Chromatin Remodeling Protein ATRX Research Articles

Nuclear Distribution of the Chromatin-Remodeling Protein ATRX in Mouse Early Embryos during Normal Development and Developmental Arrest In Vitro.

The chromatin-remodeling protein ATRX, which is currently recognized as one of the key genome caretakers, plays an important role in oogenesis and early embryogenesis in mammals. ATRX distribution in the nuclei of mouse embryos developing in vivo and in vitro, including when the embryos are arrested at the two-cell stage-the so-called two-cell block in vitro-was studied using immunofluorescent labeling and FISH. In normally developing two- and four-cell embryos, ATRX was found to be closely colocalized with pericentromeric DNA sequences detected with a probe to the mouse major satellite DNA. The association of ATRX with pericentromeric heterochromatin is mediated by nuclear actin and reduced after the treatment of embryos with latrunculin B. When culturing embryos in vitro, the distribution pattern of ATRX changes, leading to a decrease in the association of this protein with major satellite DNA especially under the two-cell block in vitro. Taken together, our data suggest that the intranuclear distribution of ATRX reflects the viability of mouse embryos and their probability of successful preimplantation development.

The dynamics of DAXX protein distribution in the nucleus of mouse early embryos

Nuclear distribution of Death-associated protein 6 (Daxx) was studied using fluorescent and electron microscopy in mouse embryos at different stages of development in vivo, from zygote to morula. Daxx was found in association with transcriptionally silent chromatin predominantly with a heterochromatin rim surrounding the nucleolus precursor bodies (NPBs) at all stages studied. At the zygote stage, Daxx was detected only at the periphery of NPBs both in male and female pronuclei. At the late two-cell stage, Daxx was localized not only in the heterochromatin rim at the periphery of NPBs but also in heterochromatin zones not associated with NPBs. At the morula stage, a diffuse distribution of Daxx prevailed. Scarce Daxx-positive zones were detected only in some embryos at the nucleolar periphery. Thus, Daxx is noticeably redistributed during mouse embryo cleavage, and the most conspicuous areas of Daxx concentration are observed at the end of two-cell stage. Daxx is found colocalized with the chromatin-remodeling protein ATRX exclusively in two-cell embryos, but the heterochromatin areas containing either Daxx or ATRX individually are also observed at this stage. However, most zones containing both Daxx and ATRX demonstrated a low FRET-efficiency. This suggest that two molecules are not approached sufficiently close for molecular interactions to occur. Our data suggests that Daxx may function without cooperation with ATRX at least at some stages of early mouse development.

Loss of atrx cooperates with p53-deficiency to promote the development of sarcomas and other malignancies.

The SWI/SNF-family chromatin remodeling protein ATRX is a tumor suppressor in sarcomas, gliomas and other malignancies. Its loss of function facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells, while it also affects Polycomb repressive complex 2 (PRC2) silencing of its target genes. To further define the role of inactivating ATRX mutations in carcinogenesis, we knocked out atrx in our previously reported p53/nf1-deficient zebrafish line that develops malignant peripheral nerve sheath tumors and gliomas. Complete inactivation of atrx using CRISPR/Cas9 was lethal in developing fish and resulted in an alpha-thalassemia-like phenotype including reduced alpha-globin expression. In p53/nf1-deficient zebrafish neither peripheral nerve sheath tumors nor gliomas showed accelerated onset in atrx+/- fish, but these fish developed various tumors that were not observed in their atrx+/+ siblings, including epithelioid sarcoma, angiosarcoma, undifferentiated pleomorphic sarcoma and rare types of carcinoma. These cancer types are included in the AACR Genie database of human tumors associated with mutant ATRX, indicating that our zebrafish model reliably mimics a role for ATRX-loss in the early pathogenesis of these human cancer types. RNA-seq of p53/nf1- and p53/nf1/atrx-deficient tumors revealed that down-regulation of telomerase accompanied ALT-mediated lengthening of the telomeres in atrx-mutant samples. Moreover, inactivating mutations in atrx disturbed PRC2-target gene silencing, indicating a connection between ATRX loss and PRC2 dysfunction in cancer development.

Identification of a novel gene fusion in ALT positive osteosarcoma.

The Alternative Lengthening of Telomeres (ALT) pathway stimulates telomere elongation and prevents cellular senescence in approximately 60% of osteosarcoma. While the precise mechanism underlying activation of the ALT pathway is unclear, mutations in the chromatin remodeling protein ATRX, histone chaperone DAXX, and the histone variant H3.3 correlate with ALT status. ATRX and DAXX facilitate deposition of the histone variant H3.3 within heterochromatic regions suggesting that loss of ATRX, DAXX, and/or H3.3 lead to defects in the stability of telomeric heterochromatin. Genetic mutations in ATRX, DAXX, and H3.3 have been detected in ALT positive cancers, however, a subset of ALT samples show loss of ATRX or DAXX protein expression or localization without evidence of genetic alterations suggesting additional uncharacterized defects in ATRX/DAXX/H3.3 function. Here, using Next Generation Sequencing we identified a novel gene fusion event between DAXX and the kinesin motor protein, KIFC3, leading to the translation of a chimeric DAXX-KIFC3 fusion protein. Moreover, we demonstrate that the fusion of KIFC3 to DAXX causes defects in DAXX function likely promoting ALT activity. These data highlight a potentially unrecognized mechanism of DAXX inactivation in ALT positive osteosarcoma and provide rationale for thorough and comprehensive analyses of ATRX/DAXX/H3.3 proteins in ALT positive cancers.

Localization of the chromatin-remodeling protein ATRX in the oocyte nucleus of some insects

A comparative study of nuclear distribution of the chromatin-remodeling protein ATRX in the oocytes of three species of insects in which the oocyte nucleus at the diplotene stage differs in structure, has been carried out using fluorescent and immunoelectron microscopy. In tohe oocyte nucleus of the tenebrionid beetles, Tribolium castaneum and Tenebrio molitor, ATRX preferably associates with the karyosphere (karyosome) that represents a result of concentration of the condensed chromosomes in a limited volume of the nucleus. In the oocyte nucleus of the house cricket, Acheta domesticus, in which a karyosphere does not form, the protein ATRX is distributed in the entire nuclear volume in association with the chromatin. The fact of ATRX presence in the extrachromosomal structures of the insect oocyte nucleus, the karyosphere capsule and specific nuclear bodies, is documented for the first time.

Abstract 1932: Mutations in ATRX increase genetic instability and sensitivity to PARP inhibitors in paediatric glioblastoma cells

Abstract Paediatric glioblastomas (pGBM) are amongst the most common causes of cancer-related deaths in children, and are defined by highly recurrent mutations in H3 histones. Mutations affecting the chromatin remodeling protein ATRX have been reported in 30% of pGBM cases, and are strongly associated with the alternative lengthening of telomeres (ALT) phenotype, but their precise interaction with histone mutations and their role in tumorigenesis remain unclear. We collected sequence data from 262 published and 64 unpublished cases of pGBM and identified somatic ATRX mutations in 54/326 (17%) of cases. ATRX mutations are mainly loss of function mutations, with the majority of frameshift mutations (37/54, 68,5%) found upstream of the helicase domain resulting in truncation of the main functional domain of ATRX. Missense mutations (16/54, 29,6%) reside almost exclusively in the helicase domain (11/54, 20,4%), whereas nonsense mutations are a less common event (7/54, 13%) but present in both the helicase (4/7, 57,1%) and ADD domains (3/7, 42,9%). ATRX mutations commonly co-segregate with H3.3 G34 (16/54) and TP53 (42/54) mutations, and define a subgroup of patients with a longer overall survival (16 months median overall survival in mutant ATRX cases versus 11 months in wild-type ATRX cases, COXPH p = 0.079), though with a greater number of somatic mutations (MWU p = 0.023) and copy number alterations (MWU p = 0.0011) than wild-type cases. We screened a series of 21 primary patient-derived pGBM cell cultures for histone and ATRX mutation status in addition to ATRX protein expression and ALT, and subjected the panel to a high-throughput in vitro cell viability screen of >400 chemotherapeutics and small molecules. We identified a specific genetic dependency for ATRX mutation and sensitivity to distinct PARP inhibitor chemotypes, including olaparib and rucaparib (PARP catalytic inhibitors), and talazoparib (PARP trapper inhibitor). These data were validated using CRISPR-Cas9-engineered ATRX knockout, targeting either the ADD or helicase domain, in SF188 pGBM cells. Gene editing was confirmed by IonTorrent sequencing and Western blot. ATRX mutant clones were also more sensitive to drugs targeting DNA damage response pathways such as bleomycin and sapacitabine. Gene expression analysis of ATRX mutant pGBM samples confirmed an intact homologous recombination pathway and overexpression of PARP1, suggesting an underlying mechanism distinct from that observed in BRCA-mutant breast and ovarian cancers. Ongoing work is aimed at unravelling the specific pathways involved, and evaluating the utility of PARP inhibition in orthotopic pGBM xenografts in vivo. These data suggest a synthetic lethality for PARP inhibitors in ATRX-deficient pGBM cells, and may represent a novel therapeutic strategy for these highly aggressive tumours in children. Citation Format: Janat Fazal-Salom, Mara Vinci, Diana Carvalho, Helen Pemberton, Stephen J. Pettitt, Christopher J. Lord, Alan Mackay, Lynn Bjerke, Chris Jones. Mutations in ATRX increase genetic instability and sensitivity to PARP inhibitors in paediatric glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1932. doi:10.1158/1538-7445.AM2017-1932

Nuclear distribution of the chromatin-remodeling protein ATRX in mouse early embryogenesis

The nucleus of mammalian embryos differs by transcriptional activity at different stages of early development. Here, we studied nuclear distribution of the chromatin-remodeling protein ATRX in pre-implantation mouse embryos. Immunofluorescent staining revealed the changes of ATRX nuclear distribution at the initial stages of early mouse development. At the stage of early zygote, a diffuse ATRX distribution pattern was prevalent. During the course of zygotic genome activation (ZGA), zones of increased ATRX concentration are observed, and they are most expressed in the nuclei of late 2-cell embryos. In the morula stage, the ATRX distribution becomes diffuse again. In zygotes, the patterns of ATRX distribution differ between male and female pronuclei. At all the stages, ATRX concentrates in the DAPI-positive areas of condensed chromatin. The level of colocalization between ATRX and heterochromatin was found the highest at the late 2-cell stage. When transcription was artificially suppressed, the pattern of intranuclear ATRX distribution was mostly determined by the mechanism of inhibitor action rather than the decreased level of transcriptional activity. Thus, the obvious changes of ATRX distribution occur and partially correlate with the main stages of ZGA during mouse early development, but these changes seem to be determined by other processes of structural and functional rearrangements of blastomere nuclei.

ATRX contributes to epigenetic asymmetry and silencing of major satellite transcripts in the maternal genome of the mouse embryo.

A striking proportion of human cleavage-stage embryos exhibit chromosome instability (CIN). Notably, until now, no experimental model has been described to determine the origin and mechanisms of complex chromosomal rearrangements. Here, we examined mouse embryos deficient for the chromatin remodeling protein ATRX to determine the cellular mechanisms activated in response to CIN. We demonstrate that ATRX is required for silencing of major satellite transcripts in the maternal genome, where it confers epigenetic asymmetry to pericentric heterochromatin during the transition to the first mitosis. This stage is also characterized by a striking kinetochore size asymmetry established by differences in CENP-C protein between the parental genomes. Loss of ATRX results in increased centromeric mitotic recombination, a high frequency of sister chromatid exchanges and double strand DNA breaks, indicating the formation of mitotic recombination break points. ATRX-deficient embryos exhibit a twofold increase in transcripts for aurora kinase B, the centromeric cohesin ESCO2, DNMT1, the ubiquitin-ligase (DZIP3) and the histone methyl transferase (EHMT1). Thus, loss of ATRX activates a pathway that integrates epigenetic modifications and DNA repair in response to chromosome breaks. These results reveal the cellular response of the cleavage-stage embryo to CIN and uncover a mechanism by which centromeric fission induces the formation of large-scale chromosomal rearrangements. Our results have important implications to determine the epigenetic origins of CIN that lead to congenital birth defects and early pregnancy loss, as well as the mechanisms involved in the oocyte to embryo transition.

The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure

The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-222) contains supplementary material, which is available to authorized users.

ATRX Dysfunction Induces Replication Defects in Primary Mouse Cells

The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells.

In silico analysis of detrimental mutations in ADD domain of chromatin remodeling protein ATRX that cause ATR-X syndrome: X-linked disorder

Many biological functions involve specific interactions of proteins. Mutations in ATRX gene can change the sequence and structure of a protein thereby impairing its function. Thus, the dysfunction of chromatin remodeling protein ATRX as a result of amino acid substitution in ADD domain often underlies the human disease, ATR-X syndrome. In general, it is mainly caused by amino acid substitution that interfered with the interactions of interest at the interface level. Hence, the study of protein–protein interactions and the interface that mediate the interactions stand important for understanding of biological function. In this work, we address the loss of function in chromatin remodeling protein ATRX due to loss of structural stability that affect the functional activity in mutant ADD domain by 17 missense mutations viz., G175E, N179S, P190A, P190L, P190S, L192F, V194I, C200S, Q219P, C220R, C220Y, W222S, C243F, R246C, R246L, G249C, and G249D. Furthermore, the loss of binding affinity of ADD domain with their interacting partner namely histone H3-peptide were investigated by (1) computing the RMSD (root mean square deviation) for the ADD domain of native with all the 17 mutants, (2) computing intra-molecular interactions in ADD domain of native with all the mutants, (3) computing binding affinity of native and mutant structures of ADD domain with histone H3-peptide through docking studies, and (4) cross validating the loss of function on binding affinity through inter-molecular interactions and normal mode analysis. Finally, as from our computational result, we concluded that all the parameters mentioned above used for studying ADD domain of mutant structures showed the decreased potential values compared to native structure that underlie ATR-X syndrome.

ATRX in chromatin assembly and genome architecture during development and disease

The regulation of genome architecture is essential for a variety of fundamental cellular phenomena that underlie the complex orchestration of mammalian development. The ATP-dependent chromatin remodeling protein ATRX is emerging as a key regulatory component of nucleosomal dynamics and higher order chromatin conformation. Here we provide an overview of the role of ATRX at chromatin and during development, and discuss recent studies exposing a repertoire of ATRX functions at heterochromatin, in gene regulation, and during mitosis and meiosis. Exciting new progress on several fronts suggest that ATRX operates in histone variant deposition and in the modulation of higher order chromatin structure. Not surprisingly, dysfunction or absence of ATRX protein has devastating consequences on embryonic development and leads to human disease.

Defective survival of proliferating Sertoli cells and androgen receptor function in a mouse model of the ATR-X syndrome

X-linked ATR-X (alpha thalassemia, mental retardation, X-linked) syndrome in males is characterized by mental retardation, facial dysmorphism, alpha thalassemia and urogenital abnormalities, including small testes. It is unclear how mutations in the chromatin-remodeling protein ATRX cause these highly specific clinical features, since ATRX is widely expressed during organ development. To investigate the mechanisms underlying the testicular defects observed in ATR-X syndrome, we generated ScAtrxKO (Sertoli cell Atrx knockout) mice with Atrx specifically inactivated in the supporting cell lineage (Sertoli cells) of the mouse testis. ScAtrxKO mice developed small testes and discontinuous tubules, due to prolonged G2/M phase and apoptosis of proliferating Sertoli cells during fetal life. Apoptosis might be a consequence of the cell cycle defect. We also found that the onset of spermatogenesis was delayed in postnatal mice, with a range of spermatogenesis defects evident in adult ScAtrxKO mice. ATRX and the androgen receptor (AR) physically interact in the testis and in the Sertoli cell line TM4 and co-operatively activate the promoter of Rhox5, an important direct AR target. We also demonstrate that ATRX directly binds to the Rhox5 promoter in TM4 cells. Finally, gene expression of Rhox5 and of another AR-dependent gene, Spinlw1, was reduced in ScAtrxKO testes. These data suggest that ATRX can directly enhance the expression of androgen-dependent genes through physical interaction with AR. Recruitment of ATRX by DNA sequence-specific transcription factors could be a general mechanism by which ATRX achieves tissue-specific transcriptional regulation which could explain the highly specific clinical features of ATR-X syndrome when ATRX is mutated.

ATRX: Put Me on Repeat

Mutations in the chromatin-remodeling protein ATRX cause alpha thalassaemia and mental retardation, but the severity of the disorder is independent of the specific mutation. In this issue of Cell, Law etal. (2010) demonstrate that ATRX alters gene expression by binding to G-rich tandem repeats, and the degree of transcriptional silencing caused by ATRX mutations correlates with the number of repeats.

Human Cytomegalovirus Protein pp71 Induces Daxx SUMOylation

Proteins that participate in a diverse array of cellular processes can be modified covalently and reversibly on lysine residues by the small ubiquitin-like modifier proteins termed SUMOs. In some instances, such modification profoundly affects protein function, but the biological significance of many SUMOylation events remains unknown. Protein SUMOylation is modulated during many viral infections. Here we demonstrate that the human cytomegalovirus (HCMV) pp71 protein promotes the SUMOylation of its cellular substrate, Daxx. A component of promyelocytic leukemia nuclear bodies, Daxx is a transcriptional corepressor that silences the expression of viral immediate-early (IE) genes at the start of both lytic and quiescent HCMV infections. pp71 is a tegument component delivered directly to cells by infecting HCMV virions. At the start of lytic infections, it travels to the nucleus and stimulates viral IE gene expression by displacing the chromatin remodeling protein ATRX from Daxx and by mediating Daxx degradation through a rare ubiquitin-independent, proteasome-dependent process. Here we report that pp71 also substantially increases the basal level of SUMOylated Daxx observed in cells. To date, consequences of Daxx SUMOylation have not been observed for cellular promoters, and we detected no qualitative change in viral IE gene expression in the absence of pp71-induced Daxx SUMOylation. Thus, while pp71 enhances the basal level of SUMOylated Daxx, the role that this modification plays in regulating Daxx activity in uninfected or HCMV-infected cells remains an enigma.

ATRX marks the inactive X chromosome (Xi) in somatic cells and during imprinted X chromosome inactivation in trophoblast stem cells

Mammalian X chromosome inactivation (XCI) is an essential mechanism to compensate for dosage imbalances between male and female embryos. Although the molecular pathways are not fully understood, heterochromatinization of the Xi requires the coordinate recruitment of multiple epigenetic marks. Using fluorescence in situ hybridization analysis combined with immunocytochemistry, we demonstrate that the chromatin remodeling protein ATRX decorates the chromatids of a single, late replicating X chromosome in female somatic cells and co-localizes with the bona fide marker of the Xi, macroH2A1.2. Chromatin immunoprecipitation using somatic, embryonic stem (ES) cells and trophoblast stem (TS) cells as model for random and imprinted XCI, respectively, revealed that, in somatic and TS cells, ATRX exhibits a specific association with sequences located within the previously described H3K9me2-hotspot, a region 5' to the X inactive-specific transcript (Xist) locus. While no ATRX-Xi interaction was detectable in undifferentiated ES cells, an enrichment of ATRX was observed after 8 days of differentiation, indicating that ATRX associates with the Xi following the onset of random XCI, consistent with a potential role in maintenance of XCI. These results have important implications regarding a previously described escape from imprinted XCI in ATRX-deficient mice as well as cases of skewed XCI in patients with ATRX syndrome.

The SWI/SNF protein ATRX co-regulates pseudoautosomal genes that have translocated to autosomes in the mouse genome

BackgroundPseudoautosomal regions (PAR1 and PAR2) in eutherians retain homologous regions between the X and Y chromosomes that play a critical role in the obligatory X-Y crossover during male meiosis. Genes that reside in the PAR1 are exceptional in that they are rich in repetitive sequences and undergo a very high rate of recombination. Remarkably, murine PAR1 homologs have translocated to various autosomes, reflecting the complex recombination history during the evolution of the mammalian X chromosome.ResultsWe now report that the SNF2-type chromatin remodeling protein ATRX controls the expression of eutherian ancestral PAR1 genes that have translocated to autosomes in the mouse. In addition, we have identified two potentially novel mouse PAR1 orthologs.ConclusionWe propose that the ancestral PAR1 genes share a common epigenetic environment that allows ATRX to control their expression.

Association of ATRX with pericentric heterochromatin and the Y chromosome of neonatal mouse spermatogonia

BackgroundEstablishment of chromosomal cytosine methylation and histone methylation patterns are critical epigenetic modifications required for heterochromatin formation in the mammalian genome. However, the nature of the primary signal(s) targeting DNA methylation at specific genomic regions is not clear. Notably, whether histone methylation and/or chromatin remodeling proteins play a role in the establishment of DNA methylation during gametogenesis is not known. The chromosomes of mouse neonatal spermatogonia display a unique pattern of 5-methyl cytosine staining whereby centromeric heterochromatin is hypo-methylated whereas chromatids are strongly methylated. Thus, in order to gain some insight into the relationship between global DNA and histone methylation in the germ line we have used neonatal spermatogonia as a model to determine whether these unique chromosomal DNA methylation patterns are also reflected by concomitant changes in histone methylation.ResultsOur results demonstrate that histone H3 tri-methylated at lysine 9 (H3K9me3), a hallmark of constitutive heterochromatin, as well as the chromatin remodeling protein ATRX remained associated with pericentric heterochromatin regions in spite of their extensive hypo-methylation. This suggests that in neonatal spermatogonia, chromosomal 5-methyl cytosine patterns are regulated independently of changes in histone methylation, potentially reflecting a crucial mechanism to maintain pericentric heterochromatin silencing. Furthermore, chromatin immunoprecipitation and fluorescence in situ hybridization, revealed that ATRX as well as H3K9me3 associate with Y chromosome-specific DNA sequences and decorate both arms of the Y chromosome, suggesting a possible role in heterochromatinization and the predominant transcriptional quiescence of this chromosome during spermatogenesis.ConclusionThese results are consistent with a role for histone modifications and chromatin remodeling proteins such as ATRX in maintaining transcriptional repression at constitutive heterochromatin domains in the absence of 5-methyl cytosine and provide evidence suggesting that the establishment and/or maintenance of repressive histone and chromatin modifications at pericentric heterochromatin following genome-wide epigenetic reprogramming in the germ line may precede the establishment of chromosomal 5-methyl cytosine patterns as a genomic silencing strategy in neonatal spermatogonia.

Daxx Cooperates with the Axin/HIPK2/p53 Complex to Induce Cell Death

Daxx, a death domain-associated protein, has been implicated in proapoptosis, antiapoptosis, and transcriptional regulation. Many factors known to play critically important roles in controlling apoptosis and gene transcription have been shown to associate with Daxx, including the Ser/Thr protein kinase HIPK2, promyelocytic leukemia protein, histone deacetylases, and the chromatin remodeling protein ATRX. Although it is clear that Daxx may exert multiple functions, the underlying mechanisms remain far from clear. Here, we show that Axin, originally identified for its scaffolding role to control beta-catenin levels in Wnt signaling, strongly associates with Daxx at endogenous levels. The Daxx/Axin complex formation is enhanced by UV irradiation. Axin tethers Daxx to the tumor suppressor p53, and cooperates with Daxx, but not DaxxDeltaAxin, which is unable to interact with Axin, to stimulate HIPK2-mediated Ser(46) phosphorylation and transcriptional activity of p53. Interestingly, Axin and Daxx seem to selectively activate p53 target genes, with strong activation of PUMA, but not p21 or Bax. Daxx-stimulated p53 transcriptional activity was significantly diminished by small interfering RNA against Axin; Daxx fails to inhibit colony formation in Axin(-/-) cells. Moreover, UV-induced cell death was attenuated by the knockdown of Axin and Daxx. All these results show that Daxx cooperates with Axin to stimulate p53, and implicate a direct role for Axin, HIPK2, and p53 in the proapoptotic function of Daxx. We have hence unraveled a novel aspect of p53 activation and shed new light on the ultimate understanding of the Daxx protein, perhaps most pertinently, in relation to stress-induced cell death.

The chromatin-remodeling protein ATRX is critical for neuronal survival during corticogenesis

Mutations in genes encoding chromatin-remodeling proteins, such as the ATRX gene, underlie a number of genetic disorders including several X-linked mental retardation syndromes; however, the role of these proteins in normal CNS development is unknown. Here, we used a conditional gene-targeting approach to inactivate Atrx, specifically in the forebrain of mice. Loss of ATRX protein caused widespread hypocellularity in the neocortex and hippocampus and a pronounced reduction in forebrain size. Neuronal “birthdating” confirmed that fewer neurons reached the superficial cortical layers, despite normal progenitor cell proliferation. The loss of cortical mass resulted from a 12-fold increase in neuronal apoptosis during early stages of corticogenesis in the mutant animals. Moreover, cortical progenitors isolated from Atrx-null mice undergo enhanced apoptosis upon differentiation. Taken together, our results indicate that ATRX is a critical mediator of cell survival during early neuronal differentiation. Thus, increased neuronal loss may contribute to the severe mental retardation observed in human patients.