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Replication timing uncovers a two-compartment nuclear architecture of interphase euchromatin

ABSTRACT: Genome replication is temporally regulated during S phase, with specific genomic regions replicating at defined times in a process that is known as replication timing (RT). Based on 3D cytology in replicating nuclei, we previously proposed a model in which maize euchromatin is subdivided into subcompartments distinguished by chromatin condensation and RT. However, whether this compartmentalization reflects a general nuclear architecture that persists throughout the cell cycle was unclear. To test this model, we conducted two orthogonal assays-Hi-C for genome-wide interaction data and 3D FISH for direct visualization of chromatin organization in maize (Zea mays L.). Hi-C analyses revealed distinct patterns of early-S regions exhibited negative insulation scores with long-range contacts, whereas middle-S regions showed the opposite. Early-S regions showed the strongest correlation with epigenomic signatures of open, transcriptionally active chromatin. 3D oligo FISH painting confirmed that early-S and middle-S replicating regions occupy adjacent but largely non-overlapping nucleoplasmic sub-territories throughout interphase stages, including G1. Together, our findings redefine the maize euchromatin "A" compartment as two spatially distinct subcompartments derived from high-frequency RT transitions between early and middle S along the linear genome. These findings have implications for chromatin-templated processes and underscore the importance of RT as a defining feature of genome organization.

ORGANISM(S): Zea mays

PROVIDER: GSE287128 | GEO | 2026/01/10

REPOSITORIES: GEO

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Project description:DNA is replicated in a defined temporal order termed the replication timing (RT) program, which is correlated with genome compartmentalization with early replicating chromatin located mainly in the A compartment and late replicating chromatin in the B compartment (Dixon et al 2012, Moindrot et al 2012, Ryba et al 2010, Yaffe et al 2010). Similarly, active histone modifications and transcriptional permissiveness are associated with early replication while a repressive chromatin state is associated with late replication (Hiratani et al 2009, Riveria-Mulia et al 2015, Lubelsky et al 2014). However, the mechanistic interplay between RT, chromatin state, and genome compartmentalization is largely unknown. Here we report RT is upstream of epigenome formation and compartmentalisation in human embryonic stem cells (hESCs) and the cancer cell line HCT116. Knockout (KO) of the conserved RT control factor RIF1, rather than causing discrete RT switches, leads 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 any other cell type to date, yet cells self-renew without signs of differentiation. Those regions that do retain RT consist of large H3K9me3 domains, which are prevalent in HCT116 but rare in hESCs, revealing two independent mechanisms of RT regulation that are used to different extents in different cell types. RIF1 KO cells also showed genome wide downregulation of H3K27ac peaks and enrichment of H3K9me3 at large domains that remained late replicating, while H3K27me3 and H3K4me3 were also re-distributed genome wide but in a cell type specific manner. Histone modification changes correlated with gene expression changes and global reorganisation of genome compartments. Some RT/compartment shifts were associated with specific TAD boundary shifts, but overall we observed a genome wide strengthening of TAD structures. Our findings support a model in which RT controlled by RIF1 plays a key role in establishing epigenetic state and genome architecture in human cells.

Project description:DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins. Replicating DNA in cultured Arabidopsis cells was labeled with BrdU for 1 hour. Cells were harvested, nuclei extracted, and the nuclei were sorted by FACS as Early, Mid or Late S phase based on DNA content (DAPI signal). Newly replicated DNA was isolated by immunoprecipitation to BrdU, amplified, and labeled with Cy5/Cy3. Input DNA served as a reference and was labeled with Cy3/Cy5. Equal amounts of DNA was hybridized to a spotted cDNA that covers Arabidopsis chr4. Each S phase sample has 3 biological replicates with a dye swap serving as a technical replicate for a total of 18 arrays.

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Replication timing uncovers a two-compartment nuclear architecture of interphase euchromatin

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