Distinct DAXX effector modules separate H3.3 nucleosome assembly from ERV repression

Endogenous retroviruses (ERVs) can compromise genome integrity when expressed, and cells have evolved chromatin-based pathways to repress their transcription. The histone H3.3 chaperone DAXX localizes to a subset of ERVs embedded in KAP1-SETDB1-associated heterochromatin and contributes to their repression. Here, we define distinct DAXX determinants that support H3.3 deposition versus ERV repression. Using complementary biochemical and genetic approaches, we identify a conserved basic patch within the DAXX histone-binding domain that engages DNA, promotes H3.3 nucleosome assembly in vitro, and is required for H3.3 enrichment at DAXX-bound ERVs in cells. Despite failure to deposit H3.3, DAXX with substitutions in this basic patch retains localization to ERVs and preserves repression, indicating that H3.3 deposition is dispensable for DAXX-dependent ERV repression. In contrast, disruption of the DAXX C-terminal SUMO-interacting motif impairs DAXX-dependent ERV repression and SUMO-dependent effector engagement, including MORC3 recruitment. These findings define separable outputs downstream from DAXX recruitment, uncoupling nucleosome assembly from ERV repression and placing SUMO-dependent effector engagement within the broader KAP1-SETDB1-centered ERV repression network.