Journal Articles

Solution structure of a DNA quadruplex containing ALS and FTD related GGGGCC repeat stabilized by 8-bromodeoxyguanosine substitution

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

A prolonged expansion of GGGGCC repeat within non-coding region of C9orf72 gene has been identified as the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are devastating neurodegenerative disorders. Formation of unusual secondary structures within expanded GGGGCC repeat, including DNA and RNA G-quadruplexes and R-loops was proposed to drive ALS and FTD pathogenesis. Initial NMR investigation on DNA oligonucleotides with four repeat units as the shortest model with the ability to form an unimolecular G-quadruplex indicated their folding into multiple G-quadruplex structures in the presence of K+ ions. Single dG to 8Br-dG substitution at position 21 in oligonucleotide d[(G4C2)3G4] and careful optimization of folding conditions enabled formation of mostly a single G-quadruplex species, which enabled determination of a high-resolution structure with NMR. G-quadruplex structure adopted by d[(G4C2)3GGBrGG] is composed of four G-quartets, which are connected by three edgewise C-C loops. All four strands adopt antiparallel orientation to one another and have alternating syn-anti progression of glycosidic conformation of guanine residues. One of the cytosines in every loop is stacked upon the G-quartet contributing to a very compact and stable structure.

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Structural basis for selective targeting of leishmanial ribosomes: aminoglycoside derivatives as promising therapeutics

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania, resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)—the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We also evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3, as a prospective therapeutic candidate for the treatment of VL.

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Nucleic Acids Research - Tue, 09/29/2015 - 01:41
Categories: Journal Articles

LARVA: an integrative framework for large-scale analysis of recurrent variants in noncoding annotations

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

In cancer research, background models for mutation rates have been extensively calibrated in coding regions, leading to the identification of many driver genes, recurrently mutated more than expected. Noncoding regions are also associated with disease; however, background models for them have not been investigated in as much detail. This is partially due to limited noncoding functional annotation. Also, great mutation heterogeneity and potential correlations between neighboring sites give rise to substantial overdispersion in mutation count, resulting in problematic background rate estimation. Here, we address these issues with a new computational framework called LARVA. It integrates variants with a comprehensive set of noncoding functional elements, modeling the mutation counts of the elements with a β-binomial distribution to handle overdispersion. LARVA, moreover, uses regional genomic features such as replication timing to better estimate local mutation rates and mutational hotspots. We demonstrate LARVA's effectiveness on 760 whole-genome tumor sequences, showing that it identifies well-known noncoding drivers, such as mutations in the TERT promoter. Furthermore, LARVA highlights several novel highly mutated regulatory sites that could potentially be noncoding drivers. We make LARVA available as a software tool and release our highly mutated annotations as an online resource (larva.gersteinlab.org).

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Optimizing RNA structures by sequence extensions using RNAcop

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

A key aspect of RNA secondary structure prediction is the identification of novel functional elements. This is a challenging task because these elements typically are embedded in longer transcripts where the borders between the element and flanking regions have to be defined. The flanking sequences impact the folding of the functional elements both at the level of computational analyses and when the element is extracted as a transcript for experimental analysis. Here, we analyze how different flanking region lengths impact folding into a constrained structure by computing probabilities of folding for different sizes of flanking regions. Our method, RNAcop (RNA context optimization by probability), is tested on known and de novo predicted structures. In vitro experiments support the computational analysis and suggest that for a number of structures, choosing proper lengths of flanking regions is critical. RNAcop is available as web server and stand-alone software via http://rth.dk/resources/rnacop.

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Local sequence assembly reveals a high-resolution profile of somatic structural variations in 97 cancer genomes

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

Genomic structural variations (SVs) are pervasive in many types of cancers. Characterizing their underlying mechanisms and potential molecular consequences is crucial for understanding the basic biology of tumorigenesis. Here, we engineered a local assembly-based algorithm (laSV) that detects SVs with high accuracy from paired-end high-throughput genomic sequencing data and pinpoints their breakpoints at single base-pair resolution. By applying laSV to 97 tumor-normal paired genomic sequencing datasets across six cancer types produced by The Cancer Genome Atlas Research Network, we discovered that non-allelic homologous recombination is the primary mechanism for generating somatic SVs in acute myeloid leukemia. This finding contrasts with results for the other five types of solid tumors, in which non-homologous end joining and microhomology end joining are the predominant mechanisms. We also found that the genes recursively mutated by single nucleotide alterations differed from the genes recursively mutated by SVs, suggesting that these two types of genetic alterations play different roles during cancer progression. We further characterized how the gene structures of the oncogene JAK1 and the tumor suppressors KDM6A and RB1 are affected by somatic SVs and discussed the potential functional implications of intergenic SVs.

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Cis-acting signals modulate the efficiency of programmed DNA elimination in Paramecium tetraurelia

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

In Paramecium, the regeneration of a functional somatic genome at each sexual event relies on the elimination of thousands of germline DNA sequences, known as Internal Eliminated Sequences (IESs), from the zygotic nuclear DNA. Here, we provide evidence that IESs’ length and sub-terminal bases jointly modulate IES excision by affecting DNA conformation in P. tetraurelia. Our study reveals an excess of complementary base pairing between IESs’ sub-terminal and contiguous sites, suggesting that IESs may form DNA loops prior to cleavage. The degree of complementary base pairing between IESs’ sub-terminal sites (termed Cin-score) is positively associated with IES length and is shaped by natural selection. Moreover, it escalates abruptly when IES length exceeds 45 nucleotides (nt), indicating that only sufficiently large IESs may form loops. Finally, we find that IESs smaller than 46 nt are favored targets of the cellular surveillance systems, presumably because of their relatively inefficient excision. Our findings extend the repertoire of cis-acting determinants for IES recognition/excision and provide unprecedented insights into the distinct selective pressures that operate on IESs and somatic DNA regions. This information potentially moves current models of IES evolution and of mechanisms of IES recognition/excision forward.

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The mRNA related ceRNA-ceRNA landscape and significance across 20 major cancer types

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

Cross-talk between competitive endogenous RNAs (ceRNAs) through shared miRNAs represents a novel layer of gene regulation that plays important roles in the physiology and development of cancers. However, a global view of their system-level properties across various types of cancers is still unknown. Here, we constructed the mRNA related ceRNA–ceRNA interaction landscape across 20 cancer types by systematically analyzing molecular profiles of 5203 tumors and miRNA regulations. Our study highlights the conserved features shared by pan-cancer and higher similarity within similar origin cell type. Moreover, a core ceRNA network was identified. Function analysis identified a common theme of cancer hallmarks, however they exhibit phenotype-specific connectivity patterns. Besides, we found a marked rewiring in the ceRNA program between various cancers, and further revealed conserved and rewired network ceRNA hubs in each cancer, which were tensely competitive interactions to constitute conserved and cancer-specific modules. By providing mechanistic linkage between known cancer miRNAs, their mediated ceRNA–ceRNA interactions, and the associations with known cancer hallmarks, the inferred cancer ceRNA–ceRNA interaction landscape will serve as a powerful public resource for further biological discoveries of tumorigenesis.

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The transcription factors SOX9 and SOX5/SOX6 cooperate genome-wide through super-enhancers to drive chondrogenesis

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

SOX9 is a transcriptional activator required for chondrogenesis, and SOX5 and SOX6 are closely related DNA-binding proteins that critically enhance its function. We use here genome-wide approaches to gain novel insights into the full spectrum of the target genes and modes of action of this chondrogenic trio. Using the RCS cell line as a faithful model for proliferating/early prehypertrophic growth plate chondrocytes, we uncover that SOX6 and SOX9 bind thousands of genomic sites, frequently and most efficiently near each other. SOX9 recognizes pairs of inverted SOX motifs, whereas SOX6 favors pairs of tandem SOX motifs. The SOX proteins primarily target enhancers. While binding to a small fraction of typical enhancers, they bind multiple sites on almost all super-enhancers (SEs) present in RCS cells. These SEs are predominantly linked to cartilage-specific genes. The SOX proteins effectively work together to activate these SEs and are required for in vivo expression of their associated genes. These genes encode key regulatory factors, including the SOX trio proteins, and all essential cartilage extracellular matrix components. Chst11, Fgfr3, Runx2 and Runx3 are among many other newly identified SOX trio targets. SOX9 and SOX5/SOX6 thus cooperate genome-wide, primarily through SEs, to implement the growth plate chondrocyte differentiation program.

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The interplay between DNA methylation and sequence divergence in recent human evolution

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

Despite the increasing knowledge about DNA methylation, the understanding of human epigenome evolution is in its infancy. Using whole genome bisulfite sequencing we identified hundreds of differentially methylated regions (DMRs) in humans compared to non-human primates and estimated that ~25% of these regions were detectable throughout several human tissues. Human DMRs were enriched for specific histone modifications and the majority were located distal to transcription start sites, highlighting the importance of regions outside the direct regulatory context. We also found a significant excess of endogenous retrovirus elements in human-specific hypomethylated.

We reported for the first time a close interplay between inter-species genetic and epigenetic variation in regions of incomplete lineage sorting, transcription factor binding sites and human differentially hypermethylated regions. Specifically, we observed an excess of human-specific substitutions in transcription factor binding sites located within human DMRs, suggesting that alteration of regulatory motifs underlies some human-specific methylation patterns. We also found that the acquisition of DNA hypermethylation in the human lineage is frequently coupled with a rapid evolution at nucleotide level in the neighborhood of these CpG sites. Taken together, our results reveal new insights into the mechanistic basis of human-specific DNA methylation patterns and the interpretation of inter-species non-coding variation.

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Chromosomal position shift of a regulatory gene alters the bacterial phenotype

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

Recent studies strongly suggest that in bacterial cells the order of genes along the chromosomal origin-to-terminus axis is determinative for regulation of the growth phase-dependent gene expression. The prediction from this observation is that positional displacement of pleiotropic genes will affect the genetic regulation and hence, the cellular phenotype. To test this prediction we inserted the origin-proximal dusB-fis operon encoding the global regulator FIS in the vicinity of replication terminus on both arms of the Escherichia coli chromosome. We found that the lower fis gene dosage in the strains with terminus-proximal dusB-fis operons was compensated by increased fis expression such that the intracellular concentration of FIS was homeostatically adjusted. Nevertheless, despite unchanged FIS levels the positional displacement of dusB-fis impaired the competitive growth fitness of cells and altered the state of the overarching network regulating DNA topology, as well as the cellular response to environmental stress, hazardous substances and antibiotics. Our finding that the chromosomal repositioning of a regulatory gene can determine the cellular phenotype unveils an important yet unexplored facet of the genetic control mechanisms and paves the way for novel approaches to manipulate bacterial physiology.

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ZNF555 protein binds to transcriptional activator site of 4qA allele and ANT1: potential implication in Facioscapulohumeral dystrophy

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

Facioscapulohumeral dystrophy (FSHD) is an epi/genetic satellite disease associated with at least two satellite sequences in 4q35: (i) D4Z4 macrosatellite and (ii) β-satellite repeats (BSR), a prevalent part of the 4qA allele. Most of the recent FSHD studies have been focused on a DUX4 transcript inside D4Z4 and its tandem contraction in FSHD patients. However, the D4Z4-contraction alone is not pathological, which would also require the 4qA allele. Since little is known about BSR, we investigated the 4qA BSR functional role in the transcriptional control of the FSHD region 4q35. We have shown that an individual BSR possesses enhancer activity leading to activation of the Adenine Nucleotide Translocator 1 gene (ANT1), a major FSHD candidate gene. We have identified ZNF555, a previously uncharacterized protein, as a putative transcriptional factor highly expressed in human primary myoblasts that interacts with the BSR enhancer site and impacts the ANT1 promoter activity in FSHD myoblasts. The discovery of the functional role of the 4qA allele and ZNF555 in the transcriptional control of ANT1 advances our understanding of FSHD pathogenesis and provides potential therapeutic targets.

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Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here we investigated the properties and mechanisms of clag3 mutual exclusion using transgenic parasite lines with extra copies of clag3 promoters located either in stable episomes or integrated in the parasite genome. We found that the additional clag3 promoters in these transgenic lines are silenced by default, but under strong selective pressure parasites with more than one clag3 promoter simultaneously active are observed, demonstrating that clag3 mutual exclusion is strongly favored but it is not strict. We show that silencing of clag3 genes is associated with the repressive histone mark H3K9me3 even in parasites with unusual clag3 expression patterns, and we provide direct evidence for heterochromatin spreading in P. falciparum. We also found that expression of a neighbor ncRNA correlates with clag3.1 expression. Altogether, our results reveal a scenario where fitness costs and non-deterministic molecular processes that favor mutual exclusion shape the expression patterns of this important gene family.

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Splicing inhibition decreases phosphorylation level of Ser2 in Pol II CTD

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

Phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II (Pol II), especially Ser2 and Ser5 residues, plays important roles in transcription and mRNA processing, including 5' end capping, splicing and 3' end processing. These phosphorylation events stimulate mRNA processing, however, it is not clear whether splicing activity affects the phosphorylation status of Pol II. In this study, we found that splicing inhibition by potent splicing inhibitors spliceostatin A (SSA) and pladienolide B or by antisense oligos against snRNAs decreased phospho-Ser2 level, but had little or no effects on phospho-Ser5 level. In contrast, transcription and translation inhibitors did not decrease phospho-Ser2 level, therefore inhibition of not all the gene expression processes cause the decrease of phospho-Ser2. SSA treatment caused early dissociation of Pol II and decrease in phospho-Ser2 level of chromatin-bound Pol II, suggesting that splicing inhibition causes downregulation of phospho-Ser2 through at least these two mechanisms.

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ChIP-seq reveals the global regulator AlgR mediating cyclic di-GMP synthesis in Pseudomonas aeruginosa

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

AlgR is a key transcriptional regulator required for the expression of multiple virulence factors, including type IV pili and alginate in Pseudomonas aeruginosa. However, the regulon and molecular regulatory mechanism of AlgR have yet to be fully elucidated. Here, among 157 loci that were identified by a ChIP-seq assay, we characterized a gene, mucR, which encodes an enzyme that synthesizes the intracellular second messenger cyclic diguanylate (c-di-GMP). A algR strain produced lesser biofilm than did the wild-type strain, which is consistent with a phenotype controlled by c-di-GMP. AlgR positively regulates mucR via direct binding to its promoter. A algRmucR double mutant produced lesser biofilm than did the single algR mutant, demonstrating that c-di-GMP is a positive regulator of biofilm formation. AlgR controls the levels of c-di-GMP synthesis via direct regulation of mucR. In addition, the cognate sensor of AlgR, FimS/AlgZ, also plays an important role in P. aeruginosa virulence. Taken together, this study provides new insights into the AlgR regulon and reveals the involvement of c-di-GMP in the mechanism underlying AlgR regulation.

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The RNA-binding protein HOS5 and serine/arginine-rich proteins RS40 and RS41 participate in miRNA biogenesis in Arabidopsis

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

MicroRNAs are a class of small regulatory RNAs that are generated from primary miRNA (pri-miRNA) transcripts with a stem-loop structure. Accuracy of the processing of pri-miRNA into mature miRNA in plants can be enhanced by SERRATE (SE) and HYPONASTIC LEAVES 1 (HYL1). HYL1 activity is regulated by the FIERY2 (FRY2)/RNA polymerase II C-terminal domain phosphatase-like 1 (CPL1). Here, we discover that HIGH OSMOTIC STRESS GENE EXPRESSION 5 (HOS5) and two serine/arginine-rich splicing factors RS40 and RS41, previously shown to be involved in pre-mRNA splicing, affect the biogenesis of a subset of miRNA. These proteins are required for correct miRNA strand selection and the maintenance of miRNA levels. FRY2 dephosphorylates HOS5 whose phosphorylation status affects its subnuclear localization. HOS5 and the RS proteins bind both intronless and intron-containing pri-miRNAs. Importantly, all of these splicing-related factors directly interact with both HYL1 and SE in nuclear splicing speckles. Our results indicate that these splicing factors are directly involved in the biogenesis of a group of miRNA.

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The yeast genome undergoes significant topological reorganization in quiescence

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

We have examined the three-dimensional organization of the yeast genome during quiescence by a chromosome capture technique as a means of understanding how genome organization changes during development. For exponentially growing cells we observe high levels of inter-centromeric interaction but otherwise a predominance of intrachromosomal interactions over interchromosomal interactions, consistent with aggregation of centromeres at the spindle pole body and compartmentalization of individual chromosomes within the nucleoplasm. Three major changes occur in the organization of the quiescent cell genome. First, intrachromosomal associations increase at longer distances in quiescence as compared to growing cells. This suggests that chromosomes undergo condensation in quiescence, which we confirmed by microscopy by measurement of the intrachromosomal distances between two sites on one chromosome. This compaction in quiescence requires the condensin complex. Second, inter-centromeric interactions decrease, consistent with prior data indicating that centromeres disperse along an array of microtubules during quiescence. Third, inter-telomeric interactions significantly increase in quiescence, an observation also confirmed by direct measurement. Thus, survival during quiescence is associated with substantial topological reorganization of the genome.

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In vivo detection and replication studies of {alpha}-anomeric lesions of 2'-deoxyribonucleosides

Nucleic Acids Research - Tue, 09/29/2015 - 01:41

DNA damage, arising from endogenous metabolism or exposure to environmental agents, may perturb the transmission of genetic information by blocking DNA replication and/or inducing mutations, which contribute to the development of cancer and likely other human diseases. Hydroxyl radical attack on the C1', C3' and C4' of 2-deoxyribose can give rise to epimeric 2-deoxyribose lesions, for which the in vivo occurrence and biological consequences remain largely unexplored. Through independent chemical syntheses of all three epimeric lesions of 2'-deoxyguanosine (dG) and liquid chromatography-tandem mass spectrometry analysis, we demonstrated unambiguously the presence of substantial levels of the α-anomer of dG (α-dG) in calf thymus DNA and in DNA isolated from mouse pancreatic tissues. We further assessed quantitatively the impact of all four α-dN lesions on DNA replication in Escherichia coli by employing a shuttle-vector method. We found that, without SOS induction, all α-dN lesions except α-dA strongly blocked DNA replication and, while replication across α-dA was error-free, replicative bypass of α-dC and α-dG yielded mainly C->A and G->A mutations. In addition, SOS induction could lead to markedly elevated bypass efficiencies for the four α-dN lesions, abolished the G->A mutation for α-dG, pronouncedly reduced the C->A mutation for α-dC and triggered T->A mutation for α-dT. The preferential misincorporation of dTMP opposite the α-dNs could be attributed to the unique base-pairing properties of the nucleobases elicited by the inversion of the configuration of the N-glycosidic linkage. Our results also revealed that Pol V played a major role in bypassing α-dC, α-dG and α-dT in vivo. The abundance of α-dG in mammalian tissue and the impact of the α-dNs on DNA replication demonstrate for the first time the biological significance of this family of DNA lesions.

Categories: Journal Articles
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