SUMMARY Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects.

The genome encodes information to program an organism's development and maintenance, and its decoding begins with regulated transcription of genomic DNA into RNA. Transcription and its control can be tracked indirectly by measuring stable RNAs, or directly by measuring nascent RNAs. The latter reveals the immediate regulatory changes in response to developmental, environmental, disease, and metabolic signals. Multiple complementary methods have been developed to quantitatively track nascent transcription genome-wide at nucleotide-resolution, providing novel insights to mechanisms of gene regulation and transcription-coupled RNA processing. Here, we critically evaluate the array of strategies used for investigating nascent transcription and discuss recent conceptual advances they provide.

Eukaryotic RNA Polymerase II has been found at both promoters and distal enhancers, suggesting additional functions beyond mRNA production. To understand this role, we sequenced nascent RNAs at single-molecule resolution to unravel the interplay between Pol2 initiation, capping, and pausing genome-wide. Our analyses reveal two pause classes that are associated with different RNA capping profiles. More proximal pausing is associated with less complete capping, less elongation, and a more enhancer-like complement of transcription factors than later pausing. Unexpectedly, Transcription Start Sites (TSSes) are predominantly found in constellations composed of multiple divergent pairs. TSS clusters are intimately associated with precise arrays of nucleosomes, and correspond with boundaries of transcription factor binding and chromatin modification at promoters and enhancers. TSS architecture is remarkably similar after the dramatic transcriptional changes caused by heat‬ stress. Together, our results suggest that promoter- and enhancer-associated Pol2 is a regulatory nexus for integrating information across TSS ensembles.

Following the discovery of widespread enhancer transcription, enhancers and promoters have been found to be far more similar than previously thought. In this issue of , two studies (Henriques and colleagues [pp. 26-41] and Mikhaylichenko and colleagues [pp. 42-57]) shine new light on the transcriptional nature of promoters and enhancers in Together, these studies support recent work in mammalian cells that indicates that most active enhancers drive local transcription using factors and mechanisms similar to those of promoters. Intriguingly, enhancer transcription is shown to be coordinated by SPT5- and P-TEFb-mediated pause-release, but the pause half-life is shorter, and termination is more rapid at enhancers than at promoters. Moreover, bidirectional transcription from promoters is associated with enhancer activity, lending further credence to models in which regulatory elements exist along a spectrum of promoter-ness and enhancer-ness. We propose a general unified model to explain possible functions of transcription at enhancers.