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BACKGROUND - Heterotrimeric G proteins are fundamental signaling proteins composed of three subunits, Gα and a Gβγ dimer. The role of Gα as a molecular switch is critical for transmitting and amplifying intracellular signaling cascades initiated by an activated G protein Coupled Receptor (GPCR). Despite their biochemical and therapeutic importance, the study of G protein evolution has been limited to the scope of a few model organisms. Furthermore, of the five primary Gα subfamilies, the underlying gene structure of only two families has been thoroughly investigated outside of Mammalia evolution. Therefore our understanding of Gα emergence and evolution across phylogeny remains incomplete.
RESULTS - We have computationally identified the presence and absence of every Gα gene (GNA-) across all major branches of Deuterostomia and evaluated the conservation of the underlying exon-intron structures across these phylogenetic groups. We provide evidence of mutually exclusive exon inclusion through alternative splicing in specific lineages. Variations of splice site conservation and isoforms were found for several paralogs which coincide with conserved, putative motifs of DNA-/RNA-binding proteins. In addition to our curated gene annotations, within Primates, we identified 15 retrotranspositions, many of which have undergone pseudogenization. Most importantly, we find numerous deviations from previous findings regarding the presence and absence of individual GNA- genes, nuanced differences in phyla-specific gene copy numbers, novel paralog duplications and subsequent intron gain and loss events.
CONCLUSIONS - Our curated annotations allow us to draw more accurate inferences regarding the emergence of all Gα family members across Metazoa and to present a new, updated theory of Gα evolution. Leveraging this, our results are critical for gaining new insights into the co-evolution of the Gα subunit and its many protein binding partners, especially therapeutically relevant G protein - GPCR signaling pathways which radiated in Vertebrata evolution.
We performed a whole-genome scan of genetic variants in splicing regulatory elements (SREs) and evaluated the extent to which natural selection has shaped extant patterns of variation in SREs. We investigated the degree of differentiation of single nucleotide polymorphisms (SNPs) in SREs among human populations and applied long-range haplotype- and multilocus allelic differentiation-based methods to detect selection signatures. We describe an approach, sampling a large number of loci across the genome from functional classes and using the consensus from multiple tests, for identifying candidates for selection signals. SRE SNPs in various SNP functional classes show different patterns of population differentiation compared with their non-SRE counterparts. Intronic regions display a greater enrichment for extreme population differentiation among the potentially tissue-dependent transcript ratio quantitative trait loci (trQTLs) than SRE SNPs in general and includ outlier trQTLs for cross-population composite likelihood ratio, suggesting that incorporation of context annotation for regulatory variation may lead to improved detection of signature of selection on these loci. The proportion of extremely rare SNPs disrupting SREs is significantly higher in European than in African samples. The approach developed here will be broadly useful for studies of function and disease-associated variation in the human genome.
BACKGROUND - Enhancers are DNA regulatory elements that influence gene expression. There is substantial diversity in enhancers' activity patterns: some enhancers drive expression in a single cellular context, while others are active across many. Sequence characteristics, such as transcription factor (TF) binding motifs, influence the activity patterns of regulatory sequences; however, the regulatory logic through which specific sequences drive enhancer activity patterns is poorly understood. Recent analysis of Drosophila enhancers suggested that short dinucleotide repeat motifs (DRMs) are general enhancer sequence features that drive broad regulatory activity. However, it is not known whether the regulatory role of DRMs is conserved across species.
RESULTS - We performed a comprehensive analysis of the relationship between short DNA sequence patterns, including DRMs, and human enhancer activity in 38,538 enhancers across 411 different contexts. In a machine-learning framework, the occurrence patterns of short sequence motifs accurately predicted broadly active human enhancers. However, DRMs alone were weakly predictive of broad enhancer activity in humans and showed different enrichment patterns than in Drosophila. In general, GC-rich sequence motifs were significantly associated with broad enhancer activity, and consistent with this enrichment, broadly active human TFs recognize GC-rich motifs.
CONCLUSIONS - Our results reveal the importance of specific sequence motifs in broadly active human enhancers, demonstrate the lack of evolutionary conservation of the role of DRMs, and provide a computational framework for investigating the logic of enhancer sequences.
Inactivation of the Pancreatic and Duodenal Homeobox 1 (PDX1) gene causes pancreatic agenesis, which places PDX1 high atop the regulatory network controlling development of this indispensable organ. However, little is known about the identity of PDX1 transcriptional targets. We simulated pancreatic development by differentiating human embryonic stem cells (hESCs) into early pancreatic progenitors and subjected this cell population to PDX1 chromatin immunoprecipitation sequencing (ChIP-seq). We identified more than 350 genes bound by PDX1, whose expression was upregulated on day 17 of differentiation. This group included known PDX1 targets and many genes not previously linked to pancreatic development. ChIP-seq also revealed PDX1 occupancy at hepatic genes. We hypothesized that simultaneous PDX1-driven activation of pancreatic and repression of hepatic programs underlie early divergence between pancreas and liver. In HepG2 cells and differentiating hESCs, we found that PDX1 binds and suppresses expression of endogenous liver genes. These findings rebrand PDX1 as a context-dependent transcriptional repressor and activator within the same cell type.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
A cationic 7-aminomethyl-7-deaza-2'-deoxyguanosine (7amG) was incorporated site-specifically into the self-complementary duplex d(G¹A²G³A⁴X⁵C⁶G⁷C⁸T⁹C¹⁰T¹¹C¹²)₂ (X = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetry-related guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed that the tethered cationic amines were in plane with the modified base pairs. The tethered amines did not form salt bridges to the phosphodiester backbone. There was also no indication of the amines being capable of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function of temperature revealed that the X⁵ imino resonance remained sharp at 55 °C. Additionally, two 5'-neighboring base pairs, A⁴:T⁹ and G³:C¹⁰, were stabilized with respect to the exchange of their imino protons with solvent. The equilibrium constant for base pair opening at the A⁴:T⁹ base pair determined by magnetization transfer from water in the absence and presence of added ammonia base catalyst decreased for the modified duplex compared to that of the A⁴:T⁹ base pair in the unmodified duplex, which confirmed that the overall fraction of the A⁴:T⁹ base pair in the open state of the modified duplex decreased. This was also observed for the G³:C¹⁰ base pair, where αK(op) for the G³:C¹⁰ base pair in the modified duplex was 3.0 × 10⁶ versus 4.1 × 10⁶ for the same base pair in the unmodified duplex. In contrast, equilibrium constants for base pair opening at the X⁵:C⁸ and C⁶:G⁷ base pairs did not change at 15 °C. These results argue against the notion that electrostatic interactions with DNA are entirely entropic and suggest that major groove cations can stabilize DNA via enthalpic contributions to the free energy of duplex formation.
The human genome is pervasively transcribed, yet only a small fraction is coding. Here we address whether this non-coding transcription arises at promoters, and detail the interactions of initiation factors TATA box binding protein (TBP), transcription factor IIB (TFIIB) and RNA polymerase (Pol) II. Using ChIP-exo (chromatin immunoprecipitation with lambda exonuclease digestion followed by high-throughput sequencing), we identify approximately 160,000 transcription initiation complexes across the human K562 genome, and more in other cancer genomes. Only about 5% associate with messenger RNA genes. The remainder associates with non-polyadenylated non-coding transcription. Regardless, Pol II moves into a transcriptionally paused state, and TBP and TFIIB remain at the promoter. Remarkably, the vast majority of locations contain the four core promoter elements- upstream TFIIB recognition element (BREu), TATA, downstream TFIIB recognition element (BREd), and initiator element (INR)-in constrained positions. All but the INR also reside at Pol III promoters, where TBP makes similar contacts. This comprehensive and high-resolution genome-wide detection of the initiation machinery produces a consolidated view of transcription initiation events from yeast to humans at Pol II/III TATA-containing/TATA-less coding and non-coding genes.
BACKGROUND - Helicobacter pylori infection is a risk factor for the development of gastric cancer, and the bacterial oncoprotein CagA contributes to gastric carcinogenesis.
METHODS - We analyzed H. pylori isolates from persons in Colombia and observed that there was marked variation among strains in levels of CagA expression. To elucidate the basis for this variation, we analyzed sequences upstream from the CagA translational initiation site in each strain.
RESULTS - A DNA motif (AATAAGATA) upstream of the translational initiation site of CagA was associated with high levels of CagA expression. Experimental studies showed that this motif was necessary but not sufficient for high-level CagA expression. H. pylori strains from a region of Colombia with high gastric cancer rates expressed higher levels of CagA than did strains from a region with lower gastric cancer rates, and Colombian strains of European phylogeographic origin expressed higher levels of CagA than did strains of African origin. Histopathologic analysis of gastric biopsy specimens revealed that strains expressing high levels of CagA or containing the AATAAGATA motif were associated with more advanced precancerous lesions than those found in persons infected with strains expressing low levels of CagA or lacking the AATAAGATA motif.
CONCLUSIONS - CagA expression varies greatly among H. pylori strains. The DNA motif identified in this study is associated with high levels of CagA expression, and may be a useful biomarker to predict gastric cancer risk.
IMPACT - These findings help to explain why some persons infected with cagA-positive H. pylori develop gastric cancer and others do not.