A mouse model of ATRX deficiency with cognitive deficits and autistic traits.

BACKGROUND: ATRX is an ATP-dependent chromatin remodeling protein with essential roles in safeguarding genome integrity and modulating gene expression. Deficiencies in this protein cause ATR-X syndrome, a condition characterized by intellectual disability and an array of developmental abnormalities, including features of autism. Previous studies demonstrated that deleting ATRX in mouse forebrain excitatory neurons postnatally resulted in male-specific memory deficits, but no apparent autistic-like behaviours. METHODS: We generated mice with an earlier embryonic deletion of ATRX in forebrain excitatory neurons and characterized their behaviour using a series of memory and autistic-related paradigms. RESULTS: We found that mutant mice displayed a broader spectrum of impairments, including fear memory, decreased anxiety-like behaviour, hyperactivity, as well as self-injurious and repetitive grooming. Sex-specific alterations were also observed, including male-specific aggression, sensory gating impairments, and decreased social memory. CONCLUSIONS: Collectively, the findings indicate that early developmental abnormalities arising from ATRX deficiency in forebrain excitatory neurons contribute to the presentation of fear memory deficits as well as autistic-like behaviours.

Systemic and intrinsic functions of ATRX in glial cell fate and CNS myelination in male mice.

Myelin, an extension of the oligodendrocyte plasma membrane, wraps around axons to facilitate nerve conduction. Myelination is compromised in ATR-X intellectual disability syndrome patients, but the causes are unknown. We show that loss of ATRX leads to myelination deficits in male mice that are partially rectified upon systemic thyroxine administration. Targeted ATRX inactivation in either neurons or oligodendrocyte progenitor cells (OPCs) reveals OPC-intrinsic effects on myelination. OPCs lacking ATRX fail to differentiate along the oligodendrocyte lineage and acquire a more plastic state that favors astrocytic differentiation in vitro and in vivo. ATRX chromatin occupancy in OPCs greatly overlaps with that of the chromatin remodelers CHD7 and CHD8 as well as H3K27Ac, a mark of active enhancers. Overall, our data indicate that ATRX regulates the onset of myelination systemically via thyroxine, and by promoting OPC differentiation and suppressing astrogliogenesis. These functions of ATRX identified in mice could explain white matter pathogenesis observed in ATR-X syndrome patients.

Selective isolation of mouse glial nuclei optimized for reliable downstream omics analyses.

BACKGROUND: Isolation of cell types of interest from the brain for molecular applications presents several challenges, including cellular damage during tissue dissociation or enrichment procedures, and low cell number in the tissue in some cases. Techniques have been developed to enrich distinct cell populations using immunopanning or fluorescence activated cell/nuclei sorting. However, these techniques often involve fixation, immunolabeling and DNA staining steps, which could potentially influence downstream omics applications. NEW METHOD: Taking advantage of readily available genetically modified mice with fluorescent-tagged nuclei, we describe a technique for the purification of cell-type specific brain nuclei, optimized to decrease sample preparation time and to limit potential artefacts for downstream omics applications. We demonstrate the applicability of this approach for the purification of glial cell nuclei and show that the resulting cell-type specific nuclei obtained can be used effectively for omics applications, including ATAC-seq and RNA-seq. RESULTS: We demonstrate excellent enrichment of fluorescently-tagged glial nuclei, yielding high quality RNA and chromatin. We identify several critical steps during nuclei isolation that help limit nuclei rupture and clumping, including quick homogenization, dilution before filtration and loosening of the pellet before resuspension, thus improving yield. Sorting of fluorescent nuclei can be achieved without fixation, antibody labelling, or DAPI staining, reducing potential artifactual results in RNA-seq and ATAC-seq analyses. We show that reproducible glial cell type-specific profiles can be obtained in transcriptomic and chromatin accessibility assays using this rapid protocol. COMPARISON WITH EXISTING METHODS: Our method allows for rapid enrichment of glial nuclei populations from the mouse brain with minimal processing steps, while still providing high quality RNA and chromatin required for reliable omics analyses. CONCLUSIONS: We provide a reproducible method to obtain nucleic material from glial cells in the mouse brain with a quick and limited sample preparation.

Atrx Deletion in Neurons Leads to Sexually Dimorphic Dysregulation of miR-137 and Spatial Learning and Memory Deficits.

ATRX gene mutations have been identified in syndromic and non-syndromic intellectual disabilities in humans. ATRX is known to maintain genomic stability in neuroprogenitor cells, but its function in differentiated neurons and memory processes remains largely unresolved. Here, we show that the deletion of neuronal Atrx in mice leads to distinct hippocampal structural defects, fewer presynaptic vesicles, and an enlarged postsynaptic area at CA1 apical dendrite-axon junctions. We identify male-specific impairments in long-term contextual memory and in synaptic gene expression, linked to altered miR-137 levels. We show that ATRX directly binds to the miR-137 locus and that the enrichment of the suppressive histone mark H3K27me3 is significantly reduced upon the loss of ATRX. We conclude that the ablation of ATRX in excitatory forebrain neurons leads to sexually dimorphic effects on miR-137 expression and on spatial memory, identifying a potential therapeutic target for neurological defects caused by ATRX dysfunction.

Effects of a postnatal Atrx conditional knockout in neurons on autism-like behaviours in male and female mice.

BACKGROUND: Alpha-thalassemia/mental retardation, X-linked, or ATRX, is an autism susceptibility gene that encodes a chromatin remodeler. Mutations of ATRX result in the ATR-X intellectual disability syndrome and have been identified in autism spectrum disorder (ASD) patients. The mechanisms by which ATRX mutations lead to autism and autistic-like behaviours are not yet known. To address this question, we generated mice with postnatal Atrx inactivation in excitatory neurons of the forebrain and performed a battery of behavioural assays that assess autistic-like behaviours. METHODS: Male and female mice with a postnatal conditional ablation of ATRX were generated using the Cre/lox system under the control of the αCaMKII gene promoter. These mice were tested in a battery of behavioural tests that assess autistic-like features. We utilized paradigms that measure social behaviour, repetitive, and stereotyped behaviours, as well as sensory gating. Statistics were calculated by two-way repeated measures ANOVA with Sidak's multiple comparison test or unpaired Student's t tests as indicated. RESULTS: The behaviour tests revealed no significant differences between Atrx-cKO and control mice. We identified sexually dimorphic changes in odor habituation and discrimination; however, these changes did not correlate with social deficits. CONCLUSION: The postnatal knockout of Atrx in forebrain excitatory neurons does not lead to autism-related behaviours in male or female mice.

Histone deacetylase 1 and 2 drive differentiation and fusion of progenitor cells in human placental trophoblasts.

Cell fusion occurs when several cells combine to form a multinuclear aggregate (syncytium). In human placenta, a syncytialized trophoblast (syncytiotrophoblast) layer forms the primary interface between maternal and fetal tissue, facilitates nutrient and gas exchange, and produces hormones vital for pregnancy. Syncytiotrophoblast development occurs by differentiation of underlying progenitor cells called cytotrophoblasts, which then fuse into the syncytiotrophoblast layer. Differentiation is associated with chromatin remodeling and specific changes in gene expression mediated, at least in part, by histone acetylation. However, the epigenetic regulation of human cytotrophoblast differentiation and fusion is poorly understood. In this study, we found that human syncytiotrophoblast development was associated with deacetylation of multiple core histone residues. Chromatin immunoprecipitation sequencing revealed chromosomal regions that exhibit dynamic alterations in histone H3 acetylation during differentiation. These include regions containing genes classically associated with cytotrophoblast differentiation (TEAD4, TP63, OVOL1, CGB), as well as near genes with novel regulatory roles in trophoblast development and function, such as LHX4 and SYDE1. Prevention of histone deacetylation using both pharmacological and genetic approaches inhibited trophoblast fusion, supporting a critical role of this process for trophoblast differentiation. Finally, we identified the histone deacetylases (HDACs) HDAC1 and HDAC2 as the critical mediators driving cytotrophoblast differentiation. Collectively, these findings provide novel insights into the epigenetic mechanisms underlying trophoblast fusion during human placental development.

Inactivation of ATRX in forebrain excitatory neurons affects hippocampal synaptic plasticity.

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.

CTCF Governs the Identity and Migration of MGE-Derived Cortical Interneurons.

The CCCTC-binding factor (CTCF) is a central regulator of chromatin topology recently linked to neurodevelopmental disorders such as intellectual disability, autism, and schizophrenia. The aim of this study was to identify novel roles of CTCF in the developing mouse brain. We provide evidence that CTCF is required for the expression of the LIM homeodomain factor LHX6 involved in fate determination of cortical interneurons (CINs) that originate in the medial ganglionic eminence (MGE). Conditional Ctcf ablation in the MGE of mice of either sex leads to delayed tangential migration, abnormal distribution of CIN in the neocortex, a marked reduction of CINs expressing parvalbumin and somatostatin (Sst), and an increased number of MGE-derived cells expressing Lhx8 and other markers of basal forebrain projection neurons. Likewise, Ctcf-null MGE cells transplanted into the cortex of wild-type hosts generate fewer Sst-expressing CINs and exhibit lamination defects that are efficiently rescued upon reexpression of LHX6. Collectively, these data indicate that CTCF regulates the dichotomy between Lhx6 and Lhx8 to achieve correct specification and migration of MGE-derived CINs.SIGNIFICANCE STATEMENT This work provides evidence that CCCTC-binding factor (CTCF) controls an early fate decision point in the generation of cortical interneurons mediated at least in part by Lhx6. Importantly, the abnormalities described could reflect early molecular and cellular events that contribute to human neurological disorders previously linked to CTCF, including schizophrenia, autism, and intellectual disability.

Inactivation of hepatic ATRX in Atrx Foxg1cre mice prevents reversal of aging-like phenotypes by thyroxine.

ATRX is an ATP-dependent chromatin remodeler required for the maintenance of genomic integrity. We previously reported that conditional Atrx ablation in the mouse embryonic forebrain and anterior pituitary using the Foxg1cre driver causes reduced health and lifespan. In these mice, premature aging-like phenotypes were accompanied by low circulating levels of insulin-like growth factor 1 (IGF-1) and thyroxine (T4), hormones that maintain stem cell pools and normal metabolic profiles, respectively. Based on emerging evidence that T4 stimulates expression of IGF-1 in pre-pubertal mice, we tested whether T4 supplementation in Atrx Foxg1cre mice could restore IGF-1 levels and ameliorate premature aging-like phenotypes. Despite restoration of normal serum T4 levels, we did not observe improvements in circulating IGF-1. In the liver, thyroid hormone target genes were differentially affected upon T4 treatment, with Igf1 and several other thyroid hormone responsive genes failing to recover normal expression levels. These findings hinted at Cre-mediated Atrx inactivation in the liver of Atrx Foxg1cre mice, which we confirmed. We conclude that the phenotypes observed in the Atrx Foxg1cre mice can be explained in part by a role of ATRX in the liver to promote T4-mediated Igf1 expression, thus explaining the inefficacy of T4 therapy observed in this study.

Epigenetic Etiology of Intellectual Disability.

Intellectual disability (ID) is a prevailing neurodevelopmental condition associated with impaired cognitive and adaptive behaviors. Many chromatin-modifying enzymes and other epigenetic regulators have been genetically associated with ID disorders (IDDs). Here we review how alterations in the function of histone modifiers, chromatin remodelers, and methyl-DNA binding proteins contribute to neurodevelopmental defects and altered brain plasticity. We also discuss how progress in human genetics has led to the generation of mouse models that unveil the molecular etiology of ID, and outline the direction in which this field is moving to identify therapeutic strategies for IDDs. Importantly, because the chromatin regulators linked to IDDs often target common downstream genes and cellular processes, the impact of research in individual syndromes goes well beyond each syndrome and can also contribute to the understanding and therapy of other IDDs. Furthermore, the investigation of these disorders helps us to understand the role of chromatin regulators in brain development, plasticity, and gene expression, thereby answering fundamental questions in neurobiology.

Identification of epigenetic signature associated with alpha thalassemia/mental retardation X-linked syndrome.

BACKGROUND: Alpha thalassemia/mental retardation X-linked syndrome (ATR-X) is caused by a mutation at the chromatin regulator gene ATRX. The mechanisms involved in the ATR-X pathology are not completely understood, but may involve epigenetic modifications. ATRX has been linked to the regulation of histone H3 and DNA methylation, while mutations in the ATRX gene may lead to the downstream epigenetic and transcriptional effects. Elucidating the underlying epigenetic mechanisms altered in ATR-X will provide a better understanding about the pathobiology of this disease, as well as provide novel diagnostic biomarkers. RESULTS: We performed genome-wide DNA methylation assessment of the peripheral blood samples from 18 patients with ATR-X and compared it to 210 controls. We demonstrated the evidence of a unique and highly specific DNA methylation "epi-signature" in the peripheral blood of ATRX patients, which was corroborated by targeted bisulfite sequencing experiments. Although genomically represented, differentially methylated regions showed evidence of preferential clustering in pericentromeric and telometric chromosomal regions, areas where ATRX has multiple functions related to maintenance of heterochromatin and genomic integrity. CONCLUSION: Most significant methylation changes in the 14 genomic loci provide a unique epigenetic signature for this syndrome that may be used as a highly sensitive and specific diagnostic biomarker to support the diagnosis of ATR-X, particularly in patients with phenotypic complexity and in patients with ATRX gene sequence variants of unknown significance.

Mosaic expression of Atrx in the mouse central nervous system causes memory deficits.

The rapid modulation of chromatin organization is thought to play a crucial role in cognitive processes such as memory consolidation. This is supported in part by the dysregulation of many chromatin-remodelling proteins in neurodevelopmental and psychiatric disorders. A key example is ATRX, an X-linked gene commonly mutated in individuals with syndromic and nonsyndromic intellectual disability. The consequences of Atrx inactivation for learning and memory have been difficult to evaluate because of the early lethality of hemizygous-null animals. In this study, we evaluated the outcome of brain-specific Atrx deletion in heterozygous female mice. These mice exhibit a mosaic pattern of ATRX protein expression in the central nervous system attributable to the location of the gene on the X chromosome. Although the hemizygous male mice die soon after birth, heterozygous females survive to adulthood. Body growth is stunted in these animals, and they have low circulating concentrations of insulin growth factor 1. In addition, they are impaired in spatial, contextual fear and novel object recognition memory. Our findings demonstrate that mosaic loss of ATRX expression in the central nervous system leads to endocrine defects and decreased body size and has a negative impact on learning and memory.

Emerging roles of ATRX in cancer.

ATRX was identified over 20 years ago as the gene responsible for a rare developmental disorder characterized by α-thalassemia and intellectual disability. Similarities to the sucrose nonfermentable SNF2 type chromatin remodelers initially suggested a role in transcriptional regulation. However, over the last years, our knowledge of the epigenetic activities of ATRX has expanded steadily. Recent exciting discoveries have propelled ATRX into the limelight of chromatin and telomere biology, development and cancer research. This review summarizes recent breakthroughs in understanding ATRX function in heterochromatin structure, genome stability and its frequent dysregulation in a variety of cancers.

ATRX promotes gene expression by facilitating transcriptional elongation through guanine-rich coding regions.

ATRX is a chromatin remodeling protein involved in deposition of the histone variant H3.3 at telomeres and pericentromeric heterochromatin. It also influences the expression level of specific genes; however, deposition of H3.3 at transcribed genes is currently thought to occur independently of ATRX. We focused on a set of genes, including the autism susceptibility gene Neuroligin 4 (Nlgn4), that exhibit decreased expression in ATRX-null cells to investigate the mechanisms used by ATRX to promote gene transcription. Overall TERRA levels, as well as DNA methylation and histone modifications at ATRX target genes are not altered and thus cannot explain transcriptional dysregulation. We found that ATRX does not associate with the promoter of these genes, but rather binds within regions of the gene body corresponding to high H3.3 occupancy. These intragenic regions consist of guanine-rich DNA sequences predicted to form non-B DNA structures called G-quadruplexes during transcriptional elongation. We demonstrate that ATRX deficiency corresponds to reduced H3.3 incorporation and stalling of RNA polymerase II at these G-rich intragenic sites. These findings suggest that ATRX promotes the incorporation of histone H3.3 at particular transcribed genes and facilitates transcriptional elongation through G-rich sequences. The inability to transcribe genes such as Nlgn4 could cause deficits in neuronal connectivity and cognition associated with ATRX mutations in humans.

ATRX is required for maintenance of the neuroprogenitor cell pool in the embryonic mouse brain.

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.

Analysis of neonatal brain lacking ATRX or MeCP2 reveals changes in nucleosome density, CTCF binding and chromatin looping.

ATRX and MeCP2 belong to an expanding group of chromatin-associated proteins implicated in human neurodevelopmental disorders, although their gene-regulatory activities are not fully resolved. Loss of ATRX prevents full repression of an imprinted gene network in the postnatal brain and in this study we address the mechanistic aspects of this regulation. We show that ATRX binds many imprinted domains individually but that transient co-localization between imprinted domains in the nuclei of neurons does not require ATRX. We demonstrate that MeCP2 is required for ATRX recruitment and that deficiency of either ATRX or MeCP2 causes decreased frequency of long-range chromatin interactions associated with altered nucleosome density at CTCF-binding sites and reduced CTCF occupancy. These findings indicate that MeCP2 and ATRX regulate gene expression at a subset of imprinted domains by maintaining a nucleosome configuration conducive to CTCF binding and to the maintenance of higher order chromatin structure.

Implantation failure in female Kiss1-/- mice is independent of their hypogonadic state and can be partially rescued by leukemia inhibitory factor.

The hypothalamic kisspeptin signaling system is a major positive regulator of the reproductive neuroendocrine axis, and loss of Kiss1 in the mouse results in infertility, a condition generally attributed to its hypogonadotropic hypogonadism. We demonstrate that in Kiss1(-/-) female mice, acute replacement of gonadotropins and estradiol restores ovulation, mating, and fertilization; however, these mice are still unable to achieve pregnancy because embryos fail to implant. Progesterone treatment did not overcome this defect. Kiss1(+/-) embryos transferred to a wild-type female mouse can successfully implant, demonstrating the defect is due to maternal factors. Kisspeptin and its receptor are expressed in the mouse uterus, and we suggest that it is the absence of uterine kisspeptin signaling that underlies the implantation failure. This absence, however, does not prevent the closure of the uterine implantation chamber, proper alignment of the embryo, and the ability of the uterus to undergo decidualization. Instead, the loss of Kiss1 expression specifically disrupts embryo attachment to the uterus. We observed that on the day of implantation, leukemia inhibitory factor (Lif), a cytokine that is absolutely required for implantation in mice, is weakly expressed in Kiss1(-/-) uterine glands and that the administration of exogenous Lif to hormone-primed Kiss1(-/-) female mice is sufficient to partially rescue implantation. Taken together, our study reveals that uterine kisspeptin signaling regulates glandular Lif levels, thereby identifying a novel and critical role for kisspeptin in regulating embryo implantation in the mouse. This study provides compelling reasons to explore this role in other species, particularly livestock and humans.

Dual effect of CTCF loss on neuroprogenitor differentiation and survival.

An increasing number of proteins involved in genome organization have been implicated in neurodevelopmental disorders, highlighting the importance of chromatin architecture in the developing CNS. The CCCTC-binding factor (CTCF) is a zinc finger DNA binding protein involved in higher-order chromatin organization, and mutations in the human CTCF gene cause an intellectual disability syndrome associated with microcephaly. However, information on CTCF function in vivo in the developing brain is lacking. To address this gap, we conditionally inactivated the Ctcf gene at early stages of mouse brain development. Cre-mediated Ctcf deletion in the telencephalon and anterior retina at embryonic day 8.5 triggered upregulation of the p53 effector PUMA (p53 upregulated modulator of apoptosis), resulting in massive apoptosis and profound ablation of telencephalic structures. Inactivation of Ctcf several days later at E11 also resulted in PUMA upregulation and increased apoptotic cell death, and the Ctcf-null forebrain was hypocellular and disorganized at birth. Although deletion of both Ctcf and Puma in the embryonic brain efficiently rescued Ctcf-null progenitor cell apoptosis, it failed to improve neonatal hypocellularity due to decreased proliferative capacity of rescued apical and outer radial glia progenitor cells. This was exacerbated by an independent effect of CTCF loss that resulted in depletion of the progenitor pool due to premature neurogenesis earlier in development. Our findings demonstrate that CTCF activities are required for two distinct events in early cortex formation: first, to correctly regulate the balance between neuroprogenitor cell proliferation and differentiation, and second, for the survival of neuroprogenitor cells, providing new clues regarding the contributions of CTCF in microcephaly/intellectual disability syndrome pathologies.

Three dimensional dual labelled DNA fluorescent in situ hybridization analysis in fixed tissue sections.

Emerging studies demonstrate that three-dimensional organization of chromatin in the nucleus plays a vital role in regulating the genome. DNA fluorescent in situ hybridization (FISH) is a common molecular technique used to visualize the location of DNA sequences. The vast majority of DNA FISH studies are conducted on cultured cells due to the technical difficulties encountered using fixed tissue sections. However, the use of cultured cells poses important limitations that could yield misleading results, making in vivo analysis a far superior approach. Here we present a protocol for multiplexed three dimensional DNA FISH in mouse brain sections, which is also applicable to other tissues. Paraffin-embedded tissues could be used but the embedding and preparation of the samples is time-consuming and often associated with poor antigenicity. To overcome this problem we:•developed a FISH technique using fixed, frozen cryosections;•provide specific instructions for tissue processing for proper fixation and freezing, including equilibration in sucrose gradients to maintain proper cellular structure;•include optimized permeabilization and washing steps to achieve specific signal and to limit background fluorescence in tissue sections.

Targeted loss of the ATR-X syndrome protein in the limb mesenchyme of mice causes brachydactyly.

ATR-X syndrome is a rare genetic disorder caused by mutations in the ATRX gene. Affected individuals are cognitively impaired and display a variety of developmental abnormalities, including skeletal deformities. To investigate the function of ATRX during skeletal development, we selectively deleted the gene in the developing forelimb mesenchyme of mice. The absence of ATRX in the limb mesenchyme resulted in shorter digits, or brachydactyly, a defect also observed in a subset of ATR-X patients. This phenotype persisted until adulthood, causing reduced grip strength and altered gait in mutant mice. Examination of the embryonic ATRX-null forelimbs revealed a significant increase in apoptotic cell death, which could explain the reduced digit length. In addition, staining for the DNA damage markers γ-histone 2A family member X (γ-H2AX) and 53BP1 demonstrated a significant increase in the number of cells with DNA damage in the embryonic ATRX-null forepaw. Strikingly, only one large bright DNA damage event was observed per nucleus in proliferating cells. These large γ-H2AX foci were located in close proximity to the nuclear lamina and remained largely unresolved after cell differentiation. In addition, ATRX-depleted forelimb mesenchymal cells did not exhibit hypersensitivity to DNA fork-stalling compounds, suggesting that the nature as well as the response to DNA damage incurred by loss of ATRX in the developing limb fundamentally differs from other tissues. Our data suggest that DNA damage-induced apoptosis is a novel cellular mechanism underlying brachydactyly that might be relevant to additional skeletal syndromes.