSalzbergSL2019Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotypeNature Biotechnology3790791510.1038/s41587-019-0201-431375807LaboratoryDA2017Coordinating Center -Analysis Working G, Statistical Methods groups-Analysis Working Genetic effects on gene expression across human tissuesNature550204213LangmeadBSalzbergSL2012Fast gapped-read alignment with Bowtie 2Nature Methods935735910.1038/nmeth.192322388286LevyDEDarnellJE2002Stats: transcriptional control and biological impactNature Reviews. Molecular Cell Biology365166210.1038/nrm90912209125LiHHandsakerBWysokerAFennellTRuanJHomerNMarthGAbecasisGDurbinR1000 Genome Project Data Processing Subgroup2009The Sequence Alignment/Map format and SAMtoolsBioinformatics (Oxford, England)252078207910.1093/bioinformatics/btp35219505943LiQSeoJ-HStrangerBMcKennaAPe’erILaframboiseTBrownMTyekuchevaSFreedmanML2013Integrative eQTL-based analyses reveal the biology of breast cancer risk lociCell15263364110.1016/j.cell.2012.12.03423374354MachielaMJChanockSJ2015LDlink: a web-based application for exploring population-specific haplotype structure and linking correlated alleles of possible functional variantsBioinformatics (Oxford, England)313555355710.1093/bioinformatics/btv40226139635MadouxFKoenigMNelsonEChowdhurySCameronMMercerB2010Modulators of STAT Transcription Factors for the Targeted Therapy of Cancer (STAT3 Activators)National Center for Biotechnology Information (US)MorrisDLShengYZhangYWangY-FZhuZTomblesonPChenLCunninghame GrahamDSBenthamJRobertsALChenRZuoXWangTWenLYangCLiuLYangLLiFHuangYYinXYangSRönnblomLFürnrohrBGVollRESchettGCostedoat-ChalumeauNGaffneyPMLauYLZhangXYangWCuiYVyseTJ2016Genome-wide association meta-analysis in Chinese and European individuals identifies ten new loci associated with systemic lupus erythematosusNature Genetics4894094610.1038/ng.360327399966ParkerSCJStitzelMLTaylorDLOrozcoJMErdosMRAkiyamaJAvan BuerenKLChinesPSNarisuNBlackBLViselAPennacchioLACollinsFSNISC Comparative Sequencing ProgramNational Institutes of Health Intramural Sequencing Center Comparative Sequencing Program AuthorsNISC Comparative Sequencing Program Authors2013Chromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variantsPNAS110179211792610.1073/pnas.131702311024127591PastinenTHudsonTJ2004Cis-acting regulatory variation in the human genomeScience (New York, N.Y.)30664765010.1126/science.110165915499010PollardKSSerreDWangXTaoHGrundbergEHudsonTJClarkAGFrazerK2008A genome-wide approach to identifying novel-imprinted genesHuman Genetics12262563410.1007/s00439-007-0440-117955261RahmanAIsenbergDA2008Systemic lupus erythematosusThe New England Journal of Medicine35892993910.1056/NEJMra07129718305268RaoSSPHuntleyMHDurandNCStamenovaEKBochkovIDRobinsonJTSanbornALMacholIOmerADLanderESAidenEL2014A 3D map of the human genome at kilobase resolution reveals principles of chromatin loopingCell1591665168010.1016/j.cell.2014.11.02125497547SaijoKSchmedtCSuI-HKarasuyamaHLowellCARethMAdachiTPatkeASantanaATarakhovskyA2003Essential role of Src-family protein tyrosine kinases in NF-kappaB activation during B cell developmentNature Immunology427427910.1038/ni89312563261ScharerCDBlalockELMiTBarwickBGJenksSADeguchiTCashmanKSNearyBEPattersonDGHicksSLKhosroshahiAEun-Hyung LeeFWeiCSanzIBossJM2019Epigenetic programming underpins B cell dysfunction in human SLENature Immunology201071108210.1038/s41590-019-0419-931263277ShiJZhangYZhengWMichailidouKGhoussainiMBollaMKWangQDennisJLushMMilneRLShuX-OBeesleyJKarSAndrulisILAnton-CulverHArndtVBeckmannMWZhaoZGuoXBenitezJBeeghly-FadielABlotWBogdanovaNVBojesenSEBrauchHBrennerHBrintonLBroeksABrüningTBurwinkelBCaiHCanisiusSChang-ClaudeJChoiJ-YCouchFJCoxACrossSSCzeneKDarabiHDevileePDroitADorkTFaschingPAFletcherOFlygerHFostiraFGaborieauVGarcía-ClosasMGilesGGMervi GripGuenelPHaimanCAHamannUHartmanMMiaoHHollestelleAHopperJLHsiungC-NkConFab InvestigatorsItoHJakubowskaAJohnsonNTorresDKabischMKangDKhanSKnightJAKosmaV-MLambrechtsDLiJLindblomALophatananonALubinskiJMannermaaAManoukianSLe MarchandLMargolinSMarmeFMatsuoKMcLeanCMeindlAMuirKNeuhausenSLNevanlinnaHNordSBørresen-DaleA-LOlsonJEOrrNvan den OuwelandAMWPeterlongoPPuttiTCRudolphASangrajrangSSawyerEJSchmidtMKSchmutzlerRKShenC-YHouM-FShrubsoleMJSoutheyMCSwerdlowATeoSHThienpontBTolandAETollenaarRAEMTomlinsonITruongTTsengC-CWenWWinqvistRWuAHYipCHZamoraPMZhengYFlorisGChengC-YHooningMJMartensJWMSeynaeveCKristensenVNHallPPharoahPDPSimardJChenevix-TrenchGDunningAMAntoniouACEastonDFCaiQLongJ2016Fine-scale mapping of 8q24 locus identifies multiple independent risk variants for breast cancerInternational Journal of Cancer1391303131710.1002/ijc.3015027087578SkibolaCFBracciPMHalperinENietersAHubbardAPaynterRASkibolaDRAganaLBeckerNTresslerPForrestMSSankararamanSCondeLHollyEASmithMT2008Polymorphisms in the estrogen receptor 1 and vitamin C and matrix metalloproteinase gene families are associated with susceptibility to lymphomaPLOS ONE3e281610.1371/journal.pone.000281618636124SunCMolinerosJELoogerLLZhouX-JKimKOkadaYMaJQiY-YKim-HowardXMotgharePBhattaraiKAdlerABangS-YLeeH-SKimT-HKangYMSuhC-HChungWTParkY-BChoeJ-YShimSCKochiYSuzukiAKuboMSumidaTYamamotoKLeeS-SKimYJHanB-GDozmorovMKaufmanKMWrenJDHarleyJBShenNChuaKHZhangHBaeS-CNathSK2016High-density genotyping of immune-related loci identifies new SLE risk variants in individuals with Asian ancestryNature Genetics4832333010.1038/ng.349626808113TisseverasingheALimSGreenwoodCUrowitzMGladmanDFortinPR2006Association between serum total cholesterol level and renal outcome in systemic lupus erythematosusArthritis and Rheumatism542211221910.1002/art.2192916802357TozawaRIshibashiSOsugaJYagyuHOkaTChenZOhashiKPerreySShionoiriFYahagiNHaradaKGotodaTYazakiYYamadaN1999Embryonic lethality and defective neural tube closure in mice lacking squalene synthaseThe Journal of Biological Chemistry274308433084810.1074/jbc.274.43.3084310521476VahediGKannoYFurumotoYJiangKParkerSCJErdosMRDavisSRRoychoudhuriRRestifoNPGadinaMTangZRuanYCollinsFSSartorelliVO’SheaJJ2015Super-enhancers delineate disease-associated regulatory nodes in T cellsNature52055856210.1038/nature1415425686607Van NessBRamosCHaznadarMHoeringAHaesslerJCrowleyJJacobusSOkenMRajkumarVGreippPBarlogieBDurieBKatzMAtluriGFangGGuptaRSteinbachMKumarVMushlinRJohnsonDMorganG2008Genomic variation in myeloma: design, content, and initial application of the Bank On A Cure SNP Panel to detect associations with progression-free survivalBMC Medicine62610.1186/1741-7015-6-2618778477WardLDKellisM2016HaploReg v4: systematic mining of putative causal variants, cell types, regulators and target genes for human complex traits and diseaseNucleic Acids Research44D877D88110.1093/nar/gkv134026657631WestraHJ2013Systematic identification of trans eQTLs as putative drivers of known disease associationsNature Genetics451238124310.1038/ng.275624013639WhyteWAOrlandoDAHniszDAbrahamBJLinCYKageyMHRahlPBLeeTIYoungRA2013Master transcription factors and mediator establish super-enhancers at key cell identity genesCell15330731910.1016/j.cell.2013.03.03523582322WingettSEwelsPFurlan-MagarilMNaganoTSchoenfelderSFraserPAndrewsS2015HiCUP: pipeline for mapping and processing Hi-C dataF1000Research4131010.12688/f1000research.7334.126835000YanHYuanWVelculescuVEVogelsteinBKinzlerKW2002Allelic variation in human gene expressionScience (New York, N.Y.)297114310.1126/science.107254512183620YinXKimKSuetsuguHBangS-YWenLKoidoMHaELiuLSakamotoYJoSLengR-XOtomoNLaurynenkaVKwonY-CShengYSuganoNHwangMYLiWMukaiMYoonKCaiMIshigakiKChungWTHuangHTakahashiDLeeS-SWangMKarinoKShimS-CZhengXMiyamuraTKangYMYeDNakamuraJSuhC-HTangYMotomuraGParkY-BDingHKurodaTChoeJ-YLiCNiiroHParkYShenCMiyamotoTAhnG-YFeiWTakeuchiTShinJ-MLiKKawaguchiYLeeY-KWangYAmanoKParkDJYangWTadaYYamajiKShimizuMAtsumiTSuzukiASumidaTOkadaYMatsudaKMatsuoKKochiYJapanese Research Committee on Idiopathic Osteonecrosis of the Femoral HeadKottyanLCWeirauchMTParameswaranSEswarSSalimHChenXYamamotoKHarleyJBOhmuraKKimT-HYangSYamamotoTKimB-JShenNIkegawaSLeeH-SZhangXTeraoCCuiYBaeS-C2021Meta-analysis of 208370 East Asians identifies 113 susceptibility loci for systemic lupus erythematosusAnnals of the Rheumatic Diseases8063264010.1136/annrheumdis-2020-21920933272962YuHPardollDJoveR2009STATs in cancer inflammation and immunity: a leading role for STAT3Nature Reviews. Cancer979880910.1038/nrc273419851315ZhangYDelahantyRGuoXZhengWLongJ2015Integrative genomic analysis reveals functional diversification of APOBEC gene family in breast cancerHuman Genomics93410.1186/s40246-015-0056-926682542ZhangYWagnerEKGuoXMayICaiQZhengWHeCLongJ2016Long intergenic non-coding RNA expression signature in human breast cancerScientific Reports63782110.1038/srep3782127897201ZhangYLiXGibsonAEdbergJKimberlyRPAbsherDM2020aSkewed allelic expression on X chromosome associated with aberrant expression of XIST on systemic lupus erythematosus lymphocytesHuman Molecular Genetics292523253410.1093/hmg/ddaa13132628254ZhangSZhangHZhouYQiaoMZhaoSKozlovaAShiJSandersARWangGLuoKSenguptaSWestSQianSStreitMAvramopoulosDCowanCAChenMPangZPGejmanPVHeXDuanJ2020bAllele-specific open chromatin in human iPSC neurons elucidates functional disease variantsScience (New York, N.Y.)36956156510.1126/science.aay398332732423ZhangY2021Allelic analysisswh:1:rev:f0db42af8fed130ebbfe0b46abf992300dadddd6Software Heritagehttps://archive.softwareheritage.org/swh:1:dir:9dec9e4c202c8a7374019f40ad49270021248752;origin=https://github.com/youngorchuang/Allelic-imbalance-analysis;visit=swh:1:snp:47cdfdd9b150ea9a0f0f34eaf59be0e3ab838694;anchor=swh:1:rev:f0db42af8fed130ebbfe0b46abf992300dadddd6
10.7554/eLife.72837.sa0Editor's evaluationGuoXingyiReviewing Editorhttps://ror.org/02vm5rt34Division of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center, and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of MedicineUnited States

Through extensive computational data analyses and functional experiments, Zhang and colleagues reported a novel functional germline variant at the risk locus 8p23 for systemic lupus erythematosus. They provided evidence that the observed risk association in this locus may be mediated through the variant regulating a nearby susceptibility gene. This study advances the understanding of the genetic susceptibility and biology underlying systemic lupus erythematosus.

10.7554/eLife.72837.sa1Decision letterGuoXingyiReviewing Editorhttps://ror.org/02vm5rt34Division of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center, and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of MedicineUnited States

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "STAT3-mediated allelic imbalance of novel genetic variant rs1047643 and B cell specific super-enhancer in association with systemic lupus erythematosus" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Betty Diamond as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) The authors should explore large-scale GWAS data or provide any other statistical evidence to strengthen their discovery of the risk association signal of rs1047643 (such as summary statistics of GWAS data of European descendants).

2) To improve and clarify the statical analyses as suggested by reviewers.

Reviewer #1 (Recommendations for the authors):

This is a well conducted study aiming to identify novel risk variants for systemic lupus erythematosus risk and to further explore potential mechanisms through a comprehensive analysis of functional genomic analyses and functional assays. The authors have identified a putative novel risk variant, rs1047643, for the disease risk, and further revealed underlying disease mechanisms of a known susceptibility factor, STAT3, mediated Super enhancer activity and cis-regulatory effects of SNP rs1047643. Their discovered mechanisms are novel, providing new insights into the potential mechanism underlying other GWAS-identified variants located in the Super enhancer element to mediate the disease risk. This work could be very important to advance the understanding of the genetic susceptibility and biology of systemic lupus erythematosus risk. I hope my comments below could help authors improve their work in their revision.

1) The authors could try to explore large-scale GWAS data to strengthen their discovery of the risk association signal of rs1047643 (such as summary statistics of GWAS data of European descendant). The authors should present the information of Linkage Disequilibrium between the identified variant and previously reported GWAS-identified risk variants. The novel risk variant, rs1047643, may not be an independent risk signal (may be in LD with rs17807624).

2) Need to clarify some statistical analyses. For example, for formula presented in the line 121, it's unclear for the "disease" variable (referring to case/control status? how to link SNP information?). If referring to case/control status, it could be a response variable? The authors need to provide detailed description about how to select the risk variant rs1047643 for your focus (only this SNP satisfied your analysis a two-stage study presented in Figure 1?).

In the line 132, not sure why populations is needed to be adjusted here (only focusing on Hispanic population). The authors need to include sentence to describe about how to perform additional analysis and how to perform "AI of RNA transcripts" (the line 233/234). "Statistical analysis" section may not be necessary (including above analysis).

3) Some limitations to be included in the Discussion sections: some functional data may not be generated from the disease target cells (i.e. Bcells). The authors need to discuss some of their allelic imbalance findings about other GWAS-identified risk variants at the locus, 8p23.

4) Need to fix some typos and other minor issues. In the abstract, "SLE "- need to spell out; "Open chromatin" may change to "chromatin accessibility". A typo 'tying' (line 60); RRBS (line 87) and "SD" (line 202) need to be spelled out. The sentence about "The differences" is unclear (line 204)

Reviewer #2 (Recommendations for the authors):

Page 7, line 121. Formula looks awkward. An equivalent way to write this model may be Disease ~ \α + \β Allelic ratio +\epsilon. However, after formatting the model into this shape, it appears that a logistic regression for binary trait should be used instead.

Figure 2A: For your Allelic Imbalance analysis, how many SNPs have you tested in order to identify rs1047643? Should we conduct multiple test correction? I can't see any descriptions from line 108 to 124. If you have done this already (hence the p-value listed in the figure is the after-correction value), would you be clear? What is the meaning of "summary" in the last row?

Page 12 -13, the paragraph for association analysis indeed mentioned an "adjustment". I am a bit confused here: (1) If your analysis starts with Allelic Imbalance analysis, then the next follow-up association test doesn't need a multiple-test adjustment. (2) based on my question above, would you clarify what "adjustment" you are talking about and what is the mathematical procedure? In the event you did some rigorous multiple-test correction here, but not in the AI analysis, your flow of the discovery might be reversely presented.

Page 14, Line 276-284. The Hi-C loop looks interesting, however little details are provided here. E.g., what is the sequencing coverage evidencing this loop?

Figure 4 indeed shows the difference between SLE and controls. However, to make the discovery more convincing, would you check how significant is the difference with the background distribution? E.g., randomly select a hundred other sites and see whether there are also difference between SLE and controls? Or the background looks similar, putting the focal discovery in a significant (or at least promising) position? Similar concern may also apply to Figures 5 and 6: it may be nice to see how likely such difference will show up in randomly selected candidates.

Reviewer #3 (Recommendations for the authors):

Overall, the report by Jin Men et al. provided supportive evidence that by mining multi-omics profiles to develop and indicate a novel functional variant, the mechanism of STAT3 medication. The analytical and statistical framework is appropriate, although not fully described. In addition, additional experiments should be performed.

1. From the results presented (line 224), it is not clear the result of significance for other SNPs in the comparison. Because it has no comprehensive results, which looked like a little confusing. It's necessary to provide the list of potential significant SNPs.

2. The authors report that 'statistical significance' (line 244) about association of rs1047643 with SLE risk, and adjusted P should be 0.023 according to Figure 3. And next the choice of two SNPs for haplotype analysis, the description of the results is inadequate.

3. In Figure 6, the authors performed chip assay to confirm stat3 binding to SE and rs1047643 regions. In addition, they used the stat3 DNA binding inhibitor. However, the inhibitor didn't affect stat3 DNA binding efficiency too much. Please explain why. It's necessary to perform time-course and concentration-dependent experiments to confirm the binding specificity. Also, the authors should perform chip-PCR assay using non-specific primers. Furthermore, the authors should design the experiments to confirm whether rs1047643 affects stat3 binding.

4. The results of Figure 6b,c is confused.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "STAT3-mediated allelic imbalance of novel genetic variant rs1047643 and B cell specific super-enhancer in association with systemic lupus erythematosus" for further consideration by eLife. Your revised article has been evaluated by Betty Diamond (Senior Editor), a Reviewing Editor, and the original reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed: please provide a response to the comments of Reviewer #2.

Reviewer #1 (Recommendations for the authors):

The authors have adequately addressed all of my concerns/comments.

Reviewer #2 (Recommendations for the authors):

In the revised manuscript, most of my comments have been satisfactorily addressed, with the following exceptions:

Q1: you have not responded to my comments on why putting the disease status on the right-hand side of the model.

Q2: disagree that one doesn't have to conduct multiple test adjustment simply due to the sample size being small.

Reviewer #3 (Recommendations for the authors):

I am satisfied with the revised manuscript and suggest the manuscript to be accepted for publication.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "STAT3-mediated allelic imbalance of novel genetic variant rs1047643 and B cell specific super-enhancer in association with systemic lupus erythematosus" for further consideration by eLife. Your revised article has been evaluated by Betty Diamond (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Please address reviewer's Q1: "you have not responded to my comments on why putting the disease status on the right-hand side of the model"

The editor agrees with the validity of the reviewer's comments and would suggest using a logistic regression with disease status as a dependent variable.

10.7554/eLife.72837.sa2Author response

Essential revisions:

1) The authors should explore large-scale GWAS data or provide any other statistical evidence to strengthen their discovery of the risk association signal of rs1047643 (such as summary statistics of GWAS data of European descendants).

2) To improve and clarify the statical analyses as suggested by reviewers.

We thank two editors for giving us an opportunity to submit the revised paper. In the revised version of manuscript, we have made a significant improvement to address these two essential comments (Page 21, and Methods section), and have now met the high standard of your journal for publication. Please see below are the point-by-point response to the reviewers’ comments.

Reviewer #1 (Recommendations for the authors):

This is a well conducted study aiming to identify novel risk variants for systemic lupus erythematosus risk and to further explore potential mechanisms through a comprehensive analysis of functional genomic analyses and functional assays. The authors have identified a putative novel risk variant, rs1047643, for the disease risk, and further revealed underlying disease mechanisms of a known susceptibility factor, STAT3, mediated Super enhancer activity and cis-regulatory effects of SNP rs1047643. Their discovered mechanisms are novel, providing new insights into the potential mechanism underlying other GWAS-identified variants located in the Super enhancer element to mediate the disease risk. This work could be very important to advance the understanding of the genetic susceptibility and biology of systemic lupus erythematosus risk. I hope my comments below could help authors improve their work in their revision.

1) The authors could try to explore large-scale GWAS data to strengthen their discovery of the risk association signal of rs1047643 (such as summary statistics of GWAS data of European descendant). The authors should present the information of Linkage Disequilibrium between the identified variant and previously reported GWAS-identified risk variants. The novel risk variant, rs1047643, may not be an independent risk signal (may be in LD with rs17807624).

We added these results in the revised manuscript (Page 21, page 7: lines 123-130).

2) Need to clarify some statistical analyses. For example, for formula presented in the line 121, it's unclear for the "disease" variable (referring to case/control status? how to link SNP information?). If referring to case/control status, it could be a response variable? The authors need to provide detailed description about how to select the risk variant rs1047643 for your focus (only this SNP satisfied your analysis a two-stage study presented in Figure 1?).

In the line 132, not sure why populations is needed to be adjusted here (only focusing on Hispanic population). The authors need to include sentence to describe about how to perform additional analysis and how to perform "AI of RNA transcripts" (the line 233/234). "Statistical analysis" section may not be necessary (including above analysis).

In the revised paper, we provide detailed description about AI analysis implemented in a linear model (Page 20-21), and re-clarify the adjustment of five countries of the Hispanic population (Page 21: lines 396-400), as well as the method section (Page 21).

3) Some limitations to be included in the Discussion sections: some functional data may not be generated from the disease target cells (i.e. Bcells). The authors need to discuss some of their allelic imbalance findings about other GWAS-identified risk variants at the locus, 8p23.

Thanks for reviewer’s suggestion. In the revised paper, we discussed the limitations (Page 16, lines 329-332) and the allelic imbalance results of 13 GWAS SNPs (Page 15, lines 307-308).

4) Need to fix some typos and other minor issues. In the abstract, "SLE "- need to spell out; "Open chromatin" may change to "chromatin accessibility". A typo 'tying' (line 60); RRBS (line 87) and "SD" (line 202) need to be spelled out. The sentence about "The differences" is unclear (line 204)

Thank reviewer for pointing out these issues. We corrected and defined these terms in the revised paper, except for ‘tying’ that is correct, not a typo.

Reviewer #2 (Recommendations for the authors):

Page 7, line 121. Formula looks awkward. An equivalent way to write this model may be Disease ~ \α + \β Allelic ratio +\epsilon. However, after formatting the model into this shape, it appears that a logistic regression for binary trait should be used instead.

In the revised paper, we rephrased the description about AI analysis implemented in a linear model (Page 20, lines 379-385)

Figure 2A: For your Allelic Imbalance analysis, how many SNPs have you tested in order to identify rs1047643? Should we conduct multiple test correction? I can't see any descriptions from line 108 to 124. If you have done this already (hence the p-value listed in the figure is the after-correction value), would you be clear? What is the meaning of "summary" in the last row?

In the revised paper, we provided more detailed description and re-clarified the statistical analyses (Page 6, lines 93-102).

Page 12 -13, the paragraph for association analysis indeed mentioned an "adjustment". I am a bit confused here: (1) If your analysis starts with Allelic Imbalance analysis, then the next follow-up association test doesn't need a multiple-test adjustment. (2) based on my question above, would you clarify what "adjustment" you are talking about and what is the mathematical procedure? In the event you did some rigorous multiple-test correction here, but not in the AI analysis, your flow of the discovery might be reversely presented.

Thanks for reviewer’s helpful comments. In the revised paper, we provided detailed description and re-clarified the statistical analyses (Page 34, lines 682-684, and Figure 3A-B).

Page 14, Line 276-284. The Hi-C loop looks interesting, however little details are provided here. E.g., what is the sequencing coverage evidencing this loop?

In the revised paper, we provided more details about read supports and statistical significance for DNA loops in the Methods section (Page 23, lines 443-444).

Figure 4 indeed shows the difference between SLE and controls. However, to make the discovery more convincing, would you check how significant is the difference with the background distribution? E.g., randomly select a hundred other sites and see whether there are also difference between SLE and controls? Or the background looks similar, putting the focal discovery in a significant (or at least promising) position? Similar concern may also apply to Figures 5 and 6: it may be nice to see how likely such difference will show up in randomly selected candidates.

In the revised paper, we presented these results (Page 9-10, lines 178-180; Page 11, lines 208-212, and Figure 4—figure supplement 1and Figure 5—figure supplement 1).

Reviewer #3 (Recommendations for the authors):

Overall, the report by Jin Men et al. provided supportive evidence that by mining multi-omics profiles to develop and indicate a novel functional variant, the mechanism of STAT3 medication. The analytical and statistical framework is appropriate, although not fully described. In addition, additional experiments should be performed.

1. From the results presented (line 224), it is not clear the result of significance for other SNPs in the comparison. Because it has no comprehensive results, which looked like a little confusing. It's necessary to provide the list of potential significant SNPs.

Because of small sample size, we just found three potential significant SNPs. In the revised paper, we reported these SNPs (Page 6, lines 93-102, and Figure 2—figure supplement 1).

2. The authors report that 'statistical significance' (line 244) about association of rs1047643 with SLE risk, and adjusted P should be 0.023 according to Figure 3. And next the choice of two SNPs for haplotype analysis, the description of the results is inadequate.

In the revised paper, we provided detailed description and re-clarified the statistical analyses (Page 7, lines 120-130).

3. In Figure 6, the authors performed chip assay to confirm stat3 binding to SE and rs1047643 regions. In addition, they used the stat3 DNA binding inhibitor. However, the inhibitor didn't affect stat3 DNA binding efficiency too much. Please explain why. It's necessary to perform time-course and concentration-dependent experiments to confirm the binding specificity. Also, the authors should perform chip-PCR assay using non-specific primers. Furthermore, the authors should design the experiments to confirm whether rs1047643 affects stat3 binding.

Thanks for reviewer’s insightful comments. Please see below are detailed description.

For the question about “the authors should perform chip-PCR assay using non-specific primers”, we designed a pair of primers as negative control which has been previously used elsewhere as ChIP-qPCR quality control (Page 25, lines 474-476). We presented these new ChIP-qPCR results in the revised manuscript (Page 12, line 228, and Figure 6—figure supplement 1).

For the question about “However, the inhibitor didn't affect stat3 DNA binding efficiency too much”, we did more experiment by increasing S3I concentration to 100 μM. The results don’t change dramatically (Figure 6C). One reason may be that the rs1047643 region is co-bound by multiple STAT family members. Because in our latest experiment where we also did STAT1 ChIP assay under S3I treatment in GM11997 cells, to our surprise is that we observed an increase of STAT1 binding on rs1047643-T allele under the S3I treatment. That is to say, as classic JAK-STAT signaling pathway shows, there may exist three different sorts of dimers (two homo-dimers, STAT1-STAT1 and STAT3-STAT3, one hetero-dimer, STAT1-STAT3) that co-regulate this locus in a complicated manner. Therefore, inhibition of STAT3 may result in a mild effect on the rs1047643 allelic binding.

For the remaining questions, we would like to logically depict our study design on this project. At the beginning, our strategy was focused on loss-of-function of STAT3 by using siRNA and shRNA methods. However, due to the extremely low transfection efficiency (<5%) in such tough suspension cells, the experiments failed. Meanwhile, we attempted to test the impact of the rs1047643 on the STAT3 binding by transfection-associated assays, but were unsuccessful, presumably due to the difficulty of transfection in lymphoblastic cells. Eventually, we changed our strategies and switched to use STAT3 drug inhibitors and activators to determine the allelic binding performance.

In addition, we have expanded this project and acquired several novel interesting findings during the last nine months. In the revised manuscript, we present the relevant results to confirm the results by controlling upstream factors in the JAK-STAT3 signaling pathway (Page 12-13, lines 244-249, and Figure 6—figure supplement 3).

4. The results of Figure 6b,c is confused.

Thanks for reviewer’s helpful comment. To make the results more readable and understandable, we re-organized the Figure 6 and split into two Figures (Figure 6 and 7) in the revised manuscript.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed: please provide a response to the comments of Reviewer #2.

Reviewer #2 (Recommendations for the authors):

In the revised manuscript, most of my comments have been satisfactorily addressed, with the following exceptions:

Q1: you have not responded to my comments on why putting the disease status on the right-hand side of the model.

To test for associations between allelic ratio and SLE disease state (case/control comparison) for each heterozygous SNP, we regarded the case/control disease status as categorical variable (control and case are coded as 0 and 1, respectively) and put it on the right-hand side implemented in regression model, the analysis has been used in other studies (1, 2). In the revised manuscript, we re-phrased the regression analysis (Page 20, lines 382-389).

Q2: disagree that one doesn't have to conduct multiple test adjustment simply due to the sample size being small.

Thanks for reviewer’s suggestion. In the revised manuscript, we re-phrased this sentence (Page 6, line 95).

1. Adoue V, Schiavi A, Light N, Almlof JC, Lundmark P, Ge B, et al. Allelic expression mapping across cellular lineages to establish impact of non-coding SNPs. Mol Syst Biol. 2014;10:754.

2. Absher DM, Li X, Waite LL, Gibson A, Roberts K, Edberg J, et al. Genome-wide DNA methylation analysis of systemic lupus erythematosus reveals persistent hypomethylation of interferon genes and compositional changes to CD4+ T-cell populations. PLoS Genet. 2013;9(8):e1003678.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Please address reviewer's Q1: "you have not responded to my comments on why putting the disease status on the right-hand side of the model"

We re-analyzed the data based on the logistic regression with disease status as a dependent variable. Due to small sample size, particularly just six samples carrying heterozygous SNP rs1047643 in one dataset (GSE71338), there is no statistical power to identify the significance when analyzing with the logistic regression model. In the revised manuscript, we discuss this limitation (Page 17, lines 334-340).