Replication Timing Uncovers a Novel Two-Compartment Arrangement of Maize Interphase Euchromatin.

The time within S phase when a given genomic region replicates is measurable and referred to as Replication Timing (RT). We previously proposed a "mini-domain" chromatin fiber RT model for maize euchromatin's spatial architecture. 3D quantitative cytology showed that euchromatin is subdivided into two compartments during S-phase, distinguished by chromatin condensation and replication timing: Early-S and Middle-S. A key gap remains in understanding whether this compartmentalization is a general feature throughout the cell cycle, which could greatly impact our knowledge of genome architecture and gene regulation. To investigate this, we conducted two orthogonal assays, Hi-C for genome-wide interaction data and 3D FISH for direct visualization of chromatin organization. The Hi-C-derived eigenvalues and insulation scores significantly concord with Early-S and Middle-S regions. Early-S regions showed negative insulation scores with more long-range contacts, whereas Middle-S regions displayed the opposite, positive insulation scores with fewer long-range contacts. Compared to Middle-S regions, the Early-S regions showed much stronger correlations with epigenomic signatures of open and transcriptionally active chromatin. Oligo FISH painting with RT-specific probes demonstrated that Early-S and Middle-S regions occupied adjacent but largely non-overlapping nucleoplasmic regions throughout all stages of interphase, including G1. These findings validate our model while establishing that the global "A" compartment of euchromatin actually consists of two spatially and epigenetically distinct substructures – now referred to as Early-S and Middle-S compartments. Given the strong correlation between replication timing and Hi-C data from root tips, we examined the conservation of Hi-C architecture in both root tip and earshoot tissues and found them to be remarkably similar. Overall, our findings highlight the importance of replication timing as a conserved feature of chromatin architecture, reflecting broader principles of genome organization. Overall design: Hi-C experiment in maize root tip and earshoot