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.

Beyond A and B Compartments: how major nuclear locales define nuclear genome organization and function.

Models of nuclear genome organization often propose a binary division into active versus inactive compartments, yet they overlook nuclear bodies. Here we integrated analysis of sequencing and image-based data to compare genome organization in four human cell types relative to three different nuclear locales: the nuclear lamina, nuclear speckles, and nucleoli. Whereas gene expression correlates mostly with nuclear speckle proximity, DNA replication timing correlates with proximity to multiple nuclear locales. Speckle attachment regions emerge as DNA replication initiation zones whose replication timing and gene composition vary with their attachment frequency. Most facultative LADs retain a partially repressed state as iLADs, despite their positioning in the nuclear interior. Knock out of two lamina proteins, Lamin A and LBR, causes a shift of H3K9me3-enriched LADs from lamina to nucleolus, and a reciprocal relocation of H3K27me3-enriched partially repressed iLADs from nucleolus to lamina. Thus, these partially repressed iLADs appear to compete with LADs for nuclear lamina attachment with consequences for replication timing. The nuclear organization in adherent cells is polarized with nuclear bodies and genomic regions segregating both radially and relative to the equatorial plane. Together, our results underscore the importance of considering genome organization relative to nuclear locales for a more complete understanding of the spatial and functional organization of the human genome.

The genetic regulatory architecture and epigenomic basis for age-related changes in rattlesnake venom.

Developmental phenotypic changes can evolve under selection imposed by age- and size-related ecological differences. Many of these changes occur through programmed alterations to gene expression patterns, but the molecular mechanisms and gene-regulatory networks underlying these adaptive changes remain poorly understood. Many venomous snakes, including the eastern diamondback rattlesnake (Crotalus adamanteus), undergo correlated changes in diet and venom expression as snakes grow larger with age, providing models for identifying mechanisms of timed expression changes that underlie adaptive life history traits. By combining a highly contiguous, chromosome-level genome assembly with measures of expression, chromatin accessibility, and histone modifications, we identified cis-regulatory elements and trans-regulatory factors controlling venom ontogeny in the venom glands of C. adamanteus. Ontogenetic expression changes were significantly correlated with epigenomic changes within genes, immediately adjacent to genes (e.g., promoters), and more distant from genes (e.g., enhancers). We identified 37 candidate transcription factors (TFs), with the vast majority being up-regulated in adults. The ontogenetic change is largely driven by an increase in the expression of TFs associated with growth signaling, transcriptional activation, and circadian rhythm/biological timing systems in adults with corresponding epigenomic changes near the differentially expressed venom genes. However, both expression activation and repression contributed to the composition of both adult and juvenile venoms, demonstrating the complexity and potential evolvability of gene regulation for this trait. Overall, given that age-based trait variation is common across the tree of life, we provide a framework for understanding gene-regulatory-network-driven life-history evolution more broadly.

Linear interaction between replication and transcription shapes DNA break dynamics at recurrent DNA break Clusters.

Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unravel the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA reveals co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity uncovers that head-to-head interactions between replication and transcription machinery result in 60% DSB contribution to the head-on compared to 40% for co-directional. Taken together we reveal TTR as a fragile class and show how the linear interaction between transcription and replication impacts genome stability.

Linear Interaction Between Replication and Transcription Shapes DNA Break Dynamics at Recurrent DNA Break Clusters.

Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unraveled the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA revealed co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity revealed that head-to-head interactions between replication and transcription machinery resulted in 60% DSB contribution to the head-on compared to 40% for co-directional. Our findings revealed TTR as a novel fragile class and highlighted how the linear interaction between transcription and replication impacts genome stability.

Emergence of replication timing during early mammalian development.

DNA replication enables genetic inheritance across the kingdoms of life. Replication occurs with a defined temporal order known as the replication timing (RT) programme, leading to organization of the genome into early- or late-replicating regions. RT is cell-type specific, is tightly linked to the three-dimensional nuclear organization of the genome1,2 and is considered an epigenetic fingerprint3. In spite of its importance in maintaining the epigenome4, the developmental regulation of RT in mammals in vivo has not been explored. Here, using single-cell Repli-seq5, we generated genome-wide RT maps of mouse embryos from the zygote to the blastocyst stage. Our data show that RT is initially not well defined but becomes defined progressively from the 4-cell stage, coinciding with strengthening of the A and B compartments. We show that transcription contributes to the precision of the RT programme and that the difference in RT between the A and B compartments depends on RNA polymerase II at zygotic genome activation. Our data indicate that the establishment of nuclear organization precedes the acquisition of defined RT features and primes the partitioning of the genome into early- and late-replicating domains. Our work sheds light on the establishment of the epigenome at the beginning of mammalian development and reveals the organizing principles of genome organization.

RIF1 regulates early replication timing in murine B cells.

The mammalian DNA replication timing (RT) program is crucial for the proper functioning and integrity of the genome. The best-known mechanism for controlling RT is the suppression of late origins of replication in heterochromatin by RIF1. Here, we report that in antigen-activated, hypermutating murine B lymphocytes, RIF1 binds predominantly to early-replicating active chromatin and promotes early replication, but plays a minor role in regulating replication origin activity, gene expression and genome organization in B cells. Furthermore, we find that RIF1 functions in a complementary and non-epistatic manner with minichromosome maintenance (MCM) proteins to establish early RT signatures genome-wide and, specifically, to ensure the early replication of highly transcribed genes. These findings reveal additional layers of regulation within the B cell RT program, driven by the coordinated activity of RIF1 and MCM proteins.

Nucleolus and centromere TSA-Seq reveals variable localization of heterochromatin in different cell types.

Genome differential positioning within interphase nuclei remains poorly explored. We extended and validated TSA-seq to map genomic regions near nucleoli and pericentric heterochromatin in four human cell lines. Our study confirmed that smaller chromosomes localize closer to nucleoli but further deconvolved this by revealing a preference for chromosome arms below 36-46 Mbp in length. We identified two lamina associated domain subsets through their differential nuclear lamina versus nucleolar positioning in different cell lines which showed distinctive patterns of DNA replication timing and gene expression across all cell lines. Unexpectedly, active, nuclear speckle-associated genomic regions were found near typically repressive nuclear compartments, which is attributable to the close proximity of nuclear speckles and nucleoli in some cell types, and association of centromeres with nuclear speckles in hESCs. Our study points to a more complex and variable nuclear genome organization than suggested by current models, as revealed by our TSA-seq methodology.

SHIELD: a platform for high-throughput screening of barrier-type DNA elements in human cells.

Chromatin boundary elements contribute to the partitioning of mammalian genomes into topological domains to regulate gene expression. Certain boundary elements are adopted as DNA insulators for safe and stable transgene expression in mammalian cells. These elements, however, are ill-defined and less characterized in the non-coding genome, partially due to the lack of a platform to readily evaluate boundary-associated activities of putative DNA sequences. Here we report SHIELD (Site-specific Heterochromatin Insertion of Elements at Lamina-associated Domains), a platform tailored for the high-throughput screening of barrier-type DNA elements in human cells. SHIELD takes advantage of the high specificity of serine integrase at heterochromatin, and exploits the natural heterochromatin spreading inside lamina-associated domains (LADs) for the discovery of potent barrier elements. We adopt SHIELD to evaluate the barrier activity of 1000 DNA elements in a high-throughput manner and identify 8 candidates with barrier activities comparable to the core region of cHS4 element in human HCT116 cells. We anticipate SHIELD could facilitate the discovery of novel barrier DNA elements from the non-coding genome in human cells.

The location and development of Replicon Cluster Domains in early replicating DNA.

Background: It has been known for many years that in metazoan cells, replication origins are organised into clusters where origins within each cluster fire near-synchronously. Despite clusters being a fundamental organising principle of metazoan DNA replication, the genomic location of origin clusters has not been documented. Methods: We synchronised human U2OS by thymidine block and release followed by L-mimosine block and release to create a population of cells progressing into S phase with a high degree of synchrony. At different times after release into S phase, cells were pulsed with EdU; the EdU-labelled DNA was then pulled down, sequenced and mapped onto the human genome. Results: The early replicating DNA showed features at a range of scales. Wavelet analysis showed that the major feature of the early replicating DNA was at a size of 500 kb, consistent with clusters of replication origins. Over the first two hours of S phase, these Replicon Cluster Domains broadened in width, consistent with their being enlarged by the progression of replication forks at their outer boundaries. The total replication signal associated with each Replicon Cluster Domain varied considerably, and this variation was reproducible and conserved over time. We provide evidence that this variability in replication signal was at least in part caused by Replicon Cluster Domains being activated at different times in different cells in the population. We also provide evidence that adjacent clusters had a statistical preference for being activated in sequence across a group, consistent with the 'domino' model of replication focus activation order observed by microscopy. Conclusions: We show that early replicating DNA is organised into Replicon Cluster Domains that behave as expected of replicon clusters observed by DNA fibre analysis. The coordinated activation of different Replicon Cluster Domains can generate the replication timing programme by which the genome is duplicated.

Spatial and temporal organization of the genome: Current state and future aims of the 4D nucleome project.

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.

Transposase expression, element abundance, element size, and DNA repair determine the mobility and heritability of PIF/Pong/Harbinger transposable elements.

Introduction: Class II DNA transposable elements account for significant portions of eukaryotic genomes and contribute to genome evolution through their mobilization. To escape inactivating mutations and persist in the host genome over evolutionary time, these elements must be mobilized enough to result in additional copies. These elements utilize a "cut and paste" transposition mechanism that does not intrinsically include replication. However, elements such as the rice derived mPing element have been observed to increase in copy number over time. Methods: We used yeast transposition assays to test several parameters that could affect the excision and insertion of mPing and its related elements. This included development of novel strategies for measuring element insertion and sequencing insertion sites. Results: Increased transposase protein expression increased the mobilization frequency of a small (430 bp) element, while overexpression inhibition was observed for a larger (7,126 bp) element. Smaller element size increased both the frequency of excision and insertion of these elements. The effect of yeast ploidy on element excision, insertion, and copy number provided evidence that homology dependent repair allows for replicative transposition. These elements were found to preferentially insert into yeast rDNA repeat sequences. Discussion: Identifying the parameters that influence transposition of these elements will facilitate their use for gene discovery and genome editing. These insights in to the behavior of these elements also provide important clues into how class II transposable elements have shaped eukaryotic genomes.

Replication timing and transcriptional control: beyond cause and effect - part IV.

Decades of work on the spatiotemporal organization of mammalian DNA replication timing (RT) continues to unveil novel correlations with aspects of transcription and chromatin organization but, until recently, mechanisms regulating RT and the biological significance of the RT program had been indistinct. We now know that the RT program is both influenced by and necessary to maintain chromatin structure, forming an epigenetic positive feedback loop. Moreover, the discovery of specific cis-acting elements regulating mammalian RT at both the domain and the whole-chromosome level has revealed multiple cell-type-specific and developmentally regulated mechanisms of RT control. We review recent evidence for diverse mechanisms employed by different cell types to regulate their RT programs and the biological significance of RT regulation during development.

SARS-CoV-2 restructures host chromatin architecture.

Some viruses restructure host chromatin, influencing gene expression, with implications for disease outcome. Whether this occurs for SARS-CoV-2, the virus causing COVID-19, is largely unknown. Here we characterized the 3D genome and epigenome of human cells after SARS-CoV-2 infection, finding widespread host chromatin restructuring that features widespread compartment A weakening, A-B mixing, reduced intra-TAD contacts and decreased H3K27ac euchromatin modification levels. Such changes were not found following common-cold-virus HCoV-OC43 infection. Intriguingly, the cohesin complex was notably depleted from intra-TAD regions, indicating that SARS-CoV-2 disrupts cohesin loop extrusion. These altered 3D genome/epigenome structures correlated with transcriptional suppression of interferon response genes by the virus, while increased H3K4me3 was found in the promoters of pro-inflammatory genes highly induced during severe COVID-19. These findings show that SARS-CoV-2 acutely rewires host chromatin, facilitating future studies of the long-term epigenomic impacts of its infection.

Brd2 is dispensable for genome compartmentalization and replication timing.

Replication Timing (RT) refers to the temporal order in which the genome is replicated during S phase. Early replicating regions correlate with the transcriptionally active, accessible euchromatin (A) compartment, while late replicating regions correlate with the heterochromatin (B) compartment and repressive histone marks. Previously, widespread A/B genome compartmentalization changes were reported following Brd2 depletion. Since RT and A/B compartmentalization are two of the most highly correlated chromosome properties, we evaluated the effects of Brd2 depletion on RT. We performed E/L Repli-Seq following Brd2 depletion in the previously described Brd2 conditional degron cell line and found no significant alterations in RT after Brd2 KD. This finding prompted us to re-analyze the Micro-C data from the previous publication. We report that we were unable to detect any compartmentalization changes in Brd2 depleted cells compared to DMSO control using the same data. Taken together, our findings demonstrate that Brd2 depletion alone does not affect A/B compartmentalization or RT in mouse embryonic stem cells.

Dynamic chromosomal interactions and control of heterochromatin positioning by Ki-67.

Ki-67 is a chromatin-associated protein with a dynamic distribution pattern throughout the cell cycle and is thought to be involved in chromatin organization. The lack of genomic interaction maps has hampered a detailed understanding of its roles, particularly during interphase. By pA-DamID mapping in human cell lines, we find that Ki-67 associates with large genomic domains that overlap mostly with late-replicating regions. Early in interphase, when Ki-67 is present in pre-nucleolar bodies, it interacts with these domains on all chromosomes. However, later in interphase, when Ki-67 is confined to nucleoli, it shows a striking shift toward small chromosomes. Nucleolar perturbations indicate that these cell cycle dynamics correspond to nucleolar maturation during interphase, and suggest that nucleolar sequestration of Ki-67 limits its interactions with larger chromosomes. Furthermore, we demonstrate that Ki-67 does not detectably control chromatin-chromatin interactions during interphase, but it competes with the nuclear lamina for interaction with late-replicating DNA, and it controls replication timing of (peri)centromeric regions. Together, these results reveal a highly dynamic choreography of genome interactions and roles for Ki-67 in heterochromatin organization.

Epigenetic control of chromosome-associated lncRNA genes essential for replication and stability.

ASARs are long noncoding RNA genes that control replication timing of entire human chromosomes in cis. The three known ASAR genes are located on human chromosomes 6 and 15, and are essential for chromosome integrity. To identify ASARs on all human chromosomes we utilize a set of distinctive ASAR characteristics that allow for the identification of hundreds of autosomal loci with epigenetically controlled, allele-restricted behavior in expression and replication timing of coding and noncoding genes, and is distinct from genomic imprinting. Disruption of noncoding RNA genes at five of five tested loci result in chromosome-wide delayed replication and chromosomal instability, validating their ASAR activity. In addition to the three known essential cis-acting chromosomal loci, origins, centromeres, and telomeres, we propose that all mammalian chromosomes also contain "Inactivation/Stability Centers" that display allele-restricted epigenetic regulation of protein coding and noncoding ASAR genes that are essential for replication and stability of each chromosome.

Cohesin-mediated loop anchors confine the locations of human replication origins.

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)3-6, subTADs7 and loops8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.