Summary 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 (ES) 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.

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 α-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.he assembly of chromosomal DNA into nucleosomes represents the most fundamental step in the formation of eukaryotic chromatin structure. The deposition, remodeling, and eviction of nucleosomes have been shown to be important for a variety of DNA-templated processes such as replication, repair, and transcription. Histone deposition pathways are thought to play a critical role in the establishment and maintenance of epigenetic information encoded by histone modifications, nucleosome positioning, and higher-order chromatin structure (1-3). An additional layer of epigenetic regulation is achieved by the use of histone variants: paralogs of the core histones genes H3, H4, H2A, and H2B that have diverged from their canonical counterparts in primary structure and function.In addition to a centromeric version, mammalian genomes encode three H3 variants. Histones H3.1 and H3.2 are primarily expressed in S-phase, whereas the H3.3 variant is expressed throughout the cell cycle. For its universal role in proliferating and nondividing cells, the function of H3.3 has been studied in a wide range of cell types and organisms (4). Differences in H3.3 and the canonical H3 species are confined to one residue in the histone tail and a cluster of three residues in the core histone fold. The three amino acid variations in the histone fold have been shown to be necessary for H3.3 replication-independent incorporation into chromatin (5, 6). Higher eukaryotes utilize separate chaperones and deposition pathways for the different histone H3 variants, and previous work identified two major pathways: replication-coupled deposition of H3.1/H3.2 by the CAF1 complex, and replication-independent deposition of H3.3 by the HIRA complex (6-8)While originally associated with euchromatic sites of active transcription, H3.3 has recently been found associated with regulatory elements and constitutive heterochromatin at telomeres (9-12). We previously found that HIRA is required for localization of H3.3 t...

Although the biological significance of protein phosphorylation in cellular signaling is widely appreciated, methods to directly detect these post-translational modifications in situ are lacking. Here we introduce the application of high-resolution NMR spectroscopy for observing de novo protein phosphorylation in vitro and in Xenopus laevis egg extracts and whole live oocyte cells. We found that the stepwise modification of adjacent casein kinase 2 (CK2) substrate sites within the viral SV40 large T antigen regulatory region proceeded in a defined order and through intermediate substrate release. This kinase mechanism contrasts with a more intuitive mode of CK2 action in which the kinase would remain substrate bound to perform both modification reactions without intermediate substrate release. For cellular signaling pathways, the transient availability of partially modified CK2 substrates could exert important switch-like regulatory functions.