Strand-resolved mutagenicity of DNA damage and repair.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.

An integrative ENCODE resource for cancer genomics.

ENCODE comprises thousands of functional genomics datasets, and the encyclopedia covers hundreds of cell types, providing a universal annotation for genome interpretation. However, for particular applications, it may be advantageous to use a customized annotation. Here, we develop such a custom annotation by leveraging advanced assays, such as eCLIP, Hi-C, and whole-genome STARR-seq on a number of data-rich ENCODE cell types. A key aspect of this annotation is comprehensive and experimentally derived networks of both transcription factors and RNA-binding proteins (TFs and RBPs). Cancer, a disease of system-wide dysregulation, is an ideal application for such a network-based annotation. Specifically, for cancer-associated cell types, we put regulators into hierarchies and measure their network change (rewiring) during oncogenesis. We also extensively survey TF-RBP crosstalk, highlighting how SUB1, a previously uncharacterized RBP, drives aberrant tumor expression and amplifies the effect of MYC, a well-known oncogenic TF. Furthermore, we show how our annotation allows us to place oncogenic transformations in the context of a broad cell space; here, many normal-to-tumor transitions move towards a stem-like state, while oncogene knockdowns show an opposing trend. Finally, we organize the resource into a coherent workflow to prioritize key elements and variants, in addition to regulators. We showcase the application of this prioritization to somatic burdening, cancer differential expression and GWAS. Targeted validations of the prioritized regulators, elements and variants using siRNA knockdowns, CRISPR-based editing, and luciferase assays demonstrate the value of the ENCODE resource.

3D genome organization contributes to genome instability at fragile sites.

Common fragile sites (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features were suggested to underlie CFS instability, however, these features are prevalent across the genome. Therefore, the molecular mechanisms underlying CFS instability remain unclear. Here, we explore the transcriptional profile and DNA replication timing (RT) under mild replication stress in the context of the 3D genome organization. The results reveal a fragility signature, comprised of a TAD boundary overlapping a highly transcribed large gene with APH-induced RT-delay. This signature enables precise mapping of core fragility regions in known CFSs and identification of novel fragile sites. CFS stability may be compromised by incomplete DNA replication and repair in TAD boundaries core fragility regions leading to genomic instability. The identified fragility signature will allow for a more comprehensive mapping of CFSs and pave the way for investigating mechanisms promoting genomic instability in cancer.

High-resolution Repli-Seq defines the temporal choreography of initiation, elongation and termination of replication in mammalian cells.

BACKGROUND: DNA replication in mammalian cells occurs in a defined temporal order during S phase, known as the replication timing (RT) programme. Replication timing is developmentally regulated and correlated with chromatin conformation and local transcriptional potential. Here, we present RT profiles of unprecedented temporal resolution in two human embryonic stem cell lines, human colon carcinoma line HCT116, and mouse embryonic stem cells and their neural progenitor derivatives. RESULTS: Fine temporal windows revealed a remarkable degree of cell-to-cell conservation in RT, particularly at the very beginning and ends of S phase, and identified 5 temporal patterns of replication in all cell types, consistent with varying degrees of initiation efficiency. Zones of replication initiation (IZs) were detected throughout S phase and interacted in 3D space preferentially with other IZs of similar firing time. Temporal transition regions were resolved into segments of uni-directional replication punctuated at specific sites by small, inefficient IZs. Sites of convergent replication were divided into sites of termination or large constant timing regions consisting of many synchronous IZs in tandem. Developmental transitions in RT occured mainly by activating or inactivating individual IZs or occasionally by altering IZ firing time, demonstrating that IZs, rather than individual origins, are the units of developmental regulation. Finally, haplotype phasing revealed numerous regions of allele-specific and allele-independent asynchronous replication. Allele-independent asynchronous replication was correlated with the presence of previously mapped common fragile sites. CONCLUSIONS: Altogether, these data provide a detailed temporal choreography of DNA replication in mammalian cells.

Replication timing alterations in leukemia affect clinically relevant chromosome domains.

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.

Integrative detection and analysis of structural variation in cancer genomes.

Structural variants (SVs) can contribute to oncogenesis through a variety of mechanisms. Despite their importance, the identification of SVs in cancer genomes remains challenging. Here, we present a framework that integrates optical mapping, high-throughput chromosome conformation capture (Hi-C), and whole-genome sequencing to systematically detect SVs in a variety of normal or cancer samples and cell lines. We identify the unique strengths of each method and demonstrate that only integrative approaches can comprehensively identify SVs in the genome. By combining Hi-C and optical mapping, we resolve complex SVs and phase multiple SV events to a single haplotype. Furthermore, we observe widespread structural variation events affecting the functions of noncoding sequences, including the deletion of distal regulatory sequences, alteration of DNA replication timing, and the creation of novel three-dimensional chromatin structural domains. Our results indicate that noncoding SVs may be underappreciated mutational drivers in cancer genomes.

DNA replication timing alterations identify common markers between distinct progeroid diseases.

Progeroid syndromes are rare genetic disorders that phenotypically resemble natural aging. Different causal mutations have been identified, but no molecular alterations have been identified that are in common to these diseases. DNA replication timing (RT) is a robust cell type-specific epigenetic feature highly conserved in the same cell types from different individuals but altered in disease. Here, we characterized DNA RT program alterations in Hutchinson-Gilford progeria syndrome (HGPS) and Rothmund-Thomson syndrome (RTS) patients compared with natural aging and cellular senescence. Our results identified a progeroid-specific RT signature that is common to cells from three HGPS and three RTS patients and distinguishes them from healthy individuals across a wide range of ages. Among the RT abnormalities, we identified the tumor protein p63 gene (TP63) as a gene marker for progeroid syndromes. By using the redifferentiation of four patient-derived induced pluripotent stem cells as a model for the onset of progeroid syndromes, we tracked the progression of RT abnormalities during development, revealing altered RT of the TP63 gene as an early event in disease progression of both HGPS and RTS. Moreover, the RT abnormalities in progeroid patients were associated with altered isoform expression of TP63 Our findings demonstrate the value of RT studies to identify biomarkers not detected by other methods, reveal abnormal TP63 RT as an early event in progeroid disease progression, and suggest TP63 gene regulation as a potential therapeutic target.

Stability of patient-specific features of altered DNA replication timing in xenografts of primary human acute lymphoblastic leukemia.

Genome-wide DNA replication timing (RT) profiles reflect the global three-dimensional chromosome architecture of cells. They also provide a comprehensive and unique megabase-scale picture of cellular epigenetic state. Thus, normal differentiation involves reproducible changes in RT, and transformation generally perturbs these, although the potential effects of altered RT on the properties of transformed cells remain largely unknown. A major challenge to interrogating these issues in human acute lymphoid leukemia (ALL) is the low proliferative activity of most of the cells, which may be further reduced in cryopreserved samples and difficult to overcome in vitro. In contrast, the ability of many human ALL cell populations to expand when transplanted into highly immunodeficient mice is well documented. To examine the stability of DNA RT profiles of serially passaged xenografts of primary human B- and T-ALL cells, we first devised a method that circumvents the need for bromodeoxyuridine incorporation to distinguish early versus late S-phase cells. Using this and more standard protocols, we found consistently strong retention in xenografts of the original patient-specific RT features. Moreover, in a case in which genomic analyses indicated changing subclonal dynamics in serial passages, the RT profiles tracked concordantly. These results indicate that DNA RT is a relatively stable feature of human ALLs propagated in immunodeficient mice. In addition, they suggest the power of this approach for future interrogation of the origin and consequences of altered DNA RT in ALL.

Replication timing: a fingerprint for cell identity and pluripotency.

Many types of epigenetic profiling have been used to classify stem cells, stages of cellular differentiation, and cancer subtypes. Existing methods focus on local chromatin features such as DNA methylation and histone modifications that require extensive analysis for genome-wide coverage. Replication timing has emerged as a highly stable cell type-specific epigenetic feature that is regulated at the megabase-level and is easily and comprehensively analyzed genome-wide. Here, we describe a cell classification method using 67 individual replication profiles from 34 mouse and human cell lines and stem cell-derived tissues, including new data for mesendoderm, definitive endoderm, mesoderm and smooth muscle. Using a Monte-Carlo approach for selecting features of replication profiles conserved in each cell type, we identify "replication timing fingerprints" unique to each cell type and apply a k nearest neighbor approach to predict known and unknown cell types. Our method correctly classifies 67/67 independent replication-timing profiles, including those derived from closely related intermediate stages. We also apply this method to derive fingerprints for pluripotency in human and mouse cells. Interestingly, the mouse pluripotency fingerprint overlaps almost completely with previously identified genomic segments that switch from early to late replication as pluripotency is lost. Thereafter, replication timing and transcription within these regions become difficult to reprogram back to pluripotency, suggesting these regions highlight an epigenetic barrier to reprogramming. In addition, the major histone cluster Hist1 consistently becomes later replicating in committed cell types, and several histone H1 genes in this cluster are downregulated during differentiation, suggesting a possible instrument for the chromatin compaction observed during differentiation. Finally, we demonstrate that unknown samples can be classified independently using site-specific PCR against fingerprint regions. In sum, replication fingerprints provide a comprehensive means for cell characterization and are a promising tool for identifying regions with cell type-specific organization.