title: A large permissive regulatory domain exclusively controls Tbx3 expression in the cardiac conduction system.
abstract: BACKGROUND
  The evolutionary conserved Tbx3/Tbx5 gene cluster encodes T-box transcription factors that play crucial roles in the development and homeostasis of the cardiac conduction system in human and mouse. Both genes are expressed in overlapping patterns and function in strictly tissue-specific and dose-dependent manners, yet, their regulation is poorly understood.
  
  
  OBJECTIVE
  To analyze the mechanism underlying the complex regulation of the Tbx3/Tbx5 cluster.
  
  
  RESULTS
  By probing the 3-dimensional architecture of the Tbx3/Tbx5 cluster using high-resolution circular chromosome conformation capture sequencing in vivo, we found that its regulatory landscape is in a preformed conformation similar in embryonic heart, limbs, and brain. Tbx3 and its flanking gene desert form a 1 Mbp loop between CCCTC-binding factor (CTCF)-binding sites that is separated from the neighboring Tbx5 loop. However, Ctcf inactivation did not result in transcriptional regulatory interaction between Tbx3 and Tbx5. Multiple sites within the Tbx3 locus contact the promoter, including sites corresponding to regions known to contain variations in the human genome influencing conduction. We identified an atrioventricular-specific enhancer and a pan-cardiac enhancer that contact the promoter and each other and synergize to activate transcription in the atrioventricular conduction system.
  
  
  CONCLUSIONS
  We provide a high-resolution model of the 3-dimensional structure and function of the Tbx3/Tbx5 locus and show that the locus is organized in a preformed, permissive structure. The Tbx3 locus forms a CTCF-independent autonomous regulatory domain with multiple combinatorial regulatory elements that control the precise pattern of Tbx3 in the cardiac conduction system.
authors: From the Department of Anatomy, Embryology, and Physiology, Academic Medical Center, Amsterdam, The Netherlands (J.H.v.W., I.B., M.v.d.B., S.S., P.B., V.M.C.); Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands (H.J.G.v.d.W., W.d.L.); Department of Cell Biology, Erasmus MC Rotterdam, Rotterdam, The Netherlands (H.J.G.v.d.W.); and Cardiovascular Development and Repair Department, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (M.G.-V., C.B.-C., M.M.).
authors: Jan Hendrik
authors: JH
authors: van Weerd
authors: From the Department of Anatomy, Embryology, and Physiology, Academic Medical Center, Amsterdam, The Netherlands (J.H.v.W., I.B., M.v.d.B., S.S., P.B., V.M.C.); Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands (H.J.G.v.d.W., W.d.L.); Department of Cell Biology, Erasmus MC Rotterdam, Rotterdam, The Netherlands (H.J.G.v.d.W.); and Cardiovascular Development and Repair Department, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (M.G.-V., C.B.-C., M.M.).
authors: Ileana
authors: I
authors: Badi
authors: From the Department of Anatomy, Embryology, and Physiology, Academic Medical Center, Amsterdam, The Netherlands (J.H.v.W., I.B., M.v.d.B., S.S., P.B., V.M.C.); Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands (H.J.G.v.d.W., W.d.L.); Department of Cell Biology, Erasmus MC Rotterdam, Rotterdam, The Netherlands (H.J.G.v.d.W.); and Cardiovascular Development and Repair Department, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (M.G.-V., C.B.-C., M.M.).
authors: Malou
authors: M
authors: van den Boogaard
authors: From the Department of Anatomy, Embryology, and Physiology, Academic Medical Center, Amsterdam, The Netherlands (J.H.v.W., I.B., M.v.d.B., S.S., P.B., V.M.C.); Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands (H.J.G.v.d.W., W.d.L.); Department of Cell Biology, Erasmus MC Rotterdam, Rotterdam, The Netherlands (H.J.G.v.d.W.); and Cardiovascular Development and Repair Department, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (M.G.-V., C.B.-C., M.M.).
authors: Sonia
authors: S
authors: Stefanovic
authors: From the Department of Anatomy, Embryology, and Physiology, Academic Medical Center, Amsterdam, The Netherlands (J.H.v.W., I.B., M.v.d.B., S.S., P.B., V.M.C.); Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands (H.J.G.v.d.W., W.d.L.); Department of Cell Biology, Erasmus MC Rotterdam, Rotterdam, The Netherlands (H.J.G.v.d.W.); and Cardiovascular Development and Repair Department, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (M.G.-V., C.B.-C., M.M.).
authors: Harmen J G
authors: HJ
authors: van de Werken
authors: From the Department of Anatomy, Embryology, and Physiology, Academic Medical Center, Amsterdam, The Netherlands (J.H.v.W., I.B., M.v.d.B., S.S., P.B., V.M.C.); Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands (H.J.G.v.d.W., W.d.L.); Department of Cell Biology, Erasmus MC Rotterdam, Rotterdam, The Netherlands (H.J.G.v.d.W.); and Cardiovascular Development and Repair Department, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (M.G.-V., C.B.-C., M.M.).
authors: Melisa
authors: M
authors: Gomez-Velazquez
authors: From the Department of Anatomy, Embryology, and Physiology, Academic Medical Center, Amsterdam, The Netherlands (J.H.v.W., I.B., M.v.d.B., S.S., P.B., V.M.C.); Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands (H.J.G.v.d.W., W.d.L.); Department of Cell Biology, Erasmus MC Rotterdam, Rotterdam, The Netherlands (H.J.G.v.d.W.); and Cardiovascular Development and Repair Department, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (M.G.-V., C.B.-C., M.M.).
authors: Claudio
authors: C
authors: Badia-Careaga
authors: From the Department of Anatomy, Embryology, and Physiology, Academic Medical Center, Amsterdam, The Netherlands (J.H.v.W., I.B., M.v.d.B., S.S., P.B., V.M.C.); Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands (H.J.G.v.d.W., W.d.L.); Department of Cell Biology, Erasmus MC Rotterdam, Rotterdam, The Netherlands (H.J.G.v.d.W.); and Cardiovascular Development and Repair Department, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (M.G.-V., C.B.-C., M.M.).
authors: Miguel
authors: M
authors: Manzanares
authors: From the Department of Anatomy, Embryology, and Physiology, Academic Medical Center, Amsterdam, The Netherlands (J.H.v.W., I.B., M.v.d.B., S.S., P.B., V.M.C.); Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands (H.J.G.v.d.W., W.d.L.); Department of Cell Biology, Erasmus MC Rotterdam, Rotterdam, The Netherlands (H.J.G.v.d.W.); and Cardiovascular Development and Repair Department, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (M.G.-V., C.B.-C., M.M.).
authors: Wouter
authors: W
authors: de Laat
authors: From the Department of Anatomy, Embryology, and Physiology, Academic Medical Center, Amsterdam, The Netherlands (J.H.v.W., I.B., M.v.d.B., S.S., P.B., V.M.C.); Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands (H.J.G.v.d.W., W.d.L.); Department of Cell Biology, Erasmus MC Rotterdam, Rotterdam, The Netherlands (H.J.G.v.d.W.); and Cardiovascular Development and Repair Department, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (M.G.-V., C.B.-C., M.M.).
authors: Phil
authors: P
authors: Barnett
authors: From the Department of Anatomy, Embryology, and Physiology, Academic Medical Center, Amsterdam, The Netherlands (J.H.v.W., I.B., M.v.d.B., S.S., P.B., V.M.C.); Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands (H.J.G.v.d.W., W.d.L.); Department of Cell Biology, Erasmus MC Rotterdam, Rotterdam, The Netherlands (H.J.G.v.d.W.); and Cardiovascular Development and Repair Department, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (M.G.-V., C.B.-C., M.M.). v.m.christoffels@amc.uva.nl.
authors: Vincent M
authors: VM
authors: Christoffels
mesh_terms: D024509:3' Flanking Region; D000818:Animals; D001665:Binding Sites; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D000076246:CCCTC-Binding Factor; D022202:Chromosomes, Artificial, Bacterial; D004270:DNA, Circular / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006329:Heart Conduction System / Q000196:embryology / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009024:Morphogenesis; D005810:Multigene Family; D012097:Repressor Proteins / Q000172:deficiency / Q000235:genetics; D015398:Signal Transduction; D020825:T-Box Domain Proteins / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D019485:Bone Morphogenetic Proteins; D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; C000619698:Ctcf protein, mouse; D004270:DNA, Circular; D012097:Repressor Proteins; D020825:T-Box Domain Proteins; C103172:T-box transcription factor 5; C491460:Tbx3 protein, mouse; D049452:Green Fluorescent Proteins
keywords: anatomy & histology; heart conduction system