Regulation of TET protein stability by calpains - PubMed (original) (raw)

Regulation of TET protein stability by calpains

Yu Wang et al. Cell Rep. 2014.

Abstract

DNA methylation at the fifth position of cytosine (5mC) is an important epigenetic modification that affects chromatin structure and gene expression. Recent studies have established a critical function of the Ten-eleven translocation (Tet) family of proteins in regulating DNA methylation dynamics. Three Tet genes have been identified in mammals, and they all encode for proteins capable of oxidizing 5mC as part of the DNA demethylation process. Although regulation of Tet expression at the transcriptional level is well documented, how TET proteins are regulated at posttranslational level is poorly understood. In this study, we report that all three TET proteins are direct substrates of calpains, a family of calcium-dependent proteases. Specifically, calpain1 mediates TET1 and TET2 turnover in mouse ESCs, and calpain2 regulates TET3 level during differentiation. This study provides evidence that TET proteins are subject to calpain-mediated degradation.

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1

Figure 1. Regulation of TET protein levels by transcription and protein stability

(A) RT-qPCR analysis of Tet mRNA levels during mESC to NPC differentiation. While Tet1 and Tet2 levels decrease during differentiation, Tet3 level is significantly up-regulated. Data represent the mean of three independent experiments, and Tet levels in mESCs are set as 1. (B, C) Representative Western blot (B) and quantification of three repeats (C) demonstrate that TET protein levels generally follow mRNA levels during NPC differentiation. (D, E) Representative Western blot analysis of TET1 and TET2 levels in mESCs treated with chloroquine, calpeptin, Z-VAD-FAM and MG132 for 24h. Quantification of three independent experiments was shown in panel E. (F, G) Calpeptin increases the half-life of Flag-TET2 protein. Western blot (F) and quantification (G) of the Flag-TET2 levels in the presence or absence of calpeptin upon inhibition of protein translation by cycloheximide. (H, I) Representative Western blot analysis of TET3 in day 7 embryoid body (EB) treated with chloroquine, calpeptin, Z-VAD-FAM and MG132 for 24h, and the results were quantified in (I). (J) Calpain activity is detectable in mESCs and during their differentiation. Western blot analysis of mESC lysate with a spectrin antibody identified both full length (arrow) and cleaved spectrin (*), a marker for calpain activity. Spectrin cleavage is detectable during mESC differentiation (lanes 4, 5), and was prevented by calpeptin treatment (compare lane 1 and 2).

Figure 2

Figure 2. Tet proteins are direct substrates of calpain1 and calpain2

(A, B) Representative Western blot analysis (A) and quantification of three independent repeats (B) demonstrate that exogenously expressed TET protein levels can be reduced by co-expression of calpain1 or calpain2 in 293T cells. (C) Western blot analysis demonstrates that both calpain 1 and calpain 2 can cleave all three Tet proteins in vitro. Purified Flag-Tet1, Tet2 and Tet3 were incubated with buffer alone, or purified Flag-calpain1 or calpain2 at room temperature for 30min before Western blot analysis using Flag antibody. Cleaved products are indicated by (*), full length TET (arrow), calpains (arrow head). (D, E) RT-qPCR (D) and Western blot (E) analysis demonstrate that calpain1 and calpain2 are reversely expressed in mESCs and NPCs. Data represent the mean of three independent experiments, and value from mESC is normalized as 1. (F) Western blot analysis demonstrates that both TET1 and TET2 levels are increased in calpain1 knockout mESCs, while calpain2 knockout have little effect. Calpain1 and calpain2 knockout mESC were generated by CRISPR. (G) Western blot analysis of the TET3 levels in day 8 EB demonstrates calpain2 knockout increases TET3 levels, while the effect of calpain1 knockout is modest. (H) Quantification of three independent experiments (F and G), value in WT cells is set as 1.

Figure 3

Figure 3. Effects of calpain-mediated TET cleavage on gene expression and NPC differentiation

(A) RT-qPCR analysis demonstrates that calpain1 or calpain2 knockout in mESCs does not affect pluripotent gene expression. Data represent the mean of three independent experiments, and value in WT mESC is set as 1. (B, C) Dot blot analysis (B) and densitometry quantification of three repeats (C) demonstrate that calpain1 knockout, but not calpain2, increased 5hmC levels in mESCs. (D) RT-qPCR analysis demonstrates that Tet1 knockdown in mESC enhances trophectoderm lineage genes (Cdx2 and Eomes) expression, and inhibits Lefty1. Knockout of calpain1 opposes this tendency, which is rescued by Tet1 knockdown. Data represent the mean of three independent experiments, and value in WT mESC is set as 1. (E) RT-qPCR analysis demonstrates that during differentiation to NPC (EB day 8), Tet3 knockout significantly reduces the expression of neuronal markers Ngn2 and Pax6, while calpain2 knockout enhanced their expression, which is reversed by Tet3 knockdown. In contrast, β3-tubulin expression is not affected by either Tet3 or calpain2. Value in WT EB is set as 1. (F) Immunostaining demonstrates generation of Nestin and Sox2 double positive NPCs. After EB disassociation and a 48h adherent culture, Nestin and Sox2 positive NPC were successfully generated from all WT and knockout cells. (G) TET3 and calpain2 have opposite effect on mESC differentiation to NPC. While Tet3−/− significantly reduced NPC generation, CAPN2−/− enhanced the differentiation efficiency, which is abolished by Tet3 knockdown. Number from WT cells is normalized to 1. P<0.05 (*), P<0.01 (**).

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