Conditional c-MYC activation in catecholaminergic cells drives distinct neuroendocrine tumors: neuroblastoma vs somatostatinoma.

The MYC proto-oncogenes (c-MYC, MYCN , MYCL ) are among the most deregulated oncogenic drivers in human malignancies including high-risk neuroblastoma, 50% of which are MYCN -amplified. Genetically engineered mouse models (GEMMs) based on the MYCN transgene have greatly expanded the understanding of neuroblastoma biology and are powerful tools for testing new therapies. However, a lack of c-MYC-driven GEMMs has hampered the ability to better understand mechanisms of neuroblastoma oncogenesis and therapy development given that c-MYC is also an important driver of many high-risk neuroblastomas. In this study, we report two transgenic murine neuroendocrine models driven by conditional c-MYC induction in tyrosine hydroxylase (Th) and dopamine ß-hydroxylase (Dbh)-expressing cells. c-MYC induction in Th-expressing cells leads to a preponderance of Pdx1 + somatostatinomas, a type of pancreatic neuroendocrine tumor (PNET), resembling human somatostatinoma with highly expressed gene signatures of δ cells and potassium channels. In contrast, c-MYC induction in Dbh-expressing cells leads to onset of neuroblastomas, showing a better transforming capacity than MYCN in a comparable C57BL/6 genetic background. The c-MYC murine neuroblastoma tumors recapitulate the pathologic and genetic features of human neuroblastoma, express GD2, and respond to anti-GD2 immunotherapy. This model also responds to DFMO, an FDA-approved inhibitor targeting ODC1, which is a known MYC transcriptional target. Thus, establishing c-MYC-overexpressing GEMMs resulted in different but related tumor types depending on the targeted cell and provide useful tools for testing immunotherapies and targeted therapies for these diseases.

PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS.

The plant homeodomain zinc-finger protein, PHF6, is a transcriptional regulator, and PHF6 germline mutations cause the X-linked intellectual disability (XLID) Börjeson-Forssman-Lehmann syndrome (BFLS). The mechanisms by which PHF6 regulates transcription and how its mutations cause BFLS remain poorly characterized. Here, we show genome-wide binding of PHF6 in the developing cortex in the vicinity of genes involved in central nervous system development and neurogenesis. Characterization of BFLS mice harbouring PHF6 patient mutations reveals an increase in embryonic neural stem cell (eNSC) self-renewal and a reduction of neural progenitors. We identify a panel of Ephrin receptors (EphRs) as direct transcriptional targets of PHF6. Mechanistically, we show that PHF6 regulation of EphR is impaired in BFLS mice and in conditional Phf6 knock-out mice. Knockdown of EphR-A phenocopies the PHF6 loss-of-function defects in altering eNSCs, and its forced expression rescues defects of BFLS mice-derived eNSCs. Our data indicate that PHF6 directly promotes Ephrin receptor expression to control eNSC behaviour in the developing brain, and that this pathway is impaired in BFLS.

Personal journeys to and in human genetics and dysmorphology.

Genetics has become a critical component of medicine over the past five to six decades. Alongside genetics, a relatively new discipline, dysmorphology, has also begun to play an important role in providing critically important diagnoses to individuals and families. Both have become indispensable to unraveling rare diseases. Almost every medical specialty relies on individuals experienced in these specialties to provide diagnoses for patients who present themselves to other doctors. Additionally, both specialties have become reliant on molecular geneticists to identify genes associated with human disorders. Many of the medical geneticists, dysmorphologists, and molecular geneticists traveled a circuitous route before arriving at the position they occupied. The purpose of collecting the memoirs contained in this article was to convey to the reader that many of the individuals who contributed to the advancement of genetics and dysmorphology since the late 1960s/early 1970s traveled along a journey based on many chances taken, replying to the necessities they faced along the way before finding full enjoyment in the practice of medical and human genetics or dysmorphology. Additionally, and of equal importance, all exhibited an ability to evolve with their field of expertise as human genetics became human genomics with the development of novel technologies.

Pathogenic variants in SMARCA1 cause an X-linked neurodevelopmental disorder modulated by NURF complex composition.

Pathogenic variants in ATP-dependent chromatin remodeling proteins are a recurrent cause of neurodevelopmental disorders (NDDs). The NURF complex consists of BPTF and either the SNF2H (SMARCA5) or SNF2L (SMARCA1) ISWI-chromatin remodeling enzyme. Pathogenic variants in BPTF and SMARCA5 were previously implicated in NDDs. Here, we describe 40 individuals from 30 families with de novo or maternally inherited pathogenic variants in SMARCA1. This novel NDD was associated with mild to severe ID/DD, delayed or regressive speech development, and some recurrent facial dysmorphisms. Individuals carrying SMARCA1 loss-of-function variants exhibited a mild genome-wide DNA methylation profile and a high penetrance of macrocephaly. Genetic dissection of the NURF complex using Smarca1, Smarca5, and Bptfsingle and double mouse knockouts revealed the importance of NURF composition and dosage for proper forebrain development. Finally, we propose that genetic alterations affecting different NURF components result in a NDD with a broad clinical spectrum.

A new mouse model of ATR-X syndrome carrying a common patient mutation exhibits neurological and morphological defects.

ATRX is a chromatin remodelling ATPase that is involved in transcriptional regulation, DNA damage repair and heterochromatin maintenance. It has been widely studied for its role in ALT-positive cancers, but its role in neurological function remains elusive. Hypomorphic mutations in the X-linked ATRX gene cause a rare form of intellectual disability combined with alpha-thalassemia called ATR-X syndrome in hemizygous males. Clinical features also include facial dysmorphism, microcephaly, short stature, musculoskeletal defects and genital abnormalities. As complete deletion of ATRX in mice results in early embryonic lethality, the field has largely relied on conditional knockout models to assess the role of ATRX in multiple tissues. Given that null alleles are not found in patients, a more patient-relevant model was needed. Here, we have produced and characterized the first patient mutation knock-in model of ATR-X syndrome, carrying the most common causative mutation, R246C. This is one of a cluster of missense mutations located in the chromatin-binding domain and disrupts its function. The knock-in mice recapitulate several aspects of the patient disorder, including craniofacial defects, microcephaly, reduced body size and impaired neurological function. They provide a powerful model for understanding the molecular mechanisms underlying ATR-X syndrome and testing potential therapeutic strategies.

Novel genetically engineered H3.3G34R model reveals cooperation with ATRX loss in upregulation of Hoxa cluster genes and promotion of neuronal lineage.

Background: Pediatric high-grade gliomas (pHGGs) are aggressive pediatric CNS tumors and an important subset are characterized by mutations in H3F3A, the gene that encodes Histone H3.3 (H3.3). Substitution of Glycine at position 34 of H3.3 with either Arginine or Valine (H3.3G34R/V), was recently described and characterized in a large cohort of pHGG samples as occurring in 5-20% of pHGGs. Attempts to study the mechanism of H3.3G34R have proven difficult due to the lack of knowledge regarding the cell-of-origin and the requirement for co-occurring mutations for model development. We sought to develop a biologically relevant animal model of pHGG to probe the downstream effects of the H3.3G34R mutation in the context of vital co-occurring mutations. Methods: We developed a genetically engineered mouse model (GEMM) that incorporates PDGF-A activation, TP53 loss and the H3.3G34R mutation both in the presence and loss of Alpha thalassemia/mental retardation syndrome X-linked (ATRX), which is commonly mutated in H3.3G34 mutant pHGGs. Results: We demonstrated that ATRX loss significantly increases tumor latency in the absence of H3.3G34R and inhibits ependymal differentiation in the presence of H3.3G34R. Transcriptomic analysis revealed that ATRX loss in the context of H3.3G34R upregulates Hoxa cluster genes. We also found that the H3.3G34R overexpression leads to enrichment of neuronal markers but only in the context of ATRX loss. Conclusions: This study proposes a mechanism in which ATRX loss is the major contributor to many key transcriptomic changes in H3.3G34R pHGGs. Accession number: GSE197988.

VGF is required for recovery after focal stroke.

The high incidence of ischemic stroke worldwide and poor efficacy of neuroprotective drugs has increased the need for novel therapies in stroke recovery. Transcription of the neurosecretory protein VGF (non-acronym) is enhanced following ischemic stroke and proposed to be important for stroke recovery. To determine the requirement for VGF in recovery, we created Vgffl/fl:Nestin-Cre conditional knockout (Vgf cKO) mice and induced a photothrombotic focal ischemic stroke. Naïve Vgf cKO mice had significant less body weight in the absence of gross defects in brain size, cortical lamination, or deficits in locomotor activity compared to wildtype controls. Following a focal stroke, the Vgf cKO mice had greater deficits including impaired recovery of forepaw motor deficits at 2- and 4-weeks post stroke. The increase in deficits occurred in the absence of any difference in lesion size and was accompanied by a striking loss of stroke-induced migration of SVZ-derived immature neurons to the peri-infarct region. Importantly, exogenous adenoviral delivery of VGF (AdVGF) significantly improved recovery in the Vgf cKO mice and was able to rescue the immature neuron migration defect observed. Taken together, our results define a requirement for VGF in post stroke recovery and identify VGF peptides as a potential future therapeutic.

Generation of a mouse model of the neurodevelopmental disorder with dysmorphic facies and distal limb anomalies syndrome.

Heterozygous variants in bromodomain and plant homeodomain containing transcription factor (BPTF) cause the neurodevelopmental disorder with dysmorphic facies and distal limb anomalies (NEDDFL) syndrome (MIM#617755) characterized by intellectual disability, speech delay and postnatal microcephaly. BPTF functions within nucleosome and remodeling factor (NURF), a complex comprising sucrose non-fermenting like (SNF2L), an Imitation SWItching (ISWI) chromatin remodeling protein encoded by the SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 1 (SMARCA1) gene. Surprisingly, ablation of Smarca1 resulted in mice with enlarged brains, a direct contrast to the phenotype of NEDDFL patients. To model the NEDDFL syndrome, we generated forebrain-specific Bptf knockout (Bptf conditional Knockout (cKO)) mice. The Bptf cKO mice were born in normal Mendelian ratios, survived to adulthood but were smaller in size with severe cortical hypoplasia. Prolonged progenitor cell cycle length and a high incidence of cell death reduced the neuronal output. Cortical lamination was also disrupted with reduced proportions of deep layer neurons, and neuronal maturation defects that impaired the acquisition of distinct cell fates (e.g. COUP-TF-interacting protein 2 (Ctip2)+ neurons). RNAseq and pathway analysis identified altered expression of fate-determining transcription factors and the biological pathways involved in neural development, apoptotic signaling and amino acid biosynthesis. Dysregulated genes were enriched for MYC Proto-Oncogene, BHLH Transcription Factor (Myc)-binding sites, a known BPTF transcriptional cofactor. We propose the Bptf cKO mouse as a valuable model for further study of the NEDDFL syndrome.

Impaired SNF2L Chromatin Remodeling Prolongs Accessibility at Promoters Enriched for Fos/Jun Binding Sites and Delays Granule Neuron Differentiation.

Chromatin remodeling proteins utilize the energy from ATP hydrolysis to mobilize nucleosomes often creating accessibility for transcription factors within gene regulatory elements. Aberrant chromatin remodeling has diverse effects on neuroprogenitor homeostasis altering progenitor competence, proliferation, survival, or cell fate. Previous work has shown that inactivation of the ISWI genes, Smarca5 (encoding Snf2h) and Smarca1 (encoding Snf2l) have dramatic effects on brain development. Smarca5 conditional knockout mice have reduced progenitor expansion and severe forebrain hypoplasia, with a similar effect on the postnatal growth of the cerebellum. In contrast, Smarca1 mutants exhibited enlarged forebrains with delayed progenitor differentiation and increased neuronal output. Here, we utilized cerebellar granule neuron precursor (GNP) cultures from Smarca1 mutant mice (Ex6DEL) to explore the requirement for Snf2l on progenitor homeostasis. The Ex6DEL GNPs showed delayed differentiation upon plating that was not attributed to changes in the Sonic Hedgehog pathway but was associated with overexpression of numerous positive effectors of proliferation, including targets of Wnt activation. Transcriptome analysis identified increased expression of Fosb and Fosl2 while ATACseq experiments identified a large increase in chromatin accessibility at promoters many enriched for Fos/Jun binding sites. Nonetheless, the elevated proliferation index was transient and the Ex6DEL cultures initiated differentiation with a high concordance in gene expression changes to the wild type cultures. Genes specific to Ex6DEL differentiation were associated with an increased activation of the ERK signaling pathway. Taken together, this data provides the first indication of how Smarca1 mutations alter progenitor cell homeostasis and contribute to changes in brain size.

Transgenic mice with an R342X mutation in Phf6 display clinical features of Börjeson-Forssman-Lehmann Syndrome.

The PHF6 mutation c.1024C > T; p.R342X, is a recurrent cause of Börjeson-Forssman-Lehmann Syndrome (BFLS), a neurodevelopmental disorder characterized by moderate-severe intellectual disability, truncal obesity, gynecomastia, hypogonadism, long tapering fingers and large ears (MIM#301900). Here, we generated transgenic mice with the identical substitution (R342X mice) using CRISPR technology. We show that the p.R342X mutation causes a reduction in PHF6 protein levels, in both human and mice, from nonsense-mediated decay and nonsense-associated alternative splicing, respectively. Magnetic resonance imaging studies indicated that R342X mice had a reduced brain volume on a mixed genetic background but developed hydrocephaly and a high incidence of postnatal death on a C57BL/6 background. Cortical development proceeded normally, while hippocampus and hypothalamus relative brain volumes were altered. A hypoplastic anterior pituitary was also observed that likely contributes to the small size of the R342X mice. Behavior testing demonstrated deficits in associative learning, spatial memory and an anxiolytic phenotype. Taken together, the R342X mice represent a good preclinical model of BFLS that will allow further dissection of PHF6 function and disease pathogenesis.

Sensory Experience Modulates Atrx-mediated Neuronal Integrity in the Mouse Retina.

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 (OP) 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.

Neurodevelopmental Disorders Caused by Defective Chromatin Remodeling: Phenotypic Complexity Is Highlighted by a Review of ATRX Function.

The ability to determine the genetic etiology of intellectual disability (ID) and neurodevelopmental disorders (NDD) has improved immensely over the last decade. One prevailing metric from these studies is the large percentage of genes encoding epigenetic regulators, including many members of the ATP-dependent chromatin remodeling enzyme family. Chromatin remodeling proteins can be subdivided into five classes that include SWI/SNF, ISWI, CHD, INO80, and ATRX. These proteins utilize the energy from ATP hydrolysis to alter nucleosome positioning and are implicated in many cellular processes. As such, defining their precise roles and contributions to brain development and disease pathogenesis has proven to be complex. In this review, we illustrate that complexity by reviewing the roles of ATRX on genome stability, replication, and transcriptional regulation and how these mechanisms provide key insight into the phenotype of ATR-X patients.

Correction to: C3aR signaling and gliosis in response to neurodevelopmental damage in the cerebellum.

Following publication of the original article, the authors noticed missing labels in Fig. 1a.

ATRX affects the repair of telomeric DSBs by promoting cohesion and a DAXX-dependent activity.

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.

Snf2h Drives Chromatin Remodeling to Prime Upper Layer Cortical Neuron Development.

Alterations in the homeostasis of either cortical progenitor pool, namely the apically located radial glial (RG) cells or the basal intermediate progenitors (IPCs) can severely impair cortical neuron production. Such changes are reflected by microcephaly and are often associated with cognitive defects. Genes encoding epigenetic regulators are a frequent cause of intellectual disability and many have been shown to regulate progenitor cell growth, including our inactivation of the Smarca1 gene encoding Snf2l, which is one of two ISWI mammalian orthologs. Loss of the Snf2l protein resulted in dysregulation of Foxg1 and IPC proliferation leading to macrocephaly. Here we show that inactivation of the closely related Smarca5 gene encoding the Snf2h chromatin remodeler is necessary for embryonic IPC expansion and subsequent specification of callosal projection neurons. Telencephalon-specific Smarca5 cKO embryos have impaired cell cycle kinetics and increased cell death, resulting in fewer Tbr2+ and FoxG1+ IPCs by mid-neurogenesis. These deficits give rise to adult mice with a dramatic reduction in Satb2+ upper layer neurons, and partial agenesis of the corpus callosum. Mice survive into adulthood but molecularly display reduced expression of the clustered protocadherin genes that may further contribute to altered dendritic arborization and a hyperactive behavioral phenotype. Our studies provide novel insight into the developmental function of Snf2h-dependent chromatin remodeling processes during brain development.

C3aR signaling and gliosis in response to neurodevelopmental damage in the cerebellum.

BACKGROUND: Conditional ablation of the Smarca5 gene in mice severely impairs the postnatal growth of the cerebellum and causes an ataxic phenotype. Comparative gene expression studies indicated that complement-related proteins were upregulated in the cerebellum of Smarca5 mutant mice. Complement proteins play critical roles within innate immune signaling pathways and, in the brain, are produced by glial cells under both normal and pathological conditions. The C3 complement protein-derived signaling peptide, C3a, has been implicated in contributing to both tissue damage and repair in conditions such as multiple sclerosis and stroke. Here, we investigated whether C3a receptor (C3aR) signaling promoted damage or repair in the developing cerebellum of Smarca5 mutant mice. METHODS: Brain and cerebellum lysates from single Smarca5 conditional knockout (Smarca5 cKO) mice, C3aR1 KO mice, or double mutant mice were used for qRT-PCR and immunoblotting to assess the contribution of C3aR to the Smarca5 cKO brain pathology. Immunohistochemistry was used to characterize alterations to astroglia and phagocyte cells in the developing cerebellum of each of the genotypes. RESULTS: C3aR signaling was observed to limit gliosis and promote granule neuron survival during postnatal cerebellar development. In Smarca5 cKO mice, disorganized astroglia with increased GFAP expression develops concurrently with cerebellar granule neuron loss and phagocyte invasion over the first 10 days following birth. Potential ligand precursors of C3aR-VGF and C3-were found to have upregulated expression and/or altered processing during this time. Phagocytes (microglia and macrophages) in both the control and Smarca5 mutant mice were the only cells observed to express C3aR. Loss of C3aR in the Smarca5 cKO cerebellum resulted in increased numbers of apoptotic cells and early phagocyte invasion into the external granule cell layer, as well as an exacerbated disorganization of the Bergmann glia. The loss of C3aR expression also attenuated an increase in the expression of the efferocytosis-related protein, MerTK, whose transcript was upregulated ~ 2.5-fold in the Smarca5 mutant cerebellum at P10. CONCLUSIONS: This data indicates that C3aR can play an important role in limiting astrogliosis and regulating phagocyte phenotypes following developmental cell loss in the brain.

Atrx inactivation drives disease-defining phenotypes in glioma cells of origin through global epigenomic remodeling.

Mutational inactivation of the SWI/SNF chromatin regulator ATRX occurs frequently in gliomas, the most common primary brain tumors. Whether and how ATRX deficiency promotes oncogenesis by epigenomic dysregulation remains unclear, despite its recent implication in both genomic instability and telomere dysfunction. Here we report that Atrx loss recapitulates characteristic disease phenotypes and molecular features in putative glioma cells of origin, inducing cellular motility although also shifting differentiation state and potential toward an astrocytic rather than neuronal histiogenic profile. Moreover, Atrx deficiency drives widespread shifts in chromatin accessibility, histone composition, and transcription in a distribution almost entirely restricted to genomic sites normally bound by the protein. Finally, direct gene targets of Atrx that mediate specific Atrx-deficient phenotypes in vitro exhibit similarly selective misexpression in ATRX-mutant human gliomas. These findings demonstrate that ATRX deficiency and its epigenomic sequelae are sufficient to induce disease-defining oncogenic phenotypes in appropriate cellular and molecular contexts.

The role of ISWI chromatin remodeling complexes in brain development and neurodevelopmental disorders.

The mammalian ISWI (Imitation Switch) genes SMARCA1 and SMARCA5 encode the ATP-dependent chromatin remodeling proteins SNF2L and SNF2H. The ISWI proteins interact with BAZ (bromodomain adjacent to PHD zinc finger) domain containing proteins to generate eight distinct remodeling complexes. ISWI complex-mediated nucleosome positioning within genes and gene regulatory elements is proving important for the transition from a committed progenitor state to a differentiated cell state. Genetic studies have implicated the involvement of many ATP-dependent chromatin remodeling proteins in neurodevelopmental disorders (NDDs), including SMARCA1. Here we review the characterization of mice inactivated for ISWI and their interacting proteins, as it pertains to brain development and disease. A better understanding of chromatin dynamics during neural development is a prerequisite to understanding disease pathologies and the development of therapeutics for these complex disorders.

Recovery from impaired muscle growth arises from prolonged postnatal accretion of myonuclei in Atrx mutant mice.

Reduced muscle mass due to pathological development can occur through several mechanisms, including the loss or reduced proliferation of muscle stem cells. Muscle-specific ablation of the α-thalassemia mental retardation syndrome mutant protein, Atrx, in transgenic mice results in animals with a severely reduced muscle mass at three weeks of age; yet this muscle mass reduction resolves by adult age. Here, we explore the cellular mechanism underlying this effect. Analysis of Atrx mutant mice included testing for grip strength and rotorod performance. Muscle fiber length, fiber volume and numbers of myofiber-associated nuclei were determined from individual EDL or soleus myofibers isolated at three, five, or eight weeks. Myofibers from three week old Atrx mutant mice are smaller with fewer myofiber-associated nuclei and reduced volume compared to control animals, despite similar fiber numbers. Nonetheless, the grip strength of Atrx mutant mice was comparable to control mice when adjusted for body weight. Myofiber volume remained smaller at five weeks, becoming comparable to controls by 8 weeks of age. Concomitantly, increased numbers of myofiber-associated nuclei and Ki67+ myoblasts indicated that the recovery of muscle mass likely arises from the prolonged accretion of new myonuclei. This suggests that under disease conditions the muscle satellite stem cell niche can remain in a prolonged active state, allowing for the addition of a minimum number of myonuclei required to achieve a normal muscle size.