Genome-Wide Screens for In Vivo Tinman Binding Sites Identify Cardiac Enhancers with Diverse Functional Architectures
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. The Drosophila homeodomain protein Tinman was the first transcription factor found to control the development and differentiation of the heart in any species. In spite of that, our knowledge of the number, identities, and mode of regulation of the downstream target genes of Tinman that are necessary to exert its cardiogenic functions is still very incomplete. To address these issues, we have performed a genome-wide analysis of DNA regions associated with Tinman-binding in embryos and the genes linked to them. The combined data from our in-depth in vivo assays of sequence elements with high Tinman occupancy allow the following general conclusions: (1) The majority of such sequences are active as regulatory elements (called enhancers) in mesodermal tissues that include Tinman-expressing cells. (2) The enhancers active in the heart progenitor cells and the heart generally are dependent on tinman gene activity, whereas those active in non-cardiac mesoderm are often bound neutrally by Tinman. (3) Tinman binding motifs in most cases are essential for cardiac enhancer activity, but in some cases they can be functionally-redundant with those of other cardiogenic factors. (4) Tinman-occupied cardiac enhancers are enriched for a newly discovered binding motif for an unknown factor that is functional in vivo.