Publications by authors named "Amnon Koren"

41 Publications

Cohesin Core Complex Gene Dosage Contributes to Germinal Center Derived Lymphoma Phenotypes and Outcomes.

Front Immunol 2021 21;12:688493. Epub 2021 Sep 21.

Division of Hematology and Medical Oncology, Department of Medicine, Weill Cornell Medicine, New York, NY, United States.

The cohesin complex plays critical roles in genomic stability and gene expression through effects on 3D architecture. Cohesin core subunit genes are mutated across a wide cross-section of cancers, but not in germinal center (GC) derived lymphomas. In spite of this, haploinsufficiency of cohesin ATPase subunit was shown to contribute to malignant transformation of GC B-cells in mice. Herein we explored potential mechanisms and clinical relevance of deficiency in GC lymphomagenesis. Transcriptional profiling of haploinsufficient murine lymphomas revealed downregulation of genes repressed by loss of epigenetic tumor suppressors and . Profiling 3D chromosomal interactions in lymphomas revealed impaired enhancer-promoter interactions affecting genes like , which was aberrantly downregulated in deficient lymphomas. plays important roles in B-cell exit from the GC reaction, and single cell RNA-seq profiles and phenotypic trajectory analysis in mutant mice revealed a specific defect in commitment to the final steps of plasma cell differentiation. Although deficiency resulted in structural abnormalities in GC B-cells, there was no increase of somatic mutations or structural variants in haploinsufficient lymphomas, suggesting that cohesin deficiency largely induces lymphomas through disruption of enhancer-promoter interactions of terminal differentiation and tumor suppressor genes. Strikingly, the presence of the haploinsufficient GC B-cell transcriptional signature in human patients with GC-derived diffuse large B-cell lymphoma (DLBCL) was linked to inferior clinical outcome and low expression of cohesin core subunits. Reciprocally, reduced expression of cohesin subunits was an independent risk factor for worse survival int DLBCL patient cohorts. Collectively, the data suggest that functions as a bona fide tumor suppressor for lymphomas through non-genetic mechanisms, and drives disease by disrupting the commitment of GC B-cells to the plasma cell fate.
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http://dx.doi.org/10.3389/fimmu.2021.688493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8490713PMC
September 2021

TIGER: inferring DNA replication timing from whole-genome sequence data.

Bioinformatics 2021 Mar 11. Epub 2021 Mar 11.

Department of Molecular Biology and Genetics, Cornell University, Ithaca NY 14850 USA.

Motivation: Genomic DNA replicates according to a reproducible spatiotemporal program, with some loci replicating early in S phase while others replicate late. Despite being a central cellular process, DNA replication timing studies have been limited in scale due to technical challenges.

Results: We present TIGER (Timing Inferred from Genome Replication), a computational approach for extracting DNA replication timing information from whole genome sequence data obtained from proliferating cell samples. The presence of replicating cells in a biological specimen leads to non-uniform representation of genomic DNA that depends on the timing of replication of different genomic loci. Replication dynamics can hence be observed in genome sequence data by analyzing DNA copy number along chromosomes while accounting for other sources of sequence coverage variation. TIGER is applicable to any species with a contiguous genome assembly and rivals the quality of experimental measurements of DNA replication timing. It provides a straightforward approach for measuring replication timing and can readily be applied at scale.

Availability And Implementation: TIGER is available at https://github.com/TheKorenLab/TIGER.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btab166DOI Listing
March 2021

Human DDK rescues stalled forks and counteracts checkpoint inhibition at unfired origins to complete DNA replication.

Mol Cell 2021 02;81(3):426-441.e8

Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes.
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http://dx.doi.org/10.1016/j.molcel.2021.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211091PMC
February 2021

Mutation Rate Variability across Human Y-Chromosome Haplogroups.

Mol Biol Evol 2021 03;38(3):1000-1005

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY.

A common assumption in dating patrilineal events using Y-chromosome sequencing data is that the Y-chromosome mutation rate is invariant across haplogroups. Previous studies revealed interhaplogroup heterogeneity in phylogenetic branch length. Whether this heterogeneity is caused by interhaplogroup mutation rate variation or nongenetic confounders remains unknown. Here, we analyzed whole-genome sequences from cultured cells derived from >1,700 males. We confirmed the presence of branch length heterogeneity. We demonstrate that sex-chromosome mutations that appear within cell lines, which likely occurred somatically or in vitro (and are thus not influenced by nongenetic confounders) are informative for germline mutational processes. Using within-cell-line mutations, we computed a relative Y-chromosome somatic mutation rate, and uncovered substantial variation (up to 83.3%) in this proxy for germline mutation rate among haplogroups. This rate positively correlates with phylogenetic branch length, indicating that interhaplogroup mutation rate variation is a likely cause of branch length heterogeneity.
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http://dx.doi.org/10.1093/molbev/msaa268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7947773PMC
March 2021

Positive and Negative Regulation of DNA Replication Initiation.

Trends Genet 2020 11 29;36(11):868-879. Epub 2020 Jul 29.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA. Electronic address:

Genomic DNA is replicated every cell cycle by the programmed activation of replication origins at specific times and chromosomal locations. The factors that define the locations of replication origins and their typical activation times in eukaryotic cells are poorly understood. Previous studies highlighted the role of activating factors and epigenetic modifications in regulating replication initiation. Here, we review the role that repressive pathways - and their alleviation - play in establishing the genomic landscape of replication initiation. Several factors mediate this repression, in particular, factors associated with inactive chromatin. Repression can support organized, yet stochastic, replication initiation, and its absence could explain instances of rapid and random replication or re-replication.
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http://dx.doi.org/10.1016/j.tig.2020.06.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572746PMC
November 2020

Molecular signatures of aneuploidy-driven adaptive evolution.

Nat Commun 2020 01 30;11(1):588. Epub 2020 Jan 30.

Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, 02115, USA.

Alteration of normal ploidy (aneuploidy) can have a number of opposing effects, such as unbalancing protein abundances and inhibiting cell growth but also accelerating genetic diversification and rapid adaptation. The interplay of these detrimental and beneficial effects remains puzzling. Here, to understand how cells develop tolerance to aneuploidy, we subject disomic (i.e. with an extra chromosome copy) strains of yeast to long-term experimental evolution under strong selection, by forcing disomy maintenance and daily population dilution. We characterize mutations, karyotype alterations and gene expression changes, and dissect the associated molecular strategies. Cells with different extra chromosomes accumulated mutations at distinct rates and displayed diverse adaptive events. They tended to evolve towards normal ploidy through chromosomal DNA loss and gene expression changes. We identify genes with recurrent mutations and altered expression in multiple lines, revealing a variant that improves growth under genotoxic stresses. These findings support rapid evolvability of disomic strains that can be used to characterize fitness effects of mutations under different stress conditions.
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http://dx.doi.org/10.1038/s41467-019-13669-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992709PMC
January 2020

Genomic methods for measuring DNA replication dynamics.

Chromosome Res 2020 03 17;28(1):49-67. Epub 2019 Dec 17.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, 14853, USA.

Genomic DNA replicates according to a defined temporal program in which early-replicating loci are associated with open chromatin, higher gene density, and increased gene expression levels, while late-replicating loci tend to be heterochromatic and show higher rates of genomic instability. The ability to measure DNA replication dynamics at genome scale has proven crucial for understanding the mechanisms and cellular consequences of DNA replication timing. Several methods, such as quantification of nucleotide analog incorporation and DNA copy number analyses, can accurately reconstruct the genomic replication timing profiles of various species and cell types. More recent developments have expanded the DNA replication genomic toolkit to assays that directly measure the activity of replication origins, while single-cell replication timing assays are beginning to reveal a new level of replication timing regulation. The combination of these methods, applied on a genomic scale and in multiple biological systems, promises to resolve many open questions and lead to a holistic understanding of how eukaryotic cells replicate their genomes accurately and efficiently.
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http://dx.doi.org/10.1007/s10577-019-09624-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7131883PMC
March 2020

Replication timing alterations in leukemia affect clinically relevant chromosome domains.

Blood Adv 2019 11;3(21):3201-3213

Department of Biological Science, Florida State University, Tallahassee, FL.

Human B-cell precursor acute lymphoid leukemias (BCP-ALLs) comprise a group of genetically and clinically distinct disease entities with features of differentiation arrest at known stages of normal B-lineage differentiation. We previously showed that BCP-ALL cells display unique and clonally heritable, stable DNA replication timing (RT) programs (ie, programs describing the variable order of replication and subnuclear 3D architecture of megabase-scale chromosomal units of DNA in different cell types). To determine the extent to which BCP-ALL RT programs mirror or deviate from specific stages of normal human B-cell differentiation, we transplanted immunodeficient mice with quiescent normal human CD34+ cord blood cells and obtained RT signatures of the regenerating B-lineage populations. We then compared these with RT signatures for leukemic cells from a large cohort of BCP-ALL patients with varied genetic subtypes and outcomes. The results identify BCP-ALL subtype-specific features that resemble specific stages of B-cell differentiation and features that seem to be associated with relapse. These results suggest that the genesis of BCP-ALL involves alterations in RT that reflect biologically significant and potentially clinically relevant leukemia-specific epigenetic changes.
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http://dx.doi.org/10.1182/bloodadvances.2019000641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855107PMC
November 2019

Insight Into Late Wilting Disease of Cucumber Demonstrates the Complexity of the Phenomenon in Fluctuating Environments.

Plant Dis 2019 Nov 6;103(11):2877-2883. Epub 2019 Sep 6.

Department of Plant Pathology and Weed Sciences, Agricultural Research Organization, The Volcani Center, Rishon Lezion 7528809, Israel.

Some diseases are caused by coinfection of several pathogens in the same plant. However, studies on the complexity of these coinfection events under different environmental conditions are scarce. Our ongoing research involves late wilting disease of cucumber caused by coinfection of (CGMMV) and spp. We specifically investigated the role of various temperatures (18, 25, 32°C) on the coinfection by CGMMV and two predominant species occurring in cucumber greenhouses under Middle Eastern climatic conditions. During the summer months, was most common, whereas predominated during the winter-spring period. preferred higher temperatures while preferred low temperatures and caused very low levels of disease at 32°C when the 6-day-old seedlings were infected with alone. Nevertheless, after applying a later coinfection with CGMMV on the 14-day-old plants, a synergistic effect was detected for both species at optimal and suboptimal temperatures, with causing high mortality incidence even at 32°C. The symptoms caused by CGMMV infection appeared earlier as the temperature increased. However, within each temperature, no significant influence of the combined infection was detected. Our results demonstrate the complexity of coinfection in changing environmental conditions and indicate its involvement in disease development and severity as compared with infection by each of the pathogens alone.
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http://dx.doi.org/10.1094/PDIS-12-18-2141-REDOI Listing
November 2019

Germline Structural Variations Are Preferential Sites of DNA Replication Timing Plasticity during Development.

Genome Biol Evol 2019 06;11(6):1663-1678

Department of Molecular Biology and Genetics, Cornell University.

The DNA replication timing program is modulated throughout development and is also one of the main factors influencing the distribution of mutation rates across the genome. However, the relationship between the mutagenic influence of replication timing and its developmental plasticity remains unexplored. Here, we studied the distribution of copy number variations (CNVs) and single nucleotide polymorphisms across the zebrafish genome in relation to changes in DNA replication timing during embryonic development in this model vertebrate species. We show that CNV sites exhibit strong replication timing plasticity during development, replicating significantly early during early development but significantly late during more advanced developmental stages. Reciprocally, genomic regions that changed their replication timing during development contained a higher proportion of CNVs than developmentally constant regions. Developmentally plastic CNV sites, in particular those that become delayed in their replication timing, were enriched for the clustered protocadherins, a set of genes important for neuronal development that have undergone extensive genetic and epigenetic diversification during zebrafish evolution. In contrast, single nucleotide polymorphism sites replicated consistently early throughout embryonic development, highlighting a unique aspect of the zebrafish genome. Our results uncover a hitherto unrecognized interface between development and evolution.
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http://dx.doi.org/10.1093/gbe/evz098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582765PMC
June 2019

Next-Generation Sequencing Enables Spatiotemporal Resolution of Human Centromere Replication Timing.

Genes (Basel) 2019 04 2;10(4). Epub 2019 Apr 2.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.

Centromeres serve a critical function in preserving genome integrity across sequential cell divisions, by mediating symmetric chromosome segregation. The repetitive, heterochromatic nature of centromeres is thought to be inhibitory to DNA replication, but has also led to their underrepresentation in human reference genome assemblies. Consequently, centromeres have been excluded from genomic replication timing analyses, leaving their time of replication unresolved. However, the most recent human reference genome, hg38, included models of centromere sequences. To establish the experimental requirements for achieving replication timing profiles for centromeres, we sequenced G₁- and S-phase cells from five human cell lines, and aligned the sequence reads to hg38. We were able to infer DNA replication timing profiles for the centromeres in each of the five cell lines, which showed that centromere replication occurs in mid-to-late S phase. Furthermore, we found that replication timing was more variable between cell lines in the centromere regions than expected, given the distribution of variation in replication timing genome-wide. These results suggest the potential of these, and future, sequence models to enable high-resolution studies of replication in centromeres and other heterochromatic regions.
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http://dx.doi.org/10.3390/genes10040269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6523654PMC
April 2019

Combined Infection with Cucumber green mottle mosaic virus and Pythium Species Causes Extensive Collapse in Cucumber Plants.

Plant Dis 2018 Apr 13;102(4):753-759. Epub 2018 Feb 13.

Department of Plant Pathology and Weed Sciences, Agricultural Research Organization, The Volcani Center.

In the last decade, the phenomenon of late-wilting has increased in cucumber greenhouses during Cucumber green mottle mosaic virus (CGMMV) epidemics. Because the wilting appears in defined patches accompanied by root rot, it was hypothesized that the phenomenon is caused by coinfection of soilborne pathogens and CGMMV. A field survey showed that 69% of the collapsed plants were infected with both Pythium spp. and CGMMV, whereas only 20 and 6.6% were singly infected with Pythium spp. or CGMMV, respectively. Artificial inoculations in controlled-environmental growth chambers and glasshouse experiments showed that coinfection with Pythium spinosum and CGMMV leads to a strong synergistic wilting effect and reduces growth parameters. The synergy values of the wilting effect were not influenced by the time interval between P. spinosum and CGMMV infection. However, dry mass synergy values were decreased with longer intervals between infections. The results obtained in this study support the complexity of the wilting phenomenon described in commercial cucumber grown in protected structures during infection of Pythium spp. on the background of a vast CGMMV epidemic. They encourage a wider perspective of the complexity of agricultural diseases to apply the most suitable disease management.
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http://dx.doi.org/10.1094/PDIS-07-17-1124-REDOI Listing
April 2018

Quantification of somatic mutation flow across individual cell division events by lineage sequencing.

Genome Res 2018 12 20;28(12):1901-1918. Epub 2018 Nov 20.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.

Mutation data reveal the dynamic equilibrium between DNA damage and repair processes in cells and are indispensable to the understanding of age-related diseases, tumor evolution, and the acquisition of drug resistance. However, available genome-wide methods have a limited ability to resolve rare somatic variants and the relationships between these variants. Here, we present lineage sequencing, a new genome sequencing approach that enables somatic event reconstruction by providing quality somatic mutation call sets with resolution as high as the single-cell level in subject lineages. Lineage sequencing entails sampling single cells from a population and sequencing subclonal sample sets derived from these cells such that knowledge of relationships among the cells can be used to jointly call variants across the sample set. This approach integrates data from multiple sequence libraries to support each variant and precisely assigns mutations to lineage segments. We applied lineage sequencing to a human colon cancer cell line with a DNA polymerase epsilon () proofreading deficiency (HT115) and a human retinal epithelial cell line immortalized by constitutive telomerase expression (RPE1). Cells were cultured under continuous observation to link observed single-cell phenotypes with single-cell mutation data. The high sensitivity, specificity, and resolution of the data provide a unique opportunity for quantitative analysis of variation in mutation rate, spectrum, and correlations among variants. Our data show that mutations arrive with nonuniform probability across sublineages and that DNA lesion dynamics may cause strong correlations between certain mutations.
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http://dx.doi.org/10.1101/gr.238543.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280753PMC
December 2018

Germline DNA replication timing shapes mammalian genome composition.

Nucleic Acids Res 2018 09;46(16):8299-8310

Department of Microbiology and Molecular Genetics, IMRIC, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

Mammalian DNA replication is a highly organized and regulated process. Large, Mb-sized regions are replicated at defined times along S-phase. Replication Timing (RT) is thought to play a role in shaping the mammalian genome by affecting mutation rates. Previous analyses relied on somatic RT profiles. However, only germline mutations are passed on to offspring and affect genomic composition. Therefore, germ cell RT information is necessary to evaluate the influences of RT on the mammalian genome. We adapted the RT mapping technique for limited amounts of cells, and measured RT from two stages in the mouse germline - primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). RT in germline cells exhibited stronger correlations to both mutation rate and recombination hotspots density than those of RT in somatic tissues, emphasizing the importance of using correct tissues-of-origin for RT profiling. Germline RT maps exhibited stronger correlations to additional genetic features including GC-content, transposable elements (SINEs and LINEs), and gene density. GC content stratification and multiple regression analysis revealed independent contributions of RT to SINE, gene, mutation, and recombination hotspot densities. Together, our results establish a central role for RT in shaping multiple levels of mammalian genome composition.
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http://dx.doi.org/10.1093/nar/gky610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6144785PMC
September 2018

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System.

J Vis Exp 2018 04 30(134). Epub 2018 Apr 30.

Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation; Department of Cell Biology, University of Oklahoma Health Sciences Center;

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.
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http://dx.doi.org/10.3791/57146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101039PMC
April 2018

A local strain of Paprika mild mottle virus breaks L resistance in peppers and is accelerated in Tomato brown rugose fruit virus-infected Tm-2-resistant tomatoes.

Virus Genes 2018 Apr 10;54(2):280-289. Epub 2018 Feb 10.

Department of Plant Pathology, ARO The Volcani Center, HaMaccabim Road 68, 7528809, P.O.B 15159, Bet Dagan, Israel.

During October 2014, unfamiliar mild mosaic and mottling symptoms were identified on leaves of pepper (Capsicum chinense cv. Habanero) seedlings grown in the Arava valley in Israel 2-3 weeks post planting. Symptomatic plants were tested positive by ELISA using laboratory-produced antisera for tobamovirus species. Typical tobamovirus rod-shaped morphology was observed by transmission electron microscopy (TEM) analysis of purified virion preparation that was used for mechanical inoculation of laboratory test plants for the completion of Koch's postulates. The complete viral genome was sequenced from small interfering RNA purified from symptomatic pepper leaves and fruits by next-generation sequencing (NGS) using Illumina MiSeq platform. The contigs generated by the assembly covered 80% of the viral genome. RT-PCR amplification and Sanger sequencing were employed in order to validate the sequence generated by NGS technology. The nucleotide sequence of the complete viral genome was 99% identical to the complete genome of Paprika mild mottle virus isolate from Japan (PaMMV-J), and the deduced amino acid sequence was 99% identical to PaMMV-J protein. Amplicons from seed RNA showed 100% identity to the viral isolate from the collected symptomatic pepper plants. Partial host range analysis revealed a slow development of systemic infection in inoculated tomato plants (Lycopersicon esculentum). Interestingly, double inoculation of susceptible wild-type tomato plants and Tm-2-resistant tomato plants with the PaMMV-IL and Tomato brown rugose fruit virus (ToBRFV) resulted in accelerated viral expression in the plants.
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http://dx.doi.org/10.1007/s11262-018-1539-2DOI Listing
April 2018

Mismatch repair prefers exons.

Nat Genet 2017 11;49(12):1673-1674

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.

A new analysis of cancer genomes identifies a decrease in the mutation burden of exons, but not introns, as compared to expectation. This difference can be explained by preferential recruitment of the DNA mismatch repair machinery to a protein modification that marks exons.
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http://dx.doi.org/10.1038/ng.3993DOI Listing
November 2017

Analysis of somatic microsatellite indels identifies driver events in human tumors.

Nat Biotechnol 2017 Oct 11;35(10):951-959. Epub 2017 Sep 11.

Massachusetts General Hospital Center for Cancer Research, Charlestown, Massachusetts, USA.

Microsatellites (MSs) are tracts of variable-length repeats of short DNA motifs that exhibit high rates of mutation in the form of insertions or deletions (indels) of the repeated motif. Despite their prevalence, the contribution of somatic MS indels to cancer has been largely unexplored, owing to difficulties in detecting them in short-read sequencing data. Here we present two tools: MSMuTect, for accurate detection of somatic MS indels, and MSMutSig, for identification of genes containing MS indels at a higher frequency than expected by chance. Applying MSMuTect to whole-exome data from 6,747 human tumors representing 20 tumor types, we identified >1,000 previously undescribed MS indels in cancer genes. Additionally, we demonstrate that the number and pattern of MS indels can accurately distinguish microsatellite-stable tumors from tumors with microsatellite instability, thus potentially improving classification of clinically relevant subgroups. Finally, we identified seven MS indel driver hotspots: four in known cancer genes (ACVR2A, RNF43, JAK1, and MSH3) and three in genes not previously implicated as cancer drivers (ESRP1, PRDM2, and DOCK3).
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http://dx.doi.org/10.1038/nbt.3966DOI Listing
October 2017

DNA replication timing during development anticipates transcriptional programs and parallels enhancer activation.

Genome Res 2017 08 16;27(8):1406-1416. Epub 2017 May 16.

Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.

In dividing cells, DNA replication occurs in a precise order, but many questions remain regarding the mechanisms of replication timing establishment and regulation. We now have generated genome-wide, high-resolution replication timing maps throughout zebrafish development. Unexpectedly, in the rapid cell cycles preceding the midblastula transition, a defined timing program was present that predicted the initial wave of zygotic transcription. Replication timing was thereafter progressively and continuously remodeled across the majority of the genome, and epigenetic changes involved in enhancer activation frequently paralleled developmental changes in replication timing. The long arm of Chromosome 4 underwent a dramatic developmentally regulated switch to late replication during gastrulation, reminiscent of mammalian X Chromosome inactivation. This study reveals that replication timing is dynamic and tightly linked to epigenetic and transcriptional changes throughout early zebrafish development. These data provide insight into the regulation and functions of replication timing and will enable further mechanistic studies.
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http://dx.doi.org/10.1101/gr.218602.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538556PMC
August 2017

Mutational Strand Asymmetries in Cancer Genomes Reveal Mechanisms of DNA Damage and Repair.

Cell 2016 Jan 21;164(3):538-49. Epub 2016 Jan 21.

Massachusetts General Hospital Cancer Center and Department of Pathology, 55 Fruit Street, Boston, MA 02114, USA; Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA; Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA. Electronic address:

Mutational processes constantly shape the somatic genome, leading to immunity, aging, cancer, and other diseases. When cancer is the outcome, we are afforded a glimpse into these processes by the clonal expansion of the malignant cell. Here, we characterize a less explored layer of the mutational landscape of cancer: mutational asymmetries between the two DNA strands. Analyzing whole-genome sequences of 590 tumors from 14 different cancer types, we reveal widespread asymmetries across mutagenic processes, with transcriptional ("T-class") asymmetry dominating UV-, smoking-, and liver-cancer-associated mutations and replicative ("R-class") asymmetry dominating POLE-, APOBEC-, and MSI-associated mutations. We report a striking phenomenon of transcription-coupled damage (TCD) on the non-transcribed DNA strand and provide evidence that APOBEC mutagenesis occurs on the lagging-strand template during DNA replication. As more genomes are sequenced, studying and classifying their asymmetries will illuminate the underlying biological mechanisms of DNA damage and repair.
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http://dx.doi.org/10.1016/j.cell.2015.12.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753048PMC
January 2016

HUMORAL IMMUNITY. T cell help controls the speed of the cell cycle in germinal center B cells.

Science 2015 Aug 16;349(6248):643-6. Epub 2015 Jul 16.

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA. Howard Hughes Medical Institute (HHMI), The Rockefeller University, New York, NY 10065, USA.

The germinal center (GC) is a microanatomical compartment wherein high-affinity antibody-producing B cells are selectively expanded. B cells proliferate and mutate their antibody genes in the dark zone (DZ) of the GC and are then selected by T cells in the light zone (LZ) on the basis of affinity. Here, we show that T cell help regulates the speed of cell cycle phase transitions and DNA replication of GC B cells. Genome sequencing and single-molecule analyses revealed that T cell help shortens S phase by regulating replication fork progression, while preserving the relative order of replication origin activation. Thus, high-affinity GC B cells are selected by a mechanism that involves prolonged dwell time in the DZ where selected cells undergo accelerated cell cycles.
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http://dx.doi.org/10.1126/science.aac4919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4809261PMC
August 2015

Genome-wide patterns and properties of de novo mutations in humans.

Nat Genet 2015 Jul 18;47(7):822-826. Epub 2015 May 18.

Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

Mutations create variation in the population, fuel evolution and cause genetic diseases. Current knowledge about de novo mutations is incomplete and mostly indirect. Here we analyze 11,020 de novo mutations from the whole genomes of 250 families. We show that de novo mutations in the offspring of older fathers are not only more numerous but also occur more frequently in early-replicating, genic regions. Functional regions exhibit higher mutation rates due to CpG dinucleotides and show signatures of transcription-coupled repair, whereas mutation clusters with a unique signature point to a new mutational mechanism. Mutation and recombination rates independently associate with nucleotide diversity, and regional variation in human-chimpanzee divergence is only partly explained by heterogeneity in mutation rate. Finally, we provide a genome-wide mutation rate map for medical and population genetics applications. Our results provide new insights and refine long-standing hypotheses about human mutagenesis.
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http://dx.doi.org/10.1038/ng.3292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485564PMC
July 2015

Cell-of-origin chromatin organization shapes the mutational landscape of cancer.

Nature 2015 Feb;518(7539):360-364

Division of Genetics, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, 02115.

Cancer is a disease potentiated by mutations in somatic cells. Cancer mutations are not distributed uniformly along the human genome. Instead, different human genomic regions vary by up to fivefold in the local density of cancer somatic mutations, posing a fundamental problem for statistical methods used in cancer genomics. Epigenomic organization has been proposed as a major determinant of the cancer mutational landscape. However, both somatic mutagenesis and epigenomic features are highly cell-type-specific. We investigated the distribution of mutations in multiple independent samples of diverse cancer types and compared them to cell-type-specific epigenomic features. Here we show that chromatin accessibility and modification, together with replication timing, explain up to 86% of the variance in mutation rates along cancer genomes. The best predictors of local somatic mutation density are epigenomic features derived from the most likely cell type of origin of the corresponding malignancy. Moreover, we find that cell-of-origin chromatin features are much stronger determinants of cancer mutation profiles than chromatin features of matched cancer cell lines. Furthermore, we show that the cell type of origin of a cancer can be accurately determined based on the distribution of mutations along its genome. Thus, the DNA sequence of a cancer genome encompasses a wealth of information about the identity and epigenomic features of its cell of origin.
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http://dx.doi.org/10.1038/nature14221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405175PMC
February 2015

Genetic variation in human DNA replication timing.

Cell 2014 Nov 13;159(5):1015-1026. Epub 2014 Nov 13.

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Genomic DNA replicates in a choreographed temporal order that impacts the distribution of mutations along the genome. We show here that DNA replication timing is shaped by genetic polymorphisms that act in cis upon megabase-scale DNA segments. In genome sequences from proliferating cells, read depth along chromosomes reflected DNA replication activity in those cells. We used this relationship to analyze variation in replication timing among 161 individuals sequenced by the 1000 Genomes Project. Genome-wide association of replication timing with genetic variation identified 16 loci at which inherited alleles associate with replication timing. We call these "replication timing quantitative trait loci" (rtQTLs). rtQTLs involved the differential use of replication origins, exhibited allele-specific effects on replication timing, and associated with gene expression variation at megabase scales. Our results show replication timing to be shaped by genetic polymorphism and identify a means by which inherited polymorphism regulates the mutability of nearby sequences.
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http://dx.doi.org/10.1016/j.cell.2014.10.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359889PMC
November 2014

Abnormal dosage of ultraconserved elements is highly disfavored in healthy cells but not cancer cells.

PLoS Genet 2014 Oct 23;10(10):e1004646. Epub 2014 Oct 23.

Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

Ultraconserved elements (UCEs) are strongly depleted from segmental duplications and copy number variations (CNVs) in the human genome, suggesting that deletion or duplication of a UCE can be deleterious to the mammalian cell. Here we address the process by which CNVs become depleted of UCEs. We begin by showing that depletion for UCEs characterizes the most recent large-scale human CNV datasets and then find that even newly formed de novo CNVs, which have passed through meiosis at most once, are significantly depleted for UCEs. In striking contrast, CNVs arising specifically in cancer cells are, as a rule, not depleted for UCEs and can even become significantly enriched. This observation raises the possibility that CNVs that arise somatically and are relatively newly formed are less likely to have established a CNV profile that is depleted for UCEs. Alternatively, lack of depletion for UCEs from cancer CNVs may reflect the diseased state. In support of this latter explanation, somatic CNVs that are not associated with disease are depleted for UCEs. Finally, we show that it is possible to observe the CNVs of induced pluripotent stem (iPS) cells become depleted of UCEs over time, suggesting that depletion may be established through selection against UCE-disrupting CNVs without the requirement for meiotic divisions.
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http://dx.doi.org/10.1371/journal.pgen.1004646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207606PMC
October 2014

Origin replication complex binding, nucleosome depletion patterns, and a primary sequence motif can predict origins of replication in a genome with epigenetic centromeres.

mBio 2014 Sep 2;5(5):e01703-14. Epub 2014 Sep 2.

Unlabelled: Origins of DNA replication are key genetic elements, yet their identification remains elusive in most organisms. In previous work, we found that centromeres contain origins of replication (ORIs) that are determined epigenetically in the pathogenic yeast Candida albicans. In this study, we used origin recognition complex (ORC) binding and nucleosome occupancy patterns in Saccharomyces cerevisiae and Kluyveromyces lactis to train a machine learning algorithm to predict the position of active arm (noncentromeric) origins in the C. albicans genome. The model identified bona fide active origins as determined by the presence of replication intermediates on nondenaturing two-dimensional (2D) gels. Importantly, these origins function at their native chromosomal loci and also as autonomously replicating sequences (ARSs) on a linear plasmid. A "mini-ARS screen" identified at least one and often two ARS regions of ≥100 bp within each bona fide origin. Furthermore, a 15-bp AC-rich consensus motif was associated with the predicted origins and conferred autonomous replicating activity to the mini-ARSs. Thus, while centromeres and the origins associated with them are epigenetic, arm origins are dependent upon critical DNA features, such as a binding site for ORC and a propensity for nucleosome exclusion.

Importance: DNA replication machinery is highly conserved, yet the definition of exactly what specifies a replication origin differs in different species. Here, we utilized computational genomics to predict origin locations in Candida albicans by combining locations of binding sites for the conserved origin replication complex, necessary for replication initiation, together with chromatin organization patterns. We identified predicted sequences that exhibited bona fide origin function and developed a linear plasmid assay to delimit the DNA fragments necessary for origin function. Additionally, we found that a short AC-rich motif, which is enriched in predicted origins, is required for origin function. Thus, we demonstrated a new machine learning paradigm for identification of potential origins from a genome with no prior information. Furthermore, this work suggests that C. albicans has two different types of origins: "hard-wired" arm origins that rely upon specific sequence motifs and "epigenetic" centromeric origins that are recruited to kinetochores in a sequence-independent manner.
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http://dx.doi.org/10.1128/mBio.01703-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4173791PMC
September 2014

Thiol peroxidase deficiency leads to increased mutational load and decreased fitness in Saccharomyces cerevisiae.

Genetics 2014 Nov 29;198(3):905-17. Epub 2014 Aug 29.

Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (∆8) is viable. In this study, we employed two independent ∆8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ∆8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ∆8 lines showed a significant increase in nonrecurrent point mutations and indels. The original ∆8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all ∆8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ∆8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness.
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http://dx.doi.org/10.1534/genetics.114.169243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4224179PMC
November 2014

DNA replication timing: Coordinating genome stability with genome regulation on the X chromosome and beyond.

Authors:
Amnon Koren

Bioessays 2014 Oct 19;36(10):997-1004. Epub 2014 Aug 19.

Department of Genetics, Harvard Medical School, Boston, MA, USA; Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Recent studies based on next-generation DNA sequencing have revealed that the female inactive X chromosome is replicated in a rapid, unorganized manner, and undergoes increased rates of mutation. These observations link the organization of DNA replication timing to gene regulation on one hand, and to the generation of mutations on the other hand. More generally, the exceptional biology of the inactive X chromosome highlights general principles of genome replication. Cells may control replication timing by a combination of intrinsic replication origin properties, local chromatin states and global levels of replication factors, leading to a functional separation between the activity of genes and their mutation.
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http://dx.doi.org/10.1002/bies.201400077DOI Listing
October 2014

Random replication of the inactive X chromosome.

Genome Res 2014 Jan 24;24(1):64-9. Epub 2013 Sep 24.

Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA;

In eukaryotic cells, genomic DNA replicates in a defined temporal order. The inactive X chromosome (Xi), the most extensive instance of facultative heterochromatin in mammals, replicates later than the active X chromosome (Xa), but the replication dynamics of inactive chromatin are not known. By profiling human DNA replication in an allele-specific, chromosomally phased manner, we determined for the first time the replication timing along the active and inactive chromosomes (Xa and Xi) separately. Replication of the Xi was different from that of the Xa, varied among individuals, and resembled a random, unstructured process. The Xi replicated rapidly and at a time largely separable from that of the euchromatic genome. Late-replicating, transcriptionally inactive regions on the autosomes also replicated in an unstructured manner, similar to the Xi. We conclude that DNA replication follows two strategies: slow, ordered replication associated with transcriptional activity, and rapid, random replication of silent chromatin. The two strategies coexist in the same cell, yet are segregated in space and time.
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http://dx.doi.org/10.1101/gr.161828.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3875862PMC
January 2014

Mutational heterogeneity in cancer and the search for new cancer-associated genes.

Nature 2013 Jul 16;499(7457):214-218. Epub 2013 Jun 16.

The Broad Institute of MIT and Harvard, Cambridge, MA, 02141, USA.

Major international projects are underway that are aimed at creating a comprehensive catalogue of all the genes responsible for the initiation and progression of cancer. These studies involve the sequencing of matched tumour-normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false-positive findings that overshadow true driver events. We show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumour-normal pairs and discover extraordinary variation in mutation frequency and spectrum within cancer types, which sheds light on mutational processes and disease aetiology, and in mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and enable the identification of genes truly associated with cancer.
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http://dx.doi.org/10.1038/nature12213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919509PMC
July 2013
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