Jain, A. Y.; Hoelper, D.; Rashoff, A. Q.; Lewis, P. W.

Endogenous retroviruses (ERVs) compromise genome integrity when expressed, and cells have evolved chromatin-based pathways to silence their transcription. The histone H3.3 chaperone DAXX localizes to a subset of ERVs and enforces their silencing through incompletely defined mechanisms. 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 silencing, indicating that histone H3.3 is dispensable for DAXX-mediated repression of ERVs. By contrast, ERV silencing requires the DAXX C-terminal SUMO-interacting motif, which mediates recruitment of SUMOylated repressors, including MORC3. These findings define modular outputs downstream of DAXX recruitment that uncouple nucleosome assembly from ERV silencing and highlight SUMO-dependent effector recruitment as the primary mechanism of silencing.