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Genome-Wide Screens for In Vivo Tinman Binding Sites Identify Cardiac Enhancers with Diverse Functional Architectures

Jin, Hong and Stojnic, Robert and Adryan, Boris and Ozdemir, Anil and Stathopoulos, Angelike and Frasch, Manfred (2013) Genome-Wide Screens for In Vivo Tinman Binding Sites Identify Cardiac Enhancers with Diverse Functional Architectures. PLoS Genetics, 9 (1). Art. No. e1003195 . ISSN 1553-7390. PMCID PMC3542182. doi:10.1371/journal.pgen.1003195.

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The NK homeodomain factor Tinman is a crucial regulator of early mesoderm patterning and, together with the GATA factor Pannier and the Dorsocross T-box factors, serves as one of the key cardiogenic factors during specification and differentiation of heart cells. Although the basic framework of regulatory interactions driving heart development has been worked out, only about a dozen genes involved in heart development have been designated as direct Tinman target genes to date, and detailed information about the functional architectures of their cardiac enhancers is lacking. We have used immunoprecipitation of chromatin (ChIP) from embryos at two different stages of early cardiogenesis to obtain a global overview of the sequences bound by Tinman in vivo and their linked genes. Our data from the analysis of ~50 sequences with high Tinman occupancy show that the majority of such sequences act as enhancers in various mesodermal tissues in which Tinman is active. All of the dorsal mesodermal and cardiac enhancers, but not some of the others, require tinman function. The cardiac enhancers feature diverse arrangements of binding motifs for Tinman, Pannier, and Dorsocross. By employing these cardiac and non-cardiac enhancers in machine learning approaches, we identify a novel motif, termed CEE, as a classifier for cardiac enhancers. In vivo assays for the requirement of the binding motifs of Tinman, Pannier, and Dorsocross, as well as the CEE motifs in a set of cardiac enhancers, show that the Tinman sites are essential in all but one of the tested enhancers; although on occasion they can be functionally redundant with Dorsocross sites. The enhancers differ widely with respect to their requirement for Pannier, Dorsocross, and CEE sites, which we ascribe to their different position in the regulatory circuitry, their distinct temporal and spatial activities during cardiogenesis, and functional redundancies among different factor binding sites.

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Stathopoulos, Angelike0000-0001-6597-2036
Additional Information:© 2013 Jin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: August 26, 2012; Accepted: November 8, 2012; Published: January 10, 2013. BA is a Royal Society University Research Fellow ( and RS wishes to acknowledge the support of a Cambridge International Fellowship (​entregistry/fees/funding/ciss). MF was partially funded by NIH HD30832/HD/NICHD ( for this work. AO was supported by NIH GM077668 granted to AS ( The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would like to thank Xiao-Yong Li and Mark Biggin for generously providing us with unpublished protocols and ChIP–chip data, John Manak for support with early access to the Affymetrix microarrays, the Mount Sinai Microarray Core Facility for performing the hybridizations, Manoj Samanta for performing the initial array analysis, and Tuomas Brock for technical support. Author Contributions: Conceived and designed the experiments: HJ RS BA AO AS MF. Performed the experiments: HJ RS AO. Analyzed the data: HJ RS BA AO AS MF. Wrote the paper: HJ RS MF.
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Cambridge International FellowshipUNSPECIFIED
Issue or Number:1
PubMed Central ID:PMC3542182
Record Number:CaltechAUTHORS:20130307-111819240
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Official Citation:Jin H, Stojnic R, Adryan B, Ozdemir A, Stathopoulos A, et al. (2013) Genome-Wide Screens for In Vivo Tinman Binding Sites Identify Cardiac Enhancers with Diverse Functional Architectures. PLoS Genet 9(1): e1003195. doi:10.1371/journal.pgen.1003195
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:37371
Deposited On:07 Mar 2013 21:14
Last Modified:09 Nov 2021 23:28

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