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Genome Research

Daesik Kim, Jin-Soo Kim
To investigate whether and how CRISPR-Cas9 on-target and off-target activities are affected by chromatin in eukaryotic cells, we first identified a series of identical endogenous DNA sequences present in both open and closed chromatin regions and then measured mutation frequencies at these sites in human cells using Cas9 complexed with matched or mismatched sgRNAs. Unlike matched sgRNAs, mismatched sgRNAs were highly sensitive to chromatin states, suggesting that off-target but not on-target DNA cleavage is hindered by chromatin...
November 9, 2018: Genome Research
Xinyang Yu, Michael J Buck
Accurate gene expression requires the targeting of transcription factors (TFs) to regulatory sequences often occluded within nucleosomes. The ability to target a transcription factor binding site (TFBS) within a nucleosome has been the defining characteristic for a special class of TFs known as pioneer factors. Recent studies suggest TP53 functions as a pioneer factor that can target its TFBS within nucleosomes, but it remains unclear how TP53 binds to nucleosomal DNA. To comprehensively examine TP53 nucleosome binding we competitively bound TP53 to multiple in vitro formed nucleosomes containing a high or low-affinity TP53 TFBS located at differing translational and rotational positions within the nucleosome...
November 8, 2018: Genome Research
Kaitlin U Laverty, Jake M Stout, Mitchell J Sullivan, Hardik Shah, Navdeep Gill, Larry Holbrook, Gintaras Deikus, Robert Sebra, Timothy R Hughes, Jonathan E Page, Harm van Bakel
Cannabis sativa is widely cultivated for medicinal, food, industrial, and recreational use, but much remains unknown regarding its genetics, including the molecular determinants of cannabinoid content. Here, we describe a combined physical and genetic map derived from a cross between the drug-type strain 'Purple Kush' and the hemp variety 'Finola'. The map reveals that cannabinoid biosynthesis genes are generally unlinked, but that aromatic prenyltransferase (AP), which produces the substrate for THCA and CBDA synthases (THCAS and CBDAS), is tightly linked to a known marker for total cannabinoid content...
November 8, 2018: Genome Research
Jia Lv, Wenqian Jiao, Haobing Guo, Pingping Liu, Ruijia Wang, Lingling Zhang, Qifan Zeng, Xiaoli Hu, Zhenmin Bao, Shi Wang
Targeted genotyping of transcriptome-scale genetic markers is highly attractive for genetic, ecological and evolutionary studies, but achieving this goal in a cost-effective manner remains a major challenge especially for laboratories working on non-model organisms. Here, we develop a high-throughput, sequencing-based GoldenGate approach (called HD-Marker), which addresses the array-related issues of original GoldenGate methodology, and allows for highly multiplexed and flexible targeted genotyping of over 12,000 loci in a single-tube assay (in contrast to fewer than 3,100 in the original GoldenGate assay)...
November 8, 2018: Genome Research
Nevena Cvetesic, Harry G Leitch, Malgorzata Borkowska, Ferenc Müller, Piero Carninci, Petra Hajkova, Boris Lenhard
Cap analysis of gene expression (CAGE) is a methodology for genome-wide quantitative mapping of mRNA 5' ends to precisely capture transcription start sites at a single nucleotide resolution. In combination with high-throughput sequencing, CAGE has revolutionized our understanding of rules of transcription initiation, led to discovery of new core promoter sequence features and discovered transcription initiation at enhancers genome-wide. The biggest limitation of CAGE is that even the most recently improved version (nAnT-iCAGE) still requires large amounts of total cellular RNA (5 micrograms), preventing its application to scarce biological samples such as those from early embryonic development or rare cell types...
November 7, 2018: Genome Research
Julien Soudet, Jatinder Kaur Gill, Françoise Stutz
In Eukaryotic organisms, replication initiation follows a temporal program. Among the parameters that regulate this program in Saccharomyces cerevisiae, chromatin structure has been at the center of attention without considering the contribution of transcription. Here, we revisit the replication initiation program in the light of widespread genomic noncoding transcription. We find that noncoding RNA transcription termination in the vicinity of ARS (Autonomously Replicating Sequences) shields replication initiation from transcriptional readthrough...
November 6, 2018: Genome Research
Wilfried M Guiblet, Marzia A Cremona, Monika Cechova, Robert S Harris, Iva Kejnovská, Eduard Kejnovsky, Kristin Eckert, Francesca Chiaromonte, Kateryna D Makova
DNA conformation may deviate from the classical B-form in ∼13% of the human genome. Non-B DNA regulates many cellular processes; however, its effects on DNA polymerization speed and accuracy have not been investigated genome-wide. Such an inquiry is critical for understanding neurological diseases and cancer genome instability. Here, we present the first simultaneous examination of DNA polymerization kinetics and errors in the human genome sequenced with Single-Molecule Real-Time (SMRT) technology. We show that polymerization speed differs between non-B and B-DNA: It decelerates at G-quadruplexes and fluctuates periodically at disease-causing tandem repeats...
November 6, 2018: Genome Research
Amy Leung, Candi Trac, Hiroyuki Kato, Kevin R Costello, Zhaoxia Chen, Rama Natarajan, Dustin E Schones
Endogenous retroviruses (ERVs) are ancient viral elements that have accumulated in the genome through retrotransposition events. Although they have lost their ability to transpose, many of the long terminal repeats (LTRs) that originally flanked full-length ERVs maintain the ability to regulate transcription. While these elements are typically repressed in somatic cells, they can function as transcriptional enhancers and promoters when this repression is lost. Epstein-Barr virus (EBV), which transforms primary B cells into continuously proliferating cells, is a tumor virus associated with lymphomas...
October 31, 2018: Genome Research
Wesley A Brashear, Terje Raudsepp, William Murphy
Despite claims that the human Y chromosome is on a path to extinction, comparative sequence analysis of primate Y chromosomes has shown the decay of the ancestral single copy genes has all but ceased in this eutherian lineage. The suite of single copy Y-linked genes is highly conserved among the majority of eutherian Y chromosomes due to strong purifying selection to retain dosage sensitive genes. By contrast, the ampliconic regions of the Y chromosome, which contain testis-specific genes that encode the majority of the transcripts on eutherian Y chromosomes, are rapidly evolving and are thought to undergo rapid species-specific turnover...
October 31, 2018: Genome Research
Michael A Schon, Max J Kellner, Alexandra Plotnikova, Falko Hofmann, Michael D Nodine
Diverse RNA 5' ends are generated through both transcriptional and post-transcriptional processes. These important modes of gene regulation often vary across cell types, and can contribute to the diversification of transcriptomes and thus cellular differentiation. Therefore, the identification of primary and processed 5' ends of RNAs is important for their functional characterization. Methods have been developed to profile either RNA 5' ends from primary transcripts or the products of RNA degradation genome-wide...
October 24, 2018: Genome Research
Steffen Erkelenz, Stephan Theiss, Wolfgang Kaisers, Johannes Ptok, Lara Walotka, Lisa Müller, Frank Hillebrand, Anna-Lena Brillen, Michael Sladek, Heiner Schaal
Most human pathogenic mutations in 5' splice sites affect the canonical GT in positions +1 and +2, leading to noncanonical dinucleotides. On the other hand, noncanonical dinucleotides are observed under physiological conditions in ∼1% of all human 5'ss. It is therefore a challenging task to understand the pathogenic mutation mechanisms underlying the conditions under which noncanonical 5'ss are used. In this work, we systematically examined noncanonical 5' splice site selection, both experimentally using splicing competition reporters and by analyzing a large RNA-seq data set of 54 fibroblast samples from 27 subjects containing a total of 2...
October 24, 2018: Genome Research
Antoine Hocher, Myriam Ruault, Petra Kaferle, Marc Descrimes, Mickaël Garnier, Antonin Morillon, Angela Taddei
The eukaryotic genome is divided into chromosomal domains of heterochromatin and euchromatin. Transcriptionally silent heterochromatin is found at subtelomeric regions, leading to the telomeric position effect (TPE) in yeast fly and human. Heterochromatin generally initiates and spreads from defined loci, and diverse mechanisms prevent the ectopic spread of heterochromatin into euchromatin. Here, we overexpressed the silencing factor Sir3 at varying levels in yeast and found that Sir3 spreads into Extended Silent Domains (ESDs), eventually reaching saturation at subtelomeres...
October 24, 2018: Genome Research
Geoffrey J Maher, Hannah K Ralph, Zhihao Ding, Nils Koelling, Hana Mlcochova, Eleni Giannoulatou, Pawan Dhami, Dirk S Paul, Stefan H Stricker, Stephan Beck, Gil McVean, Andrew Om Wilkie, Anne Goriely
Mosaic mutations present in the germline have important implications for reproductive risk and disease transmission. We previously demonstrated a phenomenon occurring in the male germline, whereby specific mutations arising spontaneously in stem cells (spermatogonia) lead to clonal expansion, resulting in elevated mutation levels in sperm over time. This process, termed selfish spermatogonial selection, explains the high spontaneous birth prevalence and strong paternal age-effect of disorders such as achondroplasia, Apert, Noonan and Costello syndromes, with direct experimental evidence currently available for specific positions of six genes (FGFR2, FGFR3, RET, PTPN11, HRAS and KRAS)...
October 24, 2018: Genome Research
Yoon Jung Kim, Peng Xie, Lian Cao, Michael Q Zhang, Tae Hoon Kim
Active enhancers of the human genome generate long noncoding transcripts known as enhancer RNAs (eRNAs). How dynamic transcriptional changes of eRNAs are physically and functionally linked with target gene transcription remains unclear. To investigate the dynamic functional relationships among eRNAs and target promoters, we obtained a dense time series of GRO-seq and ChIP-seq data to generate a time-resolved enhancer activity map of a cell undergoing an innate antiviral immune response. Dynamic changes in eRNA and pre-mRNA transcription activities suggest distinct regulatory roles of enhancers...
October 23, 2018: Genome Research
Joseph D Coolon, C Joel McManus, Kraig R Stevenson, Brenton R Graveley, Patricia J Wittkopp
No abstract text is available yet for this article.
November 2018: Genome Research
Fu-Hui Xiao, Xiao-Qiong Chen, Qin Yu, Yunshuang Ye, Yao-Wen Liu, Dongjing Yan, Li-Qin Yang, Guijun Chen, Rong Lin, Liping Yang, Xiaoping Liao, Wen Zhang, Wei Zhang, Nelson Leung-Sang Tang, Xiao-Fan Wang, Jumin Zhou, Wang-Wei Cai, Yong-Han He, Qing-Peng Kong
Centenarians (CENs) are excellent subjects to study the mechanisms of human longevity and healthy aging. Here, we analyzed the transcriptomes of 76 centenarians, 54 centenarian-children, and 41 spouses of centenarian-children by RNA sequencing and found that, among the significantly differentially expressed genes (SDEGs) exhibited by CENs, the autophagy-lysosomal pathway is significantly up-regulated. Overexpression of several genes from this pathway, CTSB , ATP6V0C , ATG4D , and WIPI1 , could promote autophagy and delay senescence in cultured IMR-90 cells, while overexpression of the Drosophila homolog of WIPI1 , Atg18a , extended the life span in transgenic flies...
November 2018: Genome Research
Mehrdad Bakhtiari, Sharona Shleizer-Burko, Melissa Gymrek, Vikas Bansal, Vineet Bafna
Whole-genome sequencing is increasingly used to identify Mendelian variants in clinical pipelines. These pipelines focus on single-nucleotide variants (SNVs) and also structural variants, while ignoring more complex repeat sequence variants. Here, we consider the problem of genotyping Variable Number Tandem Repeats (VNTRs), composed of inexact tandem duplications of short (6-100 bp) repeating units. VNTRs span 3% of the human genome, are frequently present in coding regions, and have been implicated in multiple Mendelian disorders...
November 2018: Genome Research
Armande Ang Houle, Heather Gibling, Fabien C Lamaze, Hilary A Edgington, David Soave, Marie-Julie Fave, Mawusse Agbessi, Vanessa Bruat, Lincoln D Stein, Philip Awadalla
The binding of PRDM9 to chromatin is a key step in the induction of DNA double-strand breaks associated with meiotic recombination hotspots; it is normally expressed solely in germ cells. We interrogated 1879 cancer samples in 39 different cancer types and found that PRDM9 is unexpectedly expressed in 20% of these tumors even after stringent gene homology correction. The expression levels of PRDM9 in tumors are significantly higher than those found in healthy neighboring tissues and in healthy nongerm tissue databases...
November 2018: Genome Research
Anand Mayakonda, De-Chen Lin, Yassen Assenov, Christoph Plass, H Phillip Koeffler
Numerous large-scale genomic studies of matched tumor-normal samples have established the somatic landscapes of most cancer types. However, the downstream analysis of data from somatic mutations entails a number of computational and statistical approaches, requiring usage of independent software and numerous tools. Here, we describe an R Bioconductor package, Maftools, which offers a multitude of analysis and visualization modules that are commonly used in cancer genomic studies, including driver gene identification, pathway, signature, enrichment, and association analyses...
November 2018: Genome Research
Mikhail Kolmogorov, Joel Armstrong, Brian J Raney, Ian Streeter, Matthew Dunn, Fengtang Yang, Duncan Odom, Paul Flicek, Thomas M Keane, David Thybert, Benedict Paten, Son Pham
Despite the rapid development of sequencing technologies, the assembly of mammalian-scale genomes into complete chromosomes remains one of the most challenging problems in bioinformatics. To help address this difficulty, we developed Ragout 2, a reference-assisted assembly tool that works for large and complex genomes. By taking one or more target assemblies (generated from an NGS assembler) and one or multiple related reference genomes, Ragout 2 infers the evolutionary relationships between the genomes and builds the final assemblies using a genome rearrangement approach...
November 2018: Genome Research
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