Mutations in chromatin modifier genes are frequently associated with neurodevelopmental diseases. We herein demonstrate that the chromodomain helicase DNA-binding protein 7 (Chd7), frequently ...associated with CHARGE syndrome, is indispensable for normal cerebellar development. Genetic inactivation of Chd7 in cerebellar granule neuron progenitors leads to cerebellar hypoplasia in mice, due to the impairment of granule neuron differentiation, induction of apoptosis and abnormal localization of Purkinje cells, which closely recapitulates known clinical features in the cerebella of CHARGE patients. Combinatory molecular analyses reveal that Chd7 is required for the maintenance of open chromatin and thus activation of genes essential for granule neuron differentiation. We further demonstrate that both Chd7 and Top2b are necessary for the transcription of a set of long neuronal genes in cerebellar granule neurons. Altogether, our comprehensive analyses reveal a mechanism with chromatin remodellers governing brain development via controlling a core transcriptional programme for cell-specific differentiation.
One key advantage of the CRISPR/Cas9 system in comparison with other gene editing approaches lies in its potential for multiplexing. Here, we describe an elaborate procedure that allows the assembly ...of multiple gRNA expression cassettes into a vector of choice within a single step, termed ASAP(Adaptable System for Assembly of multiplexed Plasmids)-cloning. We demonstrate the utility of ASAP-cloning for multiple CRISPR-mediated applications, including efficient multiplex gene editing, robust transcription activation and convenient analysis of Cas9 activity in the presence of multiple gRNAs.
Familial Dysautonomia (FD) is an autosomal recessive disorder caused by a splice site mutation in the gene ELP1, which disproportionally affects neurons. While classically characterized by deficits ...in sensory and autonomic neurons, neuronal defects in the central nervous system have also been described. Although ELP1 expression remains high in the normal developing and adult cerebellum, its role in cerebellar development is unknown. To explore the role of Elp1 in the cerebellum, we knocked out Elp1 in cerebellar granule cell progenitors (GCPs) and examined the outcome on animal behavior and cellular composition. We found that GCP-specific conditional knockout of Elp1 (Elp1cKO) resulted in ataxia by 8 weeks of age. Cellular characterization showed that the animals had smaller cerebella with fewer granule cells. This defect was already apparent as early as 7 days after birth, when Elp1cKO animals also had fewer mitotic GCPs and shorter Purkinje dendrites. Through molecular characterization, we found that loss of Elp1 was associated with an increase in apoptotic cell death and cell stress pathways in GCPs. Our study demonstrates the importance of ELP1 in the developing cerebellum, and suggests that loss of Elp1 in the GC lineage may also play a role in the progressive ataxia phenotypes of FD patients.
Display omitted
•High expression of ELP1/Elp1 in the developing cerebellum.•Proliferating granule cell progenitors (GCPs) express high levels of ELP1.•GCP-specific knockout of Elp1 increases apoptosis and cell cycle inhibition.•GCP-specific knockout of Elp1 reduces cerebellar size.•GCP-specific knockout of Elp1 induces ataxic phenotype.
In vivo functional investigation of oncogenes using somatic gene transfer has been successfully exploited to validate their role in tumorigenesis. For tumour suppressor genes this has proven more ...challenging due to technical aspects. To provide a flexible and effective method for investigating somatic loss-of-function alterations and their influence on tumorigenesis, we have established CRISPR/Cas9-mediated somatic gene disruption, allowing for in vivo targeting of TSGs. Here we demonstrate the utility of this approach by deleting single (Ptch1) or multiple genes (Trp53, Pten, Nf1) in the mouse brain, resulting in the development of medulloblastoma and glioblastoma, respectively. Using whole-genome sequencing (WGS) we characterized the medulloblastoma-driving Ptch1 deletions in detail and show that no off-targets were detected in these tumours. This method provides a fast and convenient system for validating the emerging wealth of novel candidate tumour suppressor genes and the generation of faithful animal models of human cancer.
Abstract
Genomic analyzes identified a subgroup of gliomas – infant-type hemispheric gliomas – that are driven by oncogenic fusions of, amongst others, ROS1 receptor tyrosine kinase with a variety of ...fusion partners. These fusions result in ligand independent constitutively active oncogenic kinases. In the present study we aim to identify the, yet poorly characterized, alterations in cellular signaling driven by ROS1 fusions leading to tumor formation to enhance our understanding of IHG formation and to identify complementary targets for therapy. To answer these questions, we deploy a combination of biochemical characterization of cellular models as well as in vivo experiments and transcriptomic and phosphoproteomic analyzes. Expression of different ROS1-fusions results in robust tumor formation in two different mouse models, proving their tumorigenic potential. The impact of the fusion partner, which dictates subcellular localization, as shown by immunofluorescence microscopy, on differences in tumor formation has not yet been established. Transcriptomic and phosphoproteomic analyses of ROS1-fusion cellular models revealed diverging effects of the fusions on transcriptomic and proteomic alterations. A common denominator, however, is a significant hyper-phosphorylation of the tyrosine phosphatase SHP2, which we established to be a direct substrate of ROS1. ROS1 fusions, irrespective of the fusion partner, drive non-canonical STAT3 signaling, indicated by direct phosphorylation of STAT3 by ROS1 and upregulation of STAT3 target genes. Characterizing the main signaling pathways using immunoblots did not detect alterations in the PI3K/AKT/mTOR or MAPK pathways. STAT3 signaling is a well characterized pathway for promoting pro-survival, proliferation and stemness cues in development and cancer, making it a paramount candidate for ROS1 driven tumorigenesis as well as an excellent target for targeted therapies.
Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have ...collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an "intrinsic" spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting
(
= 31),
(
= 21),
(
= 9), and
(
= 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management. SIGNIFICANCE: Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of
, or
gene fusions. Kinase fusion-positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype.
.
.
Advanced biological technologies allowing for genetic manipulation of the genome are increasingly being used to unravel the molecular pathogenesis of human diseases. The clustered regulatory ...interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) technology started a revolution of this field owing to its flexibility and relative ease of use. Recently, application of the CRISPR/Cas9 system has been extended to in vivo approaches, leveraging its potential for human disease modeling. Particularly in oncological research, where genetic defects in somatic cells are tightly linked to etiology and pathological phenotypes, the CRISPR/Cas technology is being used to recapitulate various types of genetic aberrations. Here we review murine cancer models that have been developed via combining the CRISPR/Cas9 technology with in vivo somatic gene transfer approaches. Exploiting these methodological advances will further accelerate detailed investigations of tumor etiology and treatment.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Glioma is the most common pediatric central nervous system tumor, with high-grade gliomas (HGG) having one of the worst prognoses of all human cancers. In order to develop better diagnostics ...and therapies, it is essential to use faithful disease models. Currently, highly passaged patient-derived xenograft (PDX) models are most widely used in preclinical studies, although alternative immunocompetent models are becoming increasingly available. Here, we compare several in vivo glioma models on a single-cell level and investigate their similarity to primary human tumors. Single-nucleus sequencing was used to analyze >125,000 nuclei of primary patient samples, xenografts, autochthonous mouse tumors and related allografts including early and late in vivo passages – with a focus on MET-fusion-driven as well as H3 K27M-mutant HGG. Transcriptomic profiles of single tumor cells and associated stromal/immune components reveal insights into model-specific intratumoral heterogeneity, tumor evolution, and similarities between mouse models and patient samples. In addition, matched tumors engrafted into immunocompromised and immunocompetent animals are used to examine tumor-immune crosstalk and the modifying role of the tumor microenvironment. This improved understanding of how the analyzed model systems evolve over time and how they reflect patient’s tumor compositions will allow to prioritize and refine modern preclinical trials.
YAP1 fusion-positive supratentorial ependymomas predominantly occur in infants, but the molecular mechanisms of oncogenesis are unknown. Here we show YAP1-MAMLD1 fusions are sufficient to drive ...malignant transformation in mice, and the resulting tumors share histo-molecular characteristics of human ependymomas. Nuclear localization of YAP1-MAMLD1 protein is mediated by MAMLD1 and independent of YAP1-Ser127 phosphorylation. Chromatin immunoprecipitation-sequencing analyses of human YAP1-MAMLD1-positive ependymoma reveal enrichment of NFI and TEAD transcription factor binding site motifs in YAP1-bound regulatory elements, suggesting a role for these transcription factors in YAP1-MAMLD1-driven tumorigenesis. Mutation of the TEAD binding site in the YAP1 fusion or repression of NFI targets prevents tumor induction in mice. Together, these results demonstrate that the YAP1-MAMLD1 fusion functions as an oncogenic driver of ependymoma through recruitment of TEADs and NFIs, indicating a rationale for preclinical studies to block the interaction between YAP1 fusions and NFI and TEAD transcription factors.
PDF
