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Emily Bernstein - One of the best experts on this subject based on the ideXlab platform.
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ATRX binds to atypical chromatin domains at the 3 exons of zinc finger genes to preserve h3k9me3 enrichment
Epigenetics, 2016Co-Authors: David Vallegarcia, Zulekha A. Qadeer, Dan Hasson, Michael A. Dyer, Domhnall S. Mchugh, Flavia G. Ghiraldini, Asif Chowdhury, Felix Recillastarga, Emily BernsteinAbstract:ATRX is a SWI/SNF chromatin remodeler proposed to govern genomic stability through the regulation of repetitive sequences, such as rDNA, retrotransposons, and pericentromeric and telomeric repeats. However, few direct ATRX target genes have been identified and high-throughput genomic approaches are currently lacking for ATRX. Here we present a comprehensive ChIP-sequencing study of ATRX in multiple human cell lines, in which we identify the 3' exons of zinc finger genes (ZNFs) as a new class of ATRX targets. These 3' exonic regions encode the zinc finger motifs, which can range from 1-40 copies per ZNF gene and share large stretches of sequence similarity. These regions often contain an atypical chromatin signature: they are transcriptionally active, contain high levels of H3K36me3, and are paradoxically enriched in H3K9me3. We find that these ZNF 3' exons are co-occupied by SETDB1, TRIM28, and ZNF274, which form a complex with ATRX. CRISPR/Cas9-mediated loss-of-function studies demonstrate (i) a reduction of H3K9me3 at the ZNF 3' exons in the absence of ATRX and ZNF274 and, (ii) H3K9me3 levels at atypical chromatin regions are particularly sensitive to ATRX loss compared to other H3K9me3-occupied regions. As a consequence of ATRX or ZNF274 depletion, cells with reduced levels of H3K9me3 show increased levels of DNA damage, suggesting that ATRX binds to the 3' exons of ZNFs to maintain their genomic stability through preservation of H3K9me3.
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ATRX binds to atypical chromatin domains at the 3′ exons of zinc finger genes to preserve H3K9me3 enrichment
Epigenetics, 2016Co-Authors: David Valle-garcia, Zulekha A. Qadeer, Dan Hasson, Michael A. Dyer, Domhnall S. Mchugh, Flavia G. Ghiraldini, Asif Chowdhury, Félix Recillas-targa, Emily BernsteinAbstract:ATRX is a SWI/SNF chromatin remodeler proposed to govern genomic stability through the regulation of repetitive sequences, such as rDNA, retrotransposons, and pericentromeric and telomeric repeats. However, few direct ATRX target genes have been identified and high-throughput genomic approaches are currently lacking for ATRX. Here we present a comprehensive ChIP-sequencing study of ATRX in multiple human cell lines, in which we identify the 3' exons of zinc finger genes (ZNFs) as a new class of ATRX targets. These 3' exonic regions encode the zinc finger motifs, which can range from 1-40 copies per ZNF gene and share large stretches of sequence similarity. These regions often contain an atypical chromatin signature: they are transcriptionally active, contain high levels of H3K36me3, and are paradoxically enriched in H3K9me3. We find that these ZNF 3' exons are co-occupied by SETDB1, TRIM28, and ZNF274, which form a complex with ATRX. CRISPR/Cas9-mediated loss-of-function studies demonstrate (i) a reduction of H3K9me3 at the ZNF 3' exons in the absence of ATRX and ZNF274 and, (ii) H3K9me3 levels at atypical chromatin regions are particularly sensitive to ATRX loss compared to other H3K9me3-occupied regions. As a consequence of ATRX or ZNF274 depletion, cells with reduced levels of H3K9me3 show increased levels of DNA damage, suggesting that ATRX binds to the 3' exons of ZNFs to maintain their genomic stability through preservation of H3K9me3.
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The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure
SpringerPlus, 2014Co-Authors: David Valle-garcia, Lyra Griffiths, Michael A. Dyer, Emily Bernstein, Félix Recillas-targaAbstract: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.
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ATRX: the case of a peculiar chromatin remodeler.
Epigenetics, 2012Co-Authors: Kajan Ratnakumar, Emily BernsteinAbstract:The SWI/SNF-like chromatin remodeler ATRX has recently garnered renewed attention. ATRX mutations were first identified in patients bearing the syndrome after which it is named, alpha thalassemia/mental retardation, X-linked. While ATRX has long been implicated in transcriptional regulation through multiple mechanisms, recent studies have identified a role for ATRX in the regulation of histone variant deposition. In addition, current reports describe ATRX to be mutated at high percentages in multiple tumor types, suggestive of a potential ‘driver’ role in cancer. Here we discuss the numerous and seemingly diverse roles for ATRX in transcriptional regulation and histone deposition and suggest that ATRX’s effects are mediated by its regulation of histones within the chromatin template.
David J. Picketts - One of the best experts on this subject based on the ideXlab platform.
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Sensory Experience Modulates ATRX-mediated Neuronal Integrity in the Mouse Retina.
Neuroscience, 2020Co-Authors: Pamela S. Lagali, Brandon Y. H. Zhao, Keqin Yan, Adam N. Baker, Stuart G. Coupland, Catherine Tsilfidis, David J. PickettsAbstract:Abstract Mutation of the α-thalassemia/mental retardation syndrome X-linked protein, ATRX, causes intellectual disability and is associated with pleiotropic defects including ophthalmological abnormalities. We have previously demonstrated that ATRX deficiency in the mouse retina leads to the selective loss of inhibitory interneurons and inner retinal dysfunction. Onset of the amacrine cell neurodegenerative phenotype in ATRX-deficient retinas occurs postnatally after neuronal specification, and coincides with eye opening. Given this timing, we sought to interrogate the influence of light-dependent visual signaling on ATRX-mediated neuronal survival and function in the mouse retina. Retina-specific ATRX conditional knockout (cKO) mice were subjected to light deprivation using two different paradigms: 1) a dark-rearing regime, and 2) genetic deficiency of metabotropic glutamate receptor 6 (mGluR6) to block the ON retinal signaling pathway. Scotopic electroretinography was performed for adult dark-reared ATRX cKO mice and controls to measure retinal neuron function in vivo. Retinal immunohistochemistry and enumeration of amacrine cells were performed for both light deprivation paradigms. We observed milder normalized a-wave, b-wave and oscillatory potential deficits in electroretinograms of dark-reared ATRX cKO mice compared to light-exposed counterparts. In addition, amacrine cell loss was partially limited by genetic restriction of retinal signaling through the ON pathway. Our results suggest that the temporal features of the ATRX cKO phenotype are likely due to a combined effect of light exposure upon eye opening and coincident developmental processes impacting the retinal circuitry. In addition, this study reveals a novel activity-dependent role for ATRX in mediating post-replicative neuronal integrity in the CNS.
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ATRX affects the repair of telomeric DSBs by promoting cohesion and a DAXX-dependent activity
PLoS biology, 2020Co-Authors: Courtney A. Lovejoy, David J. Picketts, Michael S. Huh, Kaori K. Takai, Titia De LangeAbstract:Alpha thalassemia/mental retardation syndrome X-linked chromatin remodeler (ATRX), a DAXX (death domain-associated protein) interacting protein, is often lost in cells using the alternative lengthening of telomeres (ALT) pathway, but it is not known how ATRX loss leads to ALT. We report that ATRX deletion from mouse cells altered the repair of telomeric double-strand breaks (DSBs) and induced ALT-like phenotypes, including ALT-associated promyelocytic leukemia (PML) bodies (APBs), telomere sister chromatid exchanges (T-SCEs), and extrachromosomal telomeric signals (ECTSs). Mechanistically, we show that ATRX affects telomeric DSB repair by promoting cohesion of sister telomeres and that loss of ATRX in ALT cells results in diminished telomere cohesion. In addition, we document a role for DAXX in the repair of telomeric DSBs. Removal of telomeric cohesion in combination with DAXX deficiency recapitulates all telomeric DSB repair phenotypes associated with ATRX loss. The data reveal that ATRX has an effect on telomeric DSB repair and that this role involves both telomere cohesion and a DAXX-dependent pathway.
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GENE-34. THERAPEUTICALLY TARGETING EPIGENOMIC AND TRANSCRIPTIONAL DYSFUNCTION IN ATRX-DEFICIENT GLIOMA
Neuro-Oncology, 2019Co-Authors: Carla Danussi, Anand K. Singh, Pavan Pinnamaneni, Grant Fisher, David J. Picketts, Kasthuri Kannan, Arvind Rao, Kunal Rai, Jason T. HuseAbstract:Abstract Diffusely infiltrating gliomas feature loss-of-function mutations in the chromatin remodeler gene ATRX as defining molecular alterations delineating major adult and pediatric disease subtypes. We recently reported that ATRX deficiency drives glioma-relevant phenotypes, such as increased motility and astrocytic differentiation profiles, by directly modulating epigenomic landscapes and the corresponding transcriptional profiles in glioma cells of origin. In particular, ATRX deficiency was associated with disruptions in H3.3 histone content at key genetic loci. To further understand the downstream epigenomic dysfunction induced by ATRX deficiency, we compared genome-wide chromatin-state maps of ATRX+ and ATRX- primary murine neuroepithelial progenitors (mNPCs). This ChIP–seq analysis revealed major differences in the localization of heterochromatin repressive marks H3K9me3 and H3K27me3. Specifically, we identified peculiar locations in the genome displaying H3K9me3 depletion and gain of H3K27me3 upon ATRX inactivation. Interestingly, these regions were flanked by ATRX binding sites and perfectly co-localized with Lamina-Associated Domains, known to play important roles in tissue lineage specification. To better target this dysfunction, we utilized the Broad Institute Connectivity Map (CLUE analysis) to identify compounds likely to revert the unique transcriptional perturbations induced by ATRX deficiency. We found that HDAC inhibitors, as a compound class, yielded expression profiles strongly anticorrelated to those driven by ATRX deficiency in these datasets. Further integrating existing gene expression data from our mNPCs and the TCGA LGG project with our CLUE findings highlighted SIRT2, a class III HDAC, as a top potential target. SIRT2 expression was significantly upregulated in both ATRX- mNPCs and in ATRX-mutant gliomas and its specific chemical inhibition normalized cellular motility in both ATRX- mNPCs and ATRX-mutant, patient derived glioma stem cells. These findings indicate that SIRT2 inhibition represents a viable strategy to revert the epigenetic effects of ATRX deficiency on facultative heterochromatin and their transcriptional and phenotypic consequences.
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Retinal interneuron survival requires non-cell-autonomous ATRX activity.
Human molecular genetics, 2016Co-Authors: Pamela S. Lagali, Chantal F. Medina, Brandon Y. H. Zhao, Keqin Yan, Adam N. Baker, Stuart G. Coupland, Catherine Tsilfidis, Valerie A. Wallace, David J. PickettsAbstract:ATRX is a chromatin remodeling protein that is mutated in several intellectual disability disorders including alpha-thalassemia/mental retardation, X-linked (ATR-X) syndrome. We previously reported the prevalence of ophthalmological defects in ATR-X syndrome patients, and accordingly we find morphological and functional visual abnormalities in a mouse model harboring a mutation occurring in ATR-X patients. The visual system abnormalities observed in these mice parallels the ATRX-null retinal phenotype characterized by interneuron defects and selective loss of amacrine and horizontal cells. The mechanisms that underlie selective neuronal vulnerability and neurodegeneration in the central nervous system upon ATRX mutation or deletion are unknown. To interrogate the cellular specificity of ATRX for its retinal neuroprotective functions, we employed a combination of temporal and lineage-restricted conditional ablation strategies to generate five different conditional knockout mouse models, and subsequently identified a non-cell-autonomous requirement for ATRX in bipolar cells for inhibitory interneuron survival in the retina. ATRX-deficient retinal bipolar cells exhibit functional, structural and molecular alterations consistent with impairments in neuronal activity and connectivity. Gene expression changes in the ATRX-null retina indicate defective synaptic structure and neuronal circuitry, suggest excitotoxic mechanisms of neurodegeneration, and demonstrate that common targets of ATRX in the forebrain and retina may contribute to similar neuropathological processes underlying cognitive impairment and visual dysfunction in ATR-X syndrome.
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Compromised genomic integrity impedes muscle growth after ATRX inactivation
The Journal of clinical investigation, 2012Co-Authors: Michael S. Huh, Tina Price O’dea, D. Ouazia, Bruce C. Mckay, Gianni Parise, Robin J. Parks, Michael A. Rudnicki, David J. PickettsAbstract:ATR-X syndrome is a severe intellectual disability disorder caused by mutations in the ATRX gene. Many ancillary clinical features are attributed to CNS deficiencies, yet most patients have muscle hypotonia, delayed ambulation, or kyphosis, pointing to an underlying skeletal muscle defect. Here, we identified a cell-intrinsic requirement for ATRX in postnatal muscle growth and regeneration in mice. Mice with skeletal muscle-specific ATRX conditional knockout (ATRX cKO mice) were viable, but by 3 weeks of age presented hallmarks of underdeveloped musculature, including kyphosis, 20% reduction in body mass, and 34% reduction in muscle fiber caliber. ATRX cKO mice also demonstrated a marked regeneration deficit that was not due to fewer resident satellite cells or their inability to terminally differentiate. However, activation of ATRX-null satellite cells from isolated muscle fibers resulted in a 9-fold reduction in myoblast expansion, caused by delayed progression through mid to late S phase. While in S phase, ATRX colocalized specifically to late-replicating chromatin, and its loss resulted in rampant signs of genomic instability. These observations support a model in which ATRX maintains chromatin integrity during the rapid developmental growth of a tissue.
Nathalie G Berube - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of ATRX in forebrain excitatory neurons affects hippocampal synaptic plasticity
Hippocampus, 2019Co-Authors: Radu Gugustea, Nathalie G Berube, Renee J Tamming, Nicole Martin-kenny, L. Stan LeungAbstract:α-Thalassemia X-linked intellectual disability (ATR-X) syndrome is a neurodevelopmental disorder caused by mutations in the ATRX gene that encodes a SNF2-type chromatin-remodeling protein. The ATRX protein regulates chromatin structure and gene expression in the developing mouse brain and early inactivation leads to DNA replication stress, extensive cell death, and microcephaly. However, the outcome of ATRX loss of function postnatally in neurons is less well understood. We recently reported that conditional inactivation of ATRX in postnatal forebrain excitatory neurons (ATRX-cKO) causes deficits in long-term hippocampus-dependent spatial memory. Thus, we hypothesized that ATRX-cKO mice will display impaired hippocampal synaptic transmission and plasticity. In the present study, evoked field potentials and current source density analysis were recorded from a multichannel electrode in male, urethane-anesthetized mice. Three major excitatory synapses, the Schaffer collaterals to basal dendrites and proximal apical dendrites, and the temporoammonic path to distal apical dendrites on hippocampal CA1 pyramidal cells were assessed by their baseline synaptic transmission, including paired-pulse facilitation (PPF) at 50-ms interpulse interval, and by their long-term potentiation (LTP) induced by theta-frequency burst stimulation. Baseline single-pulse excitatory response at each synapse did not differ between ATRX-cKO and control mice, but baseline PPF was reduced at the CA1 basal dendritic synapse in ATRX-cKO mice. While basal dendritic LTP of the first-pulse excitatory response was not affected in ATRX-cKO mice, proximal and distal apical dendritic LTP were marginally and significantly reduced, respectively. These results suggest that ATRX is required in excitatory neurons of the forebrain to achieve normal hippocampal LTP and PPF at the CA1 apical and basal dendritic synapses, respectively. Such alterations in hippocampal synaptic transmission and plasticity could explain the long-term spatial memory deficits in ATRX-cKO mice and provide insight into the physiological mechanisms underlying intellectual disability in ATR-X syndrome patients.
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ATRX is required for maintenance of the neuroprogenitor cell pool in the embryonic mouse brain.
Biology open, 2014Co-Authors: Kieran Ritchie, Yan Jiang, L. Ashley Watson, Benjamin Davidson, Nathalie G BerubeAbstract:ABSTRACT Mutations in the alpha-thalassemia mental retardation X-linked ( ATRX ) gene cause a spectrum of abnormalities including intellectual disability, developmental delay, seizures, and microcephaly. The ATRX protein is highly enriched at heterochromatic repetitive sequences adjacent to the centromere, and ATRX depletion results in chromosome congression, segregation, and cohesion defects. Here, we show that Cre-mediated inactivation of ATRX in the embryonic mouse ( Mus musculus ) brain results in expansion of cerebral cortical layer VI, and a concurrent thinning of layers II–IV. We observed increased cell cycle exit during early-mid neurogenesis, and a depletion of apical progenitors by late neurogenesis in the ATRX -null neocortex, explaining the disproportionate layering. Premature differentiation was associated with an increased generation of outer radial glia (oRG) and TBR2-expressing basal progenitors, as well as increased generation of early-born post-mitotic projection neurons. ATRX deletion also reduced the fidelity of mitotic spindle orientation in apical progenitors, where mutant cells were often oriented at non-parallel angles of division relative to the ventricular surface. We conclude that ATRX is required for correct lamination of the mouse neocortex by regulating the timing of neuroprogenitor cell differentiation.
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Loss of ATRX does not confer susceptibility to osteoarthritis.
PloS one, 2013Co-Authors: Lauren A. Solomon, Nathalie G Berube, Bailey A. Russell, David Makar, Frank BeierAbstract:The chromatin remodelling protein ATRX is associated with the rare genetic disorder ATR-X syndrome. This syndrome includes developmental delay, cognitive impairment, and a variety of skeletal deformities. ATRX plays a role in several basic chromatin-mediated cellular events including DNA replication, telomere stability, gene transcription, and chromosome congression and cohesion during cell division. We have used a loss-of-function approach to directly investigate the role of ATRX in the adult skeleton in three different models of selective ATRX loss. We specifically targeted deletion of ATRX to the forelimb mesenchyme, to cartilage and to bone-forming osteoblasts. We previously demonstrated that loss of ATRX in forelimb mesenchyme causes brachydactyly while deletion in chondrocytes had minimal effects during development. We now show that targeted deletion of ATRX in osteoblasts causes minor dwarfism but does not recapitulate most of the skeletal phenotypes seen in ATR-X syndrome patients. In adult mice from all three models, we find that joints lacking ATRX are not more susceptible to osteoarthritis, as determined by OARSI scoring and immunohistochemistry. These results indicate that while ATRX plays limited roles during early stages of skeletal development, deficiency of the protein in adult tissues does not confer susceptibility to osteoarthritis.
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ATRX deficiency induces telomere dysfunction endocrine defects and reduced life span
Journal of Clinical Investigation, 2013Co-Authors: Ashley L Watson, Lauren A. Solomon, Frank Beier, Yan Jiang, Matthew Edwards, Kazuo Shinya, Nathalie G BerubeAbstract:Human ATRX mutations are associated with cognitive deficits, developmental abnormalities, and cancer. We show that the ATRX-null embryonic mouse brain accumulates replicative damage at telomeres and pericentromeric heterochromatin, which is exacerbated by loss of p53 and linked to ATM activation. ATRX-deficient neuroprogenitors exhibited higher incidence of telomere fusions and increased sensitivity to replication stress–inducing drugs. Treatment of ATRX-null neuroprogenitors with the G-quadruplex (G4) ligand telomestatin increased DNA damage, indicating that ATRX likely aids in the replication of telomeric G4-DNA structures. Unexpectedly, mutant mice displayed reduced growth, shortened life span, lordokyphosis, cataracts, heart enlargement, and hypoglycemia, as well as reduction of mineral bone density, trabecular bone content, and subcutaneous fat. We show that a subset of these defects can be attributed to loss of ATRX in the embryonic anterior pituitary that resulted in low circulating levels of thyroxine and IGF-1. Our findings suggest that loss of ATRX increases DNA damage locally in the forebrain and anterior pituitary and causes tissue attrition and other systemic defects similar to those seen in aging.
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ATRX in chromatin assembly and genome architecture during development and disease.
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2011Co-Authors: Nathalie G BerubeAbstract: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.
Hsiao P.j. Voon - One of the best experts on this subject based on the ideXlab platform.
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ribosomal dna copy loss and repeat instability in ATRX mutated cancers
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Maheshi Udugama, Hsiao P.j. Voon, Elaine Sanij, Jinbae Son, Linda Hii, Jeremy D Henson, Lyn F Chan, Fiona T M Chang, Yumei LiuAbstract:ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.
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the chromatin remodelling factor ATRX suppresses r loops in transcribed telomeric repeats
EMBO Reports, 2017Co-Authors: Diu Nguyen, Hsiao P.j. Voon, Barbara Xella, Caroline Scott, David Clynes, Christian Babbs, Helena Ayyub, Jon Kerry, Jacqueline A. SharpeAbstract:Abstract ATRX is a chromatin remodelling factor found at a wide range of tandemly repeated sequences including telomeres (TTAGGG) n . ATRX mutations are found in nearly all tumours that maintain their telomeres via the alternative lengthening of telomere (ALT) pathway, and ATRX is known to suppress this pathway. Here, we show that recruitment of ATRX to telomeric repeats depends on repeat number, orientation and, critically, on repeat transcription. Importantly, the transcribed telomeric repeats form RNA–DNA hybrids (R‐loops) whose abundance correlates with the recruitment of ATRX. Here, we show loss of ATRX is also associated with increased R‐loop formation. Our data suggest that the presence of ATRX at telomeres may have a central role in suppressing deleterious DNA secondary structures that form at transcribed telomeric repeats, and this may account for the increased DNA damage, stalling of replication and homology‐directed repair previously observed upon loss of ATRX function.
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The chromatin remodelling factor ATRX suppresses R‐loops in transcribed telomeric repeats
EMBO reports, 2017Co-Authors: Diu Nguyen, Hsiao P.j. Voon, Barbara Xella, Caroline Scott, David Clynes, Christian Babbs, Helena Ayyub, Jon Kerry, Jacqueline A. Sharpe, Jackie Sloane-stanleyAbstract:Abstract ATRX is a chromatin remodelling factor found at a wide range of tandemly repeated sequences including telomeres (TTAGGG) n . ATRX mutations are found in nearly all tumours that maintain their telomeres via the alternative lengthening of telomere (ALT) pathway, and ATRX is known to suppress this pathway. Here, we show that recruitment of ATRX to telomeric repeats depends on repeat number, orientation and, critically, on repeat transcription. Importantly, the transcribed telomeric repeats form RNA–DNA hybrids (R‐loops) whose abundance correlates with the recruitment of ATRX. Here, we show loss of ATRX is also associated with increased R‐loop formation. Our data suggest that the presence of ATRX at telomeres may have a central role in suppressing deleterious DNA secondary structures that form at transcribed telomeric repeats, and this may account for the increased DNA damage, stalling of replication and homology‐directed repair previously observed upon loss of ATRX function.
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ATRX plays a key role in maintaining silencing at interstitial heterochromatic loci and imprinted genes
Cell Reports, 2015Co-Authors: Hsiao P.j. Voon, Douglas R. Higgs, Jim R Hughes, Christina Rode, Inti A De La Rosavelazquez, Thomas Jenuwein, Robert Feil, Richard J GibbonsAbstract:Histone H3.3 is a replication-independent histone variant, which replaces histones that are turned over throughout the entire cell cycle. H3.3 deposition at euchromatin is dependent on HIRA, whereas ATRX/Daxx deposits H3.3 at pericentric heterochromatin and telomeres. The role of H3.3 at heterochromatic regions is unknown, but mutations in the ATRX/Daxx/H3.3 pathway are linked to aberrant telomere lengthening in certain cancers. In this study, we show that ATRX-dependent deposition of H3.3 is not limited to pericentric heterochromatin and telomeres but also occurs at heterochromatic sites throughout the genome. Notably, ATRX/H3.3 specifically localizes to silenced imprinted alleles in mouse ESCs. ATRX KO cells failed to deposit H3.3 at these sites, leading to loss of the H3K9me3 heterochromatin modification, loss of repression, and aberrant allelic expression. We propose a model whereby ATRX-dependent deposition of H3.3 into heterochromatin is normally required to maintain the memory of silencing at imprinted loci.
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atr x syndrome protein targets tandem repeats and influences allele specific expression in a size dependent manner
Cell, 2010Co-Authors: Martin Law, Hsiao P.j. Voon, Karen M Lower, James Hughes, David Garrick, Vip Viprakasit, Matthew Mitson, Marco De Gobbi, Marco A Marra, Andrew J MorrisAbstract:ATRX is an X-linked gene of the SWI/SNF family, mutations in which cause syndromal mental retardation and downregulation of α-globin expression. Here we show that ATRX binds to tandem repeat (TR) sequences in both telomeres and euchromatin. Genes associated with these TRs can be dysregulated when ATRX is mutated, and the change in expression is determined by the size of the TR, producing skewed allelic expression. This reveals the characteristics of the affected genes, explains the variable phenotypes seen with identical ATRX mutations, and illustrates a new mechanism underlying variable penetrance. Many of the TRs are G rich and predicted to form non-B DNA structures (including G-quadruplex) in vivo. We show that ATRX binds G-quadruplex structures in vitro, suggesting a mechanism by which ATRX may play a role in various nuclear processes and how this is perturbed when ATRX is mutated.
Jason T. Huse - One of the best experts on this subject based on the ideXlab platform.
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GENE-34. THERAPEUTICALLY TARGETING EPIGENOMIC AND TRANSCRIPTIONAL DYSFUNCTION IN ATRX-DEFICIENT GLIOMA
Neuro-Oncology, 2019Co-Authors: Carla Danussi, Anand K. Singh, Pavan Pinnamaneni, Grant Fisher, David J. Picketts, Kasthuri Kannan, Arvind Rao, Kunal Rai, Jason T. HuseAbstract:Abstract Diffusely infiltrating gliomas feature loss-of-function mutations in the chromatin remodeler gene ATRX as defining molecular alterations delineating major adult and pediatric disease subtypes. We recently reported that ATRX deficiency drives glioma-relevant phenotypes, such as increased motility and astrocytic differentiation profiles, by directly modulating epigenomic landscapes and the corresponding transcriptional profiles in glioma cells of origin. In particular, ATRX deficiency was associated with disruptions in H3.3 histone content at key genetic loci. To further understand the downstream epigenomic dysfunction induced by ATRX deficiency, we compared genome-wide chromatin-state maps of ATRX+ and ATRX- primary murine neuroepithelial progenitors (mNPCs). This ChIP–seq analysis revealed major differences in the localization of heterochromatin repressive marks H3K9me3 and H3K27me3. Specifically, we identified peculiar locations in the genome displaying H3K9me3 depletion and gain of H3K27me3 upon ATRX inactivation. Interestingly, these regions were flanked by ATRX binding sites and perfectly co-localized with Lamina-Associated Domains, known to play important roles in tissue lineage specification. To better target this dysfunction, we utilized the Broad Institute Connectivity Map (CLUE analysis) to identify compounds likely to revert the unique transcriptional perturbations induced by ATRX deficiency. We found that HDAC inhibitors, as a compound class, yielded expression profiles strongly anticorrelated to those driven by ATRX deficiency in these datasets. Further integrating existing gene expression data from our mNPCs and the TCGA LGG project with our CLUE findings highlighted SIRT2, a class III HDAC, as a top potential target. SIRT2 expression was significantly upregulated in both ATRX- mNPCs and in ATRX-mutant gliomas and its specific chemical inhibition normalized cellular motility in both ATRX- mNPCs and ATRX-mutant, patient derived glioma stem cells. These findings indicate that SIRT2 inhibition represents a viable strategy to revert the epigenetic effects of ATRX deficiency on facultative heterochromatin and their transcriptional and phenotypic consequences.
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g quadruplex dna drives genomic instability and represents a targetable molecular abnormality in ATRX deficient malignant glioma
Nature Communications, 2019Co-Authors: Yuxiang Wang, Carla Danussi, Kasthuri Kannan, Timothy A Chan, Jie Yang, Aaron T Wild, Rachna Shah, Gregory J Riggins, Erik P Sulman, Jason T. HuseAbstract:Mutational inactivation of ATRX (α-thalassemia mental retardation X-linked) represents a defining molecular alteration in large subsets of malignant glioma. Yet the pathogenic consequences of ATRX deficiency remain unclear, as do tractable mechanisms for its therapeutic targeting. Here we report that ATRX loss in isogenic glioma model systems induces replication stress and DNA damage by way of G-quadruplex (G4) DNA secondary structure. Moreover, these effects are associated with the acquisition of disease-relevant copy number alterations over time. We then demonstrate, both in vitro and in vivo, that ATRX deficiency selectively enhances DNA damage and cell death following chemical G4 stabilization. Finally, we show that G4 stabilization synergizes with other DNA-damaging therapies, including ionizing radiation, in the ATRX-deficient context. Our findings reveal novel pathogenic mechanisms driven by ATRX deficiency in glioma, while also pointing to tangible strategies for drug development.
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Abstract IA12: Investigating the functional consequences of ATRX deficiency in glioma
Metabolism IDH Epigenetics, 2015Co-Authors: Jason T. HuseAbstract:Comprehensive genomic profiling in cancer continues to reveal frequent alterations in epigenetic regulators, firmly implicating chromatin biology in the oncogenic process. We and others recently found that inactivating mutations in the SWI/SNF family member ATRX represent defining molecular alterations in diffusely infiltrating gliomas, where they invariably pair with mutations in TP53 and IDH1/2. ATRX normally regulates the composition of histone monomers at a variety of sites across the genome, and may also affect the distribution of specific histone modifying complexes. A number of studies have linked ATRX deficiency to a wide spectrum of physiological dysfunction, including aberrant gene regulation, abnormal telomere maintenance, genomic instability, and aneuploidy. Nevertheless, the precise mechanisms by which ATRX deficiency promotes oncogenesis remain unknown. To investigate the effects of ATRX deficiency on chromatin structure, gene expression, and transformation, we inactivated ATRX in murine neuroepithelial progenitor cells (NPCs) also lacking Tp53. We found that, in this context, ATRX deficiency induced widespread alterations in chromatin accessibility and organization, which in turn led to dramatic shifts in gene expression. Among the altered transcriptional networks were pathways implicated in cellular motility and invasion, an established feature of diffusely infiltrating gliomas. Intriguingly, ATRX-deficient NPCs also exhibited significantly increased transwell migration. Our findings demonstrate that ATRX deficiency promotes disease-relevant biological behavior by modulating chromatin accessibility and influencing gene expression. We are now performing detailed epigenetic and biochemical studies to identify the precise transcriptional events mediating these effects. We are also investigating how IDH mutations influence the genomic distribution of ATRX, thereby modulating its function and the consequences of its deficiency. Citation Format: Jason T. Huse. Investigating the functional consequences of ATRX deficiency in glioma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr IA12.
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ELUCIDATING THE ONCOGENIC ROLE OF ATRX DEFICIENCY IN GLIOMA
Neuro-Oncology, 2014Co-Authors: Jason T. HuseAbstract:We and others recently identified loss-of-function mutations in the ATRX gene as core components of a highly recurrent molecular signature characterizing adult diffuse astrocytoma. ATRX deficiency has also been implicated in large subsets of pediatric glioma, neuroblastoma, and pancreatic neuroendocrine tumor. ATRX is a SWI/SNF chromatin remodeling factor, which appears to play a crucial role in heterochromatin maintenance, particularly at telomeres. Its dysfunction has been correlated with genomic instability and DNA replications stress, along with abnormal telomere elongation. Interestingly, germline mutations in ATRX do not cause cancer, but are instead associated with neurodegeneration and mental retardation. To further elucidate the pathogenic role of ATRX in glioma, we first assessed its expression level across different glioma subtypes. As expected, ATRX expression was lost in virtually all IDH-mutant gliomas without 1p/19q codeletion (primarily astrocytomas), but was universally maintained in IDH-wild type tumors (primarily glioblastomas), and 1p/19q co-deleted tumors (oligodendrogliomas). Interestingly, we found that ATRX-mutant gliomas were not universally negative for ATRX, with some tumor cells retaining expression. This finding, which we have confirmed by deep sequencing, suggests that ATRX mutations do not confer a strong proliferative advantage per se, but instead predispose cellular subclones to transformative events downstream, perhaps through genomic destabilization and DNA copy number alteration. To investigate issues of clonality and DNA copy number further, we have performed single cell sequencing on a number of ATRX-mutant gliomas, identifying several distinct subclones within each sample, and are in the process of integrating focused genotyping data for ATRX and other relevant genes. We are also in the process of a detailed investigation into the epigenomic and transformative consequences of ATRX deficiency using disease-relevant in vitro and in vivo modeling systems. Characterizing the pathogenic cascade induced by ATRX deficiency will provide crucial insights into therapeutic development for a number of tumor entities, including diffuse astrocytoma.