HIRA and Daxx constitute two independent histone H3.3-containing predeposition complexes.

Histone H3.3 is a universal replacement histone in metazoans that has been implicated in diverse processes ranging from gene activation to heterochromatin silencing. Here, we show that, before deposition, H3.3 exists in two biochemically distinct complexes, associated with either Daxx or HIRA, Ubinuclein-1, and Cabin-1. Although the HIRA complex is evolutionarily conserved in yeast, Daxx is a novel histone chaperone unique to metazoans. Deletion of HIRA in mouse embryonic stem cells impairs the HIRA complex integrity but does not abolish Daxx association with H3.3/H4. Similarly, HIRA interacts with H3.3/H4 in the absence of Daxx. We hypothesize that these two H3.3 chaperone systems provide separate pools of H3/H4 units for incorporation at distinct sites within the genome. We provide evidence that the association of histone H3.3 with distinct assembly systems allows it to acquire unique posttranslational modifications before deposition that might affect its role after incorporation into chromatin.