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      Phosphorylation of Tet3 by cdk5 is critical for robust activation of BRN2 during neuronal differentiation

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          Abstract

          Tet3 regulates the dynamic balance between 5-methylcyotsine (5mC) and 5-hydroxymethylcytosine (5hmC) in DNA during brain development and homeostasis. However, it remains unclear how its functions are modulated in a context-dependent manner during neuronal differentiation. Here, we show that cyclin-dependent kinase 5 (cdk5) phosphorylates Tet3 at the highly conserved serine 1310 and 1379 residues within its catalytic domain, changing its in vitro dioxygenase activity. Interestingly, when stably expressed in Tet1, 2, 3 triple-knockout mouse embryonic stem cells (ESCs), wild-type Tet3 induces higher level of 5hmC and concomitant expression of genes associated with neurogenesis whereas phosphor-mutant (S1310A/S1379A) Tet3 causes elevated 5hmC and expression of genes that are linked to metabolic processes. Consistent with this observation, Tet3-knockout mouse ESCs rescued with wild-type Tet3 have higher level of 5hmC at the promoter of neuron-specific gene BRN2 when compared to cells that expressed phosphor-mutant Tet3. Wild-type and phosphor-mutant Tet3 also exhibit differential binding affinity to histone variant H2A.Z. The differential 5hmC enrichment and H2A.Z occupancy at BRN2 promoter is correlated with higher gene expression and more efficient neuronal differentiation of ESCs that expressed wild-type Tet3. Taken together, our results suggest that cdk5-mediated phosphorylation of Tet3 is required for robust activation of neuronal differentiation program.

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          Most cited references37

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          Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome.

          The study of 5-hydroxylmethylcytosines (5hmC) has been hampered by the lack of a method to map it at single-base resolution on a genome-wide scale. Affinity purification-based methods cannot precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), that when combined with traditional bisulfite sequencing can be used for mapping 5hmC at base resolution and quantifying the relative abundance of 5hmC as well as 5mC. Application of this method to embryonic stem cells not only confirms widespread distribution of 5hmC in the mammalian genome but also reveals sequence bias and strand asymmetry at 5hmC sites. We observe high levels of 5hmC and reciprocally low levels of 5mC near but not on transcription factor-binding sites. Additionally, the relative abundance of 5hmC varies significantly among distinct functional sequence elements, suggesting different mechanisms for 5hmC deposition and maintenance. Copyright © 2012 Elsevier Inc. All rights reserved.
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            Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine.

            In contrast to 5-methylcytosine (5-mC), which has been studied extensively, little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types. Here we present a method for determining the genome-wide distribution of 5-hmC. We use the T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment and sequencing of 5-hmC-containing DNA fragments in mammalian genomes. Using this method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized. We also find a gene expression level-dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders.
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              5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging.

              DNA methylation dynamics influence brain function and are altered in neurological disorders. 5-hydroxymethylcytosine (5-hmC), a DNA base that is derived from 5-methylcytosine, accounts for ∼40% of modified cytosine in the brain and has been implicated in DNA methylation-related plasticity. We mapped 5-hmC genome-wide in mouse hippocampus and cerebellum at three different ages, which allowed us to assess its stability and dynamic regulation during postnatal neurodevelopment through adulthood. We found developmentally programmed acquisition of 5-hmC in neuronal cells. Epigenomic localization of 5-hmC-regulated regions revealed stable and dynamically modified loci during neurodevelopment and aging. By profiling 5-hmC in human cerebellum, we found conserved genomic features of 5-hmC. Finally, we found that 5-hmC levels were inversely correlated with methyl-CpG-binding protein 2 dosage, a protein encoded by a gene in which mutations cause Rett syndrome. These data suggest that 5-hmC-mediated epigenetic modification is critical in neurodevelopment and diseases.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                20 February 2020
                06 December 2019
                06 December 2019
                : 48
                : 3
                : 1225-1238
                Affiliations
                [1 ] Temasek Life Sciences Laboratory, National University of Singapore , Singapore 117604, Singapore
                [2 ] Department of Biological Sciences, National University of Singapore , Singapore 117558, Singapore
                [3 ] State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences , Shanghai 200031, China
                [4 ] Cancer Science Institute of Singapore, National University of Singapore , Singapore 117599, Singapore
                [5 ] Discipline of Genetics, Faculty of Medicine, Memorial University of Newfoundland , St. John's, NL A1B 3V6, Canada
                Author notes
                To whom correspondence should be addressed. Tel: +65 68727978; Fax: +65 68727007; Email: chintong@ 123456tll.org.sg

                The authors wish it to be known that, in their opinion, the first three authors should be regarded as Joint First Authors.

                Author information
                http://orcid.org/0000-0002-4789-8028
                http://orcid.org/0000-0002-5450-4760
                Article
                gkz1144
                10.1093/nar/gkz1144
                7026633
                31807777
                a4b228a8-f367-4a1d-9128-08e9f7aaf0d3
                © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 27 November 2019
                : 20 November 2019
                : 30 January 2019
                Page count
                Pages: 14
                Funding
                Funded by: Temasek Life Sciences Laboratory 10.13039/501100010730
                Award ID: TLL-3160
                Funded by: National Research Foundation 10.13039/100011512
                Funded by: Singapore Ministry of Education
                Categories
                Gene regulation, Chromatin and Epigenetics

                Genetics
                Genetics

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