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. 2010 Aug 10;107(32):14075-80.
doi: 10.1073/pnas.1008850107. Epub 2010 Jul 22.

Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres

Peter W Lewis  1 , Simon J Elsaesser, Kyung-Min Noh, Sonja C Stadler, C David Allis
Affiliations

Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres

V体育ios版 - Peter W Lewis et al. Proc Natl Acad Sci U S A. .

Abstract

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 VSports手机版. 3-specific chaperone and facilitates the deposition of H3. 3 at heterochromatin loci in the context of the ATRX-Daxx complex. .

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Conflict of interest statement

The authors declare no conflict of interest.

"V体育平台登录" Figures

Fig. 1.
Fig. 1.
Identification of Daxx as an H3.3-specific binding protein. (A) Silver stain of H3.1 and H3.3 associated proteins. FLAG-M2 IP was performed on the nuclear extract (lanes 1, 2). The eluate from the FLAG IP was then subjected to HA IP (lanes 3, 4). HA IP was performed on untagged HeLa nuclear extract as a control (lane 5). Protein bands from the HA affinity column were identified by mass spectrometry. Arrowhead in A and B denotes Daxx. (B) Silver stain of FLAG eluate from H3.1 and H3.3 chromatin-associated fractions. (C) Immunoblot was performed on the FLAG eluate with anti-Daxx, anti-ATRX, anti-CAF150, and anti-ASF1a/b sera. Fractionation scheme for H3.3 chromatin-associated proteins. (D) Immunoblott analysis of Mono Q fractions with with anti-Daxx, anti-FLAG, and anti-ATRX. (E) Immunoblot of anti-HA and control rabbit IgG IP of e-H3.3 from pooled Mono Q complex I fractions (left). Silver stain of input and IP material (right).
Fig. 2.
Fig. 2.
Daxx directly interacts with histone H3.3. (A) Co-IP of recombinant H3.1/H4 or H3.3/H4 with Daxx on M2-FLAG agarose. Beads were washed with buffers of indicated KCl concentrations. (B) Domain structure of human Daxx. Shaded boxes indicate highly conserved regions (dark gray: highly conserved in all metazoan Daxx; light gray: highly conserved in vertebrates). GST-fusion Daxx constructs are shown in black. (C) GST pull-down with Daxx fragments shown in B with H3.3/H4 or H3.1/H4. (D) Schematic showing amino acid differences between H3.1 and H3.3. Relevant secondary structures are indicated. Solid lines represent histone deletion constructs use in E, biotinylated peptides used in G are shown below. (E) GST pull-down with the Daxx HBD (Δ3) and H3.3/H4 tetramers with histone tail deletions and the H3.3/H4 octamer with H2A/H2B. (F) Co-IP of single H3 point mutations as described in A. (G) Peptide pull-down with biotinylated peptides of residues 80–94 or 86–97 of H3.1 and H3.3.
Fig. 3.
Fig. 3.
Daxx is a histone H3.3 chaperone. (A) In vitro reconstituted and purified Daxx-H3.3/H4 complex used for deposition assay (B) chromatin-induced supercoiling was analyzed by agarose gel electrophoresis in the absence (upper) and presence (lower) of chloroquine. (C) EMSA of tetrasome deposition on a 147 bp DNA fragment by the Daxx-H3/H4 complex. (D) Recombinant Daxx and (E) H3.1/H4 and H3.3/H4. (F) Daxx mediated H3.1/H4 or H3.3/H4 tetrosome deposition on topoisomerase I-relaxed plasmid.
Fig. 4.
Fig. 4.
ATRX–Daxx is a histone deposition and remodeling complex. (A) Recombinant ACF complex (ACF1 and ISWI), hNAP1, Daxx, H3.1/H4, H3.3/H4, and H2A/H2B. (B) SMART Superdex 200 gel filtration fractionation of ATRX–Daxx complex. ATRX–Daxx complex used in C–E was purified from free Daxx and Daxx-H3.3/H4 complex. (C) MNase digestion of chromatin assembly reactions with H3.1/H4 or H3.3/H4 alone, or in combination with NAP1 and ACF complex. (D) MNase digestion of assembly reactions with H3.1/H4 or H3.3/H4 alone, or in combination with Daxx and ATRX–Daxx complex. (E) Analysis of remodeling activity of ACF and ATRX–Daxx complexes on H3.3 mononucleosomes by native TBE PAGE.
Fig. 5.
Fig. 5.
ATRX–Daxx complex is required for H3.3-deposition at telomeres. (A) Immunoblots of nuclear extracts for Daxx, ATRX, and H3.3-HA. Tubulin immunoblot served as a loading control (B) HA-IP from the extracts in A. IP material was immunoblotted for Daxx, ATRX and H3.3-HA. (C) ChIP of HIRA, ATRX, and Daxx proteins was on 129/SvEv, Daxx-/-, ATRX–Flox, and ATRX-/- ESCs. Dot blot analysis of IP DNA using telomere and BamH1 repeat probes. (D) HA-H3.3 ChIP was performed on 129/SvEv, Daxx-/-, ATRX–Flox, and ATRX-/- ESCs. Bar graph showing the input-normalized signal from the dot blot on telomere and BamHI repeats with standard errors.
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