Tet3 regulates cellular identity and DNA methylation in neural progenitor cells

Mafalda Santiago; Claudia Antunes; Marta Guedes; Michelina Iacovino; Michael Kyba; Wolf Reik; Nuno Sousa; Luísa Pinto; Miguel R. Branco; C. Joana Marques

doi:10.1007/s00018-019-03335-7

Tet3 regulates cellular identity and DNA methylation in neural progenitor cells

Mafalda Santiago, Claudia Antunes, Marta Guedes, Michelina Iacovino, Michael Kyba, Wolf Reik, Nuno Sousa, Luísa Pinto, Miguel R. Branco, C. Joana Marques

Pediatrics - Blood/Marrow Transplant

Research output: Contribution to journal › Article › peer-review

31 Scopus citations

Abstract

TET enzymes oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), a process thought to be intermediary in an active DNA demethylation mechanism. Notably, 5hmC is highly abundant in the brain and in neuronal cells. Here, we interrogated the function of Tet3 in neural precursor cells (NPCs), using a stable and inducible knockdown system and an in vitro neural differentiation protocol. We show that Tet3 is upregulated during neural differentiation, whereas Tet1 is downregulated. Surprisingly, Tet3 knockdown led to a de-repression of pluripotency-associated genes such as Oct4, Nanog or Tcl1, with concomitant hypomethylation. Moreover, in Tet3 knockdown NPCs, we observed the appearance of OCT4-positive cells forming cellular aggregates, suggesting de-differentiation of the cells. Notably, Tet3 KD led to a genome-scale loss of DNA methylation and hypermethylation of a smaller number of CpGs that are located at neurogenesis-related genes and at imprinting control regions (ICRs) of Peg10, Zrsr1 and Mcts2 imprinted genes. Overall, our results suggest that TET3 is necessary to maintain silencing of pluripotency genes and consequently neural stem cell identity, possibly through regulation of DNA methylation levels in neural precursor cells.

Original language	English (US)
Pages (from-to)	2871-2883
Number of pages	13
Journal	Cellular and Molecular Life Sciences
Volume	77
Issue number	14
DOIs	https://doi.org/10.1007/s00018-019-03335-7
State	Published - Jul 1 2020

Bibliographical note

Funding Information:
This work was supported by the Portuguese Foundation for Science and Technology (FCT) with a project grant (PTDC/BIA-BCM/121276/2010) to C.J.M. and funded by EpiGeneSys with a Small Collaborative project to M.R.B and L.P. CJ Marques and L Pinto are the recipients of an FCT salary contracts (IF/00047/2012 and CEECIND/00371/2017 to C.J.M. and IF/01079/2014 to L.P.). C Antunes is the recipient of a PhD fellowship from the Doctoral Program PhDOC from FCT (PD/BD/106049/2015). M.R.B. is a Sir Henry Dale Fellow (101225/Z/13/Z), jointly funded by the Wellcome Trust and the Royal Society. This work has also been funded by Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER; NORTE-01-0145-FEDER-000013); FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and National Funds, through the FCT (POCI-01-0145-FEDER-007038). Acknowledgments

Publisher Copyright:
© 2019, The Author(s).

Keywords

5-hydroxymethylcytosine
Imprinted genes
Neural stem cells
Neurogenesis
Pluripotency
TET enzymes

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title = "Tet3 regulates cellular identity and DNA methylation in neural progenitor cells",

abstract = "TET enzymes oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), a process thought to be intermediary in an active DNA demethylation mechanism. Notably, 5hmC is highly abundant in the brain and in neuronal cells. Here, we interrogated the function of Tet3 in neural precursor cells (NPCs), using a stable and inducible knockdown system and an in vitro neural differentiation protocol. We show that Tet3 is upregulated during neural differentiation, whereas Tet1 is downregulated. Surprisingly, Tet3 knockdown led to a de-repression of pluripotency-associated genes such as Oct4, Nanog or Tcl1, with concomitant hypomethylation. Moreover, in Tet3 knockdown NPCs, we observed the appearance of OCT4-positive cells forming cellular aggregates, suggesting de-differentiation of the cells. Notably, Tet3 KD led to a genome-scale loss of DNA methylation and hypermethylation of a smaller number of CpGs that are located at neurogenesis-related genes and at imprinting control regions (ICRs) of Peg10, Zrsr1 and Mcts2 imprinted genes. Overall, our results suggest that TET3 is necessary to maintain silencing of pluripotency genes and consequently neural stem cell identity, possibly through regulation of DNA methylation levels in neural precursor cells.",

keywords = "5-hydroxymethylcytosine, Imprinted genes, Neural stem cells, Neurogenesis, Pluripotency, TET enzymes",

author = "Mafalda Santiago and Claudia Antunes and Marta Guedes and Michelina Iacovino and Michael Kyba and Wolf Reik and Nuno Sousa and Lu{\'i}sa Pinto and Branco, {Miguel R.} and Marques, {C. Joana}",

note = "Funding Information: This work was supported by the Portuguese Foundation for Science and Technology (FCT) with a project grant (PTDC/BIA-BCM/121276/2010) to C.J.M. and funded by EpiGeneSys with a Small Collaborative project to M.R.B and L.P. CJ Marques and L Pinto are the recipients of an FCT salary contracts (IF/00047/2012 and CEECIND/00371/2017 to C.J.M. and IF/01079/2014 to L.P.). C Antunes is the recipient of a PhD fellowship from the Doctoral Program PhDOC from FCT (PD/BD/106049/2015). M.R.B. is a Sir Henry Dale Fellow (101225/Z/13/Z), jointly funded by the Wellcome Trust and the Royal Society. This work has also been funded by Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER; NORTE-01-0145-FEDER-000013); FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and National Funds, through the FCT (POCI-01-0145-FEDER-007038). Acknowledgments Publisher Copyright: {\textcopyright} 2019, The Author(s).",

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T1 - Tet3 regulates cellular identity and DNA methylation in neural progenitor cells

AU - Santiago, Mafalda

AU - Antunes, Claudia

AU - Guedes, Marta

AU - Iacovino, Michelina

AU - Kyba, Michael

AU - Reik, Wolf

AU - Sousa, Nuno

AU - Pinto, Luísa

AU - Branco, Miguel R.

AU - Marques, C. Joana

N1 - Funding Information: This work was supported by the Portuguese Foundation for Science and Technology (FCT) with a project grant (PTDC/BIA-BCM/121276/2010) to C.J.M. and funded by EpiGeneSys with a Small Collaborative project to M.R.B and L.P. CJ Marques and L Pinto are the recipients of an FCT salary contracts (IF/00047/2012 and CEECIND/00371/2017 to C.J.M. and IF/01079/2014 to L.P.). C Antunes is the recipient of a PhD fellowship from the Doctoral Program PhDOC from FCT (PD/BD/106049/2015). M.R.B. is a Sir Henry Dale Fellow (101225/Z/13/Z), jointly funded by the Wellcome Trust and the Royal Society. This work has also been funded by Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER; NORTE-01-0145-FEDER-000013); FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and National Funds, through the FCT (POCI-01-0145-FEDER-007038). Acknowledgments Publisher Copyright: © 2019, The Author(s).

PY - 2020/7/1

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N2 - TET enzymes oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), a process thought to be intermediary in an active DNA demethylation mechanism. Notably, 5hmC is highly abundant in the brain and in neuronal cells. Here, we interrogated the function of Tet3 in neural precursor cells (NPCs), using a stable and inducible knockdown system and an in vitro neural differentiation protocol. We show that Tet3 is upregulated during neural differentiation, whereas Tet1 is downregulated. Surprisingly, Tet3 knockdown led to a de-repression of pluripotency-associated genes such as Oct4, Nanog or Tcl1, with concomitant hypomethylation. Moreover, in Tet3 knockdown NPCs, we observed the appearance of OCT4-positive cells forming cellular aggregates, suggesting de-differentiation of the cells. Notably, Tet3 KD led to a genome-scale loss of DNA methylation and hypermethylation of a smaller number of CpGs that are located at neurogenesis-related genes and at imprinting control regions (ICRs) of Peg10, Zrsr1 and Mcts2 imprinted genes. Overall, our results suggest that TET3 is necessary to maintain silencing of pluripotency genes and consequently neural stem cell identity, possibly through regulation of DNA methylation levels in neural precursor cells.

AB - TET enzymes oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), a process thought to be intermediary in an active DNA demethylation mechanism. Notably, 5hmC is highly abundant in the brain and in neuronal cells. Here, we interrogated the function of Tet3 in neural precursor cells (NPCs), using a stable and inducible knockdown system and an in vitro neural differentiation protocol. We show that Tet3 is upregulated during neural differentiation, whereas Tet1 is downregulated. Surprisingly, Tet3 knockdown led to a de-repression of pluripotency-associated genes such as Oct4, Nanog or Tcl1, with concomitant hypomethylation. Moreover, in Tet3 knockdown NPCs, we observed the appearance of OCT4-positive cells forming cellular aggregates, suggesting de-differentiation of the cells. Notably, Tet3 KD led to a genome-scale loss of DNA methylation and hypermethylation of a smaller number of CpGs that are located at neurogenesis-related genes and at imprinting control regions (ICRs) of Peg10, Zrsr1 and Mcts2 imprinted genes. Overall, our results suggest that TET3 is necessary to maintain silencing of pluripotency genes and consequently neural stem cell identity, possibly through regulation of DNA methylation levels in neural precursor cells.

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KW - Pluripotency

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Tet3 regulates cellular identity and DNA methylation in neural progenitor cells

Abstract

Bibliographical note

Keywords

UN SDGs

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