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The ATRX Syndrome Protein Forms a Chromatin-Remodeling Complex with Daxx and Localizes in Promyelocytic Leukemia Nuclear Bodies

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The ATRX Syndrome Protein Forms a Chromatin-Remodeling Complex with Daxx and Localizes in Promyelocytic Leukemia Nuclear Bodies The ATRX syndrome protein forms a chromatin-remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies Yutong Xue*, Richard Gibbons†, Zhijiang Yan*, Dafeng Yang*, Tarra L. McDowell†, Salvatore Sechi‡§, Jun Qin¶, Sharleen Zhouʈ, Doug Higgs†, and Weidong Wang*,** *Laboratory of Genetics and ‡Research Resources Branch, National Institute on Aging, National Institutes of Health, 333 Cassell Drive, TRIAD Center Room 4000, Baltimore, MD 21224; †Medical Research Council Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom; ¶Departments of Biochemistry and Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030; and ʈHoward Hughes Medical Institute, University of California, Berkeley, CA 94720 Edited by Gerald R. Crabtree, Stanford University School of Medicine, Stanford, CA, and approved July 23, 2003 (received for review December 16, 2002) ATRX syndrome is characterized by X-linked mental retardation result, the etiology of ATRX syndrome is poorly understood. associated with ␣-thalassemia. The gene mutated in this disease, Several lines of evidence hinted that ATRX protein may be part ATRX, encodes a plant homeodomain-like finger and a SWI2͞SNF2- of a chromatin-remodeling complex. First, ATRX protein not like ATPase motif, both of which are often found in chromatin- only has a SWI͞SNF2-type ATPase͞helicase motif but also has remodeling enzymes, but ATRX has not been characterized bio- a plant homeodomain-like zinc finger (10), both of which have chemically. By immunoprecipitation from HeLa extract, we found been found in molecules that modify chromatin structure. Sec- that ATRX is in a complex with transcription cofactor Daxx. The ond, ATRX in nuclear extracts fractionates as a complex be- following evidence supports that ATRX and Daxx are components tween 700 and 2,000 kDa (11), a size similar to that of other of an ATP-dependent chromatin-remodeling complex: (i) Daxx and SWI͞SNF complexes. Third, ATRX localizes at pericentromeric ATRX can be coimmunoisolated by antibodies specific for each heterochromatin (12) and has been identified in yeast two-hybrid protein; (ii) a proportion of Daxx cofractionates with ATRX as a screens by its interaction with the heterochromatin protein HP1 complex of 1 MDa by gel-filtration analysis; (iii) in extract from cells as well as a polycomb group protein EZH2 (12–14). Fourth, of a patient with ATRX syndrome, the level of the Daxx–ATRX ATRX mutations have been correlated with changes in DNA complex is correspondingly reduced; (iv) a proportion of ATRX and methylation patterns at several genomic loci (15). Here, we Daxx colocalize in promyelocytic leukemia nuclear bodies, with demonstrate that ATRX forms a complex with a transcription which Daxx had previously been located; and (v) the ATRX complex cofactor, Daxx, and this complex displays chromatin-remodeling displays ATP-dependent activities that resemble those of other activities. The results provide a step toward understanding the chromatin-remodeling complexes, including triple-helix DNA dis- physiological function of ATRX. placement and alteration of mononucleosome disruption patterns. But unlike the previously described SWI͞SNF or NURD complexes, Materials and Methods the ATRX complex does not randomize DNA phasing of the mono- Cell Culture. HeLa cells were purchased from the National Cell nucleosomes, suggesting that it may remodel chromatin differ- Culture Center (Minneapolis). The cell line derived from an ently. Taken together, the results suggest that ATRX functions in ATRX patient carries an R37X mutation (16). conjunction with Daxx in a novel chromatin-remodeling complex. The defects in ATRX syndrome may result from inappropriate Antibodies (Abs). An affinity-purified polyclonal Ab, FXNP5 expression of genes controlled by this complex. (NP5), and an ATRX mAb (23c) have been described (17). Two ATRX Abs, D19 and C16, were obtained from Santa Cruz BIOCHEMISTRY SWI͞SNF Biotechnology. Two additional rabbit Abs were raised against fusion proteins also containing maltose-binding protein (New hromatin-remodeling complexes play major roles in regula- England Biolabs) and regions of ATRX (amino acids 365–473 Ction of gene expression in eukaryotes (1, 2). These com- and 2239–2492). Two different Daxx Abs, SC7152 and SC7001, plexes can modify chromatin structure through two distinct were obtained from Santa Cruz Biotechnology. mechanisms. One is through covalent modification, including methylation, phosphorylation, and acetylation. The other is Nuclear Extract Fractionation and Immunoprecipitation. HeLa nu- through noncovalent mechanisms, which include ATP- clear extract was prepared as described (4). ATRX-associated dependent chromatin remodeling. We and others have previ- complex was isolated from HeLa nuclear extract by using an ously purified and characterized several ATP-dependent chro- immunoprecipitation protocol (C. S. Lee, Y.X., X. Zhang, and matin-remodeling complexes of the human SWI͞SNF family W.W., unpublished data). This protocol and methods for mass (3–6). In attempts to characterize additional remodeling com- spectrometry (MS) analysis, have been described in another plexes, we reasoned that because all known ATP-dependent study (18). The Superose 6 gel-filtration analysis has been remodeling complexes contain a subunit with a SWI2͞SNF2-like described (19). After fractionation, the ATRX peak fractions ATPase motif, human proteins containing this motif might were collected and incubated with or without ethidium bro- reveal unique remodeling complexes. A previous study based on mide (100 ␮g͞ml) for 1 h before immunoprecipitation. this strategy indeed identified the NURD complex (7). Here, we characterize a complex containing ATRX, a protein initially identified by mutations in patients with ATRX syndrome (8). This paper was submitted directly (Track II) to the PNAS office. Mutations in the ATRX gene are now known to cause several Abbreviation: PML, promyelocytic leukemia. X-linked mental retardation syndromes. The phenotypes include §Present address: National Institute of Diabetes and Digestive and Kidney Diseases, facial dysmorphism, urogenital defects, and ␣-thalassaemia (re- National Institutes of Health, Bethesda, MD 20892-5460. sulting from reduced ␣-globin expression) (9). However, the **To whom correspondence should be addressed. E-mail: [email protected]. ATRX protein has not been biochemically characterized. As a © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.1937626100 PNAS ͉ September 16, 2003 ͉ vol. 100 ͉ no. 19 ͉ 10635–10640 Downloaded by guest on October 1, 2021 Enzymatic Assays. The mononucleosome disruption assay (20, 21) and ATPase assay (22) followed the same procedure used for human SWI͞SNF complexes. One modification of the ATPase 32 assay is that the Pi is separated from [␥- P]ATP by TLC, and the radioactivity was quantitated by using a PhosphorImager. The triple-helix displacement assay used the same protocol and substrates used for RSC and STH1 (23). The double-helix displacement assay used assay conditions and substrates identical to those described (24). Immunofluorescence. Immunofluorescence experiments followed a previous procedure (12), except that cells were fixed in 100% methanol and then air-dried before immunostaining. Primary Abs included: mouse monoclonal anti-ATRX (23c), rabbit poly- clonal anti-Daxx (Ab-1, Oncogene Research Products), rabbit polyclonal anti-promyelocytic leukemia (PML; kindly provided by Paul Freemont, Imperial College, London). All cells were counterstained with 4Ј,6-diamidino-2-phenylindole (Sigma). Results Identification of an ATRX-Associated Complex. Our initial attempts to purify the ATRX-associated complexes used a combination of conventional and immunoaffinity chromatography, a strategy ͞ Fig. 1. Purification of an ATRX-associated complex from HeLa nuclear that successfully isolated the human SWI SNF and NURD extract. (a) Silver-stained SDS gels showing polypeptides immunoisolated by complexes. However, such approaches obtained ATRX as a three different ATRX Abs (NP5, D19, and C16) from nuclear extract. Mock single polypeptide with no associated proteins [data not shown; immunoprecipitation was done by using either protein A beads alone (lane 2) Yi Zhang’s group has independently obtained the same result or a preimmune (PI) serum (lane 5). MS has been used for identification of (Y. Zhang, personal communication)]. The ATRX-associated ATRX and its associated polypeptides in all three preparations. The number of complex was later found to be unstable in salt solutions of 0.5 M peptides that matched the indicated protein and the percentage of these or higher (see Fig. 4e). Fractionation of nuclear extract often peptides among total peptides obtained from matrix-assisted laser desorption resulted in partial or complete dissociation of the ATRX com- ionization–time-of-flight analysis are shown in parentheses for ATRX and Daxx. The other polypeptides (marked by lines) appeared to either loosely plex. For this reason, we adopted an optimized immunoprecipi- associate with ATRX or associate with ATRX through DNA by subsequent tation method (C. S. Lee, Y.X., X. Zhang, and W.W., unpub- analysis. (b) Immunoblot confirming the presence of Daxx in the polypeptides lished data) to directly isolate ATRX-associated complexes from immunoisolated by ATRX Ab. NE, nuclear extract; SN, supernatant; IP, immu- HeLa nuclear extract. This method
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