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.
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.
DNA replication initiation in eukaryotic cells is mediated by a complex system, but its spatiotemporal regulation remains poorly understood.While factors that promote replication are typically invoked to explain initiation patterns, evidence is accumulating for the involvement of a variety of repressive factors as well.Combinations of repressive histone modifications may function in synergistic ways to promote site-specific replication initiation, possibly through the activity of histone mark erasers.Considering both activation and repression as central to replication initiation could have important implications for the interpretation of DNA replication biology.
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.
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.
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.
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.
The organization of DNA replication through time: initiation from well‐defined chromosomal loci (replication origins), and efficient DNA synthesis supported by an abundance of elongation factors, give way to random initiation and sub‐optimal replication fork elongation as S phase proceeds.
Many overlapping surveillance and repair mechanisms operate in eukaryotic cells to ensure the stability of the genome. We have screened to isolate yeast mutants exhibiting increased levels of ...recombination between repeated sequences. Here we characterize one of these mutants, elg1. Strains lacking Elg1p exhibit elevated levels of recombination between homologous and nonhomologous chromosomes, as well as between sister chromatids and direct repeats. These strains also exhibit increased levels of chromosome loss. The Elg1 protein shares sequence homology with the large subunit of the clamp loader replication factor C (RFC) and with the product of two additional genes involved in checkpoint functions and genome maintenance: RAD24 and CTF18. Elg1p forms a complex with the Rfc2-5 subunits of RFC that is distinct from the previously described RFC-like complexes containing Rad24 and Ctf18. Genetic data indicate that the Elg1, Ctf18, and Rad24 RFC-like complexes work in three separate pathways important for maintaining the integrity of the genome and for coping with various genomic stresses.
The composition of a plant virome may represent spatiotemporal patterns of plant virus abundance. Using next generation sequencing, we investigated the viromes of watermelon fruits grown in two ...adjacent open fields located in Eastern Israel: Kalia and Mitzpe-Shalem. The two viromes were comprised of distinct virus species and genera. Studying spatial and temporal effects on virus occurrence, we detected the crinivirus Cucurbit yellow stunting disorder virus in Kalia and not in Mitzpe-Shalem, irrespective of collection time. A spatial effect was also observed regarding the occurrence of the begomovirus Watermelon chlorotic stunt virus, which was not detected in watermelons from Kalia, but rather in watermelon fruit in Northern Israel in 2018. A temporal effect was observed on the appearance of the ipomovirus Cucumber vein yellowing virus, which was detected in watermelons from Kalia in 2017 and was absent in the 2016 virome analysis. Importantly, regardless of temporal and spatial effects, the crinivirus Cucurbit chlorotic yellows virus, which was new to Israeli landscape, was detected in all the tested watermelon plants. These changes in viral abundance were associated with an early whitefly (Bemisia tabaci) occurrence, which might be the cause for the severe disease spread in watermelons.
Somatic mutations have important biological ramifications while exerting substantial rate, type, and genomic location heterogeneity. Yet, their sporadic occurrence makes them difficult to study at ...scale and across individuals. Lymphoblastoid cell lines (LCLs), a model system for human population and functional genomics, harbor large numbers of somatic mutations and have been extensively genotyped. By comparing 1,662 LCLs, we report that the mutational landscape of the genome varies across individuals in terms of the number of mutations, their genomic locations, and their spectra; this variation may itself be modulated by somatic trans-acting mutations. Mutations attributed to the translesion DNA polymerase η follow two different modes of formation, with one mode accounting for the hypermutability of the inactive X chromosome. Nonetheless, the distribution of mutations along the inactive X chromosome appears to follow an epigenetic memory of the active form.
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•Analysis of 885,655 mutations from 1,662 lymphoblastoid cell lines (LCLs)•Inter-individual variation in the rate and genomic distribution of mutations•BCL6 is a candidate modulator of the mutational landscape in LCLs•Hypermutation of the inactive X chromosome is attributed to DNA polymerase η
An analysis of the mutational landscape in 1,662 individuals reveals genome-wide variation in mutational loads, genomic distribution, and signatures, all of which appear to be modulated by somatic mutations in trans. The inactive X chromosome is unusual in bearing an excess of replication-timing-uncoupled DNA polymerase η-mediated mutations.
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