Wear, E. E. | bioRxiv Science

bioRxiv2025

Replication Timing Uncovers a Two-Compartment Nuclear Architecture of Interphase Euchromatin in Maize

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 "mini-domain chromatin fiber RT model" for maize euchromatin that suggested it is subdivided into early-S and middle-S compartments 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. Hi-C eigenvalues and insulation scores revealed distinct patterns of early-S regions having negative insulation scores with long-range contacts while middle-S regions showed the opposite. Early-S regions correlated more strongly with epigenomic signatures of open, transcriptionally active chromatin than middle-S regions. 3D oligo FISH painting confirmed that early-S and middle-S regions occupy adjacent but largely non-overlapping nucleoplasmic spaces during all interphase stages, including G1. Our findings redefine the maize euchromatin "A" compartment as having two distinct, closely interspersed subcompartments, early-S and middle-S, underscoring the importance of replication timing (RT) as a defining feature of chromatin architecture and genome organization.

genetics↗

bioRxiv2020

Comparing DNA replication programs reveals large timing shifts at centromeres of endocycling cells in maize roots

Plant cells undergo two types of cell cycles - the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Here, we compare sequence-based RT profiles and found that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small, with a median size of 135 kb, and shift to a later RT in the endocycle. However, we found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere in each case, which are [~]1-2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells, but primarily in late S phase of the endocycle. Strikingly, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in nuclei of different ploidies suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and reduced CENH3 enrichment after endocycle replication is consistent with the hypothesis that centromeres are being inactivated as their function is no longer needed. AUTHOR SUMMARYIn traditional cell division, or mitosis, a cells genetic material is duplicated and then split between two daughter cells. In contrast, in some specialized cell types, the DNA is duplicated a second time without an intervening division step, resulting in cells that carry twice as much DNA - a phenomenon called an endocycle, which is common during plant development. At each step, DNA replication follows an ordered program, in which highly compacted DNA is unraveled and replicated in sections at different times during the synthesis (S) phase. In plants, it is unclear whether traditional and endocycle programs are the same. Using root tips of maize, we found a small portion of the genome whose replication in the endocycle is shifted in time, usually to later in S phase. Some of these regions are scattered around the genome, and mostly coincide with active genes. However, the most prominent shifts occur in centromeres. This location is noteworthy because centromeres orchestrate the process of separating duplicated chromosomes into daughter cells, a function that is not needed in the endocycle. Our observation that centromeres replicate later in the endocycle suggests there is an important link between the time of replication and the function of centromeres.

genomics↗

bioRxiv2016

Repliscan: a tool for classifying replication timing regions

BackgroundReplication timing experiments that use label incorporation and high throughput sequencing produce peaked data similar to ChIP-Seq experiments. However, the differences in experimental design, coverage density, and possible results make traditional ChIP-Seq analysis methods inappropriate for use with replicating timing.\n\nResultsTo accurately detect and classify regions of replication across the genome, we present Repliscan. Repliscan robustly normalizes, automatically removes outlying and uninformative data points, and classifies Repli-seq signals into discrete combinations of replication signatures. The quality control steps and self-fitting methods makes Repliscan generally applicable and superior to previous methods with thresholds inapplicable to different genomes.\n\nConclusionsRepliscan is simple and effective to use on organisms with different magnitude genome sizes and sequencing coverage as low as 2.4x.

bioinformatics↗