Abstract Chromatin states and 3D architecture have been used as proxy to identify replication initiation zones (IZs) in mammalian cells. While they do often correlate, their functional interconnections remain a puzzle. Here, we dissect these relationships by focusing on the histone H3.3 chaperone HIRA, which plays a role in both early initiation zone (IZ) definition and higher-order organisation of active chromatin. We monitored in parallel early replication initiation, chromatin accessibility, histone post-translational modifications (PTMs) and 3D organisation in wild-type cells, HIRA knock-out cells and HIRA knock-out cells complemented with HIRA. In the absence of HIRA, impaired early firing at HIRA-dependent IZs does not correspond to changes in chromatin accessibility or patterns of histone H3 PTMs. With respect to 3D organisation, a small subset of early IZs initially in compartment A switched to B and lost early initiation in the absence of HIRA. Critically, HIRA complementation restores these early IZ (and H3.3 variant enrichment) without substantial compartment reversal. Thus, our work reveals that regulation of early replication initiation by HIRA can be uncoupled from accessibility, histone mark patterns and compartment organisation.