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.
Data sets and code for all conservation measures evaluated are available at http://compbio.cs.princeton.edu/conservation/
G-quadruplex (G4) DNA structures are extremely stable four-stranded secondary structures held together by non-canonical G-G base pairs. Genome-wide chromatin immuno-precipitation was used to determine the in vivo binding sites of the multi-functional S. cerevisiae Pif1 DNA helicase, a potent unwinder of G4 structures in vitro. G4 motifs were a significant subset of the high confidence Pif1 binding sites. Replication slowed in the vicinity of these motifs, and they were prone to breakage in Pif1-deficient cells, while non G4 Pif1 binding sites did not show this behavior. Introducing many copies of G4 motifs caused slow growth in replication stressed Pif1 deficient cells that was relieved by spontaneous mutations that eliminated their ability to form G4 structures, bind Pif1, slow DNA replication and stimulate DNA breakage. These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to prevent replication fork stalling and DNA breakage.
Identifying a protein's functional sites is an important step towards characterizing its molecular function. Numerous structure- and sequence-based methods have been developed for this problem. Here we introduce ConCavity, a small molecule binding site prediction algorithm that integrates evolutionary sequence conservation estimates with structure-based methods for identifying protein surface cavities. In large-scale testing on a diverse set of single- and multi-chain protein structures, we show that ConCavity substantially outperforms existing methods for identifying both 3D ligand binding pockets and individual ligand binding residues. As part of our testing, we perform one of the first direct comparisons of conservation-based and structure-based methods. We find that the two approaches provide largely complementary information, which can be combined to improve upon either approach alone. We also demonstrate that ConCavity has state-of-the-art performance in predicting catalytic sites and drug binding pockets. Overall, the algorithms and analysis presented here significantly improve our ability to identify ligand binding sites and further advance our understanding of the relationship between evolutionary sequence conservation and structural and functional attributes of proteins. Data, source code, and prediction visualizations are available on the ConCavity web site (http://compbio.cs.princeton.edu/concavity/).
Many modern human genomes retain DNA inherited from interbreeding with archaic hominins, such as Neanderthals, yet the influence of this admixture on human traits is largely unknown. We analyzed the contribution of common Neanderthal variants to over 1,000 electronic health record (EHR)-derived phenotypes in ~28,000 adults of European ancestry. We discovered and replicated associations of Neanderthal alleles with neurological, psychiatric, immunological, and dermatological phenotypes. Neanderthal alleles together explain a significant fraction of the variation in risk for depression and skin lesions resulting from sun exposure (actinic keratosis), and individual Neanderthal alleles are significantly associated with specific human phenotypes, including hypercoagulation and tobacco use. Our results establish that archaic admixture influences disease risk in modern humans, provide hypotheses about the effects of hundreds of Neanderthal haplotypes and demonstrate the utility of EHR data in evolutionary analyses.
The genetic changes underlying the dramatic differences in form and function between humans and other primates are largely unknown, although it is clear that gene regulatory changes play an important role. To identify regulatory sequences with potentially human-specific functions, we and others used comparative genomics to find non-coding regions conserved across mammals that have acquired many sequence changes in humans since divergence from chimpanzees. These regions are good candidates for performing human-specific regulatory functions. Here, we analysed the DNA sequence, evolutionary history, histone modifications, chromatin state and transcription factor (TF) binding sites of a combined set of 2649 non-coding human accelerated regions (ncHARs) and predicted that at least 30% of them function as developmental enhancers. We prioritized the predicted ncHAR enhancers using analysis of TF binding site gain and loss, along with the functional annotations and expression patterns of nearby genes. We then tested both the human and chimpanzee sequence for 29 ncHARs in transgenic mice, and found 24 novel developmental enhancers active in both species, 17 of which had very consistent patterns of activity in specific embryonic tissues. Of these ncHAR enhancers, five drove expression patterns suggestive of different activity for the human and chimpanzee sequence at embryonic day 11.5. The changes to human non-coding DNA in these ncHAR enhancers may modify the complex patterns of gene expression necessary for proper development in a human-specific manner and are thus promising candidates for understanding the genetic basis of human-specific biology.
G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs) across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs). Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.
SUMMARY Sirtuins are NAD+-dependent protein deacylases that regulate several aspects of metabolism and aging. In contrast to the other mammalian sirtuins, the primary enzymatic activity of mitochondrial sirtuin 4 (SIRT4) and its overall role in metabolic control has remained enigmatic. Using a combination of phylogenetics, structural biology, and enzymology, we show that SIRT4 removes three acyl moieties from lysine residues – methylglutaryl(MG)-, hydroxymethylglutaryl(HMG)-, and 3-methylglutaconyl(MGc)-lysine. The metabolites leading to these post-translational modifications are intermediates in leucine oxidation, and we show a primary role for SIRT4 in controlling this pathway in mice. Furthermore, we find that dysregulated leucine metabolism in SIRT4KO mice leads to elevated basal and stimulated insulin secretion, which progressively develops into glucose intolerance and insulin resistance. These findings identify a robust enzymatic activity for SIRT4, uncover a mechanism controlling branched-chain amino acid flux, and position SIRT4 as a crucial player maintaining insulin secretion and glucose homeostasis during aging.
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