{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,16]],"date-time":"2026-05-16T11:44:26Z","timestamp":1778931866047,"version":"3.51.4"},"reference-count":38,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,1,1]],"date-time":"2025-01-01T00:00:00Z","timestamp":1735689600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institutes of Health","award":["R01AI172255"],"award-info":[{"award-number":["R01AI172255"]}]},{"name":"National Institutes of Health","award":["R01AI183676"],"award-info":[{"award-number":["R01AI183676"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by widespread inflammation and autoantibody production. Its development and progression involve genetic, epigenetic, and environmental factors. Although genome-wide association studies (GWAS) have repeatedly identified a susceptibility signal at 16p13, its fine-scale source and its functional and mechanistic role in SLE remain unclear. We used bioinformatics to prioritize likely functional variants and validated the top candidate through various experimental techniques, including clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing in B cells. To assess the functional impact of the proposed causal variant in C-type lectin domain family 16, member A (CLEC16A), we compared autophagy levels between wild-type (WT) and knock-out (KO) cells. Systematic bioinformatics analysis identified the highly conserved non-coding intronic variant rs17673553, with the risk allele apparently affecting enhancer function and regulating several target genes, including CLEC16A itself. Luciferase reporter assays followed by chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) validated this enhancer activity, demonstrating that the risk allele increases the binding of enhancer histone marks (H3K27ac and H3K4me1), the CTCF-binding factor, and key immune transcription factors (GATA3 and STAT3). Knock-down of GATA3 and STAT3 via siRNA led to a significant decrease in CLEC16A expression. These regulatory effects on the target gene were further confirmed using CRISPR-based genome editing and CRISPR-dCas9-based epigenetic activation\/silencing. Functionally, WT cells exhibited higher levels of starvation-induced autophagy compared to KO cells, highlighting the role of CLEC16A and the rs17673553 locus in autophagy regulation. These findings suggest that the rs17673553 locus\u2014particularly the risk allele\u2014drives significant allele-specific chromatin modifications and binding of multiple transcription factors, thereby mechanistically regulating the expression of target autophagy-associated genes, including CLEC16A itself. This mechanism could potentially explain the association between rs17673553 and SLE, and could underlie the signal at 16p13.<\/jats:p>","DOI":"10.3390\/ijms26010314","type":"journal-article","created":{"date-parts":[[2025,1,1]],"date-time":"2025-01-01T11:24:19Z","timestamp":1735730659000},"page":"314","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Defining Mechanistic Links Between the Non-Coding Variant rs17673553 in CLEC16A and Lupus Susceptibility"],"prefix":"10.3390","volume":"26","author":[{"given":"Harikrishna Reddy","family":"Rallabandi","sequence":"first","affiliation":[{"name":"Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7019-5195","authenticated-orcid":false,"given":"Manish Kumar","family":"Singh","sequence":"additional","affiliation":[{"name":"Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Loren L.","family":"Looger","sequence":"additional","affiliation":[{"name":"Department of Neurosciences, University of California, La Jolla, San Diego, CA 92093, USA"},{"name":"Howard Hughes Medical Institute, University of California, La Jolla, San Diego, CA 92093, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6013-9328","authenticated-orcid":false,"given":"Swapan K.","family":"Nath","sequence":"additional","affiliation":[{"name":"Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2326","DOI":"10.1016\/S0140-6736(24)00398-2","article-title":"Systemic lupus erythematosus","volume":"403","author":"Hoi","year":"2024","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1381","DOI":"10.1007\/s00296-023-05346-x","article-title":"Systemic lupus erythematosus: Latest insight into etiopathogenesis","volume":"43","author":"Akhil","year":"2023","journal-title":"Rheumatol. Int."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"101971","DOI":"10.1016\/j.berh.2024.101971","article-title":"Exploring the contribution of genetics on the clinical manifestations of systemic lupus erythematosus","volume":"38","year":"2024","journal-title":"Best Pract. Res. Clin. Rheumatol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1071","DOI":"10.1002\/art.42829","article-title":"A multilayered post-GWAS analysis pipeline defines functional variants and target genes for systemic lupus erythematosus (SLE)","volume":"76","author":"Looger","year":"2024","journal-title":"Arthritis Rheumatol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1038\/nature06010","article-title":"A genome-wide association study identifies KIAA0350 as a type 1 diabetes gene","volume":"448","author":"Hakonarson","year":"2007","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1136\/jmg.2010.077859","article-title":"Polymorphisms at 16p13 are associated with systemic lupus erythematosus in the Chinese population","volume":"48","author":"Zhang","year":"2011","journal-title":"J. Med. Genet."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Pushkarev, O., van Mierlo, G., Kribelbauer, J.F., Saelens, W., Gardeux, V., and Deplancke, B. (2024). Non-coding variants impact cis-regulatory coordination in a cell type-specific manner. Genome Biol., 25.","DOI":"10.1186\/s13059-024-03333-4"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Pe\u00f1a-Mart\u00ednez, E.G., and Rodr\u00edguez-Mart\u00ednez, J.A. (2024). Decoding Non-coding Variants: Recent Approaches to Studying Their Role in Gene Regulation and Human Diseases. Front. Biosci. (Schol. Ed.), 16.","DOI":"10.31083\/j.fbs1601004"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Cano-Gamez, E., and Trynka, G. (2020). From GWAS to Function: Using Functional Genomics to Identify the Mechanisms Underlying Complex Diseases. Front. Genet., 11.","DOI":"10.3389\/fgene.2020.00424"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"265","DOI":"10.2337\/db17-0321","article-title":"Clec16a, Nrdp1, and USP8 Form a Ubiquitin-Dependent Tripartite Complex That Regulates \u03b2-Cell Mitophagy","volume":"67","author":"Pearson","year":"2018","journal-title":"Diabetes"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"6804","DOI":"10.1038\/ncomms7804","article-title":"Association of CLEC16A with human common variable immunodeficiency disorder and role in murine B cells","volume":"6","author":"Li","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1471","DOI":"10.1136\/ard.2009.114934","article-title":"A CLEC16A variant confers risk for juvenile idiopathic arthritis and anti-cyclic citrullinated peptide antibody negative rheumatoid arthritis","volume":"69","author":"Skinningsrud","year":"2010","journal-title":"Ann. Rheum. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/jhg.2009.96","article-title":"Replication of CD58 and CLEC16A as genome-wide significant risk genes for multiple sclerosis","volume":"54","author":"Hoppenbrouwers","year":"2009","journal-title":"J. Hum. Genet."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Pandey, R., Bakay, M., and Hakonarson, H. (2023). CLEC16A-An Emerging Master Regulator of Autoimmunity and Neurodegeneration. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24098224"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1577","DOI":"10.1016\/j.cell.2014.05.016","article-title":"The diabetes susceptibility gene Clec16a regulates mitophagy","volume":"157","author":"Soleimanpour","year":"2014","journal-title":"Cell"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1016\/j.intimp.2016.09.017","article-title":"The role of autophagy in the pathogenesis of systemic lupus erythematosus","volume":"40","author":"Liu","year":"2016","journal-title":"Int. Immunopharmacol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1038\/nrrheum.2016.92","article-title":"Autophagy: Controlling cell fate in rheumatic diseases","volume":"12","author":"Rockel","year":"2016","journal-title":"Nat. Rev. Rheumatol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1007\/978-981-15-4272-5_28","article-title":"Autophagy and Autoimmune Diseases","volume":"1207","author":"Jin","year":"2020","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1038\/ng.3496","article-title":"High-density genotyping of immune-related loci identifies new SLE risk variants in individuals with Asian ancestry","volume":"48","author":"Sun","year":"2016","journal-title":"Nat. Genet."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1457","DOI":"10.1038\/ng.3434","article-title":"Genetic association analyses implicate aberrant regulation of innate and adaptive immunity genes in the pathogenesis of systemic lupus erythematosus","volume":"47","author":"Bentham","year":"2015","journal-title":"Nat. Genet."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"16021","DOI":"10.1038\/ncomms16021","article-title":"Transancestral mapping and genetic load in systemic lupus erythematosus","volume":"8","author":"Langefeld","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1038\/ng.2892","article-title":"A general framework for estimating the relative pathogenicity of human genetic variants","volume":"46","author":"Kircher","year":"2014","journal-title":"Nat. Genet."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1038\/s41592-018-0048-5","article-title":"An enhanced CRISPR repressor for targeted mammalian gene regulation","volume":"15","author":"Yeo","year":"2018","journal-title":"Nat. Methods"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/nature14136","article-title":"Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex","volume":"517","author":"Konermann","year":"2015","journal-title":"Nature"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"463","DOI":"10.1007\/s10059-013-0138-2","article-title":"Nutrient regulation of the mTOR complex 1 signaling pathway","volume":"35","author":"Kim","year":"2013","journal-title":"Mol. Cells"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"304","DOI":"10.1016\/j.yexcr.2017.02.017","article-title":"Human CLEC16A regulates autophagy through modulating mTOR activity","volume":"352","author":"Tam","year":"2017","journal-title":"Exp. Cell Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3989","DOI":"10.1038\/s41388-019-0713-x","article-title":"Control of mTOR signaling by ubiquitin","volume":"38","author":"Jiang","year":"2019","journal-title":"Oncogene"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1083\/jcb.200911126","article-title":"The novel endosomal membrane protein Ema interacts with the class C Vps-HOPS complex to promote endosomal maturation","volume":"188","author":"Kim","year":"2010","journal-title":"J. Cell Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"525","DOI":"10.1080\/15548627.2022.2080383","article-title":"An intrinsically disordered protein region encoded by the human disease gene CLEC16A regulates mitophagy","volume":"19","author":"Gingerich","year":"2023","journal-title":"Autophagy"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Pandey, R., Bakay, M., Strenkowski, B.P., Hain, H.S., and Hakonarson, H. (2021). JAK\/STAT inhibitor therapy partially rescues the lipodystrophic autoimmune phenotype in Clec16a KO mice. Sci. Rep., 11.","DOI":"10.1038\/s41598-021-86493-8"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1093\/cei\/uxad007","article-title":"The ups and downs of STAT3 function: Too much, too little and human immune dysregulation","volume":"212","author":"Mackie","year":"2023","journal-title":"Clin. Exp. Immunol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1473","DOI":"10.1093\/bioinformatics\/btu048","article-title":"Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases","volume":"30","author":"Bae","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"724","DOI":"10.1038\/s41588-023-01365-3","article-title":"Annotating and prioritizing human non-coding variants with RegulomeDB v.2","volume":"55","author":"Dong","year":"2023","journal-title":"Nat. Genet."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3555","DOI":"10.1093\/bioinformatics\/btv402","article-title":"LDlink: A web-based application for exploring population-specific haplotype structure and linking correlated alleles of possible functional variants","volume":"31","author":"Machiela","year":"2015","journal-title":"Bioinformatics"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Bendl, J., Musil, M., \u0160toura\u010d, J., Zendulka, J., Damborsk\u00fd, J., and Brezovsk\u00fd, J. (2016). PredictSNP2: A Unified Platform for Accurately Evaluating SNP Effects by Exploiting the Different Characteristics of Variants in Distinct Genomic Regions. PLoS Comput. Biol., 12.","DOI":"10.1371\/journal.pcbi.1004962"},{"key":"ref_36","unstructured":"GTEx Consortium (2020). The GTEx Consortium atlas of genetic regulatory effects across human tissues. Science, 369, 1318."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1300","DOI":"10.1038\/s41588-021-00913-z","article-title":"Large-scale cis- and trans-eQTL analyses identify thousands of genetic loci and polygenic scores that regulate blood gene expression","volume":"53","author":"Claringbould","year":"2021","journal-title":"Nat. Genet."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"D886","DOI":"10.1093\/nar\/gky1016","article-title":"CADD: Predicting the deleteriousness of variants throughout the human genome","volume":"47","author":"Rentzsch","year":"2019","journal-title":"Nucleic Acids Res."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/1\/314\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,7]],"date-time":"2025-10-07T15:23:11Z","timestamp":1759850591000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/1\/314"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,1]]},"references-count":38,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,1]]}},"alternative-id":["ijms26010314"],"URL":"https:\/\/doi.org\/10.3390\/ijms26010314","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2024.12.02.24318337","asserted-by":"object"}]},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,1,1]]}}}