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NAR Top Articles - Genomics

Genomics

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March 2015


Optimization of scarless human stem cell genome editing
Yang, LH; Guell, M; Byrne, S; Yang, JL; De Los Angeles, A; Mali, P; Aach, J; Kim-Kiselak, C; Briggs, AW; Rios, X; Huang, PY; Daley, G; Church, G
Nucleic Acids Res. 2013, 41, 9049-9061
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Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7-8x higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%)...

Mapping of six somatic linker histone H1 variants in human breast cancer cells uncovers specific features of H1.2
Millan-Arino, L; Islam, AMMK; Izquierdo-Bouldstridge, A; Mayor, R; Terme, JM; Luque, N; Sancho, M; Lopez-Bigas, N; Jordan, A
Nucleic Acids Res. 2014, 42, 4474-4493
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Seven linker histone H1 variants are present in human somatic cells with distinct prevalence across cell types. Despite being key structural components of chromatin, it is not known whether the different variants have specific roles in the regulation of nuclear processes or are differentially distributed throughout the genome. Using variant-specific antibodies to H1 and hemagglutinin (HA)-tagged recombinant H1 variants expressed in breast cancer cells, we have investigated the distribution of six H1 variants in promoters and genome-wide. H1 is depleted at promoters depending on its transcriptional status and differs between variants. Notably, H1.2 is less abundant than other variants at the transcription start sites of inactive genes, and promoters enriched in H1.2 are different from those enriched in other variants and tend to be repressed. Additionally, H1.2 is enriched at chromosomal domains characterized by low guanine-cytosine (GC) content and is associated with lamina-associated domains. Meanwhile, other variants are associated with higher GC content, CpG islands and gene-rich domains...

Stability, delivery and functions of human sperm RNAs at fertilization
Sendler, E; Johnson, GD; Mao, SH; Goodrich, RJ; Diamond, MP; Hauser, R; Krawetz, SA
Nucleic Acids Res. 2013, 41, 4104-4117
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Increasing attention has focused on the significance of RNA in sperm, in light of its contribution to the birth and long-term health of a child, role in sperm function and diagnostic potential. As the composition of sperm RNA is in flux, assigning specific roles to individual RNAs presents a significant challenge. For the first time RNA-seq was used to characterize the population of coding and non-coding transcripts in human sperm. Examining RNA representation as a function of multiple methods of library preparation revealed unique features indicative of very specific and stage-dependent maturation and regulation of sperm RNA, illuminating their various transitional roles. Correlation of sperm transcript abundance with epigenetic marks suggested roles for these elements in the pre- and post-fertilization genome. Several classes of non-coding RNAs including lncRNAs, CARs, pri-miRNAs, novel elements and mRNAs have been identified which, based on factors including relative abundance, integrity in sperm, available knockout data of embryonic effect and presence or absence in the unfertilized human oocyte, are likely to be essential male factors critical to early post-fertilization...

Non-metastatic 2 (NME2)-mediated suppression of lung cancer metastasis involves transcriptional regulation of key cell adhesion factor vinculin
Thakur, RK; Yadav, VK; Kumar, A; Singh, A; Pal, K; Hoeppner, L; Saha, D; Purohit, G; Basundra, R; Kar, A; Halder, R; Kumar, P; Baral, A; Kumar, MJM; Baldi, A; Vincenzi, B; Lorenzon, L; Banerjee, R; Kumar, P; Shridhar, V; Mukhopadhyay, D; Chowdhury, S
Nucleic Acids Res. 2014, 42, 11589-11600
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Tumor metastasis refers to spread of a tumor from site of its origin to distant organs and causes majority of cancer deaths. Although >30 metastasis suppressor genes (MSGs) that negatively regulate metastasis have been identified so far, two issues are poorly understood: first, which MSGs oppose metastasis in a tumor type, and second, which molecular function of MSG controls metastasis. Herein, integrative analyses of tumor-transcriptomes (n = 382), survival data (n = 530) and lymph node metastases (n = 100) in lung cancer patients identified nonmetastatic 2 (NME2) as a key MSG from a pool of >30 metastasis suppressors. Subsequently, we generated a promoter-wide binding map for NME2 using chromatin immunoprecipitation with promoter microarrays (ChIP-chip), and transcriptome profiling. We discovered novel targets of NME2 which are involved in focal adhesion signaling...

Single-cell paired-end genome sequencing reveals structural variation per cell cycle
Voet, T; Kumar, P; Van Loo, P; Cooke, SL; Marshall, J; Lin, ML; Esteki, MZ; Van der Aa, N; Mateiu, L; McBride, DJ; Bignell, GR; McLaren, S; Teague, J; Butler, A; Raine, K; Stebbings, LA; Quail, MA; D'Hooghe, T; Moreau, Y; Futreal, PA; Stratton, MR; Vermee, JR; Campbell, PJ
Nucleic Acids Res. 2013, 41, 6119-6138
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The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants...

metaseq: a Python package for integrative genome-wide analysis reveals relationships between chromatin insulators and associated nuclear mRNA
Dale, RK; Matzat, LH; Lei, EP
Nucleic Acids Res. 2014, 42, 9158-9170
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Here we introduce metaseq, a software library written in Python, which enables loading multiple genomic data formats into standard Python data structures and allows flexible, customized manipulation and visualization of data from high-throughput sequencing studies. We demonstrate its practical use by analyzing multiple datasets related to chromatin insulators, which are DNA-protein complexes proposed to organize the genome into distinct transcriptional domains. Recent studies in Drosophila and mammals have implicated RNA in the regulation of chromatin insulator activities. Moreover, the Drosophila RNA-binding protein Shep has been shown to antagonize gypsy insulator activity in a tissue-specific manner, but the precise role of RNA in this process remains unclear. Better understanding of chromatin insulator regulation requires integration of multiple datasets, including those from chromatin-binding, RNA-binding, and gene expression experiments. We use metaseq to integrate RIP-and ChIP-seq data for Shep and the core gypsy insulator protein Su(Hw) in two different cell types, along with publicly available ChIP-chip and RNA-seq data...

A comprehensive survey of non-canonical splice sites in the human transcriptome
Parada, GE; Munita, R; Cerda, CA; Gysling, K
Nucleic Acids Res. 2014, 42, 10564-10578
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We uncovered the diversity of non-canonical splice sites at the human transcriptome using deep transcriptome profiling. We mapped a total of 3.7 billion human RNA-seq reads and developed a set of stringent filters to avoid false non-canonical splice site detections. We identified 184 splice sites with non-canonical dinucleotides and U2/U12-like consensus sequences. We selected 10 of the herein identified U2/U12-like non-canonical splice site events and successfully validated 9 of them via reverse transcriptase-polymerase chain reaction and Sanger sequencing. Analyses of the 184 U2/U12-like non- canonical splice sites indicate that 51% of them are not annotated in GENCODE. In addition, 28% of them are conserved in mouse and 76% are involved in alternative splicing events, some of them with tissue-specific alternative splicing patterns. Interestingly, our analysis identified some U2/U12-like non-canonical splice sites that are converted into canonical splice sites by RNA A-to-I editing. Moreover, the U2/U12-like non-canonical splice sites have a differential distribution of splicing regulatory sequences, which may contribute to their recognition and regulation...

The effect of tRNA levels on decoding times of mRNA codons
Dana, A; Tuller, T
Nucleic Acids Res. 2014, 42, 9171-9181
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The possible effect of transfer ribonucleic acid (tRNA) concentrations on codons decoding time is a fundamental biomedical research question; however, due to a large number of variables affecting this process and the non-direct relation between them, a conclusive answer to this question has eluded so far researchers in the field. In this study, we perform a novel analysis of the ribosome profiling data of four organisms which enables ranking the decoding times of different codons while filtering translational phenomena such as experimental biases, extreme ribosomal pauses and ribosome traffic jams. Based on this filtering, we show for the first time that there is a significant correlation between tRNA concentrations and the codons estimated decoding time both in prokaryotes and in eukaryotes in natural conditions (-0.38 to -0.66, all P values <0.006); in addition, we show that when considering tRNA concentrations, codons decoding times are not correlated with aminoacyl-tRNA levels. The reported results support the conjecture that translation efficiency is directly influenced by the tRNA levels in the cell. Thus, they should help to understand the evolution of synonymous aspects of coding sequences via the adaptation of their codons to the tRNA pool.

The complex methylome of the human gastric pathogen Helicobacter pylori
Krebes, J; Morgan, RD; Bunk, B; Sproer, C; Luong, K; Parusel, R; Anton, BP; Konig, C; Josenhans, C; Overmann, J; Roberts, RJ; Korlach, J; Suerbaum, S
Nucleic Acids Res. 2014, 42, 2415-2432
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The genome of Helicobacter pylori is remarkable for its large number of restriction-modification (R-M) systems, and strain-specific diversity in R-M systems has been suggested to limit natural transformation, the major driving force of genetic diversification in H. pylori. We have determined the comprehensive methylomes of two H. pylori strains at single base resolution, using Single Molecule Real-Time (SMRT (R)) sequencing. For strains 26695 and J99-R3, 17 and 22 methylated sequence motifs were identified, respectively. For most motifs, almost all sites occurring in the genome were detected as methylated. Twelve novel methylation patterns corresponding to nine recognition sequences were detected (26695, 3; J99-R3, 6). Functional inactivation, correction of frameshifts as well as cloning and expression of candidate methyltransferases (MTases) permitted not only the functional characterization of multiple, yet undescribed, MTases, but also revealed novel features of both Type I and Type II R-M systems, including frameshift-mediated changes of sequence specificity...

REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer
Svensson, C; Ceder, J; Iglesias-Gato, D; Chuan, YC; Pang, ST; Bjartell, A; Martinez, RM; Bott, L; Helczynski, L; Ulmert, D; Wang, Y; Niu, Y; Collins, C; Flores-Morales, A
Nucleic Acids Res. 2014, 42, 999-1015
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The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds chromatin regions containing well-characterized cis-elements known to mediate REST transcriptional repression, while cell imaging studies confirmed that REST and AR closely co-localize in vivo. Androgen-induced gene repression also involves modulation of REST protein turnover through actions on the ubiquitin ligase beta-TRCP. Androgen deprivation or AR blockage with inhibitor MDV3100 (Enzalutamide) leads to neuroendocrine (NE) differentiation, a phenomenon that is mimicked by REST inactivation. Gene expression profiling revealed that REST not only acts to repress neuronal genes but also genes involved in cell cycle progression, including Aurora Kinase A, that has previously been implicated in the growth of NE-like castration-resistant tumors...

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