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The histone variant H3.3 is implicated in the formation and maintenance of specialized chromatin structure in metazoan cells. H3.3-containing nucleosomes are assembled in a replication-independent manner by means of dedicated chaperone proteins. We previously identified the death domain associated protein (Daxx) and the alpha-thalassemia X-linked mental retardation protein (ATRX) as H3.3-associated proteins. Here, we report that the highly conserved N terminus of Daxx interacts directly with variant-specific residues in the H3.3 core. Recombinant Daxx assembles H3.3/H4 tetramers on DNA templates, and the ATRX-Daxx complex catalyzes the deposition and remodeling of H3.3-containing nucleosomes. We find that the ATRX-Daxx complex is bound to telomeric chromatin, and that both components of this complex are required for H3.3 deposition at telomeres in murine embryonic stem cells (ESCs). These data demonstrate that Daxx functions as an H3.3-specific chaperone and facilitates the deposition of H3.3 at heterochromatin loci in the context of the ATRX-Daxx complex.

Histone proteins often come in different variants serving specialized functions in addition to their fundamental role in packaging DNA. The metazoan histone H3.3 has been most closely associated with active transcription. Its role in histone replacement at active genes and promoters is conserved to the single histone H3 in yeast. However, recent genetic studies in flies have challenged its importance as a mark of active chromatin, and revealed unexpected insights into essential functions of H3.3 in the germline. With strikingly little amino acid sequence difference to the canonical H3, H3.3 therefore accomplishes a surprising variety of cellular and developmental processes., (2010 Elsevier Ltd. All rights reserved.)

The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc-finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells., ((c) 2010 Elsevier Inc. All rights reserved.)

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.