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Patient Mutations Alter ATRX Targeting to PML Nuclear Bodies

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Patient Mutations Alter ATRX Targeting to PML Nuclear Bodies European Journal of Human Genetics (2008) 16, 192–201 & 2008 Nature Publishing Group All rights reserved 1018-4813/08 $30.00 www.nature.com/ejhg ARTICLE Patient mutations alter ATRX targeting to PML nuclear bodies Nathalie G Be´rube´1,3,4, Jasmine Healy1,4, Chantal F Medina1, Shaobo Wu1, Todd Hodgson1, Magdalena Jagla1 and David J Picketts*,2 1Molecular Medicine Program, Ottawa Health Research Institute, Ottawa, Ontario, Canada; 2Departments of Medicine and Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada ATRX is a SWI/SNF-like chromatin remodeling protein mutated in several X-linked mental retardation syndromes. Gene inactivation studies in mice demonstrate that ATRX is an essential protein and suggest that patient mutations likely retain partial activity. ATRX associates with the nuclear matrix, pericentromeric heterochromatin, and promyelocytic leukemia nuclear bodies (PML-NBs) in a speckled nuclear staining pattern. Here, we used GFP–ATRX fusion proteins to identify the specific domains of ATRX necessary for subnuclear targeting and the effect of patient mutations on this localization. We identified two functional nuclear localization signals (NLSs) and two domains that target ATRX to nuclear speckles. One of the latter domains is responsible for targeting ATRX to PML-NBs. Surprisingly, this domain encompassed motifs IV–VI of the SNF2 domain suggesting that in addition to chromatin remodeling, it may also have a role in subnuclear targeting. More importantly, four different patient mutations within this domain resulted in an B80% reduction in the number of transfected cells with ATRX nuclear speckles and PML colocalization. These results demonstrate that patient mutations have a dramatic effect on subnuclear targeting to PML-NBs. Moreover, these findings support the hypothesis that ATRX patient mutations represent functional hypomorphs and suggest that loss of proper targeting to PML-NBs is an important contributor to the pathogenesis of the ATR-X syndrome. European Journal of Human Genetics (2008) 16, 192–201; doi:10.1038/sj.ejhg.5201943; published online 24 October 2007 Keywords: ATR-X syndrome; ATRX; chromatin remodeling; SWI/SNF; X-linked mental retardation; XLMR Introduction have been identified as the cause of the ATR-X syndrome,3 The ATR-X syndrome (OMIM no. 301040) is a severe other XLMR disorders with similar features but lacking X-linked mental retardation (XLMR) syndrome associated thalassemia,4 and the acquired form of a-thalassemia with a characteristic facial appearance, urogenital abnorm- myelodysplasia syndrome (ATMDS).5–8 alities and a-thalassemia.1,2 Mutations in the ATRX gene The ATRX gene encodes a chromatin remodeling protein with an ATPase/helicase domain of the SNF2 family and a plant homeodomain (PHD) zinc finger.9 ATRX mutations *Correspondence: Dr DJ Picketts, Ottawa Health Research Institute, 501 are predominantly missense mutations with most identi- Smyth Road, Ottawa, Ontario K1H 8L6, Canada. fied in the PHD (B60%) or SNF2 domains (B25%).10 While Tel: þ 613 737 8989; Fax: þ 613 737 8803; E-mail: [email protected] it is presumed that ATRX mutations are associated with 3Current address: Departments of Biochemistry and Paediatrics, Uni- reduced function, few studies have addressed structure/ versity of Western Ontario; Child Health Research Institute and Lawson function relationships. Health Research Institute, London, Ontario, Canada. Subcellular localization and initial biochemical studies 4These authors have contributed equally to this work. Received 7 June 2007; revised 22 September 2007; accepted 25 with ATRX indicate that it likely has a repressive role on September 2007; published online 24 October 2007 nuclear processes. It is localized to pericentromeric Altered ATRX targeting to PML NG Be´rube´ et al 193 heterochromatin and can physically interact with proteins Construct L9 was generated by deleting the BamHI that reside within heterochromatin, including HP1 and fragment from the 85/30 þ 8R4 vector. Constructs L18 EZH2.11 – 14 The ATRX protein also localizes to PML-NBs and L19 were obtained by isolating the BamHI and EcoRI- where it interacts with Daxx, a transcriptional and BamHI fragments from 8R4-C2, respectively and religating apoptotic regulator.12,15,16 ATRX is also involved in the downstream of the nuclear localization signal in the regulation of methylation at ribosomal DNA (rDNA) linearized L8 construct. We obtained L20 by cutting a SacI repeats.17 Given that ATRX acts at multiple locations fragment from L19 and religating to the EGFP-C3 vector. within the nucleus, some mutations are likely to alter L24 was generated by removing an AflII-SalI fragment from subnuclear localization thereby reducing function. the L16 construct. Finally, L25 was obtained by cutting the In this regard, immunoblots of protein isolated from a L16 construct with AflII and XhoI, treating with Klenow panel of ATR-X patients with unique missense mutations followed by religation. have shown that many samples have reduced ATRX 13,18 protein levels. Nonetheless, other experiments have Site-directed mutagenesis demonstrated a more direct role of mutations on protein Mutant constructs were generated by a PCR-based site- function. Baculovirus expressed ATRX protein containing directed mutagenesis method (Stratagene QuickChange engineered patient mutations within the SNF2 domain kit) using the L16, L2 and L19 GFP-tagged constructs as were shown to have attenuated ATPase activity compared templates. Template DNA (20 ng) was amplified using the 15 to the wild-type (WT) protein. Similarly, expression of appropriate primers (100 ng each forward and reverse, see the PHD domain in bacteria demonstrated that patient Table 1) using the PCR in a 50-ml reaction with the 18 mutations impaired DNA-binding activity. Collectively, following conditions: 951C for 30 s, then 951C for 30 s, these studies suggest that some mutations may interfere 551C for 1 min and 681C for 14 min for 12 cycles. The directly with function while others may have an indirect samples were then incubated for 1 h at 371C with DpnIto effect through altered protein stability or aberrant sub- digest parental non-mutated double-stranded DNA. The nuclear targeting of the protein. product (1 ml) was transformed in XL-2 Blue competent Here, we utilized GFP–fusion proteins to identify the cells. Plasmid DNA was obtained from the resulting domains responsible for nuclear localization, heterochro- colonies and screened using the new or abolished restric- matin binding and subnuclear targeting to the PML-NBs. tion enzyme sites introduced by silent mutations (see Moreover, we demonstrate that patient mutations within Table 1 below). one domain reduces the efficiency of targeting to PML-NBs. Transfections and immunofluorescence HeLa cells were grown in EMEM supplemented with 10% Materials and methods fetal bovine serum in 5% CO2 at 371C. Plasmid DNA for Construction of GFP-tagged protein subdomains each GFP deletion construct was isolated using the Midi- The full-length ATRX-EGFP construct was generated by Preparation kit from Qiagen and verified by sequence cutting the HA-ATRX-pCAGGS construct19 with XhoI and analysis. Transient transfection was performed using the SstII and ligating to EGFP-C2. EcoRI, SacI-PstI and PstI-SstII Lipofectamine 2000 reagent (Life Technologies Inc.) ac- fragments were cut from HA-ATRX-pCAGGs and ligated to cording to the manufacturer’s protocol. Depending on the the EGFP-C2 linearized vector to create the L16, 85/30 and level of cell death observed with some of the constructs, 8R/4 constructs, respectively. The 8R4-EGFPC2 construct cells were processed after 16–24 h by fixing in cold was put back in frame by cutting with XhoI and filling ethanol/methanol (3:1) and counterstaining with DAPI. the ends with Klenow enzyme. The L3, L5, L6 and L8 Immunofluorescence images were captured using a Zeiss constructs were created by cutting 85/30-C2 with SacI- Axioplan 2 microscope outfitted with an AxioCam camera HindIII, EcoRI-PstI, SacI-AflII and HindII-EcoRI, respectively. and AxioVision software. For analysis of mutated Table 1 Primers used to establish ATRX-GFP constructs with patient mutations Construct Primer sequence Mutation Silent mutationa L16R-C 50-CTGCAAGAAATGCATTCTATGCAACCTAGGTCGAAAGG-30 C4T (953) Arg-Cys AvrII L19-Ped3 50-CGAAACATTGACTATCACCGTCTAGATGGTTCCACTAC-30 T4C (6250) Tyr4His XbaI L19-Ped17 50-AGCCAGGAGCTCGATGTTAAATGAAGAGAAGCAATCTAC-30 C4T (7156) Arg4Stop SacI L19-Ped23 50-GCAGAGGAAATTGGGGTTAAAGTACTAGTTTTCAGCCAGTCC-30 A4T (6104) Asp4Val SpeI L19-Ped26 50-GTATTGACAAAACAACAGATGTGAATAAGCTGTGTTCAGCGA-30 Deln (7201) Novel Stop PvuIIb aSilent mutation introduced to create new restriction site (primer sequence underlined) for analysis. bSite is abolished. European Journal of Human Genetics Altered ATRX targeting to PML NG Be´rube´ et al 194 constructs, at least 300 cells were counted and assessed for nuclear localization signals (NLSs) in the ATRX protein localization pattern. sequence encompassing amino acids 685–697, three overlapping sites within 1126–1196, and individual sites at 1420–1426 and 1928–1938. As such, we divided Results the ATRX gene into three fragments – L16, 85/30 and 8R/4 Nuclear localization of ATRX deletion constructs for a preliminary analysis of nuclear localization. We To identify the domains responsible for nuclear and observed that both L16 and 85/30 independently localized subnuclear localization we generated a panel of GFP– to the nucleus with distinct patterns.
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