5-hydroxymethylcytosine is required for terminal differentiation of Purkinje neurons
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ABSTRACT:
PROVIDER: PRJNA700864 | ENA |
REPOSITORIES: ENA
Dataset's files
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| SRR13665300_1.fastq.gz | Fastqsanger.gz | |||
| SRR13665300_2.fastq.gz | Fastqsanger.gz | |||
| SRR13665301_1.fastq.gz | Fastqsanger.gz | |||
| SRR13665301_2.fastq.gz | Fastqsanger.gz | |||
| SRR13665302_1.fastq.gz | Fastqsanger.gz |
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Project description:Principal neurons in the mammalian brain exit cell cycle and execute a complex and prolonged differentiation program that continues into early adult life. Although high levels of 5-hydroxymethylcytosine (5hmC) accumulate in neurons, it is not known whether 5hmC can serve as an intermediate in DNA demethylation in postmitotic neurons. Here we report high resolution mapping of DNA methylation and hydroxymethylation, chromatin accessibility, and activating and repressive histone marks in developing postmitotic Purkinje cells (PCs). Our data reveal new relationships between PC transcriptional and epigenetic programs, and identify a class of late expressed genes that lose both 5mC and 5hmC during terminal differentiation. Deletion of the 5hmC writers Tet1, Tet2, and Tet3 from postmitotic Purkinje cells prevents loss of 5mC and 5hmC in regulatory domains and gene bodies and hinders transcriptional and epigenetic developmental transitions, resulting in hyper-excitability and increased susceptibility to excitotoxic drugs. Our data demonstrate that Tet-mediated active DNA demethylation occurs in vivo, and that acquisition of the precise molecular and electrophysiological properties of adult PCs requires continued oxidation of 5mC to 5hmC during the final phases of differentiation.
2021-09-01 | GSE166423 | GEO
Project description:Cellular diversification is a fundamental feature of the brain that is critical for physiological functions of the nervous system including perception, motor control, and learning and memory. Advances in single cell RNA-sequencing have led to characterization of transcriptomic profiles of distinct major types of neurons in the brain. However, how transcriptomic profiles diversify within a specific population of neurons and their links to function remain poorly understood. Purkinje neurons represent some of the most iconic cells in the brain with decades of research characterizing their anatomy, cell biology, physiology, and roles in plasticity, learning and memory, as well as in diseases of the nervous system. In this study, we deployed an approach of isolating nuclei tagged in specific cell types followed by cell sorting and single nuclear RNA sequencing to profile Purkinje neurons and their response to motor activity and learning in adult mice. We uncovered the molecular map of two major subpopulations of Purkinje neurons, identified by the hallmark genes Aldoc and Plcb4, which bear distinct transcriptomic features. Remarkably, Plcb4+, but not Aldoc+, Purkinje neurons display robust plasticity of gene expression in mice subjected to sensorimotor and learning experience with downregulation of gene clusters related to chromatin regulation and synaptic organization and upregulation of gene clusters related to neuronal activity and synaptic transmission and neuron-immune interactions. Using in vivo calcium imaging and optogenetics perturbation approaches, we show that activation of Plcb4+ Purkinje neuron plays a crucial role in associative motor learning. Upon integrating single nuclear RNA-seq datasets with weighted gene network analysis, we also identify a motor activity and learning specific gene module that includes components of the FGFR2-SOS1-MAPK signaling pathway in Plcb4+ Purkinje neurons. Knockout of FGFR2 in Plcb4+ Purkinje neurons in adult mice by a CRISPR approach dramatically disrupts motor learning. Our findings provide a platform for identification of subpopulations of neurons and define how diversification of Purkinje neurons in the cerebellum links to their responses to motor learning. Our study, and by extension similar studies in the human brain, will provide the foundation for understanding the selective vulnerability of Plcb4+ Purkinje neurons to neurological diseases.
2022-05-11 | GSE153184 | GEO
Project description:The Stromal interaction molecule 1 (STIM1) is an ER-Ca2+ sensor and an essential component of ER-Ca2+ store operated Ca2+entry (SOCE). Loss of STIM1 affects metabotropic Glutamate Receptor 1 (mGluR1) mediated synaptic transmission, neuronal Ca2+ homeostasis and intrinsic plasticity in Purkinje Neurons (PNs). Long-term changes of intracellular Ca2+ signaling in PNs lead to neurodegenerative conditions, as evident in individuals with mutations of the ER-Ca2+ channel, the Inositol, 1,4,5-triphosphate receptor (IP3R). Moreover, Changes in gene expression upon reduced SOCE in non-excitable immune cells, the developing mouse brain, Drosophila pupal neurons and human neural precursor cells have been reported. Gene expression profiles of mature differentiated neurons with loss of STIM1/nSOC have not been published to date. The study evaluated the differential gene expression in STIM1 knockout purkinje neurons compared to wild type purkinje neurons from 1 year old mice. Analysis of gene expression profiles demonstrated that STIM1 dependent Ca2+ homeostasis and signaling helps to maintain the expression of multiple key components of synaptic architecture and function in ageing animals. Our findings are significant in the context of finding new therapeutic means of alleviating the neurodegenerative changes associated with human SCAs.
2021-03-22 | GSE158513 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below. The neurodegenerative disease known as ataxia-telangiectasia (A-T) is caused by the absence of the ATM (A-T mutated) protein. A long-standing mystery surrounding A-T is why cerebellar Purkinje cells (PCs) appear uniquely vulnerable to ATM-deficiency. Here, we present that 5-hydroxymethylcytosine (5hmC), a newly recognized epigenetic marker found at high levels in neurons, is substantially reduced in human A-T and Atm-/- mouse cerebellar PCs. TET1, an enzyme that converts 5mC to 5hmC, responds to DNA damage. Manipulation of TET1 activity directly affects neuronal cell cycle reentry and cell death after the induction of DNA damage. Quantitative, genome-wide analysis of 5hmC of samples from human cerebellum showed that in ATM-deficiency there is a remarkable genome-wide reduction of 5hmC enrichment at both proximal and distal regulatory elements. These results reveal a role of TET1-mediated 5hmC in DNA damage response, and provide insights into the basis of a PC-specific DNA demethylation alteration in ATM-deficiency. Refer to individual Series
2015-10-30 | E-GEOD-61169 | biostudies-arrayexpress
Project description:The modified DNA base 5-hydroxymethylcytosine (5hmC) is enriched in neurons where it may contribute to gene function and cellular identity. To address this issue in an in vivo neuronal population, we assessed the patterning, stability, and function of the base within gene bodies in olfactory sensory neurons. We find that gene body 5hmC linearly correlates with transcriptional output and is stable in fully mature neurons and those lacking de novo methyltransferase activity. Overexpression of Tet3, which oxidizes methylated cytosines (5mC) to 5hmC, markedly alters gene body 5hmC levels and provides evidence that 5hmC facilitates transcription. This manipulation disrupts olfactory receptor expression and the targeting of axons to the olfactory bulb, key molecular and anatomical features of the olfactory system that are necessary for proper physiology. Our results support a direct, positive and physiologically significant role for gene body 5hmC in transcriptional elongation and the maintenance of cellular identity independent of its function as an intermediate to demethylation. We assessed the role of 5hmC in mature olfactory sensory neurons by assessing 5hmC levels in 2 month old neurons, olfactory epithelia lacking Dnmt3a, and mOSNs overexpressing Tet3. To determine genome-wide levels of 5hmC, we performed DNA immunoprecipitation coupled to Illumina sequencing. To determine transcript levels, we prepared and sequenced rRNA-depleted cDNA libraries.
2013-09-03 | E-GEOD-43617 | biostudies-arrayexpress
Project description:The molecular mechanism(s) leading to Purkinje neuron loss in the neurodegenerative disorder Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS) are limited by the complex morphology of this cell type. Purkinje neurons are notoriously difficult to isolate and maintain in culture presenting considerable difficultly to identify molecular changes in response to riboCGG repeat-containing mRNA that induces neurotoxicity in FXTAS. Several studies have uncovered a number of RNA binding proteins involved in translation that aberrantly interact with the toxic RNA; however, whether these interactions alter the translational profile of cells has not been investigated. Here we employ bacTRAP translational profiling to demonstrate that Purkinje neurons ectopically expressing 90 CGG repeats exhibit a dramatic change in their translational profile even prior to the onset of riboCGG-induced phenotypes. This approach identified nearly 500 transcripts that are differentially associated with ribosomes in r(CGG)90-expressing mice. Functional annotation cluster analysis revealed broad ontologies enriched in the r(CGG)90 list, including RNA binding and response to stress. Intriguingly, a transcript for the Tardbp gene, implicated in a number of other neurodegenerative disorders, exhibits altered association with ribosomes in the presence of r(CGG)90 repeats. We therefore tested and showed that reduced association of Tardbp mRNA with the ribosomes results in a loss of TDP-43 protein expression in r(CGG)90–expressing Purkinje neurons. Furthermore, we showed that TDP-43 could modulate the rCGG repeat-mediated toxicity in a Drosophila model that we developed previously. These findings together suggest translational dysregulation may be an underlying mechanism of riboCGG-induced neurotoxicity and provide insight into the pathogenicity of FXTASBAC-trap studies of Purkinje cels in normal and mutant mice Looking for cell type specific gene expression differences caused by mutation in FMR1 using BAC-Trap RNA capture assay. A cutoff value of 4.5 for mean probe_set mean across all 18 hybridizations was used to determine a cutoff for expression in Purkinje cells. This 16040 corresponded to a mean value of at least 3.4323 in the 4week time point. L7CGG90Fmr1:Pcp2 BAC (r(CGG)90 BAC) and wild-type:L7/Pcp2 BAC (WT BAC) transgenic mice used in this study have a 50% C57B6 and 50% FVB genetic background and were derived from the same parents by crossing L7CGG90Fmr1 heterozygote transgenics (100% C57B6 background) with L7/Pcp2eEGFPL10a BAC homozygous transgenics (100% FVB background). All mice reported were F1 progeny and were therefore hemizygous for one or both transgenes. Littermate wild-type mice were used for all experiments whenever possible.
2014-05-01 | E-GEOD-57034 | biostudies-arrayexpress
Project description:The modified DNA base 5-hydroxymethylcytosine (5hmC) is enriched in neurons where it may contribute to gene function and cellular identity. To address this issue in an in vivo neuronal population, we assessed the patterning, stability, and function of the base within gene bodies in olfactory sensory neurons. We find that gene body 5hmC linearly correlates with transcriptional output and is stable in fully mature neurons and those lacking de novo methyltransferase activity. Overexpression of Tet3, which oxidizes methylated cytosines (5mC) to 5hmC, markedly alters gene body 5hmC levels and provides evidence that 5hmC facilitates transcription. This manipulation disrupts olfactory receptor expression and the targeting of axons to the olfactory bulb, key molecular and anatomical features of the olfactory system that are necessary for proper physiology. Our results support a direct, positive and physiologically significant role for gene body 5hmC in transcriptional elongation and the maintenance of cellular identity independent of its function as an intermediate to demethylation.
2013-09-03 | GSE43617 | GEO
Project description:We performed gene-expression analysis of mouse Purkinje cells as a model M-bM-^@M-^\single-type neuronM-bM-^@M-^]. DNA microarray analysis detected at least 7,055 genes in Purkinje cells, most of which are classified into functional molecule categories. Our comparative analysis between Purkinje cells and the granule cell layer showed that the characteristic expression pattern in Purkinje cells was particularly represented by M-bM-^@M-^\the neural communication systemM-bM-^@M-^] components. Pukinje cells and granule cell layer of the mouse cerebellum were collected by laser microdissection for RNA extraction and hybridization on Affymetrix microarrays.
2013-01-17 | E-GEOD-26749 | biostudies-arrayexpress
Project description:The signalling protein PKCγ is a major regulator of Purkinje cell development and synaptic function. We have shown previously that increased PKCγ activity impairs dendritic development of cerebellar Purkinje cells. Mutations in the protein kinase Cγ gene (PRKCG) cause spinocerebellar ataxia type 14 (SCA14). In a transgenic mouse model of SCA14 expressing the human S361G mutation, Purkinje cell dendritic development is impaired in cerebellar slice cultures similar to pharmacological activation of PKC. The mechanisms of PKCγ-driven inhibition of dendritic growth are still unclear. Using immunoprecipitation-coupled mass spectrometry analysis we have identified Collapsin Response Mediator Protein 2 (CRMP2) as a protein interacting with constitutive active PKCγ(S361G) and confirmed the interaction with the Duolink™ proximity ligation assay. We show that in cerebellar slice cultures from PKCγ(S361G)- mice, phosphorylation of CRMP2 at the known PKC target site Thr555 is increased in Purkinje cells confirming phosphorylation of CRMP2 by PKCγ. miRNA-mediated CRMP2 knockdown decreased Purkinje cell dendritic outgrowth in dissociated cerebellar cultures as did the transfection of CRMP2 mutants with a modified Thr555 site. In contrast, dendritic development was normal after wildtype CRMP2 overexpression. In a novel knock-in mouse expressing only the phospho-defective T555A-mutant CRMP2, Purkinje cell dendritic development was reduced in dissociated cultures. This reduction could be rescued by transfecting wildtype CRMP2 but only partially by the phospho-mimetic T555D-mutant. Our findings establish CRMP2 as an important target of PKCγ phosphorylation in Purkinje cells mediating its control of dendritic development. Dynamic regulation of CRMP2 phosphorylation via PKCγ is required for its correct function.
2022-02-24 | PXD020124 | Pride
Project description:Despite the importance of epigenetic regulation in neurological disorders, little is known about neuronal chromatin. Cerebellar Purkinje neurons have large and euchromatic nuclei, whereas granule cell nuclei are small and have a more typical heterochromatin distribution. While comparing the abundance of 5-methylcytosine in Purkinje and granule cell nuclei, we detected the presence of an unusual DNA nucleotide. Using thin-layer chromatography, high-pressure liquid chromatography, and mass spectrometry, we identified the nucleotide as 5-hydroxymethyl-2'-deoxycytidine (hmdC). hmdC constitutes 0.6% of total nucleotides in Purkinje cells, 0.2% in granule cells, and is not present in cancer cell lines. hmdC is a constituent of nuclear DNA that is highly abundant in the brain, suggesting a role in epigenetic control of neuronal function.
| S-EPMC3263819 | biostudies-literature