TET3 controls the expression of the H3K27me3 demethylase Kdm6b during neural commitment

Abstract

The acquisition of cell identity is associated with developmentally regulated changes in the cellular histone methylation signatures. For instance, commitment to neural differentiation relies on the tightly controlled gain or loss of H3K27me3, a hallmark of polycomb-mediated transcriptional gene silencing, at specific gene sets. The KDM6B demethylase, which removes H3K27me3 marks at defined promoters and enhancers, is a key factor in neurogenesis. Therefore, to better understand the epigenetic regulation of neural fate acquisition, it is important to determine how Kdm6b expression is regulated. Here, we investigated the molecular mechanisms involved in the induction of Kdm6b expression upon neural commitment of mouse embryonic stem cells. We found that the increase in Kdm6b expression is linked to a rearrangement between two 3D configurations defined by the promoter contact with two different regions in the Kdm6b locus. This is associated with changes in 5-hydroxymethylcytosine (5hmC) levels at these two regions, and requires a functional ten-eleven-translocation (TET) 3 protein. Altogether, our data support a model whereby Kdm6b induction upon neural commitment relies on an intronic enhancer the activity of which is defined by its TET3-mediated 5-hmC level. This original observation reveals an unexpected interplay between the 5-hmC and H3K27me3 pathways during neural lineage commitment in mammals. It also questions to which extent KDM6B-mediated changes in H3K27me3 level account for the TET-mediated effects on gene expression.

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Acknowledgements

We thank Claire Chazaud for advice in ES cell derivation and all members of P.A.’s team for critical reading of the manuscript.

Funding

This research has been financed by the French government IDEX-ISITE initiative 16- IDEX-0001 (CAP 20–25) (Emergence, Challenge 3-recherche) (to F. C. and P. A.), the Ligue Contre le Cancer comité de l’Ardéche, du Puy de Dôme and du Cantal (to F. C. and P. A.) and “Conseil Régional d’Auvergne” (to PA). B. M. had a fellowship from Fondation pour la recherche médicale (FRM). C.J.M. is funded by the Portuguese Foundation for Science and Technology (FCT-CEECIND/00371/2017).

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PA initiated and supervised the study. BM, FC and PA designed the study. BM, JC, TB, AC, SMM, DN, CGG, SC, NA, CCH, CC, IV, CJM, FC and PA performed the experiments. FC performed the bioinformatics analyses. BM, JC, AC, DN, CGG, IV, JM, FC and PA analyzed the data. BM and FC produced the figures with JC’s and IV’s input. PA wrote the paper. All authors read and approved the final manuscript.

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Correspondence to Franck Court or Philippe Arnaud.

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Supp Figure 1: Characterization of the Kdm6b locus. a

Pou5f1, Nestin and Pax6 expression levels in ES cells (n=6) and in NP cells at day 12 (n=6) of in vitro corticogenesis. b Kdm6b expression level in ES cells (n=4) and in NP cells (n=4) at day 12 of in vitro corticogenesis. Expression was assessed with primers located in exon 11 (left panel) and with primers between exon 21 and 23 (right panel). c Race-PCR mapping along the Kdm6b locus in ES cells, neonate brain, and at D8 of in vitro corticogenesis. d CGI1 and CGI2 bisulphite-based DNA methylation analyses by COBRA and/or sequencing in ES and NP cells. For each sequenced region, the methylation patterns are symbolized by lollipops (black: methylated CpG; white: unmethylated CpG). In a and b, statistical significance was determined with the unpaired t-test (p values in the figure). Data are presented as the mean ± SEM (TIF 2047 kb)

Supp Figure 2: Genomic features of the Kdm6b locus. a

Position of the Kdm6b locus relative to the topological associated domains (TAD), as defined by Dixon et al [34]. b The main genomic features are conserved at the mouse Kdm6b and human KDM6B loci, including the CpG island (CGI3) between exons 17 and 18. c Kdm6bos expression level in ES cells (n=3) and at D8 (n=3) and D12 (NP stage; n=3) of in vitro corticogenesis. Statistical significance was determined by one-way ANOVA (comparison of each differentiation point with ES cells). Data are presented as the mean ± SEM. There was no significant difference between ES and the differentiation points. d Data-mining analysis of H3K4me1, H3K27ac, and H3K4me3 at the Kdm6b locus in ES cells, whole brain, and NP cells. The GRO-seq signals obtained from ES cell are also shown. Signal are shown for Kdm6b sense (upper panel) and antisense (lower panel) transcription (TIF 3262 kb)

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Montibus, B., Cercy, J., Bouschet, T. et al. TET3 controls the expression of the H3K27me3 demethylase Kdm6b during neural commitment. Cell. Mol. Life Sci. 78, 757–768 (2021). https://doi.org/10.1007/s00018-020-03541-8

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Keywords

  • H3K27me3
  • 5-Hydroxymethylcytosine
  • Kdm6b
  • Tet3
  • Enhancer
  • Neural stem cells
  • Neurogenesis