Genome compartments guide protamine replacement and genome stability during spermiogenesis

Compartment-scale genome organisation persists in mammalian sperm, yet how histone-protamine replacement is orchestrated in space and time during spermiogenesis remains unclear. Here we combine stage-resolved purification of mouse spermatids with spike-in-normalised ATAC-seq and PRM1 CUT&Tag to map chromatin accessibility and protamine incorporation across spermiogenesis. We uncover a transient, genome-wide hyper-accessible phase coincident with replacement that is uncoupled from transcription and suppressed in catalytic PHF7-mutant spermatids. PRM1 loading initiates within accessible A-compartment chromatin and later spreads across both A- and B-compartments, whereas protamine-null mice demonstrated that PRM1/ PRM2 deficiency selectively destabilises A-compartment closure. Sequencing of short DNA fragments from Prm1/Prm2 dosage-reduced epididymal sperm reveals early enrichment of breakage at protamine-targeted A-compartment regions, which dissipates as fragmentation becomes genome-wide during epididymal transit. Together, our data place histone-protamine replacement within a compartment-centred framework that links 3D genome domains to the timing, targeting and integrity of the sperm genome.