SUMMARY Heart development is exquisitely sensitive to the precise temporal regulation of thousands of genes that govern developmental decisions during differentiation. However, we currently lack a detailed understanding of how chromatin and gene expression patterns are coordinated during developmental transitions in the cardiac lineage. Here, we interrogated the transcriptome and several histone modifications across the genome during defined stages of cardiac differentiation. We find distinct chromatin patterns that are coordinated with stage-specific expression of functionally related genes, including many human disease-associated genes. Moreover, we discover a novel pre-activation chromatin pattern at the promoters of genes associated with heart development and cardiac function. We further identify stage-specific distal enhancer elements and find enriched DNA binding motifs within these regions that predict sets of transcription factors that orchestrate cardiac differentiation. Together, these findings form a basis for understanding developmentally regulated chromatin transitions during lineage commitment and the molecular etiology of congenital heart disease.

Dysmorphogenesis of the cardiac outflow tract (OFT) causes many congenital heart defects, including those associated with DiGeorge syndrome. Genetic manipulation in the mouse and mutational analysis in patients have shown that Tbx1, a T-box transcription factor, has a key role in the pathogenesis of this syndrome. Here, we have dissected Tbx1 function during OFT development using genetically modified mice and tissue-specific deletion, and have defined a dual role for this protein in OFT morphogenesis. We show that Tbx1 regulates cell contribution to the OFT by supporting cell proliferation in the secondary heart field, a source of cells fated to the OFT. This process might be regulated in part by Fgf10, which we show for the first time to be a direct target of Tbx1 in vitro. We also show that Tbx1 expression is required in cells expressing Nkx2.5 for the formation of the aortopulmonary septum, which divides the aorta from the main pulmonary artery. These results explain why aortic arch patterning defects and OFT defects can occur independently in individuals with DiGeorge syndrome. Furthermore, our data link, for the first time, the function of the secondary heart field to congenital heart disease.Key words: Tbx1, Mouse, Outflow tract, DiGeorge syndrome SummaryTbx1 has a dual role in the morphogenesis of the cardiac outflow tract

Transcriptional cascades responsible for initiating the formation of vertebrate embryonic structures such as limbs are not well established. Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations along the anteroposterior axis of the embryo is not known. The T-box transcription factor TBX5 is important for normal heart and limb formation, but its role in early limb development is not well defined. We report that mouse embryos lacking Tbx5 do not form forelimb buds, although the patterning of the lateral plate mesoderm into the limb field is intact. Tbx5 is not essential for an early establishment of forelimb versus hindlimb identity. In the absence of Tbx5, the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression. Tbx5 directly activates the Fgf10 gene via a conserved binding site, providing a simple and direct mechanism for limb bud initiation. Lef1/Tcf1-dependent Wnt signaling is not essential for initiation of Tbx5 or Fgf10 transcription, but is required in concert with Tbx5 for maintenance of normal levels of Fgf10 expression. We conclude that Tbx5 is not essential for the early establishment of the limb field in the lateral plate mesoderm but is a primary and direct initiator of forelimb bud formation. These data suggest common pathways for the differentiation and growth of embryonic structures downstream of T-box genes.

Dominant mutations in cardiac transcription factor genes cause human inherited congenital heart defects (CHDs); however, their molecular basis is not understood. Interactions between transcription factors and the Brg1/Brm-associated factor (BAF) chromatin remodelling complex suggest potential mechanisms; however, the role of BAF complexes in cardiogenesis is not known. In this study, we show that dosage of Brg1 is critical for mouse and zebrafish cardiogenesis. Disrupting the balance between Brg1 and disease-causing cardiac transcription factors, including Tbx5, Tbx20 and Nkx2–5, causes severe cardiac anomalies, revealing an essential allelic balance between Brg1 and these cardiac transcription factor genes. This suggests that the relative levels of transcription factors and BAF complexes are important for heart development, which is supported by reduced occupancy of Brg1 at cardiac gene promoters in Tbx5 haploinsufficient hearts. Our results reveal complex dosage-sensitive interdependence between transcription factors and BAF complexes, providing a potential mechanism underlying transcription factor haploinsufficiency, with implications for multigenic inheritance of CHDs.

Rapid electrical conduction in the His-Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification and morphogenesis of the His-Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction-forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. Irx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for Irx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart. development | electrophysiology | transcription factor

SUMMARYThe Iroquois homeobox (Irx) homeodomain transcription factors are important for several aspects of embryonic development. In the developing heart, individual Irx genes are important for certain postnatal cardiac functions, including cardiac repolarization (Irx5) and rapid ventricular conduction (Irx3). Irx genes are expressed in dynamic and partially overlapping patterns in the developing heart. Here we show in mice that Irx3 and Irx5 have redundant function in the endocardium to regulate atrioventricular canal morphogenesis and outflow tract formation. Our data suggest that direct transcriptional repression of Bmp10 by Irx3 and Irx5 in the endocardium is required for ventricular septation. A postnatal deletion of Irx3 and Irx5 in the myocardium leads to prolongation of atrioventricular conduction, due in part to activation of expression of the Na + channel protein Nav1.5. Surprisingly, combined postnatal loss of Irx3 and Irx5 results in a restoration of the repolarization gradient that is altered in Irx5 mutant hearts, suggesting that postnatal Irx3 activity can be repressed by Irx5. Our results have uncovered complex genetic interactions between Irx3 and Irx5 in embryonic cardiac development and postnatal physiology.

The accurate and comprehensive identification of functional regulatory sequences in mammalian genomes remains a major challenge. Here we describe Site-specific Integration FACS-sequencing (SIF-seq), an unbiased, medium-throughput functional assay for the discovery of distant-acting enhancers. Pluripotent cell reporter assays, targeted single-copy genomic integration, and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences. We demonstrate the utility of this method by functionally interrogating >500 kb of mouse and human sequence for enhancer activity and identifying embryonic stem (ES) cell enhancers at pluripotency loci including NANOG. We also demonstrate the effectiveness of the approach in differentiated cell populations through the identification of cardiac enhancers from cardiomyocytes and neuronal enhancers from neural progenitors. SIF-seq is a powerful and flexible method for the de novo functional identification of mammalian enhancers in a potentially wide variety of cell types.

We studied the effects of 17 beta-estradiol (E2) and PTH on alkaline phosphatase activity in SaOS-2 cells that had been passaged and grown continually in the presence (SaOS + Dex) or absence (SaOS - Dex) of 10(-8) M dexamethasone (Dex). We showed that the more differentiated SaOS + Dex cells had higher alkaline phosphatase activity and PTH-responsive adenylate cyclase than the less differentiated SaOS - Dex cells. In SaOS - Dex cells, E2 or PTH (1 x 10(-11)-1 x 10(-6) M) had no effect on alkaline phosphatase activity. On the other hand, in SaOS + Dex cells, PTH and E2 each had small but very significant stimulatory effects on alkaline phosphatase activity. The combined effects of PTH and E2 resulted in potentiation which was dose dependent for PTH and E2. 17 alpha-estradiol and tamoxifen (1 x 10(-11)-1 x 10(-6) M) had no effect on PTH-stimulated alkaline phosphatase activity in SaOS + Dex cells, but the latter inhibited the E2 effect, clearly demonstrating its specificity. In conclusion, we have shown that in more differentiated osteoblastic cells, E2 and PTH have interactive effects on alkaline phosphatase activity.