Decoding single-cell replication states from imaging data reveals locus-specific subnuclear position dependencies of replication initiation

Replication timing (RT) is a fundamental feature of genome regulation, tightly linked to chromatin state and three-dimensional (3D) nuclear organization. Yet how the reproducible population-level timing program emerges from stochastic replication decisions in individual cells remains unclear, because it requires measuring RT and 3D genome organization jointly in the same cell. Here, we introduce RepTile, the first systematic framework that infers allele-resolved, single-cell replication states directly from multiplexed FISH and chromosome tracing experiments. By extracting replication information from spot-count distributions while correcting for genomic and spatial detection biases, RepTile infers cell-cycle progression, locus-specific replication timing, cell-to-cell heterogeneity in replication timing, and replication states of individual allele-resolved locus copies within their native three-dimensional nuclear context. Using RepTile on mouse embryonic stem cells, we find that the association between replication timing and subnuclear position observed in population-level genomic experiments conceals fundamentally different locus-specific behaviors. We found two classes of chromatin regions: "position-sensitive" and "position-insensitive". Position-sensitive regions replicate at different times depending on their subnuclear location, typically firing earliest when positioned in their canonical locale. Strikingly, this holds even when the canonical locale is repressive, such as the nuclear envelope. On the other hand, position-insensitive loci replicate at their characteristic times regardless of subnuclear position. These classes map onto distinct regulatory regimes, which are particularly pronounced at replication initiation zones (IZ): position-sensitive IZs are enriched in domains that replicate constitutively early or late across cell types, whereas position-insensitive IZs are enriched in domains that switch RT developmentally and contain early replication control elements (ERCEs), cis-regulatory elements known to govern IZ firing times. Together, these results challenge the view that subnuclear location broadly defines replication timing, indicating that positional dependence is specific to constitutive domains, while developmental domains carry intrinsic cis-acting programs that decouple their replication from subnuclear location.