William F. Thompson

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.

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.

Replication timing (RT), the temporal order in which genomic regions replicate, is considered a functional feature of multiple cellular processes and chromatin organization. Two approaches to measure RT are the Repli-seq and DNA copy number (also called S/G1) methods. We previously adapted Repli-seq using 5-ethynyl-2’- deoxyuridine (EdU) pulse-labeling and bivariate flow sorting, and while the approach offers high resolution and exposes heterogeneity in timing, the S/G1 method is a simpler, faster and less resource-intensive assessment. Here we modified the S/G1 technique by using EdU labeling (EdU-S/G1) to facilitate better separation of replicating from non-replicating nuclei during flow sorting, which enables the collection of a more pure sample of G1-phase nuclei. When comparing the three methods we found that profiles from the S/G1 and EdU-S/G1 methods are highly correlated with each other and with Repli-seq profiles for early replication. We also found that the EdU-S/G1 approach offers a better representation of replication in early and late S phase than the conventional S/G1 method. However, the high reproducibility of RT profiles among all three methods indicates that considerations of cost and sample availability can drive the decision of which method to choose.

Genome replication is temporally regulated during S phase, with specific genomic regions replicating at defined times in a process 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 also 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 subcompartments—Early-S and Middle-S—and underscore the importance of replication timing as a defining feature of chromatin architecture and genome organization.

DNA replication initiation sites in plants associate most strongly with AT-rich and highly accessible chromatin, and not with genes or a particular epigenetic s

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 SUMMARY: In traditional cell division, or mitosis, a cell’s 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.

The Arabidopsis genome replicates in two noninteracting compartments during early/mid and late S phase.

The time during S phase at which different maize DNA sequences replicate reveals a complex temporal program influenced by genomic features, transcriptional acti