Outside-in progression of heterochromatin replication and exclusion of CDC45 from the PCH domain in Drosophila.

Heterochromatin replication isn't random or uniform, but occurs in a characteristic spatial and temporal pattern. Previous studies produced conflicting models for pericentric heterochromatin (PCH) replication, suggesting either that heterochromatic sequences translocate to the domain surface for replication, or that replication can also occur internally through localized decondensation. To distinguish true overlap from peripheral enrichment around an irregular PCH domain, we developed FOC-Map, a colocalization analysis approach that combines segmentation of one channel with binning of the other. Applying FOC-Map to three-dimensional Airyscan imaging of cultured Drosophila cells, we find that replication foci at the onset of late S-phase are confined to the outer boundary of the PCH domain, forming a shell-like pattern with little overlap into the HP1a-rich interior. As late S-phase progresses, replication foci are observed within the domain, localizing to low-HP1a regions interspersed between more condensed regions. We then assessed the distribution of CDC45, a rate-limiting replication initiation factor, and found that CDC45 foci are depleted from the PCH domain throughout the cell cycle. We propose that low levels of CDC45 within HP1a-rich PCH limit replication initiation to the domain periphery, giving rise to the shell-like pattern of replication foci that progressively works inward until PCH replication is complete.