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Huntington's disease (HD) is a neurodegenerative genetic disorder caused by an expansion in the CAG repeat tract of the huntingtin (HTT) gene resulting in behavioural, cognitive, and motor defects. Current knowledge of disease pathogenesis remains incomplete, and no disease course-modifying interventions are in clinical use. We have previously reported the development and characterisation of the OVT73 transgenic sheep model of HD. The 73 polyglutamine repeat is somatically stable and therefore likely captures a prodromal phase of the disease with an absence of motor symptomatology even at 5-years of age and no detectable striatal cell loss. To better understand the disease-initiating events we have undertaken a single nuclei transcriptome study of the striatum of an extensively studied cohort of 5-year-old OVT73 HD sheep and age matched wild-type controls. We have identified transcriptional upregulation of genes encoding N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in medium spiny neurons, the cell type preferentially lost early in HD. Further, we observed an upregulation of astrocytic glutamate uptake transporters and medium spiny neuron GABAA receptors, which may maintain glutamate homeostasis. Taken together, these observations support the glutamate excitotoxicity hypothesis as an early neurodegeneration cascade-initiating process but the threshold of toxicity may be regulated by several protective mechanisms. Addressing this biochemical defect early may prevent neuronal loss and avoid the more complex secondary consequences precipitated by cell death.
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Amyotrophic lateral sclerosis (ALS) is a severe motor neuron disease with uncertain genetic predisposition in most sporadic cases. The spatial architecture of cell types and gene expression are the basis of cell-cell interactions, biological function and disease pathology, but are not well investigated in the human motor cortex, a key ALS-relevant brain region. Recent studies indicated single nucleus transcriptomic features of motor neuron vulnerability in ALS motor cortex. However, the brain regional vulnerability of ALS-associated genes and the genetic link between region-specific genes and ALS risk remain largely unclear. Here, we developed an entropy-weighted differential gene expression matrix-based tool (SpatialE) to identify the spatial enrichment of gene sets in spatial transcriptomics. We benchmarked SpatialE against another enrichment tool (multimodal intersection analysis) using spatial transcriptomics data from both human and mouse brain tissues. To investigate regional vulnerability, we analysed three human motor cortex and two dorsolateral prefrontal cortex tissues for spatial enrichment of ALS-associated genes. We also used Cell2location to estimate the abundance of cell types in ALS-related cortex layers. To dissect the link of regionally expressed genes and ALS risk, we performed burden analyses of rare loss-of-function variants detected by whole-genome sequencing in ALS patients and controls, then analysed differential gene expression in the TargetALS RNA-sequencing dataset. SpatialE showed more accurate and specific spatial enrichment of regional cell type markers than multimodal intersection analysis in both mouse brain and human dorsolateral prefrontal cortex. Spatial transcriptomic analyses of human motor cortex showed heterogeneous cell types and spatial gene expression profiles. We found that 260 manually curated ALS-associated genes are significantly enriched in layer 5 of the motor cortex, with abundant expression of upper motor neurons and layer 5 excitatory neurons. Burden analyses of rare loss-of-function variants in Layer 5-associated genes nominated NOMO1 as a novel ALS-associated gene in a combined sample set of 6814 ALS patients and 3324 controls (P = 0.029). Gene expression analyses in CNS tissues revealed downregulation of NOMO1 in ALS, which is consistent with a loss-of-function disease mechanism. In conclusion, our integrated spatial transcriptomics and genomic analyses identified regional brain vulnerability in ALS and the association of a layer 5 gene (NOMO1) with ALS risk.
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Esclerosis Amiotrófica Lateral , Corteza Motora , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Humanos , Ratones , Animales , Corteza Motora/metabolismo , Corteza Motora/patología , Transcriptoma , Genómica/métodos , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , MasculinoRESUMEN
Here we present ICARUS, a web server to enable users without experience in R to undertake single cell RNA-seq analysis. The focal point of ICARUS is its intuitive tutorial-style user interface, designed to guide logical navigation through the multitude of pre-processing, analysis and visualization steps. ICARUS is easily accessible through a dedicated web server (https://launch.icarus-scrnaseq.cloud.edu.au/) and avoids installation of software on the user's computer. Notable features include the facility to apply quality control thresholds and adjust dimensionality reduction and cell clustering parameters. Data is visualized through 2D/3D UMAP and t-SNE plots and may be curated to remove potential confounders such as cell cycle heterogeneity. ICARUS offers flexible differential expression analysis with user-defined cell groups and gene set enrichment analysis to identify likely affected biological pathways. Eleven organisms including human, dog, mouse, rat, zebrafish, fruit fly, nematode, yeast, cattle, chicken and pig are currently supported. Visualization of multimodal data including those generated by CITE-seq and the 10X Genomics Multiome kit is included. ICARUS incorporates a function to save the current state of analysis avoiding computationally intensive steps during repeat analysis. The complete analysis of a typical single cell RNA-seq dataset by inexperienced users may be achieved in 1-2 h.
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Análisis de Secuencia de ARN , Análisis de la Célula Individual , Programas Informáticos , Animales , Bovinos , Perros , Humanos , Ratones , Ratas , Computadores , Genómica , Porcinos , Pez CebraRESUMEN
Lysine acetylation has recently emerged as an important post-translational modification in diverse organisms, but relatively little is known about its roles in mammalian development and stem cells. Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a multidomain histone binder and a master activator of three lysine acetyltransferases, MOZ, MORF and HBO1, which are also known as KAT6A, KAT6B and KAT7, respectively. While the MOZ and MORF genes are rearranged in leukemia, the MORF gene is also mutated in prostate and other cancers and in four genetic disorders with intellectual disability. Here we show that forebrain-specific inactivation of the mouse Brpf1 gene causes hypoplasia in the dentate gyrus, including underdevelopment of the suprapyramidal blade and complete loss of the infrapyramidal blade. We trace the developmental origin to compromised Sox2+ neural stem cells and Tbr2+ intermediate neuronal progenitors. We further demonstrate that Brpf1 loss deregulates neuronal migration, cell cycle progression and transcriptional control, thereby causing abnormal morphogenesis of the hippocampus. These results link histone binding and acetylation control to hippocampus development and identify an important epigenetic regulator for patterning the dentate gyrus, a brain structure critical for learning, memory and adult neurogenesis.
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Proteínas Portadoras/genética , Giro Dentado/metabolismo , Epigénesis Genética/genética , Histona Acetiltransferasas/metabolismo , Morfogénesis/genética , Acetilación , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/metabolismo , Diferenciación Celular/genética , Proteínas de Unión al ADN , Giro Dentado/crecimiento & desarrollo , Giro Dentado/patología , Hipocampo/crecimiento & desarrollo , Hipocampo/patología , Histona Acetiltransferasas/genética , Histonas/metabolismo , Humanos , Ratones , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Prosencéfalo/embriología , Prosencéfalo/crecimiento & desarrollo , Prosencéfalo/metabolismo , Procesamiento Proteico-Postraduccional/genética , Proteínas de Dominio T Box/genéticaRESUMEN
To interpret epigenetic information, chromatin readers utilize various protein domains for recognition of DNA and histone modifications. Some readers possess multidomains for modification recognition and are thus multivalent. Bromodomain- and plant homeodomain-linked finger-containing protein 3 (BRPF3) is such a chromatin reader, containing two plant homeodomain-linked fingers, one bromodomain and a PWWP domain. However, its molecular and biological functions remain to be investigated. Here, we report that endogenous BRPF3 preferentially forms a tetrameric complex with HBO1 (also known as KAT7) and two other subunits but not with related acetyltransferases such as MOZ, MORF, TIP60, and MOF (also known as KAT6A, KAT6B, KAT5, and KAT8, respectively). We have also characterized a mutant mouse strain with a lacZ reporter inserted at the Brpf3 locus. Systematic analysis of ß-galactosidase activity revealed dynamic spatiotemporal expression of Brpf3 during mouse embryogenesis and high expression in the adult brain and testis. Brpf3 disruption, however, resulted in no obvious gross phenotypes. This is in stark contrast to Brpf1 and Brpf2, whose loss causes lethality at E9.5 and E15.5, respectively. In Brpf3-null mice and embryonic fibroblasts, RT-quantitative PCR uncovered no changes in levels of Brpf1 and Brpf2 transcripts, confirming no compensation from them. These results indicate that BRPF3 forms a functional tetrameric complex with HBO1 but is not required for mouse development and survival, thereby distinguishing BRPF3 from its paralogs, BRPF1 and BRPF2.
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Embrión de Mamíferos/enzimología , Desarrollo Embrionario , Histona Acetiltransferasas/metabolismo , Complejos Multienzimáticos/metabolismo , Animales , Pérdida del Embrión/enzimología , Pérdida del Embrión/genética , Células HEK293 , Histona Acetiltransferasas/genética , Humanos , Ratones , Ratones Mutantes , Complejos Multienzimáticos/genéticaRESUMEN
Cerebrospinal fluid (CSF) represents a promising source of cell-free DNA (cfDNA) for tumors of the central nervous system. A CSF-based liquid biopsy may obviate the need for riskier tissue biopsies and serve as a means for monitoring tumor recurrence or response to therapy. Spinal ependymomas most commonly occur in adults, and aggressive resection must be delicately balanced with the risk of injury to adjacent normal tissue. In patients with subtotal resection, recurrence commonly occurs. A CSF-based liquid biopsy matched to the patient's spinal ependymoma mutation profile has potential to be more sensitive then surveillance MRI, but the utility has not been well characterized for tumors of the spinal cord. In this study, we collected matched blood, tumor, and CSF samples from three adult patients with WHO grade II intramedullary spinal ependymoma. We performed whole exome sequencing on matched tumor and normal DNA to design Droplet Digital™ PCR (ddPCR) probes for tumor and wild-type mutations. We then interrogated CSF samples for tumor-derived cfDNA by performing ddPCR on extracted cfDNA. Tumor cfDNA was not reliably detected in the CSF of our cohort. Anatomic sequestration and low grade of intramedullary spinal cord tumors likely limits the role of CSF liquid biopsy.
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Ácidos Nucleicos Libres de Células/líquido cefalorraquídeo , Ependimoma/genética , Ependimoma/metabolismo , Neoplasias de la Médula Espinal/genética , Neoplasias de la Médula Espinal/metabolismo , Adulto , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Estudios de Cohortes , Ependimoma/patología , Ependimoma/cirugía , Humanos , Biopsia Líquida , Masculino , Persona de Mediana Edad , Mutación , Clasificación del Tumor , Proyectos Piloto , Neoplasias de la Médula Espinal/patología , Neoplasias de la Médula Espinal/cirugía , Secuenciación del ExomaRESUMEN
With hundreds of chromatin regulators identified in mammals, an emerging issue is how they modulate biological and pathological processes. BRPF1 (bromodomain- and PHD finger-containing protein 1) is a unique chromatin regulator possessing two PHD fingers, one bromodomain and a PWWP domain for recognizing multiple histone modifications. In addition, it binds to the acetyltransferases MOZ, MORF, and HBO1 (also known as KAT6A, KAT6B, and KAT7, respectively) to promote complex formation, restrict substrate specificity, and enhance enzymatic activity. We have recently showed that ablation of the mouse Brpf1 gene causes embryonic lethality at E9.5. Here we present systematic analyses of the mutant animals and demonstrate that the ablation leads to vascular defects in the placenta, yolk sac, and embryo proper, as well as abnormal neural tube closure. At the cellular level, Brpf1 loss inhibits proliferation of embryonic fibroblasts and hematopoietic progenitors. Molecularly, the loss reduces transcription of a ribosomal protein L10 (Rpl10)-like gene and the cell cycle inhibitor p27, and increases expression of the cell-cycle inhibitor p16 and a novel protein homologous to Scp3, a synaptonemal complex protein critical for chromosome association and embryo survival. These results uncover a crucial role of Brpf1 in controlling mouse embryo development and regulating cellular and gene expression programs.
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Proteínas Portadoras/metabolismo , Cromatina/metabolismo , Desarrollo Embrionario , Proteínas Adaptadoras Transductoras de Señales , Animales , Línea Celular , Proliferación Celular , Proteínas de Unión al ADN , Femenino , Fibroblastos/citología , Hematopoyesis , Ratones , Neovascularización Fisiológica , Defectos del Tubo Neural/metabolismo , Placenta/irrigación sanguínea , Placenta/metabolismo , Embarazo , Saco Vitelino/irrigación sanguínea , Saco Vitelino/embriologíaRESUMEN
Epigenetic mechanisms are important in different neurological disorders, and one such mechanism is histone acetylation. The multivalent chromatin regulator BRPF1 (bromodomain- and plant homeodomain-linked (PHD) zinc finger-containing protein 1) recognizes different epigenetic marks and activates three histone acetyltransferases, so it is both a reader and a co-writer of the epigenetic language. The three histone acetyltransferases are MOZ, MORF, and HBO1, which are also known as lysine acetyltransferase 6A (KAT6A), KAT6B, and KAT7, respectively. The MORF gene is mutated in four neurodevelopmental disorders sharing the characteristic of intellectual disability and frequently displaying callosal agenesis. Here, we report that forebrain-specific inactivation of the mouse Brpf1 gene caused early postnatal lethality, neocortical abnormalities, and partial callosal agenesis. With respect to the control, the mutant forebrain contained fewer Tbr2-positive intermediate neuronal progenitors and displayed aberrant neurogenesis. Molecularly, Brpf1 loss led to decreased transcription of multiple genes, such as Robo3 and Otx1, important for neocortical development. Surprisingly, elevated expression of different Hox genes and various other transcription factors, such as Lhx4, Foxa1, Tbx5, and Twist1, was also observed. These results thus identify an important role of Brpf1 in regulating forebrain development and suggest that it acts as both an activator and a silencer of gene expression in vivo.
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Agenesia del Cuerpo Calloso/genética , Encéfalo/anomalías , Encéfalo/crecimiento & desarrollo , Proteínas Portadoras/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Adaptadoras Transductoras de Señales , Agenesia del Cuerpo Calloso/metabolismo , Animales , Conducta Animal , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Cuerpo Calloso/crecimiento & desarrollo , Cuerpo Calloso/metabolismo , Proteínas de Unión al ADN , Eliminación de Gen , Silenciador del Gen , Ratones , Ratones Noqueados , Neurogénesis , Activación TranscripcionalRESUMEN
α-Tubulin acetylation at Lys-40, located on the luminal side of microtubules, has been widely studied and used as a marker for stable microtubules in the cilia and other subcellular structures, but the functional consequences remain perplexing. Recent studies have shown that Mec-17 and its paralog are responsible for α-tubulin acetylation in Caenorhabditis elegans. There is one such protein known as Atat1 (α-tubulin acetyltransferase 1) per higher organism. Zebrafish Atat1 appears to govern embryo development, raising the intriguing possibility that Atat1 is also critical for development in mammals. In addition to Atat1, three other mammalian acetyltransferases, ARD1-NAT1, ELP3, and GCN5, have been shown to acetylate α-tubulin in vitro, so an important question is how these four enzymes contribute to the acetylation in vivo. We demonstrate here that Atat1 is a major α-tubulin acetyltransferase in mice. It is widely expressed in mouse embryos and tissues. Although Atat1-null animals display no overt phenotypes, α-tubulin acetylation is lost in sperm flagella and the dentate gyrus is slightly deformed. Furthermore, human ATAT1 colocalizes on bundled microtubules with doublecortin. These results thus suggest that mouse Atat1 may regulate advanced functions such as learning and memory, thereby shedding novel light on the physiological roles of α-tubulin acetylation in mammals.
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Acetiltransferasas/metabolismo , Giro Dentado/metabolismo , Tubulina (Proteína)/metabolismo , Acetilación , Acetiltransferasas/genética , Animales , Western Blotting , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Células Cultivadas , Giro Dentado/anomalías , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Proteínas de Microtúbulos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cola del Espermatozoide/metabolismo , Testículo/embriología , Testículo/crecimiento & desarrollo , Testículo/metabolismo , Factores de TiempoRESUMEN
Ectopic expression of transcription factors has been shown to reprogram somatic cells into induced pluripotent stem (iPS) cells. It remains largely unexplored how this process is regulated by post-translational modifications. Several reprogramming factors possess conserved sumoylation sites, so we investigated whether and how this modification regulates reprogramming of fibroblasts into iPS cells. Substitution of the sole sumoylation site of the Krüppel-like factor (KLF4), a well known reprogramming factor, promoted iPS cell formation. In comparison, much smaller effects on reprogramming were observed for sumoylation-deficient mutants of SOX2 and OCT4, two other classical reprogramming factors. We also analyzed KLF2, a KLF4 homolog and a member of the KLF family of transcription factors with a known role in reprogramming. KLF2 was sumoylated at two conserved neighboring motifs, but substitution of the key lysine residues only stimulated reprogramming slightly. KLF5 is another KLF member with an established link to embryonic stem cell pluripotency. Interestingly, although it was much more efficiently sumoylated than either KLF2 or KLF4, KLF5 was inactive in reprogramming, and its sumoylation was not responsible for this deficiency. Furthermore, sumoylation of KLF4 but not KLF2 or KLF5 stimulated adipocyte differentiation. These results thus demonstrate the importance KLF4 sumoylation in regulating pluripotency and adipocyte differentiation.
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Adipocitos/metabolismo , Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Sumoilación , Células 3T3-L1 , Adipocitos/citología , Animales , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/citología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
Histone deacetylases 4 (HDAC4), -5, -7, and -9 form class IIa within the HDAC superfamily and regulate diverse physiological and pathological cellular programs. With conserved motifs for phosphorylation-dependent 14-3-3 binding, these deacetylases serve as novel signal transducers that are able to modulate histone acetylation and gene expression in response to extracellular cues. Here, we report that in a PKA-sensitive manner the tumor suppressor kinase LKB1 acts through salt-inducible kinase 2 (SIK2) and SIK3 to promote nucleocytoplasmic trafficking of class IIa HDACs. Both SIK2 and SIK3 phosphorylate the deacetylases at the conserved motifs and stimulate 14-3-3 binding. SIK2 activates MEF2-dependent transcription and relieves repression of myogenesis by the deacetylases. Distinct from SIK2, SIK3 induces nuclear export of the deacetylases independent of kinase activity and 14-3-3 binding. These findings highlight the difference among members of the SIK family and indicate that LKB1-dependent SIK activation constitutes an important signaling module upstream from class IIa deacetylases for regulating cellular programs controlled by MEF2 and other transcription factors.
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Histona Desacetilasas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas 14-3-3/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP , Animales , Línea Celular Tumoral , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Genes Reporteros , Células HEK293 , Células HeLa , Humanos , Ratones , Unión Proteica , Transducción de SeñalRESUMEN
Bromodomain and plant homeodomain (PHD) finger containing protein 1 (Brpf1) is an activator and scaffold protein of a multiunit complex that includes other components involving lysine acetyltransferase (KAT) 6A/6B/7. Brpf1, KAT6A, and KAT6B mutations were identified as the causal genes of neurodevelopmental disorders leading to intellectual disability. Our previous work revealed strong and specific expression of Brpf1 in both the postnatal and adult forebrain, especially the hippocampus, which has essential roles in learning and memory. Here, we hypothesized that Brpf1 plays critical roles in the function of forebrain excitatory neurons, and that its deficiency leads to learning and memory deficits. To test this, we knocked out Brpf1 in forebrain excitatory neurons using CaMKIIa-Cre. We found that Brpf1 deficiency reduced the frequency of miniature excitatory postsynaptic currents and downregulated the expression of genes Pcdhgb1, Slc16a7, Robo3, and Rho, which are related to neural development, synapse function, and memory, thereby damaging spatial and fear memory in mice. These findings help explain the mechanisms of intellectual impairment in patients with BRPF1 mutation.
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The association of neurogenesis and gliogenesis with glioma remains unclear. By conducting single-cell RNA-seq analyses on 26 gliomas, we reported their classification into primitive oligodendrocyte precursor cell (pri-OPC)-like and radial glia (RG)-like tumors and validated it in a public cohort and TCGA glioma. The RG-like tumors exhibited wild-type isocitrate dehydrogenase and tended to carry EGFR mutations, and the pri-OPC-like ones were prone to carrying TP53 mutations. Tumor subclones only in pri-OPC-like tumors showed substantially down-regulated MHC-I genes, suggesting their distinct immune evasion programs. Furthermore, the two subgroups appeared to extensively modulate glioma-infiltrating lymphocytes in distinct manners. Some specific genes not expressed in normal immune cells were found in glioma-infiltrating lymphocytes. For example, glial/glioma stem cell markers OLIG1/PTPRZ1 and B cell-specific receptors IGLC2/IGKC were expressed in pri-OPC-like and RG-like glioma-infiltrating lymphocytes, respectively. Their expression was positively correlated with those of immune checkpoint genes (e.g., LGALS33) and poor survivals as validated by the increased expression of LGALS3 upon IGKC overexpression in Jurkat cells. This finding indicated a potential inhibitory role in tumor-infiltrating lymphocytes and could provide a new way of cancer immune evasion.
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Complex biological traits and disease often involve patterns of gene expression that can be characterised and examined. Here we present ICARUS v2.0, an update to our single cell RNA-seq analysis web server with additional tools to investigate gene networks and understand core patterns of gene regulation in relation to biological traits. ICARUS v2.0 enables gene co-expression analysis with MEGENA, transcription factor regulated network identification with SCENIC, trajectory analysis with Monocle3, and characterisation of cell-cell communication with CellChat. Cell cluster gene expression profiles may be examined against Genome Wide Association Studies with MAGMA to find significant associations with GWAS traits. Additionally, differentially expressed genes may be compared against the Drug-Gene Interaction database (DGIdb 4.0) to facilitate drug discovery. ICARUS v2.0 offers a comprehensive toolbox of the latest single cell RNA-seq analysis methodologies packed into an efficient, user friendly, tutorial style web server application (accessible at https://launch.icarus-scrnaseq.cloud.edu.au/) that enables single cell RNA-seq analysis tailored to the user's dataset.
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The bromodomain and PHD finger-containing protein1 (BRPF1) is a member of family IV of the bromodomain-containing proteins that participate in the post-translational modification of histones. It functions in the form of a tetrameric complex with a monocytic leukemia zinc finger protein (MOZ or KAT6A), MOZ-related factor (MORF or KAT6B) or HAT bound to ORC1 (HBO1 or KAT7) and two small non-catalytic proteins, the inhibitor of growth 5 (ING5) or the paralog ING4 and MYST/Esa1-associated factor 6 (MEAF6). Mounting studies have demonstrated that all the four core subunits play crucial roles in different biological processes across diverse species, such as embryonic development, forebrain development, skeletal patterning and hematopoiesis. BRPF1, KAT6A and KAT6B mutations were identified as the cause of neurodevelopmental disorders, leukemia, medulloblastoma and other types of cancer, with germline mutations associated with neurodevelopmental disorders displaying intellectual disability, and somatic variants associated with leukemia, medulloblastoma and other cancers. In this paper, we depict the molecular structures and biological functions of the BRPF1-KAT6A/KAT6B complex, summarize the variants of the complex related to neurodevelopmental disorders and cancers and discuss future research directions and therapeutic potentials.
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Background: GBM astrocytes may adopt fetal astrocyte transcriptomic signatures involved in brain development and migration programs to facilitate diffuse tumor infiltration. Our previous data show that ETS variant 6 (ETV6) is highly expressed in human GBM and fetal astrocytes compared to normal mature astrocytes. We hypothesized that ETV6 played a role in GBM tumor progression. Methods: Expression of ETV6 was first examined in two American and three Chinese tissue microarrays. The correlation between ETV6 staining intensity and patient survival was calculated, followed by validation using public databases-TCGA and REMBRANDT. The effect of ETV6 knockdown on glioma cell proliferation (EdU), viability (AnnexinV labeling), clonogenic growth (colony formation), and migration/invasion (transwell assays) in GBM cells was tested. RNA sequencing and Western blot were performed to elucidate the underlying molecular mechanisms. Results: ETV6 was highly expressed in GBM and associated with an unfavorable prognosis. ETV6 silencing in glioma cells led to increased apoptosis or decreased proliferation, clonogenicity, migration, and invasion. RNA-Seq-based gene expression and pathway analyses revealed that ETV6 knockdown in U251 cells led to the upregulation of genes involved in extracellular matrix organization, NF-κB signaling, TNF-mediated signaling, and the downregulation of genes in the regulation of cell motility, cell proliferation, PI3K-AKT signaling, and the Ras pathway. The downregulation of the PI3K-AKT and Ras-MAPK pathways were further validated by immunoblotting. Conclusion: Our findings suggested that ETV6 was highly expressed in GBM and its high expression correlated with poor survival. ETV6 silencing decreased an aggressive in vitro phenotype probably via the PI3K-AKT and Ras-MAPK pathways. The study encourages further investigation of ETV6 as a potential therapeutic target of GBM.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Glioblastoma/genética , Neoplasias Encefálicas/metabolismo , FN-kappa B/genética , Línea Celular Tumoral , Glioma/genética , FenotipoRESUMEN
Patients with monoallelic bromodomain and PHD finger-containing protein 1 (BRPF1) mutations showed intellectual disability. The hippocampus has essential roles in learning and memory. Our previous work indicated that Brpf1 was specifically and strongly expressed in the hippocampus from the perinatal period to adulthood. We hypothesized that mouse Brpf1 plays critical roles in the morphology and function of hippocampal neurons, and its deficiency leads to learning and memory deficits. To test this, we performed immunofluorescence, whole-cell patch clamp, and mRNA-Seq on shBrpf1-infected primary cultured hippocampal neurons to study the effect of Brpf1 knockdown on neuronal morphology, electrophysiological characteristics, and gene regulation. In addition, we performed stereotactic injection into adult mouse hippocampus to knock down Brpf1 in vivo and examined the learning and memory ability by Morris water maze. We found that mild knockdown of Brpf1 reduced mEPSC frequency of cultured hippocampal neurons, before any significant changes of dendritic morphology showed. We also found that Brpf1 mild knockdown in the hippocampus showed a decreasing trend on the spatial learning and memory ability of mice. Finally, mRNA-Seq analyses showed that genes related to learning, memory, and synaptic transmission (such as C1ql1, Gpr17, Htr1d, Glra1, Cxcl10, and Grin2a) were dysregulated upon Brpf1 knockdown. Our results showed that Brpf1 mild knockdown attenuated hippocampal excitatory synaptic transmission and reduced spatial learning and memory ability, which helps explain the symptoms of patients with BRPF1 mutations.
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Intellectual disability is closely related to impaired GABA neurotransmission. Brpf1 was specifically expressed in medial ganglionic eminence (MGE), a developmental niche of GABAergic interneurons, and patients with BRPF1 mutations showed intellectual disability. To test its role in the development and function of MGE-derived GABAergic interneurons, we performed immunofluorescence staining, whole-cell patch-clamp, MGE transplantation, and mRNA-Seq to understand its effect on neuronal differentiation, dendritic morphology, electrophysiology, migration, and gene regulation, using mouse MGE-derived GABAergic interneurons infected with AAV-shBrpf1. The results showed that Brpf1 knockdown had a decreasing trend, although not significant, on the differentiation of GABAergic interneurons into parvalbumin+ interneurons. Moreover, increased firing threshold, decreased number of evoked action potentials, and a reduced amplitude of miniature inhibitory postsynaptic currents were observed before any significant change of MAP2+ dendritic morphology and in vivo migration ability appeared. Finally, mRNA-Seq analysis revealed that genes related to neurodevelopment and synaptic transmission such as Map2k7 were dysregulated. Our results demonstrated a key role of Brpf1 in inhibitory neurotransmission and related gene expression of GABAergic interneurons.