Histone H3.3 is a replication-independent histone variant, which replaces canonical histone H3.1/2 outside of S phase. H3.3 is expressed throughout the cell cycle, as well as in quiescent cells and is referred to as the “replacement histone”. H3.3 is largely deposited in a DNA synthesis-independent fashion by a distinct set of chaperones proteins. H3.3 accumulates in post mitotic cells, such as cerebral cortical neurons and is incorporated at sites of UV damage where it protects against sensitivity to UV light.

H3.3 deposition occurs on DNA sequences that are transiently nucleosome-free, such as during transcription and DNA repair, therefore serving as a marker for regions of high transcriptional activity. H3.3 is also enriched in heterochromatic subtelomeric and pericentromeric regions. H3.3 plays important roles in many developmental contexts such as in stem cells, during fertilization and reproduction and during reprogramming of genomes following fertilization or somatic cell nuclear transfer. Mutations in histone H3.3 are common events in certain cancers.