Human Molecular Genetics, 2000, Vol. 9, No. 4 539-547
© 2000 Oxford University Press
Cell cycle-dependent phosphorylation of the ATRX protein correlates with changes in nuclear matrix and chromatin association
1Ottawa Hospital Research Institute, Ottawa, Ontario, Canada K1H 8L6 and 2Departments of Medicine, Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario, Canada K1H 8C8
Received 22 September 1999; Revised and Accepted 22 December 1999.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Mutations in the ATRX gene are associated with an X-linked mental retardation (XLMR) syndrome most often accompanied by
-thalassaemia (ATR-X syndrome). The ATRX gene encodes a predicted protein of 280 kDa featuring a PHD zinc finger motif and an ATPase/helicase domain of the SWI/SNF type; the vast majority of mutations in the ATRX gene fall within these two motifs. Although these domains are suggestive of a role for ATRX in transcriptional regulation by affecting chromatin structure and/or function, the precise cellular role of the ATRX protein remains undefined. Using indirect immunofluorescence and biochemical frac- tionation, we demonstrate that the ATRX protein has a punctate nuclear staining pattern and that it is tightly associated with the nuclear matrix at interphase. At the onset of M phase, the ATRX protein was associated mainly with condensed chromatin. The association of the ATRX protein with chromosomes at mitosis is concomitant with phosphorylation of the protein and its association with heterochromatin protein 1 (HP1). The phosphorylation-dependent changes in localization between the nuclear matrix and condensed chromatin are consistent with a dual role for ATRX, possibly involving gene regulation at interphase and chromo- somal segregation at mitosis. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Patients with the ATR-X syndrome have severe mental retardation associated with a characteristic facial appearance,
-thalassaemia and variable degrees of urogenital abnormalities (1). These strict diagnostic criteria were important for the establishment of linkage analysis to Xq12–q21.31 and the subsequent localization of the ATRX gene (2,3). To date, mutations in the ATRX gene have been identified in 52 individuals including patients who lack detectable -thalassaemia (2,4–7). ATRX gene mutations have also been identified in patients afflicted with Juberg–Marsidi, Carpenter–Waziri and other less well-characterized X-linked mental retardation (XLMR) syndromes (8–11). Collectively, these studies demonstrate that ATRX mutations result in a broad spectrum of phenotypes and suggest that the ATRX gene warrants careful consideration as a common cause of XLMR.
The ATRX gene encodes a predicted protein of 280 kDa that represents a novel member of the SWI2/SNF2 family of chromatin remodelling proteins, which are involved in transcriptional regulation, DNA repair and mitotic recombination (5,12,13). Members of the SWI2/SNF2 protein family contain an ATPase/helicase domain in combination with one or more chromatin interaction motifs [eg. chromodomain, bromodomain and plant homeodomain (PHD) zinc finger]. In addition to the ATPase/helicase domain, the ATRX protein contains a PHD-type zinc finger domain that most closely resembles that of the DNMT3 family of DNA methyltransferases (Fig. 1B) (14). A further functional domain is proposed to lie between the PHD and ATPase/helicase domains of ATRX, a region poorly conserved between mouse and human (15). This region of ATRX has been shown to interact with murine heterochromatin protein 1 (HP1) and the Polycomb protein EZH2 in two-hybrid screens and to contain a coiled-coil motif by sequence analysis (16,17).
|
The vast majority of mutations (~90%) in the ATRX gene occur within the PHD (~65%) and ATPase (~25%) domains, underscoring the functional importance of these two motifs in ATRX protein function. Several patients with severe genital abnormalities have mutations that result in truncation of the last 100 amino acids, implying a role for this end of the protein in urogenital development and/or function (5). Apart from the previous example, there appears to be no strong genotype–phenotype correlation with regard to ATRX mutations. This is demonstrated most clearly by the variable level of haemoglobin H inclusions in 15 unrelated individuals with the same mutation (18).
The ATRX protein has been postulated to be a transcriptional regulator since ATRX mutations appear specifically to down-regulate the expression of the -globin genes but not the closely related ß-globin genes. The specificity of this defect is likely to be a direct result of the different chromosomal environments within which these two gene clusters reside (19,20). Furthermore, ATR-X patients do not have an increased risk of malignancy, chromosome breakage or UV sensitivity in affected individuals, as might be expected with defective SWI2/SNF2 proteins involved in DNA repair (2).
Biochemical studies have shown that SNF2-like proteins exist in multiprotein complexes that range in size from 500 to 2000 kDa and are comprised of 2–15 subunits (21–28). In vitro studies have demonstrated that these purified complexes perform a variety of functions including disruption of nucleosome positioning and chromatin assembly, and the enhancement of transcription factor binding to recognition sequences (29–31). Interestingly, one complex (NuRD) has been shown to have both chromatin remodelling and histone deacetylase activity (27,28). Although it remains to be determined whether ATRX functions as part of a multiprotein complex, several splicing mutations of the ATRX gene have been identified that suggest a stoichiometric requirement for the ATRX protein, consistent with such a role (5).
Despite the biochemical purification and analysis of the catalytic properties of these multiprotein complexes in vitro, little is known about their cellular targets, biological pathways or regulation. As a starting point to help to define the normal function of ATRX and understand the abnormalities characteristic of the ATR-X syndrome, we have examined the subcellular localization of ATRX. We have shown through biochemical fractionation and indirect immunofluorescence analyses that the protein is associated with the nuclear matrix. We also present evidence that the ATRX protein is phosphorylated in a cell cycle-dependent manner, with phosphorylation in early M phase correlating with release from the nuclear matrix and association with condensed chromatin.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Immunolocalization of ATRX in interphasic and mitotic cells
To begin to assess the role of ATRX in vivo, we raised a polyclonal antibody (fxnp5) against a bacterially expressed ATRX fusion protein of 88.5 kDa comprising residues 320–730 of the human ATRX protein. Immunoblot analyses revealed that the antibody recognized two protein bands of 280 and 180 kDa in both HeLa and 293 cell extracts (Fig. 1A). Pre-incubation of the antibody with the ATRX fusion protein abrogated the signal on immunoblot, demonstrating the specificity of the antibody for the ATRX protein. The identity of the 180 kDa protein is unknown, but it probably represents a degradation or alternatively spliced product, as several ATRX-specific monoclonal antibodies raised against a portion of the protein encompassing the PHD finger also detect this band (32). Additionally, the 180 kDa band co-fractionates with the larger 280 kDa protein following gel filtration analysis, using both the fxnp5 polyclonal and the 39f monoclonal antibodies by western blot analysis (data not shown).
The fxnp5 antibody was used to investigate by indirect immunofluorescence the localization of ATRX in HeLa cells grown on coverslips. In interphasic cells, this antibody revealed a speckled pattern that was confined to the nucleus (Fig. 2A and B). Similar patterns of immunofluorescence were obtained in all cell lines tested, including 293 and Cos-1 cells, and with a mouse monoclonal antibody specific to the PHD finger domain of ATRX (data not shown; see ref. 32). However, in mitotic cells, the speckled pattern was greatly reduced and the ATRX signal was partially re-localized to condensed chromatin during metaphase and anaphase; the remainder of the signal was detected in the cytoplasm (Fig. 2C–F). The signal was co-localized entirely with chromatin by the time the cells reached telophase (Fig. 2E–J). The association of ATRX with condensed chromatin as well as in the cytoplasm during metaphase was also observed following colcemid treatment (Fig. 2C and D).
|
ATRX and HP1
co-immunoprecipitate at mitosisThe localization of ATRX to condensed chromatin at mitosis is very similar to the distribution of HP1. Interestingly, an interaction between murine ATRX (HP1-BP38) and HP1
was identified previously in a two-hybrid screen using murine HP1 as bait (17). In order to assess whether this localization is coincidental or of functional importance, we used co-immunoprecipitation to determine whether ATRX and HP1 interact at mitosis. ATRX was immunoprecipitated from mitotic HeLa cell extracts with the fxnp5 antibody and subjected to immunoblot analysis with antibodies specific for human HP1. This experiment revealed that HP1 co-immunoprecipitated with ATRX but not in control experiments with sheep IgG, demonstrating a direct association with ATRX (Fig. 3, left). To confirm these results, the converse experiment was performed whereby HP1 was first immunoprecipitated from mitotic HeLa extracts followed by detection of ATRX by immunoblot analysis. ATRX consistently co-immunoprecipitated with HP1, but always to a lesser extent than the opposite experiment, which is most likely to be a reflection of the HP1 antibody used for immunoprecipitation (Fig. 3, right). These results show that HP1 and ATRX physically interact during mitosis when bound to condensed chromatin. In light of recent results demonstrating that ATRX localizes to the centromeres of human metaphase chromosomes (32), it is likely that the interaction detected between ATRX and HP1 occurs at these sites within condensed chromatin.
|
ATRX is bound to the core nuclear matrix in interphasic cells
As many transcription factors and other SNF2 family members have been shown to bind to the core nuclear matrix, we further investigated the subnuclear distribution of ATRX in HeLa and 293 cells. Unsynchronized cells were extracted sequentially to obtain cytoplasmic, chromatin and core nuclear matrix fractions following high salt treatment (33). Immunoblot analysis with the fxnp5 antibody showed that ATRX fractionates almost exclusively with the core nuclear matrix in both cell lines (Fig. 4A). The efficacy of the fractionation procedure was ascertained by immunoblot assay with control antibodies specific to: p38, a stress-activated protein kinase found in the cytoplasm and nucleus (34); histone H1, a component of chromatin; and lamin A/C, a core nuclear matrix-associated protein (Fig. 4B–D).
|
Using a similar procedure, HeLa cells grown on coverslips were extracted in situ with detergent (CSK buffer), DNase I, ammonium sulfate and high salt, leaving behind the core nuclear matrix. The efficiency of chromatin digestion and removal was assessed by the disappearance of DNA staining with 4',6-diamidino-2-phenylindole (DAPI). The speckled pattern of ATRX was still detected after high salt treatment, indicating that a significant proportion of ATRX protein is associated with the core nuclear matrix (Fig. 5). These results do not exclude the possibility that ATRX associates with chromatin and the nuclear matrix simultaneously at interphase. However, a very small proportion of ATRX fractionates with the chromatin fraction, possibly due to a stronger interaction with the nuclear matrix components.
|
ATRX is phosphorylated at mitosis
Cell cycle-dependent changes in nuclear localization of the human SWI2/SNF2 proteins have been shown to correlate with changes in phosphorylation (35). Concomitantly, the presence of several predicted phosphorylation sites in the ATRX protein sequence prompted us to examine whether the changes that we observed in subcellular localization correlated with a change in the phosphorylation status of the protein. We examined the electrophoretic mobility of the ATRX protein by western blot analysis in interphase and mitotic HeLa cell extracts. ATRX consistently migrated more slowly in mitotic compared with interphasic extracts (Fig. 6A, lanes I and M), suggesting that post-translational modifications of the protein occur at mitosis. To determine whether the mobility shift was caused by a phosphorylation event, HeLa cells were treated with nocodazole to induce a pro-metaphase arrest, and mitotic cells were separated from interphase cells by shake-off. Mitotic cell extracts were immunoprecipitated with the fxnp5 antibody and half the sample was treated with
-phosphatase. The ATRX protein band migrated faster following phosphatase treatment, indicating that the mobility shift was caused by phosphorylation of the protein (Fig. 6B, compare +ppase with –ppase). The specificity of -phosphatase is primarily for phosphoserine and phosphothreonine residues, although it is known to dephosphorylate phosphotyrosine residues. We have shown that ATRX immunoprecipitated from mitotic cells is recognized by an anti-phosphoserine antibody (Fig. 6D), but not by anti-phosphothreonine or anti-phosphotyrosine antibodies (data not shown), demonstrating that ATRX is phosphorylated predominantly on serine residues.
|
To examine the phosphorylation status of ATRX during the cell cycle, HeLa cells were synchronized at G2–M by treatment with nocodazole, and protein extracts were collected at different time points following release from the block for immunoblot analysis. Phosphorylation of ATRX was most evident at pro-metaphase (0 h) (Fig. 6C, mitotic lane), whereas the appearance of the faster migrating form, corresponding to hypophosphorylated ATRX, occurred between 2 and 5 h after release from the block (Fig. 6C, lanes 2, 3 and 5). Dephosphorylation of ATRX seems to coincide with exit of the cells from M phase. The cells were also followed by immunofluorescence at each time point, and cytokinesis was indeed observed ~5 h following release from the block, at which point ATRX had resumed a punctate nuclear pattern (data not shown). These results are in agreement with previous studies demonstrating that HeLa cells exit the mitotic phase 3–5 h after release from a nocodazole-induced G2–M block (36). A reduction in the electrophoretic mobility is again observed 24 h following release, probably due to the subsequent phosphorylation of ATRX in cells that have once again reached the onset of mitosis. We routinely observe maximal ATRX protein levels at mitosis, possibly as a consequence of increased solubility of ATRX at that time point under the extraction conditions used (see below).
Hypophosphorylated ATRX is associated with the nuclear matrix
The cell cycle progression studies demonstrated that the hypophosphorylated form of ATRX is predominant in interphase cells and suggested that hyperphosphorylated ATRX was extracted more readily. Moreover, the biochemical and in situ fractionation studies showed that ATRX is not easily extracted at interphase due to its association with the nuclear matrix. To confirm that the hypophosphorylated form of ATRX associates with the nuclear matrix, we examined the solubility and phosphorylation state of the ATRX protein following NP-40 extraction. HeLa cells were blocked at pro-metaphase with nocodazole, extracted with the detergent NP-40, then analysed for ATRX protein. HeLa cells entering mitosis were distinguished from interphase cells by strong DAPI staining of the condensing chromosomes. We observed the nuclear speckled pattern of ATRX in interphase cells, suggesting poor solubilization of ATRX at this stage of the cell cycle (Fig. 7). However, this signal was greatly reduced or absent in cells that had entered mitosis, demonstrating the increased solubility of ATRX. To confirm that this increased ability to extract ATRX is the result of phosphorylation, we examined the electrophoretic mobility of the NP-40-soluble (supernatant) and insoluble (pellet) fractions. As expected, we observed reduced mobility of ATRX in the soluble fraction but not in the pellet (Fig. 6A, lanes P and S), suggesting that phosphorylation of ATRX is necessary for release of the protein from the nuclear matrix and that it may facilitate progression to mitosis.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
To shed light on the possible functions of the ATRX protein, we have examined its subcellular localization and phosphorylation status in human cells. We found that it is localized in distinct speckles during interphase. At the onset of M phase, this speckled pattern was no longer observed and ATRX was detected on condensed chromatin and, to a lesser extent, in the surrounding cytoplasmic region. The pattern of ATRX localization to chromatin at mitosis appears in distinct regions at metaphase but changes to a more generalized association at telophase.
The behaviour of ATRX during mitosis is similar to that of HP1, which binds centromeric DNA during metaphase and anaphase but covers whole chromosomes at telophase (37). HP1 was first identified as a non-histone chromosomal protein that binds pericentric heterochromatin and mediates position effect variegation in Drosophila. The mouse and human homologues mHP1/hHP1, M31/HP1ß and M32/HP1
bind either heterochromatin or euchromatin, with M31/HP1ß interacting mainly with pericentric heterochromatin (37–39). Previous studies have identified an association of murine ATRX with HP1 in a yeast two-hybrid assay (17). We have confirmed this interaction in mitotic HeLa cell extracts by co-immunoprecipitation experiments. These results are consistent with the finding that ATRX associates with pericentric heterochromatin of mouse and human metaphase chromosomes, as well as the p arms of human acrocentric chromosomes (32). This study also demonstrated that ATRX co-localized with M31 in murine cells, although the correlation is not as clear in human cells. It is conceivable that the distinct binding patterns that we observe for ATRX in mitotic and interphasic cells could result from specific interactions with separate HP1 isoforms.
Several transcription factors, such as YY1 (40,41), Rb (42), PML (43), AML/CBF (44), Pit1 (45), histone acetyltransferases and deacetylases (46–49) and hbrm/BRG-1 (50), are bound to the nuclear matrix and are believed to link transcriptionally active chromatin to the nuclear matrix. The nuclear matrix, a protein–RNA structure that persists after nuclease treatment and high salt extraction, is comprised of the lamin–pore complex and an internal network of poorly characterized ribonucleoprotein filaments. Its importance in the maintenance of nuclear architecture, DNA replication, RNA processing and steroid hormone action has been well documented (51–54). Genes that are actively transcribed are enriched in nuclear matrix preparations (55), suggesting that the nuclear matrix may support the formation of nuclear domains for the regulation of gene expression. We have demonstrated that ATRX associates with the core nuclear matrix. When extracted in situ from interphase HeLa cells, most of the ATRX speckles were still visible even after DNase I and high salt treatment. The importance of nuclear matrix association to achieve normal function is demonstrated by studies of tumour cells with mutated forms of Rb that could no longer associate with the nuclear matrix, suggesting that this interaction is crucial for its tumour suppressor function (56). Attachment to the nuclear matrix was also shown to be necessary for maximal activity of the AML transcription factor (44,57). Further studies on the effect of different ATRX mutations on the association of the ATRX protein with the nuclear matrix will yield valuable information on the importance of this interaction for proper function.
We examined the phosphorylation status of ATRX during the cell cycle and found a correlation with nuclear matrix attachment as well as speckle formation. ATRX is hypophosphorylated at interphase when association with the nuclear matrix is observed. Phosphorylation of ATRX at the onset of mitosis correlates with its association with condensed chromatin and its disassociation from the nuclear matrix, as demonstrated by the increased solubility of the protein in the presence of NP-40 at mitosis. These results suggest that the association of ATRX with the nuclear matrix is dynamic and could be regulated by phosphorylation. There is a precedence for the phosphorylation-dependent release of transcription factors from the nuclear matrix. Phosphorylation of Rb at the G1–S boundary has been shown to decrease its affinity for the nuclear matrix (56). Moreover, phosphorylation can influence chromatin remodelling activity. For example, phosphatase treatment of the inactive mitotic BRG1 complex results in reactivation of its chromatin remodelling activity (36). The cell cycle changes in ATRX phosphorylation are similar to those found for the human SWI/SNF proteins, although phosphorylation of hBrm and BRG1 at the onset of mitosis results in their exclusion from rather than association with condensed chromatin (35). The phosphorylation pattern of ATRX is also comparable to that of HP1 and HP1, which become hyper- phosphorylated in HeLa cells during M phase (58). Importantly, casein kinase II-directed phosphorylation of Drosophila HP1 [Su(var)2-5] has been proposed to promote an interaction with target proteins in heterochromatin, consistent with the possibility that phosphorylation of HP1 could facilitate its interaction with ATRX (59).
There is mounting evidence that the nuclear matrix plays a crucial role in the maintenance of chromosome territory organization (60–64). As a nuclear matrix-associated protein, ATRX may be involved in the compartmentalization of higher order chromatin and could affect gene expression of chromosomal domains through positional effects. The phosphorylation and re-localization of ATRX to condensed chromatin and centromeres at mitosis may reflect a change in its function at that stage of the cell cycle. The association of ATRX with HP1 may provide clues as to its function during mitosis. Human HP1 associates with the inner centromere protein (INCENP), and this interaction is necessary for proper targeting of INCENP to the mitotic spindle. Dominant-negative forms of INCENP remain bound to centromeres throughout the cell cycle, causing a failure of cells to complete cytokinesis (65). Similarly, the yeast homologues of HP1 (Swi6, clr4) are thought to be involved in kinetochore assembly, and mutation analyses link them to chromosomal segregation (66,67). The interaction of ATRX with HP1 thus suggests a role for ATRX in chromosomal inheritance and cytokinesis.
Taken together, our results are consistent with the possibility that ATRX plays more than one role, depending on the stage of the cell cycle and its phosphorylation status. Since ATRX can bind strongly to both chromatin and the nuclear matrix, it may function as a linker molecule in the establishment of specific nuclear domains at interphase to influence such processes as transcription, replication and splicing. Furthermore, in the absence of the nuclear matrix at mitosis, it may act at the centromere in tandem with HP1 and other partners to regulate chromosome segregation and proper mitotic division. The various ATRX mutations found in patients with XLMR and -thalassaemia may affect its normal binding to chromatin or to the nuclear matrix. The PHD finger is a common site of mutations in the ATRX gene product and, interestingly, the PHD domains of the AIRE gene product recently were shown to be important for correct subcellular distribution (68,69). As a consequence of aberrant localization, ATRX turnover may be increased, thereby reducing its availability for complex formation and normal function. Consistent with this possibility is the demonstration that ATRX protein levels are reduced in eight lymphoblast cell lines from patients as compared with TAFII250, a similarly sized protein (32). However, elucidation of the deleterious effects of ATRX mutations on localization or biochemical properties such as ATPase/helicase activity awaits purification of the ATRX protein.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Cell culture, synchronization and protein extraction
HeLa (human cervical carcinoma), 293 (human embryonic kidney) and Cos-1 (monkey) cells were maintained at 37°C in 5% CO2 in monolayer culture in Eagle minimum essential medium (HeLa and 293) or Dulbecco’s modified Eagle’s medium (Cos-1) (Gibco BRL, Burlington, Ontario) supplemented with 10% fetal bovine serum (CanSera, Rexdale, Ontario). For cell cycle synchronization, cells were split 1:3 one day prior to adding 0.1 µg/ml nocodazole or 100 µg/ml colchicine for 12 h to synchronize cells in pro-metaphase. Mitotic cells were separated from interphasic cells by shake-off and replated in six 100 mm dishes. For each time point, protein was extracted with RIPA buffer [150 mM NaCl, 1% NP-40, 50 mM Tris pH 8.0, 0.5% deoxycholic acid, 0.1% SDS, protease inhibitors (CompleteMini, EDTA-free; Boehringer Mannheim, Laval, Quebec), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 500 mM NaF, 30 mM Na3VO4] for 15 min at 4°C. Extracts were passed several times through a 25 gauge needle followed by DNase I treatment (170 U/ml) for 15 min at 37°C. The extracts were centrifuged for 10 min and the supernatant frozen at –80°C in aliquots. To collect interphasic cells, the cells still attached to the culture dishes were trypsinized and extracts obtained as described above.
Nuclear matrix preparation
Nuclear matrix proteins were fractionated from HeLa cells according to the method of He et al. (33). Cultured cells were washed twice in phosphate-buffered saline and sequentially treated with cytoskeletal (CSK) buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EDTA, protease inhibitors, 2 mM PMSF and 0.5% Triton X-100) for 10 min at 4°C; 100 µg/ml DNase I (Pharmacia Biotech, Baie d’Urfe, Quebec) in CSK for 15 min at 37°C; ammonium sulfate (0.25 M) for 5 min at room temperature, followed by a high salt wash (2 M NaCl in CSK buffer) for 5 min at 4°C. The final pellet (core nuclear matrix) was resuspended in 8 M urea, aliquotted and frozen at –80°C. To prepare for immunolabelling, cells grown on coverslips were extracted with the same method as described above and fixed in EtOH:MeOH (3:1) or MeOH:acetone (1:1).
Antibodies and immunoblot analysis
PCR primers XNP98 (5'-CTTGGTCGAAAGGAGTTGTCCAC-3') and Nhe/Stop (5'-GAGGATTGCTAGCATTTAATCAG-3') were designed to amplify a 1.5 kb fragment encompassing the unique restriction sites SpeI and NheI and engineering a stop codon following the NheI site. The PCR product was cloned into PCR-Script and completely sequenced before excising an SpeI–HindIII fragment for subcloning into pMAL-C2 digested with XbaI and HindIII. Clones containing the correct insert were analysed for the production of the expected fusion protein of 89 kDa after induction with 0.3 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). Fxnp5-8 was chosen for large-scale affinity purification of the fusion protein using amylose resin and elution with 10 mM maltose. The purified fusion protein was used to raise sheep polyclonal antibodies (fxnp5) and for affinity purification after coupling 3 mg to POROS-AL beads (Affinity Biologicals, Hamilton, Ontario). Finally, in an effort to remove maltose-binding protein (MBP)-specific antibodies, the affinity-purified IgG was adsorbed a total of five times to a non-specific MBP fusion protein. For both immunofluorescence and immunoblot analysis, the antibody was used at a concentration of 3 µg/ml. The mouse monoclonal antibody 39f was raised against a GST fusion protein encompassing ATRX amino acids 85–319. It was kindly provided by Dr Douglas Higgs (Institute of Medicine, Oxford, UK), and the full description of this antibody is published elsewhere (32). Other antibodies used were mouse anti-lamin A/C 131C3 antibody (a gift from Dr Y. Raymond, CHUM, Montreal, Quebec), mouse anti-p38 antibody and rabbit anti-phosphoserine (a gift from Dr L. Megeney, OHRI, University of Ottawa), mouse anti-histone H1 antibody (a gift from Dr M. McBurney, University of Ottawa), mouse anti-phosphotyrosine PY20 antibody (Transduction Laboratories, Lexington, KY) and mouse anti-HP1 (a gift from Dr W.C. Earnshaw, University of Edinburgh, UK). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-sheep antibodies (Sigma, St Louis, MO) were used for immunoblot analysis. Fluorescein-conjugated anti-sheep antibody (Sigma) and anti-mouse Alexa 594 antibody (Molecular Probes, Eugene, OR) were used for immunofluorescence analysis. For western blot analysis, protein extracts were electrophoresed by SDS–PAGE and transferred to nylon membrane (Immobilon-P; Millipore, Mississauga, Ontario) by semi-dry transfer using CAPs buffer pH 11.0. The membrane was blocked for 1 h in 5% milk in TBST (Tris-buffered saline with 0.05% Tween-20) and incubated with primary and HRP-conjugated secondary antibodies for 1 h at room temperature. Enhanced chemiluminescence reagents were used for detection. For the pre-adsorption of the ATRX polyclonal antibody, 4 µg of antibody was incubated overnight at 4°C with 3.6 µg of the fxnp5 fusion protein.
Immunofluorescence
After fixation, cells on coverslips were blocked for 10 min in 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and incubated for 1 h with the appropriate antibodies in 2% BSA in PBS (fxnp5, 1:100; anti-lamin A/C, 1:2000). Fluorescein-conjugated anti-sheep (Sigma) or Alexa 594-conjugated anti-mouse (Molecular Probes) antibodies were used for detection, and the cellular DNA was labelled with either 0.1 µg/ml DAPI for 5 min at room temperature or 0.2 µg/ml propidium iodide and 20 µg/ml RNase A for 10 min at 37°C. Coverslips were mounted with antifade (p-phenyldiamine; Sigma) and visualized with a Zeiss Axiophot photomicroscope.
NP-40 extraction
HeLa cells were incubated overnight in the presence of 0.1 µg/ml nocodazole. After washing three times with PBS, the cells were incubated in nuclear buffer [15 mM NaCl, 60 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 mM Tris pH 7.4, 0.5 mM dithiothreitol (DTT), 300 mM sucrose] with 0.3% NP-40 for 3 min at room temperature and centrifuged. The supernatant was kept as the NP-40-extractable fraction. The pellet was resuspended in solution C (20 mM HEPES, 10% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1 mM DTT) and sonicated.
Immunoprecipitation and protein phosphatase treatment
ATRX was immunoprecipitated from mitotic cell extracts with 5 µg of either fxnp5 antibody or control sheep IgG and protein G–Sepharose (Pharmacia Biotech) overnight at 4°C. The immunocomplex was washed five times with RIPA buffer. Half of the immunoprecipitated fraction was used for phosphatase treatment with 400 U of protein phosphatase (New England Biolabs, Mississauga, Ontario) at 30°C for 30 min before SDS–PAGE analysis. In other experiments, all of the washed immunocomplex was analysed by SDS–PAGE analysis.
|
ACKNOWLEDGEMENTS |
|---|
We thank Dr Valerie Wallace and Dr Lynn Megeney for review of the manuscript and helpful discussions. We also thank Dr Yves Raymond for the anti-lamin A/C antibody, Dr Lynn Megeney for the anti-p38 and anti-phosphoserine antibodies, Dr Michael McBurney for the anti-histone H1 antibody and Dr W.C. Earnshaw for the human anti-HP1
antibody. The production of the fxnp5 fusion protein and the fxnp5 antibody was performed by D.J.P. while still in the laboratory of Dr D.R. Higgs. This work was supported by grant MT-14112 from MRC Canada. D.J.P. is an MRC scholar.
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +1 613 737 8989; Fax: +1 613 737 8803; Email: dpicketts@ogh.on.ca
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Gibbons, R.J., Brueton, L., Buckle, V.J., Burn, J., Clayton-Smith, J., Davison, B.B.C., Gardner, R.J.M., Homfray, T., Kearney, L., Kingston, H.M. et al. (1995) Clinical and hematologic aspects of the X-linked
-thalassemia/mental retardation syndrome (ATR-X). Am. J. Med. Genet., 55, 288–299.[Web of Science][Medline]
2 Gibbons, R.J., Picketts, D.J., Villard, L. and Higgs, D.R. (1995) Mutations in a putative global transcriptional regulator cause X-linked mental retardation with -thalassemia (ATR-X syndrome). Cell, 80, 837–845.[Web of Science][Medline]
3 Gibbons, R.J., Suthers, G.K., Wilkie, A.O., Buckle, V.J. and Higgs, D.R. (1992) X-linked -thalassemia/mental retardation (ATR-X) syndrome: localization to Xq12–q21.31 by X inactivation and linkage analysis. Am. J. Hum. Genet., 51, 1136–1149.[Web of Science][Medline]
4 Ion, A., Telvi, L., Chaussain, J.L., Galacteros, F., Valayer, J., Fellous, M. and McElreavey, F. (1996) A novel mutation in the putative DNA helicase XH2 is responsible for male-to-female sex reversal associated with an atypical form of the ATR-X syndrome. Am. J. Hum. Genet., 58, 1185–1191.
5 Picketts, D.J., Higgs, D.R., Bachoo, S., Blake, D.J., Quarrell, O.W. and Gibbons, R.J. (1996) ATRX encodes a novel member of the SNF2 family of proteins: mutations point to a common mechanism underlying the ATR-X syndrome. Hum. Mol. Genet., 5, 899–907.
6 Villard, L., Lacombe, D. and Fontes, M. (1996) A point mutation in the XNP gene, associated with an ATR-X phenotype without -thalassemia. Eur. J. Hum. Genet., 4, 316–320.[Web of Science][Medline]
7 Villard, L., Toutain, A., Lossi, A.M., Gecz, J., Houdayer, C., Moraine, C. and Fontes, M. (1996) Splicing mutation in the ATR-X gene can lead to a dysmorphic mental retardation phenotype without -thalassemia. Am. J. Hum. Genet., 58, 499–505.[Web of Science][Medline]
8 Abidi, F., Schwartz, C.E., Carpenter, N.J., Villard, L., Fontes, M. and Curtis, M. (1999) Carpenter–Waziri syndrome results from a mutation in XNP. Am. J. Med. Genet., 85, 249–251.[Web of Science][Medline]
9 Lossi, A.M., Millan, J.M., Villard, L., Orellana, C., Cardoso, C., Prieto, F., Fontes, M. and Martinez, F. (1999) Mutation of the XNP/ATR-X gene in a family with severe mental retardation, spastic paraplegia and skewed pattern of X inactivation: demonstration that the mutation is involved in the inactivation bias. Am. J. Hum. Genet., 65, 558–562.[Web of Science][Medline]
10 Villard, L., Gecz, J., Mattei, J.F., Fontes, M., Saugier-Veber, P., Munnich, A. and Lyonnet. S. (1996) XNP mutation in a large family with Juberg–Marsidi syndrome. Nature Genet., 12, 359–360.[Web of Science][Medline]
11 Villard, L., Bonino, M.C., Abidi, F., Ragusa, A., Belougne, J., Lossi, A.M., Seaver, L., Bonnefont, J.P., Romano, C., Fichera, M. et al. (1999) Evaluation of a mutation screening strategy for sporadic cases of ATR-X syndrome. J. Med. Genet., 36, 183–186.
12 Carlson, M. and Laurent, B.C. (1994) The SNF/SWI family of global transcriptional activators. Curr. Opin. Cell Biol., 6, 396–402.[Web of Science][Medline]
13 Eisen, J.A., Sweder, K.S. and Hanawalt, P.C. (1995) Evolution of the SNF2 family of proteins: subfamilies with distinct sequences and functions. Nucleic Acids Res., 23, 2715–2723.
14 Xie, S., Wang, Z., Okano, M., Nogami, M., Li, Y., He, W.W., Okumura, K. and Li, E. (1999) Cloning, expression and chromosome locations of the human DNMT3 gene family. Gene, 236, 87–95.[Web of Science][Medline]
15 Picketts, D.J., Tastan, A.O., Higgs, D.R. and Gibbons, R.J. (1998) Comparison of the human and murine ATRX gene identifies highly conserved, functionally important domains. Mamm. Genome, 9, 400–403.[Web of Science][Medline]
16 Cardoso, C., Timsit, S., Villard, L., Khrestchatisky, M., Fontes, M. and Colleaux, L. (1998) Specific interaction between the XNP/ATR-X gene product and the SET domain of the human EZH2 protein. Hum. Mol. Genet., 7, 679–684.
17 Le Douarin, B., Nielsen, A.L., Garnier, J.M., Ichinose, H., Jeanmougin, F., Losson, R. and Chambon, P. (1996) A possible involvement of TIF1 and TIF1ß in the epigenetic control of transcription by nuclear receptors. EMBO J., 15, 6701–6715.[Web of Science][Medline]
18 Gibbons, R.J., Bachoo, S., Picketts, D.J., Aftimos, S., Asenbauer, B., Bergoffen, J., Berry, S.A., Dahl, N., Fryer, A., Keppler, K. et al. (1997) Mutations in transcriptional regulator ATRX establish the functional significance of a PHD-like domain. Nature Genet., 17, 146–148.[Web of Science][Medline]
19 Craddock, C.F., Vyas, P., Sharpe, J.A., Ayyub, H., Wood, W.G. and Higgs, D.R. (1995) Contrasting effects of - and ß-globin regulatory elements on chromatin structure may be related to their different chromosomal environments. EMBO J., 14, 1718–1726.[Web of Science][Medline]
20 Vyas, P., Vickers, M.A., Simmons, D.L., Ayyub, H., Craddock, C.F. and Higgs, D.R. (1992) Cis-acting sequences regulating expression of the human -globin cluster lie within constitutively open chromatin. Cell, 69, 781–793.[Web of Science][Medline]
21 Cairns, B.R., Kim, Y.J., Sayre, M.H., Laurent, B.C. and Kornberg, R.D. (1994) A multisubunit complex containing the SWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 gene products isolated from yeast. Proc. Natl Acad. Sci. USA, 91, 1950–1954.
22 Cairns, B.R., Lorch, Y., Li, Y., Zhang, M., Lacomis, L., Erdjument-Bromage, H., Tempst, P., Du, J., Laurent, B.C. and Kornberg. R.D. (1996) RSC, an essential, abundant chromatin-remodeling complex. Cell, 87, 1249–1260.[Web of Science][Medline]
23 Ito, T., Levenstein, M.E., Fyodorov, D.V., Kutach, A.K., Kobayashi, R. and Kadonaga, J.T. (1999) ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly. Genes Dev., 13, 1529–1539.
24 Peterson, C.L., Dingwall, A. and Scott, M.P. (1994) Five SWI/SNF gene products are components of a large multisubunit complex required for transcriptional enhancement. Proc. Natl Acad. Sci. USA, 91, 2905–2908.
25 Tsukiyama, T. and Wu, C. (1995) Purification and properties of an ATP-dependent nucleosome remodeling factor. Cell, 83, 1011–1020.[Web of Science][Medline]
26 Varga-Weisz, P.D., Wilm, M., Bonte, E., Dumas, K., Mann, M. and Becker, P.B. (1997) Chromatin-remodelling factor CHRAC contains the ATPases ISWI and topoisomerase II. Nature, 388, 598–602.[Medline]
27 Xue, Y., Wong, J., Moreno, G.T., Young, M.K., Cote, J. and Wang, W. (1998) NURD, a novel complex with both ATP-dependent chromatin-remodeling and histone deacetylase activities. Mol. Cell, 2, 851–861.[Web of Science][Medline]
28 Zhang, Y., LeRoy, G., Seelig, H.P., Lane, W.S. and Reinberg, D. (1998) The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities. Cell, 95, 279–289.[Web of Science][Medline]
29 Cote, J., Quinn, J., Workman, J.L. and Peterson, C.L. (1994) Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science, 265, 53–60.
30 Hirschhorn, J.N., Brown, S.A., Clark, C.D. and Winston, F. (1992) Evidence that SNF2/SWI2 and SNF5 activate transcription in yeast by altering chromatin structure. Genes Dev., 6, 2288–2298.
31 Ito, T., Bulger, M., Pazin, M.J., Kobayashi, R. and Kadonaga, J.T. (1997) ACF, an ISWI-containing and ATP-utilizing chromatin assembly and remodeling factor. Cell, 90, 145–155.[Web of Science][Medline]
32 McDowell, T.L., Gibbons, R.J., Sutherland, H., O’Rourke, D.M., Bickmore, W.A., Pombo, A., Turley, H., Gatter, K., Picketts, D.J., Buckle, V.J. et al. (1999) Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes. Proc. Natl Acad. Sci. USA, 96, 13983–13988.
33 He, D., Jeffrey, A., Nickerson, J. and Penman, S. (1990) Core filaments of the nuclear matrix. J. Cell Biol., 110, 569–580.
34 Han, J., Lee, J.D., Bibbs, L. and Ulevitch, R.J. (1994) A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science, 265, 808–811.
35 Muchardt, C., Reyes, J.-C., Bourachot, B., Legouy, E. and Yaniv, M. (1996) The hbrm and BRM-1 proteins, components of the human SNF–SWI complex, are phosphorylated and excluded from the condensed chromosomes during mitosis. EMBO J., 15, 3394–3402.[Web of Science][Medline]
36 Sif, S., Stukenberg, P.T., Kirschner, M.W. and Kingston, R.E. (1998) Mitotic inactivation of a human SWI/SNF chromatin remodeling complex. Genes Dev., 12, 2842–2851.
37 Furuta, K., Chan, E.K.L., Kiyosawa, K., Reimer, G., Luderschmidt, C. and Tan, E.M. (1997) Heterochromatin protein HP1Hsß (p25ß) and its localization with centromeres in mitosis. Chromosoma, 106, 11–19.[Web of Science][Medline]
38 Nicol, L. and Jeppesen, P. (1994) Human autoimmune sera recognize a conserved 26kD protein associated with mammalian heterochromatin that is homologous to heterochromatin protein 1 of Drosophila. Chromosome Res., 2, 245–253.[Medline]
39 Horsley, D., Hutchings, A., Butcher, G.W. and Singh, P.B. (1996) M32, a murine homologue of Drosophila heterochromatin protein 1 (HP1), localizes to euchromatin within interphase nuclei and is largely excluded from constitutive heterochromatin. Cytogenet. Cell Genet., 73, 308–311.[Web of Science][Medline]
40 Bushmeyer, S.M. and Atchison, M.L. (1998) Identification of YY1 sequences necessary for association with the nuclear matrix and for transcriptional repression functions. J. Cell. Biochem., 68, 484–499.[Web of Science][Medline]
41 McNeil, S., Guo, B., Stein, J.L., Lian, J.B., Bushmeyer, S., Seto, E., Atchison, M.L., Penman, S., van Wijnen, A.J. and Stein, G.S. (1998) Targeting of the YY1 transcription factor to the nucleolus and the nuclear matrix in situ: the C-terminus is a principal determinant for nuclear trafficking. J. Cell. Biochem., 68, 500–510.[Web of Science][Medline]
42 Mancini, M.A., Shan, B., Nickerson, J.A., Penman, S. and Lee, W.-H. (1994) The retinoblastoma gene product is a cell cycle-dependent nuclear matrix-associated protein. Proc. Natl Acad. Sci. USA, 91, 418–422.
43 Chang, K.-S., Fan, Y.-H., Andreeff, M., Liu, J. and Mu, Z.-M. (1995) The PML gene encodes a phosphoprotein associated with the nuclear matrix. Blood, 85, 3646–3653.
44 Zeng, C., McNeil, S., Pockwinse, S., Nickerson, J., Shopland, L., Lawrence, J.B., Penman, S., Hiebert, S., Lian, J.B., van Wijnen, A.J. et al. (1998) Intranuclear targeting of AML/CBF regulatory factors to nuclear matrix-associated transcriptional domains. Proc. Natl Acad. Sci. USA, 95, 1585–1589.
45 Mancini, M.G., Liu, B., Sharp, D. and Mancini, M.A. (1999) Subnuclear partitioning and functional regulation of the Pit-1 transcription factor. J. Cell. Biochem., 72, 322–338.[Web of Science][Medline]
46 Davie, J.F. (1997) Nuclear matrix, dynamic histone acetylation and transcriptionally active chromatin. Mol. Biol. Rep., 24, 197–207.[Web of Science][Medline]
47 Davie, J.R. (1995) The nuclear matrix and the regulation of chromatin organization and function. Int. Rev. Cytol., 162A, 191–250.
48 Hendzel, M.J., Delcuve, J.P. and Davie, J.R. (1991) Histone deacetylase is a component of the internal nuclear matrix. J. Biol. Chem., 266, 21936–21942.
49 Hendzel, M.J., Sun, J.M., Chen, H.Y., Rattner, J.B. and Davie, J.R. (1994) Histone acetyltransferase is associated with the nuclear matrix. J. Biol. Chem., 269, 22894–22901.
50 Reyes, J.C., Muchardt, C. and Yaniv, M. (1997) Components of the human SWI/SNF complex are enriched in active chromatin and are associated with the nuclear matrix. J. Cell Biol., 137, 263–274.
51 Berezney, R. and Coffey, D.S. (1975) Nuclear protein matrix: association with newly synthesized DNA. Science, 189, 291–293.
52 Barrack, E.R. (1987) Steroid hormone receptor localization in the nuclear matrix: interaction with acceptor sites. J. Steroid Biochem., 27, 115–121.[Web of Science][Medline]
53 Stein, G.S., van Wijnen, A.J., Stein, J., Lian, J.B. and Montecino, M. (1995) Contributions of nuclear architecture to transcriptional control. Int. Rev. Cytol., 162A, 251–278.
54 Fey, E.G., Krochmalnic, G. and Penman, S. (1986) The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy. J. Cell Biol., 102, 1654–1665.
55 Ciejek, E.M., Tsai, M.-J. and O’Malley, B.W. (1983) Actively transcribed genes are associated with the nuclear matrix. Nature, 306, 607–609.[Medline]
56 Mittnacht, S. and Weinberg, R.A. (1991) G1/S phosphorylation of the retinoblastoma protein is associated with an altered affinity for the nuclear compartment. Cell, 65, 381–383.[Web of Science][Medline]
57 Zeng, C., van Wijnen, A.J., Stein, J.L., Meyers, S., Sun, W., Shopland, L., Lawrence, J.B., Penman, S., Lian, J.B., Stein, G.S. and Hiebert, S.W. (1997) Identification of a nuclear matrix targeting signal in the leukemia and bone-related AML/CBF- transcription factors. Proc. Natl Acad. Sci. USA, 94, 6746–6751.
58 Minc, E., Allory, Y., Worman, H.J., Courvalin, J.C. and Buendia, B. (1999) Localization and phosphorylation of HP1 proteins during the cell cycle in mammalian cells. Chromosoma, 108, 220–234.[Web of Science][Medline]
59 Zhao, T. and Eissenberg, J.C. (1999) Phosphorylation of heterochromatin protein 1 by casein kinase II is required for efficient heterochromatin binding in Drosophila. J. Biol. Chem., 274, 15095–15100.
60 Berezney, R., Mortillaro, M., Ma, H., Meng, C., Samarabandu, J., Wei, X., Somanathan, S., Liou, W.S., Pan, S.J. and Cheng, P.C. (1996) Connecting nuclear architecture and genomic function. Cell. Biochem., 62, 223–226.
61 Nakayasu, H. and Berezney, R. (1989) Mapping replicational sites in the eucaryotic cell nucleus. Cell Biol., 108, 1–11.
62 Smith, H.C., Ochs, R.L., Fernandez, E.A. and Spector, D.L. (1986) Macromolecular domains U-snRNP protein p28: further evidence for an in situ nuclear matrix. Mol. Cell. Biochem., 70, 151–168.[Web of Science][Medline]
63 Jackson, D.A., Hassan, A.B., Errington, R.J. and Cook, P.R. (1993) Visualization of focal sites of transcription within human nuclei. EMBO J., 12, 1059–1065.[Web of Science][Medline]
64 Ma, H., Siegel, A.J. and Berezney, R. (1999) Association of chromosome territories with the nuclear matrix: disruption of human chromosome territories correlates with the release of a subset of nuclear matrix proteins. J. Cell Biol., 146, 531–542.
65 Ainsztein, A.M., Kandels-Lewis, S., Mackay, A.M. and Earnshaw, W.C. (1998) INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1. J. Cell Biol., 143, 1763–1774.
66 Allshire, R.C., Nimmo, E.R., Ekwall, K., Javerzat, J.P. and Cranstonm, G. (1995) Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation. Genes Dev., 9, 218–233.
67 Ekwall, K., Javerzat, J.P., Lorentz, A., Schmidt, H., Cranston, G. and Allshire, R. (1995) The chromodomain protein Swi6: a key component at fission yeast centromeres. Science, 269, 1429–1431.
68 Björses, P., Markku, P.-H., Jaakko, K., Aaltonen, J., Peltonen, L. and Ulmanen, I. (1999) Localization of the APECED protein in distinct nuclear structures. Hum. Mol. Genet., 8, 259–266.
69 Rinderle, C., Christensen, H.-M., Schweiger, S., Lehrach, H. and Yaspo, M.-L. (1999) AIRE encodes a nuclear protein co-localizing with cytoskeletal filaments: altered sub-cellular distribution of mutants lacking the PHD zinc fingers. Hum. Mol. Genet., 8, 277–290.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. F. Medina, C. Mazerolle, Y. Wang, N. G. Berube, S. Coupland, R. J. Gibbons, V. A. Wallace, and D. J. Picketts Altered visual function and interneuron survival in Atrx knockout mice: inference for the human syndrome Hum. Mol. Genet., March 1, 2009; 18(5): 966 - 977. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Seah, M. A. Levy, Y. Jiang, S. Mokhtarzada, D. R. Higgs, R. J. Gibbons, and N. G. Berube Neuronal Death Resulting from Targeted Disruption of the Snf2 Protein ATRX Is Mediated by p53 J. Neurosci., November 19, 2008; 28(47): 12570 - 12580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ritchie, C. Seah, J. Moulin, C. Isaac, F. Dick, and N. G. Berube Loss of ATRX leads to chromosome cohesion and congression defects J. Cell Biol., January 28, 2008; 180(2): 315 - 324. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Nan, J. Hou, A. Maclean, J. Nasir, M. J. Lafuente, X. Shu, S. Kriaucionis, and A. Bird Interaction between chromatin proteins MECP2 and ATRX is disrupted by mutations that cause inherited mental retardation PNAS, February 20, 2007; 104(8): 2709 - 2714. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Luciani, D. Depetris, Y. Usson, C. Metzler-Guillemain, C. Mignon-Ravix, M. J. Mitchell, A. Megarbane, P. Sarda, H. Sirma, A. Moncla, et al. PML nuclear bodies are highly organised DNA-protein structures with a function in heterochromatin remodelling at the G2 phase J. Cell Sci., June 15, 2006; 119(12): 2518 - 2531. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Samaniego, S. Y. Jeong, C. de la Torre, I. Meier, and S. M. Diaz de la Espina CK2 phosphorylation weakens 90 kDa MFP1 association to the nuclear matrix in Allium cepa J. Exp. Bot., January 1, 2006; 57(1): 113 - 124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hristova, D. Birse, Y. Hong, and V. Ambros The Caenorhabditis elegans Heterochronic Regulator LIN-14 Is a Novel Transcription Factor That Controls the Developmental Timing of Transcription from the Insulin/Insulin-Like Growth Factor Gene ins-33 by Direct DNA Binding Mol. Cell. Biol., December 15, 2005; 25(24): 11059 - 11072. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D Vallee, E Chevrier, G E Graham, M A Lazzaro, P A Lavigne, A G Hunter, and D J Picketts A novel PHF6 mutation results in enhanced exon skipping and mild Borjeson-Forssman-Lehmann syndrome J. Med. Genet., October 1, 2004; 41(10): 778 - 783. [Full Text] [PDF]
|
|
|
|
|
|
A. M. Ishov, O. V. Vladimirova, and G. G. Maul Heterochromatin and ND10 are cell-cycle regulated and phosphorylation-dependent alternate nuclear sites of the transcription repressor Daxx and SWI/SNF protein ATRX J. Cell Sci., August 1, 2004; 117(17): 3807 - 3820. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Tang, S. Wu, H. Liu, R. Stratt, O. G. Barak, R. Shiekhattar, D. J. Picketts, and X. Yang A Novel Transcription Regulatory Complex Containing Death Domain-associated Protein and the ATR-X Syndrome Protein J. Biol. Chem., May 7, 2004; 279(19): 20369 - 20377. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. P. Steensma, D. R. Higgs, C. A. Fisher, and R. J. Gibbons Acquired somatic ATRX mutations in myelodysplastic syndrome associated with {alpha} thalassemia (ATMDS) convey a more severe hematologic phenotype than germline ATRX mutations Blood, March 15, 2004; 103(6): 2019 - 2026. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Narang, R. Dumas, L. M. Ayer, and R. A. Gravel Reduced histone biotinylation in multiple carboxylase deficiency patients: a nuclear role for holocarboxylase synthetase Hum. Mol. Genet., January 1, 2004; 13(1): 15 - 23. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. R. Higgs Ham-Wasserman Lecture: Gene Regulation in Hematopoiesis: New Lessons from Thalassemia Hematology, January 1, 2004; 2004(1): 1 - 13. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Zaidi, D. W. Young, S. M. Pockwinse, A. Javed, J. B. Lian, J. L. Stein, A. J. van Wijnen, and G. S. Stein Mitotic partitioning and selective reorganization of tissue-specific transcription factors in progeny cells PNAS, December 9, 2003; 100(25): 14852 - 14857. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Yan, E. Cho, S. Lockett, and K. Muegge Association of Lsh, a Regulator of DNA Methylation, with Pericentromeric Heterochromatin Is Dependent on Intact Heterochromatin Mol. Cell. Biol., December 1, 2003; 23(23): 8416 - 8428. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Xue, R. Gibbons, Z. Yan, D. Yang, T. L. McDowell, S. Sechi, J. Qin, S. Zhou, D. Higgs, and W. Wang The ATRX syndrome protein forms a chromatin-remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies PNAS, September 16, 2003; 100(19): 10635 - 10640. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Christiansen, T. Stevnsner, C. Modin, P. M. Martensen, R. M. Brosh Jr, and V. A. Bohr Functional consequences of mutations in the conserved SF2 motifs and post-translational phosphorylation of the CSB protein Nucleic Acids Res., February 1, 2003; 31(3): 963 - 973. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H. Lee and D. G. Skalnik CpG-binding Protein Is a Nuclear Matrix- and Euchromatin-associated Protein Localized to Nuclear Speckles Containing Human Trithorax. IDENTIFICATION OF NUCLEAR MATRIX TARGETING SIGNALS J. Biol. Chem., October 25, 2002; 277(44): 42259 - 42267. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Dantzer, L. Luna, M. Bjoras, and E. Seeberg Human OGG1 undergoes serine phosphorylation and associates with the nuclear matrix and mitotic chromatin in vivo Nucleic Acids Res., June 1, 2002; 30(11): 2349 - 2357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. G. Berube, M. Jagla, C. Smeenk, Y. De Repentigny, R. Kothary, and D. J. Picketts Neurodevelopmental defects resulting from ATRX overexpression in transgenic mice Hum. Mol. Genet., February 1, 2002; 11(3): 253 - 261. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Cardoso, Y. Lutz, C. Mignon, E. Compe, D. Depetris, M.-G. Mattei, M. Fontes, and L. Colleaux ATR-X mutations cause impaired nuclear location and altered DNA binding properties of the XNP/ATR-X protein J. Med. Genet., October 1, 2000; 37(10): 746 - 751. [Abstract] [Full Text]
|
|
|
|
|
|
W. M. W. Cheung, A. H. Chu, P. W. K. Chu, and N. Y. Ip Cloning and Expression of a Novel Nuclear Matrix-associated Protein That Is Regulated during the Retinoic Acid-induced Neuronal Differentiation J. Biol. Chem., May 11, 2001; 276(20): 17083 - 17091. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||