Nucleic Acids Research

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Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment

Mon, 2016-10-31 07:42

Presence of excess unaltered, wild-type (WT) DNA providing no information of biological or clinical value often masks rare alterations containing diagnostic or therapeutic clues in cancer, prenatal diagnosis, infectious diseases or organ transplantation. With the surge of high-throughput technologies there is a growing demand for removing unaltered DNA over large pools-of-sequences. Here we present nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO), a single-step approach with broad genome coverage that can remove WT-DNA from numerous sequences simultaneously, prior to genomic analysis. NaME-PrO employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating WT-DNA targets and guiding nuclease digestion to these sites. Mutation-containing DNA creates probe-DNA mismatches that inhibit digestion, thus subsequent DNA-amplification magnifies DNA-alterations at all selected targets. We demonstrate several-hundred-fold mutation enrichment in diverse human samples on multiple clinically relevant targets including tumor samples and circulating DNA in 50-plex reactions. Enrichment enables routine mutation detection at 0.01% abundance while by adjusting conditions it is possible to sequence mutations down to 0.00003% abundance, or to scan tumor-suppressor genes for rare mutations. NaME-PrO introduces a simple and highly parallel process to remove un-informative DNA sequences and unmask clinically and biologically useful alterations.

Contiguous and accurate de novo assembly of metazoan genomes with modest long read coverage

Mon, 2016-10-31 07:42

Genome assemblies that are accurate, complete and contiguous are essential for identifying important structural and functional elements of genomes and for identifying genetic variation. Nevertheless, most recent genome assemblies remain incomplete and fragmented. While long molecule sequencing promises to deliver more complete genome assemblies with fewer gaps, concerns about error rates, low yields, stringent DNA requirements and uncertainty about best practices may discourage many investigators from adopting this technology. Here, in conjunction with the platinum standard Drosophila melanogaster reference genome, we analyze recently published long molecule sequencing data to identify what governs completeness and contiguity of genome assemblies. We also present a hybrid meta-assembly approach that achieves remarkable assembly contiguity for both Drosophila and human assemblies with only modest long molecule sequencing coverage. Our results motivate a set of preliminary best practices for obtaining accurate and contiguous assemblies, a ‘missing manual’ that guides key decisions in building high quality de novo genome assemblies, from DNA isolation to polishing the assembly.

SNP calling from RNA-seq data without a reference genome: identification, quantification, differential analysis and impact on the protein sequence

Mon, 2016-10-31 07:42

SNPs (Single Nucleotide Polymorphisms) are genetic markers whose precise identification is a prerequisite for association studies. Methods to identify them are currently well developed for model species, but rely on the availability of a (good) reference genome, and therefore cannot be applied to non-model species. They are also mostly tailored for whole genome (re-)sequencing experiments, whereas in many cases, transcriptome sequencing can be used as a cheaper alternative which already enables to identify SNPs located in transcribed regions. In this paper, we propose a method that identifies, quantifies and annotates SNPs without any reference genome, using RNA-seq data only. Individuals can be pooled prior to sequencing, if not enough material is available from one individual. Using pooled human RNA-seq data, we clarify the precision and recall of our method and discuss them with respect to other methods which use a reference genome or an assembled transcriptome. We then validate experimentally the predictions of our method using RNA-seq data from two non-model species. The method can be used for any species to annotate SNPs and predict their impact on the protein sequence. We further enable to test for the association of the identified SNPs with a phenotype of interest.

An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting

Mon, 2016-10-31 07:42

The CRISPR/Cas9 system is a powerful genome editing tool and has been widely used for biomedical research. However, many challenges, such as off-target effects and lack of easy solutions for multiplex targeting, are still limiting its applications. To overcome these challenges, we first developed a highly efficient doxycycline-inducible Cas9-EGFP vector. This vector allowed us to track the cells for uniform temporal control and efficient gene disruption, even in a polyclonal setting. Furthermore, the inducible CRISPR/Cas9 system dramatically decreased off-target effects with a pulse exposure of the genome to the Cas9/sgRNA complex. To target multiple genes simultaneously, we established simple one-step cloning approaches for expression of multiple sgRNAs with improved vectors. By combining our inducible and multiplex genome editing approaches, we were able to simultaneously delete Lysine Demethylase (KDM) 5A, 5B and 5C efficiently in vitro and in vivo. This user friendly and highly efficient toolbox provides a solution for easy genome editing with tight temporal control, minimal off-target effects and multiplex targeting.

FocalScan: Scanning for altered genes in cancer based on coordinated DNA and RNA change

Mon, 2016-10-31 07:42

Somatic genomic copy-number alterations can lead to transcriptional activation or inactivation of tumor driver or suppressor genes, contributing to the malignant properties of cancer cells. Selection for such events may manifest as recurrent amplifications or deletions of size-limited (focal) regions. While methods have been developed to identify such focal regions, finding the exact targeted genes remains a challenge. Algorithms are also available that integrate copy number and RNA expression data, to aid in identifying individual targeted genes, but specificity is lacking. Here, we describe FocalScan, a tool designed to simultaneously uncover patterns of focal copy number alteration and coordinated expression change, thus combining both principles. The method outputs a ranking of tentative cancer drivers or suppressors. FocalScan works with RNA-seq data, and unlike other tools it can scan the genome unaided by a gene annotation, enabling identification of novel putatively functional elements including lncRNAs. Application on a breast cancer data set suggests considerably better performance than other DNA/RNA integration tools.

A basic domain in the histone H2B N-terminal tail is important for nucleosome assembly by FACT

Mon, 2016-10-31 07:42

Nucleosome assembly in vivo requires assembly factors, such as histone chaperones, to bind to histones and mediate their deposition onto DNA. In yeast, the essential histone chaperone FACT (FAcilitates Chromatin Transcription) functions in nucleosome assembly and H2A–H2B deposition during transcription elongation and DNA replication. Recent studies have identified candidate histone residues that mediate FACT binding to histones, but it is not known which histone residues are important for FACT to deposit histones onto DNA during nucleosome assembly. In this study, we report that the histone H2B repression (HBR) domain within the H2B N-terminal tail is important for histone deposition by FACT. Deletion of the HBR domain causes significant defects in histone occupancy in the yeast genome, particularly at HBR-repressed genes, and a pronounced increase in H2A–H2B dimers that remain bound to FACT in vivo. Moreover, the HBR domain is required for purified FACT to efficiently assemble recombinant nucleosomes in vitro. We propose that the interaction between the highly basic HBR domain and DNA plays an important role in stabilizing the nascent nucleosome during the process of histone H2A–H2B deposition by FACT.

The zinc fingers of YY1 bind single-stranded RNA with low sequence specificity

Mon, 2016-10-31 07:42

Classical zinc fingers (ZFs) are traditionally considered to act as sequence-specific DNA-binding domains. More recently, classical ZFs have been recognised as potential RNA-binding modules, raising the intriguing possibility that classical-ZF transcription factors are involved in post-transcriptional gene regulation via direct RNA binding. To date, however, only one classical ZF-RNA complex, that involving TFIIIA, has been structurally characterised. Yin Yang-1 (YY1) is a multi-functional transcription factor involved in many regulatory processes, and binds DNA via four classical ZFs. Recent evidence suggests that YY1 also interacts with RNA, but the molecular nature of the interaction remains unknown. In the present work, we directly assess the ability of YY1 to bind RNA using in vitro assays. Systematic Evolution of Ligands by EXponential enrichment (SELEX) was used to identify preferred RNA sequences bound by the YY1 ZFs from a randomised library over multiple rounds of selection. However, a strong motif was not consistently recovered, suggesting that the RNA sequence selectivity of these domains is modest. YY1 ZF residues involved in binding to single-stranded RNA were identified by NMR spectroscopy and found to be largely distinct from the set of residues involved in DNA binding, suggesting that interactions between YY1 and ssRNA constitute a separate mode of nucleic acid binding. Our data are consistent with recent reports that YY1 can bind to RNA in a low-specificity, yet physiologically relevant manner.

Sir2 regulates stability of repetitive domains differentially in the human fungal pathogen Candida albicans

Mon, 2016-10-31 07:42

DNA repeats, found at the ribosomal DNA locus, telomeres and subtelomeric regions, are unstable sites of eukaryotic genomes. A fine balance between genetic variability and genomic stability tunes plasticity of these chromosomal regions. This tuning mechanism is particularly important for organisms such as microbial pathogens that utilise genome plasticity as a strategy for adaptation. For the first time, we analyse mechanisms promoting genome stability at the rDNA locus and subtelomeric regions in the most common human fungal pathogen: Candida albicans. In this organism, the histone deacetylase Sir2, the master regulator of heterochromatin, has acquired novel functions in regulating genome stability. Contrary to any other systems analysed, C. albicans Sir2 is largely dispensable for repressing recombination at the rDNA locus. We demonstrate that recombination at subtelomeric regions is controlled by a novel DNA element, the TLO Recombination Element, TRE, and by Sir2. While the TRE element promotes high levels of recombination, Sir2 represses this recombination rate. Finally, we demonstrate that, in C. albicans, mechanisms regulating genome stability are plastic as different environmental stress conditions lead to general genome instability and mask the Sir2-mediated recombination control at subtelomeres. Our data highlight how mechanisms regulating genome stability are rewired in C. albicans.

Phospho-site mutants of the RNA Polymerase II C-terminal domain alter subtelomeric gene expression and chromatin modification state in fission yeast

Mon, 2016-10-31 07:42

Eukaryotic gene expression requires that RNA Polymerase II (RNAP II) gain access to DNA in the context of chromatin. The C-terminal domain (CTD) of RNAP II recruits chromatin modifying enzymes to promoters, allowing for transcription initiation or repression. Specific CTD phosphorylation marks facilitate recruitment of chromatin modifiers, transcriptional regulators, and RNA processing factors during the transcription cycle. However, the readable code for recruiting such factors is still not fully defined and how CTD modifications affect related families of genes or regional gene expression is not well understood. Here, we examine the effects of manipulating the Y1S2P3T4S5P6S7 heptapeptide repeat of the CTD of RNAP II in Schizosaccharomyces pombe by substituting non-phosphorylatable alanines for Ser2 and/or Ser7 and the phosphomimetic glutamic acid for Ser7. Global gene expression analyses were conducted using splicing-sensitive microarrays and validated via RT-qPCR. The CTD mutations did not affect pre-mRNA splicing or snRNA levels. Rather, the data revealed upregulation of subtelomeric genes and alteration of the repressive histone H3 lysine 9 methylation (H3K9me) landscape. The data further indicate that H3K9me and expression status are not fully correlated, suggestive of CTD-dependent subtelomeric repression mechansims that act independently of H3K9me levels.

A subset of replication-dependent histone mRNAs are expressed as polyadenylated RNAs in terminally differentiated tissues

Mon, 2016-10-31 07:42

Histone proteins are synthesized in large amounts during S-phase to package the newly replicated DNA, and are among the most stable proteins in the cell. The replication-dependent (RD)-histone mRNAs expressed during S-phase end in a conserved stem-loop rather than a polyA tail. In addition, there are replication-independent (RI)-histone genes that encode histone variants as polyadenylated mRNAs. Most variants have specific functions in chromatin, but H3.3 also serves as a replacement histone for damaged histones in long-lived terminally differentiated cells. There are no reported replacement histone genes for histones H2A, H2B or H4. We report that a subset of RD-histone genes are expressed in terminally differentiated tissues as polyadenylated mRNAs, likely serving as replacement histone genes in long-lived non-dividing cells. Expression of two genes, HIST2H2AA3 and HIST1H2BC, is conserved in mammals. They are expressed as polyadenylated mRNAs in fibroblasts differentiated in vitro, but not in serum starved fibroblasts, suggesting that their expression is part of the terminal differentiation program. There are two histone H4 genes and an H3 gene that encode mRNAs that are polyadenylated and expressed at 5- to 10-fold lower levels than the mRNAs from H2A and H2B genes, which may be replacement genes for the H3.1 and H4 proteins.

Set7 mediated interactions regulate transcriptional networks in embryonic stem cells

Mon, 2016-10-31 07:42

Histone methylation by lysine methyltransferase enzymes regulate the expression of genes implicated in lineage specificity and cellular differentiation. While it is known that Set7 catalyzes mono-methylation of histone and non-histone proteins, the functional importance of this enzyme in stem cell differentiation remains poorly understood. We show Set7 expression is increased during mouse embryonic stem cell (mESC) differentiation and is regulated by the pluripotency factors, Oct4 and Sox2. Transcriptional network analyses reveal smooth muscle (SM) associated genes are subject to Set7-mediated regulation. Furthermore, pharmacological inhibition of Set7 activity confirms this regulation. We observe Set7-mediated modification of serum response factor (SRF) and mono-methylation of histone H4 lysine 4 (H3K4me1) regulate gene expression. We conclude the broad substrate specificity of Set7 serves to control key transcriptional networks in embryonic stem cells.

Different N-terminal isoforms of Oct-1 control expression of distinct sets of genes and their high levels in Namalwa Burkitt's lymphoma cells affect a wide range of cellular processes

Mon, 2016-10-31 07:42

Oct-1 transcription factor has various functions in gene regulation. Its expression level is increased in several types of cancer and is associated with poor survival prognosis. Here we identified distinct Oct-1 protein isoforms in human cells and compared gene expression patterns and functions for Oct-1A, Oct-1L, and Oct-1X isoforms that differ by their N-terminal sequences. The longest isoform, Oct-1A, is abundantly expressed and is the main Oct-1 isoform in most of human tissues. The Oct-1L and the weakly expressed Oct-1X regulate the majority of Oct-1A targets as well as additional sets of genes. Oct-1X controls genes involved in DNA replication, DNA repair, RNA processing, and cellular response to stress. The high level of Oct-1 isoforms upregulates genes related to cell cycle progression and activates proliferation both in Namalwa Burkitt's lymphoma cells and primary human fibroblasts. It downregulates expression of genes related to antigen processing and presentation, cytokine-cytokine receptor interaction, oxidative metabolism, and cell adhesion, thus facilitating pro-oncogenic processes.

Identification of the mRNA targets of tRNA-specific regulation using genome-wide simulation of translation

Mon, 2016-10-31 07:42

tRNA gene copy number is a primary determinant of tRNA abundance and therefore the rate at which each tRNA delivers amino acids to the ribosome during translation. Low-abundance tRNAs decode rare codons slowly, but it is unclear which genes might be subject to tRNA-mediated regulation of expression. Here, those mRNA targets were identified via global simulation of translation. In-silico mRNA translation rates were compared for each mRNA in both wild-type and a ${\rm{tRNA}}_{{\rm{CUG}}}^{{\rm{Gln}}}$ sup70-65 mutant, which exhibits a pseudohyphal growth phenotype and a 75% slower CAG codon translation rate. Of 4900 CAG-containing mRNAs, 300 showed significantly reduced in silico translation rates in a simulated tRNA mutant. Quantitative immunoassay confirmed that the reduced translation rates of sensitive mRNAs were ${\rm{tRNA}}_{{\rm{CUG}}}^{{\rm{Gln}}}$ concentration-dependent. Translation simulations showed that reduced ${\rm{tRNA}}_{{\rm{CUG}}}^{{\rm{Gln}}}$ concentrations triggered ribosome queues, which dissipated at reduced translation initiation rates. To validate this prediction experimentally, constitutive gcn2 kinase mutants were used to reduce in vivo translation initiation rates. This repaired the relative translational rate defect of target mRNAs in the sup70-65 background, and ameliorated sup70-65 pseudohyphal growth phenotypes. We thus validate global simulation of translation as a new tool to identify mRNA targets of tRNA-specific gene regulation.

Histone H2B mono-ubiquitylation maintains genomic integrity at stalled replication forks

Mon, 2016-10-31 07:42

Histone modifications play an important role in regulating access to DNA for transcription, DNA repair and DNA replication. A central player in these events is the mono-ubiquitylation of histone H2B (H2Bub1), which has been shown to regulate nucleosome dynamics. Previously, it was shown that H2Bub1 was important for nucleosome assembly onto nascent DNA at active replication forks. In the absence of H2Bub1, incomplete chromatin structures resulted in several replication defects. Here, we report new evidence, which shows that loss of H2Bub1 contributes to genomic instability in yeast. Specifically, we demonstrate that H2Bub1-deficient yeast accumulate mutations at a high frequency under conditions of replicative stress. This phenotype is due to an aberrant DNA Damage Tolerance (DDT) response upon fork stalling. We show that H2Bub1 normally functions to promote error-free translesion synthesis (TLS) mediated by DNA polymerase eta (Pol). Without H2Bub1, DNA polymerase zeta (Pol) is responsible for a highly mutagenic alternative mechanism. While H2Bub1 does not appear to regulate other DDT pathways, error-free DDT mechanisms are employed by H2Bub1-deficient cells as another means for survival. However, in these instances, the anti-recombinase, Srs2, is essential to prevent the accumulation of toxic HR intermediates that arise in an unconstrained chromatin environment.

Translesion synthesis of O4-alkylthymidine lesions in human cells

Mon, 2016-10-31 07:42

Environmental exposure, endogenous metabolism and cancer chemotherapy can give rise to alkylation of DNA, and the resulting alkylated thymidine (alkyldT) lesions were found to be poorly repaired and persistent in mammalian tissues. Unrepaired DNA lesions may compromise genomic integrity by inhibiting DNA replication and inducing mutations in these processes. In this study, we explored how eight O4-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, (R)-sBu and (S)-sBu, are recognized by DNA replication machinery in HEK293T human embryonic kidney cells. We found that the O4-alkyldT lesions are moderately blocking to DNA replication, with the bypass efficiencies ranging from 20 to 33% in HEK293T cells, and these lesions induced substantial frequencies T->C transition mutation. We also conducted the replication experiments in the isogenic cells where individual translesion synthesis (TLS) DNA polymerases were depleted by the CRISPR/Cas9 genome editing method. Our results showed that deficiency in Pol or Pol , but not Pol or Pol , led to pronounced drops in bypass efficiencies for all the O4-alkyldT lesions except O4-MedT. In addition, depletion of Pol resulted in significant decreases in T->C mutation frequencies for all the O4-alkyldT lesions except O4-MedT and O4-nBudT. Thus, our study provided important new knowledge about the cytotoxic and mutagenic properties of the O4-alkyldT lesions and defined the roles of TLS polymerases in bypassing these lesions in human cells.

OGT restrains the expansion of DNA damage signaling

Mon, 2016-10-31 07:42

O-linked N-acetylglucosamine linkage (O-GlcNAcylation) to serine or threonine residues regulates numerous biological processes; however, its role in DNA damage response remains elusive. Here, we found that O-GlcNAcylation is induced by DNA damage response. O-GlcNAc transferase (OGT), the solo enzyme for O-GlcNAcylation, relocates to the sites of DNA damage and induces the O-GlcNAcylation of histone H2AX and mediator of DNA damage checkpoint 1 (MDC1). The O-GlcNAcylation negatively regulates DNA double-strand break-induced phosphorylation of H2AX and MDC1 by restraining the expansion of these phosphorylation events from the sites of DNA damage. Therefore, our study reveals the molecular mechanism and biological function of OGT-dependent O-GlcNAcylation in response to DNA damage.

Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro

Mon, 2016-10-31 07:42

Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3'-cordycepin, 5'- and 3'-phosphate and also to 5'-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5'-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2'-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2',1''-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1' of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs.

Meiotic prophase roles of Rec8 in crossover recombination and chromosome structure

Mon, 2016-10-31 07:42

Rec8 is a prominent component of the meiotic prophase chromosome axis that mediates sister chromatid cohesion, homologous recombination and chromosome synapsis. Here, we explore the prophase roles of Rec8. (i) During the meiotic divisions, Rec8 phosphorylation mediates its separase-mediated cleavage. We show here that such cleavage plays no detectable role for chromosomal events of prophase. (ii) We have analyzed in detail three rec8 phospho-mutants, with 6, 24 or 29 alanine substitutions. A distinct ‘separation of function’ phenotype is revealed. In the mutants, axis formation and recombination initiation are normal, as is non-crossover recombination; in contrast, crossover (CO)-related events are defective. Moreover, the severities of these defects increase coordinately with the number of substitution mutations, consistent with the possibility that global phosphorylation of Rec8 is important for these effects. (iii) We have analyzed the roles of three kinases that phosphorylate Rec8 during prophase. Timed inhibition of Dbf4-dependent Cdc7 kinase confers defects concordant with rec8 phospho-mutant phenotypes. Inhibition of Hrr25 or Cdc5/polo-like kinase does not. Our results suggest that Rec8's prophase function, independently of cohesin cleavage, contributes to CO-specific events in conjunction with the maintenance of homolog bias at the leptotene/zygotene transition of meiotic prophase.

Identification of non-coding genetic variants in samples from hypoxemic respiratory disease patients that affect the transcriptional response to hypoxia

Mon, 2016-10-31 07:42

A wide range of diseases course with an unbalance between the consumption of oxygen by tissues and its supply. This situation triggers a transcriptional response, mediated by the hypoxia inducible factors (HIFs), that aims to restore oxygen homeostasis. Little is known about the inter-individual variation in this response and its role in the progression of disease. Herein, we sought to identify common genetic variants mapping to hypoxia response elements (HREs) and characterize their effect on transcription. To this end, we constructed a list of genome-wide HIF-binding regions from publicly available experimental datasets and studied the genetic variability in these regions by targeted re-sequencing of genomic samples from 96 chronic obstructive pulmonary disease and 144 obstructive sleep apnea patients. This study identified 14 frequent variants disrupting potential HREs. The analysis of the genomic regions containing these variants by means of reporter assays revealed that variants rs1009329, rs6593210 and rs150921338 impaired the transcriptional response to hypoxia. Finally, using genome editing we confirmed the functional role of rs6593210 in the transcriptional regulation of EGFR. In summary, we found that inter-individual variability in non-coding regions affect the response to hypoxia and could potentially impact on the progression of pulmonary diseases.