Epigenetic and transcriptional landscapes of Dnmt3a-deficient olfactory sensory neurons
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE52464 | GEO | 2014/11/05
SECONDARY ACCESSION(S): PRJNA229056
REPOSITORIES: GEO
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Project description:During differentiation, neurons experience a reorganization of DNA modification patterns within their genomes. However, the mechanisms underlying this developmental patterning and its role in defining the neuronaÂÂl state are currently unclear. Here, we find that the dÂÂe novo DNA methyltransferase Dnmt3a is necessary for elevated levels of 5-hydroxymethylcytosine (5hmC), a derivative of 5-methylcytosine (5mC), in olfactory sensory neurons (OSNs). Through an analysis of genome-wide 5mC and 5hmC distributions in isolated OSNs, we find that Dnmt3a-dependent 5mC and 5hmC occurs within regions of high accessibility, neural enhancers, and the transcription start sites of transcribed genes. Its loss results in the global disruption of gene expression patterns, including the upregulation of silent genes, the downregulation of mOSN-expressed genes, and the alteration of odorant-induced transcriptional responses of immediate early genes. Together, these results demonstrate that Dnmt3a is necessary to define the neuronal transcriptional state and may be broadly involved in refining expression profiles within differentiated cells. To determine the contributions of Dnmt3a to the DNA modification and transcriptional landscapes of a post-mitotic neuronal population, we performed DNA immunoprecipitation (DIP-seq) using antibodies specific for 5mC and 5hmC and rRNA-depleted transcriptional profiling (RNA-seq) coupled to high-throughput sequencing using genomic DNA or RNA from FACS-isolated mature olfactory sensory neurons (mOSNs) from main olfactory epithelium (MOE) of Dnmt3a wildtype (WT), heterozygous-null (Het), or homozygous-null (KO) 3-week old mice. Similarly, to compare this information with other epigenetic features of the MOE, we performed H3K4me1 (WT), H3K27ac (WT), and H3K27me3 (WT and KO) chromatin immunoprecipitation (ChIP)-seq and DNase I hypersensitivity assays (DNase-seq) using MOE nuclei from 3-week old mice. In addition, we assayed the influence of Dnmt3a-deficiency on the induction of odorant-responsive genes by exposing 3-week old Dnmt3a WT, Het, and KO mice to either water or a 1:1:1 mixture of amyl acetate:acetophenone:octanal for 1 hour and performed rRNA-depleted RNA-seq using RNA isolated from their MOEs.
2014-11-05 | E-GEOD-52464 | 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:Ten-eleven translocation (Tet) hydroxylases (Tet1-3) oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In neurons increased 5hmC levels within gene bodies correlate positively with gene expression. The mechanisms controlling Tet activity and 5hmC levels are poorly understood. In particular, it is not known how the neuronal Tet3 isoform lacking a DNA binding domain is targeted to the DNA. To identify factors binding to Tet3 we screened for proteins that co-precipitate with Tet3 from mouse retina and identified the transcriptional repressor Rest as a highly enriched Tet3-specific interactor. Rest was able to enhance Tet3 hydroxylase activity after co-expression and overexpression of Tet3 activated transcription of Rest-target genes. Moreover, we found that Tet3 also interacts with Nsd3 and two other H3K36 methyltransferases and is able to induce H3K36 trimethylation. We propose a mechanism for transcriptional activation in neurons that involves Rest-guided targeting of Tet3 to the DNA for directed 5hmC-generation and Nsd3-mediated H3K36 trimethylation.
2015-04-02 | PXD001857 | Pride
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:Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contribution of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity and may constitute another layer of epigenetic regulation.
2020-07-24 | PXD019514 | Pride
Project description:Ten-eleven translocation (Tet) hydroxylases (Tet1-3) oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In neurons increased 5hmC levels within gene bodies correlate positively with gene expression. Here, we studied 5hmC profiles (hMeDIP) during retinal maturation between postnatal week 2 and postnatal week 3 using HiSeq 2000 instrument. Study of retinal 5hmC profile dynamics in 2-week old and 3-week old wild type (WT) mouse.
2015-04-02 | E-GEOD-65860 | biostudies-arrayexpress
Project description:Ten-eleven translocation (Tet) hydroxylases (Tet1-3) oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In neurons increased 5hmC levels within gene bodies correlate positively with gene expression. Here, we studied 5hmC profiles (hMeDIP) during retinal maturation between postnatal week 2 and postnatal week 3 using HiSeq 2000 instrument.
2015-04-02 | GSE65860 | GEO
Project description:The TET family of dioxygenases catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), but their involvement in establishing normal 5mC patterns during mammalian development and their contributions to aberrant control of 5mC during cellular transformation remains largely unknown. We depleted TET1, TET2, and TET3 by siRNA in a pluripotent embryonic carcinoma cell model and examined the impact on genome-wide 5mC and 5hmC patterns. TET1 depletion yielded widespread reduction of 5hmC, while depletion of TET2 and TET3 reduced 5hmC at a subset of TET1 targets suggesting functional co-dependence. TET2 or TET3-depletion also caused increased 5hmC, suggesting they play a major role in 5hmC removal. All TETs prevent hypermethylation throughout the genome, a finding dramatically illustrated in CpG island shores, where TET depletion resulted in prolific hypermethylation. Surprisingly, TETs also promote methylation, as hypomethylation was associated with 5hmC reduction. TET function was highly specific to chromatin environment: 5hmC maintenance by all TETs occurred at polycomb-marked chromatin and genes expressed at moderate levels; 5hmC removal by TET2 is associated with highly transcribed genes enriched for H3K4me3 and H3K36me3. Importantly, genes prone to hypermethylation in cancer become depleted of 5hmC with TET deficiency, suggesting the TETs normally promote 5hmC at these loci, and all three TETs are required for 5hmC enrichment at enhancers, a condition necessary for expression of adjacent genes. These results provide novel insight into the division of labor among TET proteins and reveal an important connection of TET activity with chromatin landscape and gene expression. Methylation and hydroxymethylation profiling by affinity-based high throughput sequencing
2014-06-14 | E-GEOD-51902 | biostudies-arrayexpress
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:The TET family of dioxygenases catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), but their involvement in establishing normal 5mC patterns during mammalian development and their contributions to aberrant control of 5mC during cellular transformation remains largely unknown. We depleted TET1, TET2, and TET3 by siRNA in a pluripotent embryonic carcinoma cell model and examined the impact on genome-wide 5mC and 5hmC patterns. TET1 depletion yielded widespread reduction of 5hmC, while depletion of TET2 and TET3 reduced 5hmC at a subset of TET1 targets suggesting functional co-dependence. TET2 or TET3-depletion also caused increased 5hmC, suggesting they play a major role in 5hmC removal. All TETs prevent hypermethylation throughout the genome, a finding dramatically illustrated in CpG island shores, where TET depletion resulted in prolific hypermethylation. Surprisingly, TETs also promote methylation, as hypomethylation was associated with 5hmC reduction. TET function was highly specific to chromatin environment: 5hmC maintenance by all TETs occurred at polycomb-marked chromatin and genes expressed at moderate levels; 5hmC removal by TET2 is associated with highly transcribed genes enriched for H3K4me3 and H3K36me3. Importantly, genes prone to hypermethylation in cancer become depleted of 5hmC with TET deficiency, suggesting the TETs normally promote 5hmC at these loci, and all three TETs are required for 5hmC enrichment at enhancers, a condition necessary for expression of adjacent genes. These results provide novel insight into the division of labor among TET proteins and reveal an important connection of TET activity with chromatin landscape and gene expression. Affymetrix gene expression Human ST1.0 microarray of NCCIT human embryonic carcinoma cells (4 samples in duplicate).
2014-06-14 | E-GEOD-51901 | biostudies-arrayexpress