Rationale : 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. Methods and 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.

BackgroundRecent genome-wide association studies have uncovered genomic loci that underlie an increased risk for atrial fibrillation, the major cardiac arrhythmia in humans. The most significant locus is located in a gene desert at 4q25, approximately 170 kilobases upstream of PITX2, which codes for a transcription factor involved in embryonic left-right asymmetry and cardiac development. However, how this genomic region functionally and structurally relates to PITX2 and atrial fibrillation is unknown.ResultsTo characterise its function, we tested genomic fragments from 4q25 for transcriptional activity in a mouse atrial cardiomyocyte cell line and in transgenic mouse embryos, identifying a non-tissue-specific potentiator regulatory element. Chromosome conformation capture revealed that this region physically interacts with the promoter of the cardiac specific isoform of Pitx2. Surprisingly, this regulatory region also interacts with the promoter of the next neighbouring gene, Enpep, which we show to be expressed in regions of the developing mouse heart essential for cardiac electrical activity.ConclusionsOur data suggest that de-regulation of both PITX2 and ENPEP could contribute to an increased risk of atrial fibrillation in carriers of disease-associated variants, and show the challenges that we face in the functional analysis of genome-wide disease associations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0138-0) contains supplementary material, which is available to authorized users.

Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development.

SUMMARYThe apical ectodermal ridge (AER) is a specialized epithelium located at the distal edge of the limb bud that directs outgrowth along the proximodistal axis. Although the molecular basis for its function is well known, the cellular mechanisms that lead to its maturation are not fully understood. Here, we show that Arid3b, a member of the ARID family of transcriptional regulators, is expressed in the AER in mouse and chick embryos, and that interference with its activity leads to aberrant AER development, in which normal structure is not achieved. This happens without alterations in cell numbers or gene expression in main signalling pathways. Cells that are defective in Arid3b show an abnormal distribution of the actin cytoskeleton and decreased motility in vitro. Moreover, movements of pre-AER cells and their contribution to the AER were defective in vivo in embryos with reduced Arid3b function. Our results show that Arid3b is involved in the regulation of cell motility and rearrangements that lead to AER maturation.

Highlights d MEFs lacking STAG2 show reduced proliferation and mild cohesion defects d Stag2 loss in adult mice reduces fitness but does not increase tumor incidence d Stag2 KO embryos die by E10.5 with global developmental delay and malformed hearts

Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that Oct4 is necessary for activation of HoxB genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the HoxB cluster is coordinately regulated by OCT4 binding sites located at the 3′ end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs.

Pluripotent cells are a transient population present in the early mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while simultaneously suppressing lineage specification. Interestingly, these factors are not exclusive to uncommitted cells, but are also expressed during early phases of differentiation. However, their role in the transition from pluripotency to lineage specification is largely unknown. Using genetic models for controlled Oct4 or Nanog expression during postimplantation stages, we found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that the HoxB cluster is coordinately regulated in this way by OCT4 binding sites located at the 3' end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the pre-committed epiblast, but are also necessary for the proper deployment of subsequent developmental programs. Dual regulation of Hox genes by pluripotency factors Lopez-Jimenez et al. 3

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