{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T13:18:32Z","timestamp":1785417512508,"version":"3.56.0"},"posted":{"date-parts":[[2025,5,19]]},"group-title":"Developmental Biology","reference-count":71,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,5,19]],"date-time":"2025-05-19T00:00:00Z","timestamp":1747612800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["R01HL138659"],"award-info":[{"award-number":["R01HL138659"]}],"id":[{"id":"https:\/\/ror.org\/012pb6c26","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,5,19]]},"abstract":"ABSTRACT<\/jats:title>\n \n Congenital heart defects (CHDs) are frequently observed in the most common form of Cornelia de Lange Syndrome (CdLS), which is caused by haploinsufficiency for\n NIPBL<\/jats:italic>\n , a gene involved in chromatin looping and cis-regulatory control of gene expression. Here, we surveyed cardiac defects in mice made\n Nipbl<\/jats:italic>\n -haploinsufficient in the second heart field using two Cre drivers:\n Mef2c-Cre<\/jats:italic>\n and\n Isl1-Cre<\/jats:italic>\n . Only\n Isl1-Cre<\/jats:italic>\n -driven\n Nipbl<\/jats:italic>\n -haploinsufficiency resulted in CHDs \u2013 a finding we traced to the additional contribution of\n Isl1<\/jats:italic>\n -haploinsufficiency caused by the\n Isl1-Cre<\/jats:italic>\n allele. To test whether combined reduction of\n Nipbl<\/jats:italic>\n and\n Isl1<\/jats:italic>\n cause CHDs, we made mice globally haploinsufficient for both genes. Indeed,\n \n Nipbl\n +\/-<\/jats:sup>\n ; Isl1\n +\/-<\/jats:sup>\n <\/jats:italic>\n mice exhibited a substantially higher frequency and severity of CHDs than mice haploinsufficient for either gene alone. As a member of the LIM-homeodomain transcription factor family,\n Isl1<\/jats:italic>\n is involved in chromatin looping and enhancer-promoter communication via a mechanism distinct from that of\n Nipbl<\/jats:italic>\n . Nevertheless, when we performed RNA sequencing on E10.5 hearts from wildtype,\n \n Nipbl\n +\/-<\/jats:sup>\n <\/jats:italic>\n ,\n \n Isl1\n +\/-<\/jats:sup>\n <\/jats:italic>\n , and\n \n Nipbl\n +\/-<\/jats:sup>\n ; Isl1\n +\/-<\/jats:sup>\n <\/jats:italic>\n embryos, we observed that combined haploinsufficiency resulted in largely additive gene expression changes, including dysregulation of known cardiac regulators (\n Irx4, Tbx1, Foxo6, Heyl, Bnc1, Sox17<\/jats:italic>\n ) and novel candidates (\n Gbx1, Csdc2, Myrf, Pou6f1, Zfp579, ad Zfp763<\/jats:italic>\n ). A subset of additive changes arose from\n opposing<\/jats:italic>\n regulatory influences in single mutants that restored gene expression to WT levels in\n \n Nipbl\u207a\n \/<\/jats:sup>\n \u207b; Isl1\u207a\n \/<\/jats:sup>\n \u207b\n <\/jats:italic>\n hearts. For example,\n Hoxc4, Pitx2, Isl1<\/jats:italic>\n itself, and\n Pax6<\/jats:italic>\n (a known target of\n Isl1<\/jats:italic>\n ), were upregulated in\n \n Nipbl\n +\/-<\/jats:sup>\n <\/jats:italic>\n hearts, downregulated in\n \n Isl1\n +\/-<\/jats:sup>\n <\/jats:italic>\n hearts, but expressed at WT levels in\n \n Nipbl\u207a\n \/<\/jats:sup>\n \u207b; Isl1\u207a\n \/<\/jats:sup>\n \u207b\n <\/jats:italic>\n hearts. Since loss of\n Isl1<\/jats:italic>\n upregulation from\n \n Nipbl\n +\/-<\/jats:sup>\n <\/jats:italic>\n to\n \n Nipbl\n +\/-<\/jats:sup>\n ; Isl1\n +\/-<\/jats:sup>\n <\/jats:italic>\n hearts coincided with a marked increase in CHDs, we propose that\n Isl1<\/jats:italic>\n upregulation compensates for the loss of cis- regulatory interactions due to\n Nipbl<\/jats:italic>\n -haploinsufficiency, and protects hearts from severe CHD risk. Supporting this model, other LIM-homeodomain transcription factors (\n Lhx2, Lhx3, Lhx9<\/jats:italic>\n ) were also upregulated in\n \n Nipbl\n +\/-<\/jats:sup>\n <\/jats:italic>\n hearts, with\n Lhx3<\/jats:italic>\n and\n Lhx9<\/jats:italic>\n showing even greater upregulation in\n \n Nipbl\n +\/-<\/jats:sup>\n ; Isl1\n +\/-<\/jats:sup>\n <\/jats:italic>\n hearts. Despite this, CHDs resulting from the combined loss of\n Nipbl<\/jats:italic>\n and\n Isl1<\/jats:italic>\n were particularly severe. These findings suggest that heart development is exquisitely sensitive to small changes in gene expression, leading to synergistic phenotypic interactions when relatively modest gene expression changes are combined.\n <\/jats:p>","DOI":"10.1101\/2025.05.15.654123","type":"posted-content","created":{"date-parts":[[2025,5,19]],"date-time":"2025-05-19T20:20:32Z","timestamp":1747686032000},"source":"Crossref","is-referenced-by-count":1,"title":["Interaction between long-range chromatin regulators\n Nipbl<\/i>\n &\n Isl1<\/i>\n synergistically drives heart defects in mice"],"prefix":"10.64898","author":[{"given":"Stephenson","family":"Chea","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rosaysela","family":"Santos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martha E.","family":"Lopez-Burks","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arthur D.","family":"Lander","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anne L.","family":"Calof","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025052114251911000_2025.05.15.654123v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tcb.2023.03.006"},{"key":"2025052114251911000_2025.05.15.654123v1.2","doi-asserted-by":"publisher","DOI":"10.1093\/cercor\/bhx185"},{"key":"2025052114251911000_2025.05.15.654123v1.3","doi-asserted-by":"publisher","DOI":"10.1542\/peds.107.3.e32"},{"key":"2025052114251911000_2025.05.15.654123v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.gde.2024.102282"},{"key":"2025052114251911000_2025.05.15.654123v1.5","doi-asserted-by":"publisher","DOI":"10.1111\/cge.12499"},{"key":"2025052114251911000_2025.05.15.654123v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/nrg1710"},{"key":"2025052114251911000_2025.05.15.654123v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-15552-x"},{"key":"2025052114251911000_2025.05.15.654123v1.8","doi-asserted-by":"publisher","DOI":"10.1016\/j.stem.2015.08.007"},{"key":"2025052114251911000_2025.05.15.654123v1.9","doi-asserted-by":"crossref","first-page":"2499","DOI":"10.1002\/ajmg.a.35582","article-title":"Congenital heart disease in Cornelia de Lange syndrome: phenotype and genotype analysis","volume":"158A","year":"2012","journal-title":"Am J Med Genet A"},{"key":"2025052114251911000_2025.05.15.654123v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/S1097-2765(00)80420-7"},{"key":"2025052114251911000_2025.05.15.654123v1.11","doi-asserted-by":"publisher","DOI":"10.1242\/dev.01256"},{"key":"2025052114251911000_2025.05.15.654123v1.12","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1217812110"},{"key":"2025052114251911000_2025.05.15.654123v1.13","doi-asserted-by":"crossref","unstructured":"Ehrlich, K. C. , Lacey, M. & Ehrlich, M . 2020. Epigenetics of Skeletal Muscle- Associated Genes in the Asb, Lrrc, Tmem, and Osbpl Gene Families. Epigenomes, 4.","DOI":"10.3390\/epigenomes4010001"},{"key":"2025052114251911000_2025.05.15.654123v1.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2020.05.146"},{"key":"2025052114251911000_2025.05.15.654123v1.15","doi-asserted-by":"publisher","DOI":"10.1161\/01.RES.0000260913.95642.3b"},{"key":"2025052114251911000_2025.05.15.654123v1.16","doi-asserted-by":"crossref","unstructured":"Franco, D. , Sedmera, D. & Lozano-Velasco, E . 2017. Multiple Roles of Pitx2 in Cardiac Development and Disease. J Cardiovasc Dev Dis, 4.","DOI":"10.3390\/jcdd4040016"},{"key":"2025052114251911000_2025.05.15.654123v1.17","doi-asserted-by":"crossref","unstructured":"Gambardella, L. , Mcmanus, S. A. , Moignard, V. , Sebukhan, D. , Delaune, A. , Andrews, S. , Bernard, W. G. , Morrison, M. A. , Riley, P. R. , Gottgens, B. , Gambardella Le Novere, N. & Sinha, S. 2019. BNC1 regulates cell heterogeneity in human pluripotent stem cell-derived epicardium. Development, 146.","DOI":"10.1242\/dev.174441"},{"key":"2025052114251911000_2025.05.15.654123v1.18","doi-asserted-by":"publisher","DOI":"10.1038\/s41422-019-0168-1"},{"key":"2025052114251911000_2025.05.15.654123v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2004.07.053"},{"key":"2025052114251911000_2025.05.15.654123v1.20","doi-asserted-by":"publisher","DOI":"10.1086\/424698"},{"key":"2025052114251911000_2025.05.15.654123v1.21","doi-asserted-by":"publisher","DOI":"10.1203\/01.PDR.0000148710.69159.61"},{"key":"2025052114251911000_2025.05.15.654123v1.22","doi-asserted-by":"publisher","DOI":"10.1016\/S0735-1097(98)00259-9"},{"key":"2025052114251911000_2025.05.15.654123v1.23","doi-asserted-by":"publisher","DOI":"10.1242\/dev.145805"},{"key":"2025052114251911000_2025.05.15.654123v1.24","doi-asserted-by":"publisher","DOI":"10.1113\/JP281952"},{"key":"2025052114251911000_2025.05.15.654123v1.25","doi-asserted-by":"publisher","DOI":"10.3389\/fcell.2023.1259175"},{"key":"2025052114251911000_2025.05.15.654123v1.26","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1208690109"},{"key":"2025052114251911000_2025.05.15.654123v1.27","unstructured":"Heffner, C. & Murray, S. 2024. Whole mount \u03b2-galactosidase staining [Online]. www.jax.org: The Jackson Laboratory. Available: https:\/\/www.jax.org\/research-and-faculty\/resources\/cre-repository\/whole-mount-staining-protocol [Accessed]."},{"key":"2025052114251911000_2025.05.15.654123v1.28","doi-asserted-by":"publisher","DOI":"10.1016\/S0735-1097(02)01886-7"},{"key":"2025052114251911000_2025.05.15.654123v1.29","doi-asserted-by":"publisher","DOI":"10.1620\/tjem.244.177"},{"key":"2025052114251911000_2025.05.15.654123v1.30","doi-asserted-by":"crossref","unstructured":"Hubert, K. A. & Wellik, D. M . 2023. Hox genes in development and beyond. Development, 150.","DOI":"10.1242\/dev.192476"},{"key":"2025052114251911000_2025.05.15.654123v1.31","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1000650"},{"key":"2025052114251911000_2025.05.15.654123v1.32","doi-asserted-by":"crossref","unstructured":"Kelly, R. G. , Buckingham, M. E. & Moorman, A. F . 2014. Heart fields and cardiac morphogenesis. Cold Spring Harb Perspect Med, 4.","DOI":"10.1101\/cshperspect.a015750"},{"key":"2025052114251911000_2025.05.15.654123v1.33","doi-asserted-by":"publisher","DOI":"10.1016\/j.diff.2012.04.005"},{"key":"2025052114251911000_2025.05.15.654123v1.34","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.112.265041"},{"key":"2025052114251911000_2025.05.15.654123v1.35","doi-asserted-by":"publisher","DOI":"10.1161\/01.RES.77.2.211"},{"key":"2025052114251911000_2025.05.15.654123v1.36","doi-asserted-by":"publisher","DOI":"10.1101\/gad.239749.114"},{"key":"2025052114251911000_2025.05.15.654123v1.37","doi-asserted-by":"publisher","DOI":"10.1016\/S0925-4773(00)00459-7"},{"key":"2025052114251911000_2025.05.15.654123v1.38","doi-asserted-by":"publisher","DOI":"10.1387\/ijdb.180192sz"},{"key":"2025052114251911000_2025.05.15.654123v1.39","doi-asserted-by":"publisher","DOI":"10.1016\/j.cels.2015.12.004"},{"key":"2025052114251911000_2025.05.15.654123v1.40","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.1000119"},{"key":"2025052114251911000_2025.05.15.654123v1.41","doi-asserted-by":"publisher","DOI":"10.1002\/BDR2.1589"},{"key":"2025052114251911000_2025.05.15.654123v1.42","doi-asserted-by":"publisher","DOI":"10.1002\/humu.22430"},{"key":"2025052114251911000_2025.05.15.654123v1.43","doi-asserted-by":"publisher","DOI":"10.1161\/JAHA.122.025687"},{"key":"2025052114251911000_2025.05.15.654123v1.44","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.1001181"},{"key":"2025052114251911000_2025.05.15.654123v1.45","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1004671"},{"key":"2025052114251911000_2025.05.15.654123v1.46","doi-asserted-by":"publisher","DOI":"10.1002\/dvg.20335"},{"key":"2025052114251911000_2025.05.15.654123v1.47","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-genet-102108-134233"},{"key":"2025052114251911000_2025.05.15.654123v1.48","doi-asserted-by":"publisher","DOI":"10.1002\/dvdy.24078"},{"key":"2025052114251911000_2025.05.15.654123v1.49","doi-asserted-by":"publisher","DOI":"10.1542\/peds.2012-3435"},{"key":"2025052114251911000_2025.05.15.654123v1.50","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1005963"},{"key":"2025052114251911000_2025.05.15.654123v1.51","doi-asserted-by":"publisher","DOI":"10.2174\/157340310791162703"},{"key":"2025052114251911000_2025.05.15.654123v1.52","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.109.194464"},{"key":"2025052114251911000_2025.05.15.654123v1.53","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-48321-y"},{"key":"2025052114251911000_2025.05.15.654123v1.54","doi-asserted-by":"publisher","DOI":"10.1002\/dvg.20437"},{"key":"2025052114251911000_2025.05.15.654123v1.55","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.2000197"},{"key":"2025052114251911000_2025.05.15.654123v1.56","doi-asserted-by":"publisher","DOI":"10.1177\/0192623309335060"},{"key":"2025052114251911000_2025.05.15.654123v1.57","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.117.311147"},{"key":"2025052114251911000_2025.05.15.654123v1.58","doi-asserted-by":"publisher","DOI":"10.1113\/expphysiol.2005.031773"},{"key":"2025052114251911000_2025.05.15.654123v1.59","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-030-38062-5_2"},{"key":"2025052114251911000_2025.05.15.654123v1.60","doi-asserted-by":"publisher","DOI":"10.1038\/ncb1177"},{"key":"2025052114251911000_2025.05.15.654123v1.61","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1008774"},{"key":"2025052114251911000_2025.05.15.654123v1.62","doi-asserted-by":"publisher","DOI":"10.1016\/j.ydbio.2005.08.041"},{"key":"2025052114251911000_2025.05.15.654123v1.63","doi-asserted-by":"publisher","DOI":"10.1016\/S0070-2153(10)90001-X"},{"key":"2025052114251911000_2025.05.15.654123v1.64","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.111.259960"},{"key":"2025052114251911000_2025.05.15.654123v1.65","doi-asserted-by":"publisher","DOI":"10.1161\/01.RES.85.2.128"},{"key":"2025052114251911000_2025.05.15.654123v1.66","unstructured":"Ware, S. M. & Jefferies, J. L . 2012. New Genetic Insights into Congenital Heart Disease. J Clin Exp Cardiolog, S8."},{"key":"2025052114251911000_2025.05.15.654123v1.67","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2006.03.049"},{"key":"2025052114251911000_2025.05.15.654123v1.68","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1005655"},{"key":"2025052114251911000_2025.05.15.654123v1.69","doi-asserted-by":"publisher","DOI":"10.1242\/dev.01174"},{"key":"2025052114251911000_2025.05.15.654123v1.70","doi-asserted-by":"publisher","DOI":"10.1242\/dev.02322"},{"key":"2025052114251911000_2025.05.15.654123v1.71","first-page":"e383","article-title":"Forkhead box O6 (FoxO6) promotes cardiac pathological remodeling and dysfunction by activating Kif15-Tgf-beta1 under aggravated afterload","volume":"4","year":"2020","journal-title":"MedComm"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.05.15.654123","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T08:44:02Z","timestamp":1768466642000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.05.15.654123"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,19]]},"references-count":71,"URL":"https:\/\/doi.org\/10.1101\/2025.05.15.654123","relation":{},"subject":[],"published":{"date-parts":[[2025,5,19]]},"subtype":"preprint"}}