This study investigates the localization of histone variant H3.3 at specific genomic regions in mammalian embryonic stem cells (ESCs) and neuronal precursor cells (NPCs). Using genome-wide profiling techniques, the researchers found that H3.3 enrichment patterns are dependent on the amino acid sequence of the endogenous H3.3 gene and change with cellular differentiation. H3.3 is enriched at active and repressed genes, as well as at transcription factor binding sites (TFBS) and telomeres. The histone chaperone Hira is required for H3.3 enrichment at active and repressed genes but not at TFBS or telomeres. Surprisingly, Atrx and Daxx, which are associated with H3.3 in both the presence and absence of Hira, are specifically required for H3.3 localization at telomeres and for the repression of telomeric RNA. These findings demonstrate that multiple and distinct factors control H3.3 localization at specific genomic locations in mammalian cells.This study investigates the localization of histone variant H3.3 at specific genomic regions in mammalian embryonic stem cells (ESCs) and neuronal precursor cells (NPCs). Using genome-wide profiling techniques, the researchers found that H3.3 enrichment patterns are dependent on the amino acid sequence of the endogenous H3.3 gene and change with cellular differentiation. H3.3 is enriched at active and repressed genes, as well as at transcription factor binding sites (TFBS) and telomeres. The histone chaperone Hira is required for H3.3 enrichment at active and repressed genes but not at TFBS or telomeres. Surprisingly, Atrx and Daxx, which are associated with H3.3 in both the presence and absence of Hira, are specifically required for H3.3 localization at telomeres and for the repression of telomeric RNA. These findings demonstrate that multiple and distinct factors control H3.3 localization at specific genomic locations in mammalian cells.