Dynamic and coordinated epigenetic regulation of developmental transitions in the cardiac lineage.

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 preactivation 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.

Brg1 modulates enhancer activation in mesoderm lineage commitment.

The interplay between different levels of gene regulation in modulating developmental transcriptional programs, such as histone modifications and chromatin remodeling, is not well understood. Here, we show that the chromatin remodeling factor Brg1 is required for enhancer activation in mesoderm induction. In an embryonic stem cell-based directed differentiation assay, the absence of Brg1 results in a failure of cardiomyocyte differentiation and broad deregulation of lineage-specific gene expression during mesoderm induction. We find that Brg1 co-localizes with H3K27ac at distal enhancers and is required for robust H3K27 acetylation at distal enhancers that are activated during mesoderm induction. Brg1 is also required to maintain Polycomb-mediated repression of non-mesodermal developmental regulators, suggesting cooperativity between Brg1 and Polycomb complexes. Thus, Brg1 is essential for modulating active and repressive chromatin states during mesoderm lineage commitment, in particular the activation of developmentally important enhancers. These findings demonstrate interplay between chromatin remodeling complexes and histone modifications that, together, ensure robust and broad gene regulation during crucial lineage commitment decisions.

IgG-cleaving endopeptidase enables in vivo gene therapy in the presence of anti-AAV neutralizing antibodies.

Neutralizing antibodies to adeno-associated virus (AAV) vectors are highly prevalent in humans1,2, and block liver transduction3-5 and vector readministration6; thus, they represent a major limitation to in vivo gene therapy. Strategies aimed at overcoming anti-AAV antibodies are being studied7, which often involve immunosuppression and are not efficient in removing pre-existing antibodies. Imlifidase (IdeS) is an endopeptidase able to degrade circulating IgG that is currently being tested in transplant patients8. Here, we studied if IdeS could eliminate anti-AAV antibodies in the context of gene therapy. We showed efficient cleavage of pooled human IgG (intravenous Ig) in vitro upon endopeptidase treatment. In mice passively immunized with intravenous Ig, IdeS administration decreased anti-AAV antibodies and enabled efficient liver gene transfer. The approach was scaled up to nonhuman primates, a natural host for wild-type AAV. IdeS treatment before AAV vector infusion was safe and resulted in enhanced liver transduction, even in the setting of vector readministration. Finally, IdeS reduced anti-AAV antibody levels from human plasma samples in vitro, including plasma from prospective gene therapy trial participants. These results provide a potential solution to overcome pre-existing antibodies to AAV-based gene therapy.

Live-cell imaging reveals enhancer-dependent Sox2 transcription in the absence of enhancer proximity.

Enhancers are important regulatory elements that can control gene activity across vast genetic distances. However, the underlying nature of this regulation remains obscured because it has been difficult to observe in living cells. Here, we visualize the spatial organization and transcriptional output of the key pluripotency regulator Sox2 and its essential enhancer Sox2 Control Region (SCR) in living embryonic stem cells (ESCs). We find that Sox2 and SCR show no evidence of enhanced spatial proximity and that spatial dynamics of this pair is limited over tens of minutes. Sox2 transcription occurs in short, intermittent bursts in ESCs and, intriguingly, we find this activity demonstrates no association with enhancer proximity, suggesting that direct enhancer-promoter contacts do not drive contemporaneous Sox2 transcription. Our study establishes a framework for interrogation of enhancer function in living cells and supports an unexpected mechanism for enhancer control of Sox2 expression that uncouples transcription from enhancer proximity.

Insights into nuclear dynamics using live-cell imaging approaches.

The nucleus contains the genetic blueprint of the cell and myriad interactions within this subcellular structure are required for gene regulation. In the current scientific era, characterization of these gene regulatory networks through biochemical techniques coupled with systems-wide 'omic' approaches has become commonplace. However, these strategies are limited because they represent a mere snapshot of the cellular state. To obtain a holistic understanding of nuclear dynamics, relevant molecules must be studied in their native contexts in living systems. Live-cell imaging approaches are capable of providing quantitative assessment of the dynamics of gene regulatory interactions within the nucleus. We survey recent insights into what live-cell imaging approaches have provided the field of nuclear dynamics. In this review, we focus on interactions of DNA with other DNA loci, proteins, RNA, and the nuclear envelope. WIREs Syst Biol Med 2017, 9:e1372. doi: 10.1002/wsbm.1372 For further resources related to this article, please visit the WIREs website.

Chromatin remodelling complex dosage modulates transcription factor function in heart development.

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.

Lessons for cardiac regeneration and repair through development.

Cell-based regenerative strategies have the potential to revolutionize the way cardiovascular injury is treated, but successful therapies will require a precise understanding of the mechanisms that dictate cell fate, survival and differentiation. Recent advances in the study of cardiac development hold promise for unlocking the keys for successful therapies. Using mouse models and embryonic stem cells, researchers are uncovering cardiac progenitor cells in both embryonic and adult contexts. Furthermore, the signaling molecules and transcriptional regulators that govern these cells and their behavior are being revealed. Here, we focus on the recent advances in these areas of cardiac developmental research and their impact on the expanding field of regenerative medicine.