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Most large-scale genetic studies of autism have focused on the discovery of genes by proving an enrichment of de novo mutations (DNMs) in autism probands or characterizing polygenic risk based on the association of common variants. We present evidence in support of an oligogenic model where two or more ultrarare mutations of more modest effect are preferentially transmitted to children with autism. Such private gene-disruptive mutations are enriched in families where there are multiple affected individuals, emerged two or three generations ago, and map to genes not previously associated with autism. Although no single gene has reached statistical significance, this class of variation should be considered along with genetic and nongenetic factors to better explain the etiology of this complex trait.

Replication timing organizes epigenome The temporal order of DNA replication is conserved from yeast to humans, but its biological significance remains unclear. Klein et al. eliminated the protein RIF1, a master regulator of replication timing, in several human cell lines. RIF1 loss during the G1 phase of the cell cycle resulted in a heterogeneous, nearly random replication timing program from the first S phase that persisted even in stable RIF1-null clones. Altered replication timing was followed by replication-dependent redistribution of active and repressive histone modifications and alterations in genome architecture. These results support a model in which replication timing orchestrates the epigenetic state of newly replicated chromatin. Science , this issue p. 371

This paper develops statistical tools for testing differences in shapes of chromosomes resulting from certain gene knockouts (KO), specifically RIF1 gene KO (RKO) and the cohesin subunit RAD21 gene KO (CKO). It utilizes a two-sample test for comparing shapes of KO chromosomes with wild type (WT) at two levels: (1) Coarse shape analysis, where one compares shapes of full or large parts of chromosomes, and (2) Fine shape analysis, where chromosomes are first segmented into (TAD-based) pieces and then the corresponding pieces are compared across populations. The shape comparisons – coarse and fine – are based on an elastic shape metric for comparing shapes of 3D curves. The experiments show that the KO populations, RKO and CKO, have statistically significant differences from WT at both coarse and fine levels. Furthermore, this framework highlights local regions where these differences are most prominent.

Summary The temporal order of DNA replication is regulated during development and is highly correlated with gene expression, histone modifications and 3D genome architecture. We tracked changes in replication timing, gene expression, and chromatin conformation capture (Hi-C) A/B compartments over the first two cell cycles during differentiation of human embryonic stem cells to definitive endoderm. Remarkably, transcriptional programs were irreversibly reprogrammed within the first cell cycle and were largely but not universally coordinated with replication timing changes. Moreover, changes in A/B compartment and several histone modifications that normally correlate strongly with replication timing showed weak correlation during the early cell cycles of differentiation but showed increased alignment in later differentiation stages and in terminally differentiated cell lines. Thus, epigenetic cell fate transitions during early differentiation can occur despite dynamic and discordant changes in otherwise highly correlated genomic properties., Graphical Abstract, Highlights • Stable transcriptional reprogramming toward endoderm within one cell cycle • Replication timing can change in the first S phase of a cell fate change • Early discordance between Replication timing and Hi-C compartments • Alignment of replication timing to compartments increases in later cell cycles, The temporal order of DNA replication is regulated during development, and is highly correlated with gene expression, chromatin structure, and 3D-genome architecture. Gilbert and colleagues find that stable transcriptional changes of human embryonic stem cells to endoderm can occur within a single cell cycle, accompanied by discordance in replication timing and chromatin compartment that align in later differentiation stages.

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability across dividing cells in metazoans. It is currently unknown how the location of replication origins and the timing of their activation is determined in the human genome. Here, we dissect the role for G1 phase topologically associating domains (TADs), subTADs, and loops in the activation of replication initiation zones (IZs). We identify twelve subtypes of self-interacting chromatin domains distinguished by their degree of nesting, the presence of corner dot structures indicative of loops, and their co-localization with A/B compartments. Early replicating IZs localize to boundaries of nested corner-dot TAD/subTADs anchored by high density arrays of co-occupied CTCF+cohesin binding sites with divergently oriented motifs. By contrast, late replicating IZs localize to weak TADs/subTAD boundaries devoid of corner dots and most often anchored by singlet CTCF+cohesin sites. Upon global knock-down of cohesin-mediated loops in G1, early wave focal IZs replicate later in S phase and convert to diffuse placement along the genome. Moreover, IZs in mid-late S phase are delayed to the final minutes before entry into G2 when cohesin-mediated dot-less boundaries are ablated. We also delete a specific loop anchor and observe a sharp local delay of an early wave IZ to replication in late S phase. Our data demonstrate that cohesin-mediated loops at genetically-encoded TAD/subTAD boundaries in G1 phase are an essential determinant of the precise genomic placement of human replication origins in S phase.

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.

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.

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., Genomic maps acquired after transcriptional activation or inactivation of genes reveals how transcription affects their interaction with the nuclear lamina.

Additional file 1: Figure S1. Validation of NPC differentiation by qPCR using primers against Oct4, Dppa2, Nestin, Sox1. Figure S2. Validation of BrdU pull-down by qPCR using primers against alpha- and beta globin. Figure S3. RPM of each S phase fraction is corrected using G1 WGS. Figure S4. G1 mappability control fraction was devoid of DNA replication. Figure S5. Normalisation of Repli-Seq heatmaps preserves signal. Figure S6. Percentage of replication in S1-S16 for top 10% earliest and latest replicated E/L Repli-Seq bins in mESC, mNPC, H1, H9 and HCT116. Figure S7. Comparison between H1 hESC datasets from [9] and H1 hESC datasets from this work. Figure S8. Correlation heatmaps showing concordance between High-Resolution Repli-Seq datasets of human and mouse cell lines. Figure S9. Schematic showing the identification of replication features using BIRCH. Figure S10. OK-seq IZs are primarily early replicating. Figure S11. SNS-seq signal centred around rightward (dark green) and rightward (orange) TTRs in HCT116, H9 and mESCs. Figure S12. Mean line plots of H3K27ac, H3K4me3, H3K9me3 and H3K27me3 fold enrichment signal centred on late CTRs and termination sites (

Additional file 2 : Table S1 Primer lists for human and mouse. Table S2 Resource table stating sources of external datasets.

DNA is replicated in a defined temporal order termed the replication timing (RT) program. RT is spatially segregated in the nucleus with early/late replication corresponding to Hi-C A/B chromatin compartments, respectively. Early replication is also associated with active histone modifications and transcriptional permissiveness. However, the mechanistic interplay between RT, chromatin state, and genome compartmentalization is largely unknown. Here we report that RT is central to epigenome maintenance and compartmentalization in both human embryonic stem cells (hESCs) and cancer cell line HCT116. Knockout (KO) of the conserved RT control factor RIF1, rather than causing discrete RT switches as previously suspected, lead to dramatically increased cell to cell heterogeneity of RT genome wide, despite RIF1’s enrichment in late replicating chromatin. RIF1 KO hESCs have a nearly random RT program, unlike all prior RIF1 KO cells, including HCT116, which show localized alterations. Regions that retain RT, which are prevalent in HCT116 but rare in hESCs, consist of large H3K9me3 domains revealing two independent mechanisms of RT regulation that are used to different extents in different cell types. RIF1 KO results in a striking genome wide downregulation of H3K27ac peaks and enrichment of H3K9me3 at large domains that remain late replicating, while H3K27me3 and H3K4me3 are re-distributed genome wide in a cell type specific manner. These histone modification changes coincided with global reorganization of genome compartments, transcription changes and a genome wide strengthening of TAD structures. Inducible degradation of RIF1 revealed that disruption of RT is upstream of genome compartmentalization changes. Our findings demonstrate that disruption of RT leads to widespread epigenetic mis-regulation, supporting previously speculative models in which the timing of chromatin assembly at the replication fork plays a key role in maintaining the global epigenetic state, which in turn drives genome architecture.

DNA replication in mammalian cells occurs in a defined temporal order during S phase, known as the replication timing (RT) programme. RT 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 as well as F1 subspecies hybrid mouse embryonic stem cells and their neural progenitor derivatives. Strong enrichment of nascent DNA in fine temporal windows reveals a remarkable degree of cell to cell conservation in replication timing and patterns of replication genome-wide. We identify 5 patterns of replication in all cell types, consistent with varying degrees of initiation efficiency. Zones of replication initiation were found throughout S phase and resolved to ~50kb precision. Temporal transition regions were resolved into segments of uni-directional replication punctuated with small zones of inefficient initiation. Small and large valleys of convergent replication were consistent with either termination or broadly distributed initiation, respectively. RT correlated with chromatin compartment across all cell types but correlations of initiation time to chromatin domain boundaries and histone marks were cell type specific. Haplotype phasing revealed previously unappreciated regions of allele-specific and alleleindependent asynchronous replication. Allele-independent asynchrony was associated with large transcribed genes that resemble common fragile sites. Altogether, these data reveal a remarkably deterministic temporal choreography of DNA replication in mammalian cells.Highly homogeneous replication landscape between cells in a populationInitiation zones resolved within constant timing and timing transition regionsActive histone marks enriched within early initiation zones while enrichment of repressive marks is cell type specific.Transcribed long genes replicate asynchronously.

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 towards the nuclear interior. How this process works and how it impacts flanking chromosomal regions is poorly understood. We addressed these questions by systematic manipulation of gene activity and detailed analysis of NL interactions. Activation of genes inside LADs typically causes 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. These findings show how NL interactions can be shaped locally by transcription and point to a remarkable flexibility of interphase chromosomes.

The metabolic enzyme CTP synthase (CTPS) can form filamentous structures named cytoophidia in numerous types of cells, including follicle cells. However, the regulation of cytoophidium assembly remains elusive. The apicobasal polarity, a defining characteristic of Drosophila follicle epithelium, is established and regulated by a variety of membrane domains. Here we show that CTPS can form cytoophidia in Drosophila epithelial follicle cells. Cytoophidia localise to the basolateral side of follicle cells. If apical polarity regulators are knocked down, cytoophidia become unstable and distribute abnormally. Knockdown of basolateral polarity regulators has no significant effect on cytoophidia, even though the polarity is disturbed. Our results indicate that cytoophidia are maintained via polarised distribution on the basolateral side of Drosophila follicle epithelia, which is primarily achieved through the apical polarity regulators.

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 thenecessity 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.

DNA replication occurs in a defined temporal order during S phase, known as the replication timing programme, which is regulated not only during the cell cycle but also during the process of development and differentiation. The units of replication timing regulation, known as replication domains (RDs), frequently comprise several nearly synchronously firing replication origins. Replication domains correspond to topologically associating domains (TADs) mapped by chromatin conformation capture methods and are likely to be the molecular equivalents of replication foci observed using cytogenetic methods. Both TAD and replication foci are considered to be stable structural units of chromosomes, conserved through the cell cycle and development, and accordingly, the boundaries of RDs also appear to be stable in different cell types. During both normal development and progression of disease, distinct cell states are characterized by unique replication timing signatures, with approximately half of genomic RDs switching replication timing between these cell states. Advances in functional genomics provide hope that we can soon gain an understanding of the cause and consequence of the replication timing programme and its myriad correlations with chromatin context and gene regulation.

DNA replication occurs in a defined temporal order during S phase, known as the replication timing programme, which is regulated not only during the cell cycle but also during the process of development and differentiation. The units of replication timing regulation, known as replication domains (RDs), frequently comprise several nearly synchronously firing replication origins. Replication domains correspond to topologically associating domains (TADs) mapped by chromatin conformation capture methods and are likely to be the molecular equivalents of replication foci observed using cytogenetic methods. Both TAD and replication foci are considered to be stable structural units of chromosomes, conserved through the cell cycle and development, and accordingly, the boundaries of RDs also appear to be stable in different cell types. During both normal development and progression of disease, distinct cell states are characterized by unique replication timing signatures, with approximately half of genomic RDs switching replication timing between these cell states. Advances in functional genomics provide hope that we can soon gain an understanding of the cause and consequence of the replication timing programme and its myriad correlations with chromatin context and gene regulation.