Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication.

The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these "early replication control elements" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.

Control of DNA replication timing in the 3D genome.

The 3D organization of mammalian chromatin was described more than 30 years ago by visualizing sites of DNA synthesis at different times during the S phase of the cell cycle. These early cytogenetic studies revealed structurally stable chromosome domains organized into subnuclear compartments. Active-gene-rich domains in the nuclear interior replicate early, whereas more condensed chromatin domains that are largely at the nuclear and nucleolar periphery replicate later. During the past decade, this spatiotemporal DNA replication programme has been mapped along the genome and found to correlate with epigenetic marks, transcriptional activity and features of 3D genome architecture such as chromosome compartments and topologically associated domains. But the causal relationship between these features and DNA replication timing and the regulatory mechanisms involved have remained an enigma. The recent identification of cis-acting elements regulating the replication time and 3D architecture of individual replication domains and of long non-coding RNAs that coordinate whole chromosome replication provide insights into such mechanisms.

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.

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.

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.

Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication.

The heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation sites make mapping the location and timing of replication initiation in human cells difficult. To address this challenge, we have developed optical replication mapping (ORM), a high-throughput single-molecule approach, and used it to map early-initiation events in human cells. The single-molecule nature of our data and a total of >2,500-fold coverage of the human genome on 27 million fibers averaging ∼300 kb in length allow us to identify initiation sites and their firing probability with high confidence. We find that the distribution of human replication initiation is consistent with inefficient, stochastic activation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.

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.

4D Genome Rewiring during Oncogene-Induced and Replicative Senescence.

To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.

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.

Expanded encyclopaedias of DNA elements in the human and mouse genomes.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

Improving Sepsis Outcomes in the Era of Pay-for-Performance and Electronic Quality Measures: A Joint IDSA/ACEP/PIDS/SHEA/SHM/SIDP Position Paper.

The Centers for Medicare & Medicaid Services (CMS) introduced the Severe Sepsis/Septic Shock Management Bundle (SEP-1) as a pay-for-reporting measure in 2015 and is now planning to make it a pay-for-performance measure by incorporating it into the Hospital Value-Based Purchasing Program. This joint IDSA/ACEP/PIDS/SHEA/SHM/SIPD position paper highlights concerns with this change. Multiple studies indicate that SEP-1 implementation was associated with increased broad-spectrum antibiotic use, lactate measurements, and aggressive fluid resuscitation for patients with suspected sepsis but not with decreased mortality rates. Increased focus on SEP-1 risks further diverting attention and resources from more effective measures and comprehensive sepsis care. We recommend retiring SEP-1 rather than using it in a payment model and shifting instead to new sepsis metrics that focus on patient outcomes. CMS is developing a community-onset sepsis 30-day mortality electronic clinical quality measure (eCQM) that is an important step in this direction. The eCQM preliminarily identifies sepsis using systemic inflammatory response syndrome (SIRS) criteria, antibiotic administrations or diagnosis codes for infection or sepsis, and clinical indicators of acute organ dysfunction. We support the eCQM but recommend removing SIRS criteria and diagnosis codes to streamline implementation, decrease variability between hospitals, maintain vigilance for patients with sepsis but without SIRS, and avoid promoting antibiotic use in uninfected patients with SIRS. We further advocate for CMS to harmonize the eCQM with the Centers for Disease Control and Prevention's (CDC) Adult Sepsis Event surveillance metric to promote unity in federal measures, decrease reporting burden for hospitals, and facilitate shared prevention initiatives. These steps will result in a more robust measure that will encourage hospitals to pay more attention to the full breadth of sepsis care, stimulate new innovations in diagnosis and treatment, and ultimately bring us closer to our shared goal of improving outcomes for patients.

Local rewiring of genome-nuclear lamina interactions by transcription.

Transcriptionally inactive genes are often positioned at the nuclear lamina (NL), as part of large lamina-associated domains (LADs). Activation of such genes is often accompanied by repositioning toward the nuclear interior. How this process works and how it impacts flanking chromosomal regions are poorly understood. We addressed these questions by systematic activation or inactivation of individual genes, followed by detailed genome-wide analysis of NL interactions, replication timing, and transcription patterns. Gene activation inside LADs typically causes NL detachment of the entire transcription unit, but rarely more than 50-100 kb of flanking DNA, even when multiple neighboring genes are activated. The degree of detachment depends on the expression level and the length of the activated gene. Loss of NL interactions coincides with a switch from late to early replication timing, but the latter can involve longer stretches of DNA. Inactivation of active genes can lead to increased NL contacts. These extensive datasets are a resource for the analysis of LAD rewiring by transcription and reveal a remarkable flexibility of interphase chromosomes.

Hybrid modelling of biological systems: current progress and future prospects.

Integrated modelling of biological systems is becoming a necessity for constructing models containing the major biochemical processes of such systems in order to obtain a holistic understanding of their dynamics and to elucidate emergent behaviours. Hybrid modelling methods are crucial to achieve integrated modelling of biological systems. This paper reviews currently popular hybrid modelling methods, developed for systems biology, mainly revealing why they are proposed, how they are formed from single modelling formalisms and how to simulate them. By doing this, we identify future research requirements regarding hybrid approaches for further promoting integrated modelling of biological systems.

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.

Smoking Progression and Nicotine-Enhanced Reward Sensitivity Predicted by Resting-State Functional Connectivity in Salience and Executive Control Networks.

INTRODUCTION: The neural underpinnings underlying individual differences in nicotine-enhanced reward sensitivity and smoking progression are poorly understood. Thus, we investigated whether brain resting-state functional connectivity (rsFC) during smoking abstinence predicts nicotine-enhanced reward sensitivity and smoking progression in young light smokers. We hypothesized that high rsFC between brain areas with high densities of nicotinic receptors (insula, anterior cingulate cortex [ACC], hippocampus, thalamus) and areas involved in reward-seeking (nucleus accumbens [NAcc], prefrontal cortex [PFC]) would predict nicotine-enhanced reward sensitivity and smoking progression. METHODS: Young light smokers (N=64, age 18-24, M = 1.89 cigarettes/day) participated in the study. These individuals smoked between 5 to 35 cigarettes per week and lifetime use never exceeded 35 cigarettes per week. Their rsFC was assessed using functional magnetic resonance imaging after 14-hour nicotine-deprivation. Subjects also completed a probabilistic reward task after smoking a placebo on one day and a regular cigarette on another day. RESULTS: The probabilistic-reward-task assessed greater nicotine-enhanced reward sensitivity was associated with greater rsFC between the right anterior PFC and right NAcc, but with reduced rsFC between the ACC and left inferior prefrontal gyrus and the insula and ACC. Decreased rsFC within the salience network (ACC and insula) predicted increased smoking progression across 18 months and greater nicotine-enhanced reward sensitivity. CONCLUSIONS: These findings provide the first evidence that differences in rsFCs in young light smokers are associated with nicotine-enhanced reward sensitivity and smoking progression. IMPLICATIONS: Weaker rsFC within the salience network predicted greater nicotine-enhanced reward sensitivity and smoking progression. These findings suggest that salience network rsFC and drug-enhanced reward sensitivity may be useful tools and potential endophenotypes for reward sensitivity and drug-dependence research.

Replicating Large Genomes: Divide and Conquer.

Complete duplication of large metazoan chromosomes requires thousands of potential initiation sites, only a small fraction of which are selected in each cell cycle. Assembly of the replication machinery is highly conserved and tightly regulated during the cell cycle, but the sites of initiation are highly flexible, and their temporal order of firing is regulated at the level of large-scale multi-replicon domains. Importantly, the number of replication forks must be quickly adjusted in response to replication stress to prevent genome instability. Here we argue that large genomes are divided into domains for exactly this reason. Once established, domain structure abrogates the need for precise initiation sites and creates a scaffold for the evolution of other chromosome functions.

Nuclear Architecture Organized by Rif1 Underpins the Replication-Timing Program.

DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals.

The Tiger Rattlesnake genome reveals a complex genotype underlying a simple venom phenotype.

Variation in gene regulation is ubiquitous, yet identifying the mechanisms producing such variation, especially for complex traits, is challenging. Snake venoms provide a model system for studying the phenotypic impacts of regulatory variation in complex traits because of their genetic tractability. Here, we sequence the genome of the Tiger Rattlesnake, which possesses the simplest and most toxic venom of any rattlesnake species, to determine whether the simple venom phenotype is the result of a simple genotype through gene loss or a complex genotype mediated through regulatory mechanisms. We generate the most contiguous snake-genome assembly to date and use this genome to show that gene loss, chromatin accessibility, and methylation levels all contribute to the production of the simplest, most toxic rattlesnake venom. We provide the most complete characterization of the venom gene-regulatory network to date and identify key mechanisms mediating phenotypic variation across a polygenic regulatory network.

Hybrid modelling of biological systems using fuzzy continuous Petri nets.

Integrated modelling of biological systems is challenged by composing components with sufficient kinetic data and components with insufficient kinetic data or components built only using experts' experience and knowledge. Fuzzy continuous Petri nets (FCPNs) combine continuous Petri nets with fuzzy inference systems, and thus offer an hybrid uncertain/certain approach to integrated modelling of such biological systems with uncertainties. In this paper, we give a formal definition and a corresponding simulation algorithm of FCPNs, and briefly introduce the FCPN tool that we have developed for implementing FCPNs. We then present a methodology and workflow utilizing FCPNs to achieve hybrid (uncertain/certain) modelling of biological systems illustrated with a case study of the Mercaptopurine metabolic pathway. We hope this research will promote the wider application of FCPNs and address the uncertain/certain integrated modelling challenge in the systems biology area.