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.

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.

Rare variants and the oligogenic architecture of autism.

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 maintains the global epigenetic state in human cells.

The temporal order of DNA replication [replication timing (RT)] is correlated with chromatin modifications and three-dimensional genome architecture; however, causal links have not been established, largely because of an inability to manipulate the global RT program. We show that loss of RIF1 causes near-complete elimination of the RT program by increasing heterogeneity between individual cells. RT changes are coupled with widespread alterations in chromatin modifications and genome compartmentalization. Conditional depletion of RIF1 causes replication-dependent disruption of histone modifications and alterations in genome architecture. These effects were magnified with successive cycles of altered RT. These results support models in which the timing of chromatin replication and thus assembly plays a key role in maintaining the global epigenetic state.

Polarised maintenance of cytoophidia in Drosophila follicle epithelia.

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.

STATISTICAL COMPARISONS OF CHROMOSOMAL SHAPE POPULATIONS.

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.

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.

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.

Rapid Irreversible Transcriptional Reprogramming in Human Stem Cells Accompanied by Discordance between Replication Timing and Chromatin Compartment.

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.

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.

Replication Domains: Genome Compartmentalization into Functional Replication Units.

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.