HIRA defines early replication initiation zones independently of their genome compartment

Chromatin states and 3D architecture have been used as proxy to identify replication initiation zones (IZs) in mammalian cells, yet their functional interconnections remain a puzzle. Here, to dissect these relationships, we focus on the histone H3.3 chaperone HIRA recently implicated in early initiation zone (IZ) definition. We monitor 3D organisation, chromatin accessibility and histone post-translational modifications (PTMs) in wild-type and HIRA knock-out cells in parallel with early replication initiation. In the absence of HIRA, compartment A loses H3.3 enrichment and gains accessibility without changes in associated histone post-translational modifications (PTMs). Furthermore, impaired early firing at HIRA-dependent IZs does not correspond to changes in chromatin accessibility or patterns of histone H3 PTMs. Additionally, a small subset of early IZs initially in compartment A switch to B and lose early initiation in the absence of HIRA. Critically, HIRA complementation restores these early IZ, and H3.3 variant enrichment, without substantial compartment reversal. Thus, while HIRA contributes to compartment A features, its role in regulating early replication initiation can be uncoupled from accessibility, histone marks and compartment organisation.