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Zeb2 recruits HDAC–NuRD to inhibit Notch and controls Schwann cell differentiation and remyelination

Nature Neuroscience volume 19, pages 1060–1072 (2016)Cite this article

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Abstract

The mechanisms that coordinate and balance a complex network of opposing regulators to control Schwann cell (SC) differentiation remain elusive. Here we demonstrate that zinc-finger E-box-binding homeobox 2 (Zeb2, also called Sip1) transcription factor is a critical intrinsic timer that controls the onset of SC differentiation by recruiting histone deacetylases HDAC 1 and 2 (HDAC1/2) and nucleosome remodeling and deacetylase complex (NuRD) co-repressor complexes in mice. Zeb2 deletion arrests SCs at an undifferentiated state during peripheral nerve development and inhibits remyelination after injury. Zeb2 antagonizes inhibitory effectors including Notch and Sox2. Importantly, genome-wide transcriptome analysis reveals a Zeb2 target gene encoding the Notch effector Hey2 as a potent inhibitor for Schwann cell differentiation. Strikingly, a genetic Zeb2 variant associated with Mowat-Wilson syndrome disrupts the interaction with HDAC1/2–NuRD and abolishes Zeb2 activity for SC differentiation. Therefore, Zeb2 controls SC maturation by recruiting HDAC1/2–NuRD complexes and inhibiting a Notch–Hey2 signaling axis, pointing to the critical role of HDAC1/2–NuRD activity in peripheral neuropathies caused by ZEB2 mutations.

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Figure 1: SC-specific deletion of Zeb2 results in peripheral nerve hypomyelination.
Figure 2: Zeb2 is required for SC myelination.
Figure 3: Zeb2 deletion in SCs inhibits SC differentiation and myelination.
Figure 4: Zeb2 is required for SC differentiation during nerve repair.
Figure 5: Zeb2 represses Sox2 activity to promote SC differentiation.
Figure 6: Zeb2 regulates the SC differentiation program and inhibits Notch effector Hey2 expression.
Figure 7: Zeb2 represses a Notch–Hey2 signaling axis.
Figure 8: Zeb2 associates with HDAC1 and HDAC2 to repress SC differentiation inhibitor expression.

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  • 20 June 2016

    In the version of this article initially published online, Danny Huylebroeck was listed under affiliation 2 (Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China) instead of affiliation 3 (Department of Cell Biology, Erasmus University Medical Center, Rotterdam, the Netherlands). The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

The authors thank X. Chen and Z. Ma for technical support and initial observation of Zeb2 mutants. We thank A. Rauch for discussing ZEB2 missense mutations in MOWS patients, J. Svaren and E. Hurlock for suggestions and G. Verstappen and L. van Grunsven for initial study of Zeb2R22G–NuRD interactions. We are grateful to K.-A. Nave for communications of unpublished data. We also thank R. Kopan (University of Cincinnati) and A.J. Capiobianco (University of Miami) for the TP-1 reporter and lentiviral DN-MAML constructs and to J.W. Schneider and E.N. Olson (University of Texas Southwestern Medical Center)) for Notch-GFP reporter mice and Flag-HRT2/Hey2 vectors. This study was funded in part by grants from the US National Institute of Health R01NS072427 and R01NS075243 to Q.R.L., R01NS062796 to J.R.C. and R01AR064551 to M.P.J., and from the National Multiple Sclerosis Society (NMSS-4727) to Q.R.L. L.M.N.W. was supported by an NMSS Postdoctoral Fellowship (FA 2045A1/T). The work was also supported by Belspo grant IAP7-07 DevRepair, the Research Council of KU Leuven (GOA-11/012), FWO-V (G.0782.14), the type 3 large-infrastructure support InfraMouse by the Hercules Foundation (ZW09-03) and Erasmus MC start-up funds, to D.H.

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Authors and Affiliations

  1. Department of Pediatrics, Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA

    Lai Man Natalie Wu, Jincheng Wang, Chuntao Zhao, Haibo Wang, Liguo Zhang & Q Richard Lu

  2. Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China

    Jincheng Wang

  3. Department of Cell Biology, Erasmus University Medical Center, Rotterdam, the Netherlands

    Andrea Conidi & Danny Huylebroeck

  4. Department of Anesthesiology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA

    Zachary Ford & Michael P Jankowski

  5. Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany

    Christiane Zweier

  6. Department of Biological Science, Rutgers University, Newark, New Jersey, USA

    Brian G Ayee & Patrice Maurel

  7. KU Leuven Department of Human Genetics, VIB Center for the Biology of Disease, Leuven, Belgium

    An Zwijsen

  8. Department of Neurology, Program in Biomedical and Neurosciences, University of California, San Francisco, San Francisco, California, USA.,

    Jonah R Chan

  9. KU Leuven Department of Development and Regeneration, Laboratory of Molecular Biology (Celgen), Leuven, Belgium

    Danny Huylebroeck

  10. Key Laboratory of Birth Defects, Children's Hospital of Fudan University, Shanghai, China

    Q Richard Lu

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Contributions

L.M.N.W. and Q.R.L. designed the experiments. L.M.N.W. carried out the studies. J.W., A.C. and L.Z. assisted with coimmunoprecipitation biochemical experiments for HDAC–NuRD and BMP–Smads. C. Zhao assisted with ChIP experiments. H.W. assisted with Notch-TP1 reporter assays. C. Zweier provided ZEB2 variant identification data. Z.F. assisted with heat hypersensitivity experiments. M.P.J. assisted with CMAP recordings. B.G.A., P.M., A.Z. and J.R.C. provided input and data interpretation. D.H. provided mice with the loxP-flanked Zeb2 allele and input on the study. L.M.N.W. and Q.R.L. wrote the manuscript.

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Correspondence to Q Richard Lu.

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Integrated supplementary information

Supplementary Figure 1 Zeb2loxP/+;Dhh-cre peripheral nerves are comparable with wildtype or Zeb2loxP/loxP nerves

(a) Immunostaining showing Ki67 and Oct6 expression in P7 wildtype and Zeb2loxP/+;Dhh-cre longitudinal sciatic nerves. n = 3 animals/genotype. Scale bar: 50 µm. (b) Quantification of the proportions of Oct6+ (left) and Ki67+ (right) nuclei in P7 wildtype and Zeb2loxP/+;Dhh-cre sciatic nerves. Data are mean ± S.E.M.; P(Oct6) = 0.9896 t = 0.01390 df = 4; P(Ki67) = 0.7743 t = 0.3068 df = 4; Two-tailed unpaired Student’s t-test; n = 3 animals/genotype. (c) EM images showing unmyelinated axons in Remak bundles intercalated by SC processes in sciatic nerves of Zeb2loxP/loxP and Zeb2loxP/+;Dhh-cre mice in contrast to unsorted axon bundles in Zeb2loxP/loxP;Dhh-cre mice at 3 weeks. Scale bar: 2 µm. (d) A scatter plot showing g-ratio of myelinated axons of Zeb2loxP/loxP and Zeb2loxP/+;Dhh-cre sciatic nerves at 3 weeks. At least 100 axons per animal were counted. n = 3 animals/genotype. (e) g-ratios between Zeb2loxP/loxP and Zeb2loxP/+;Dhh-cre sciatic nerves at 3 weeks. n = 3 animals/genotype; data are mean ± S.E.M., P = 0.9272 t = 0.09727 df = 4; Two-tailed unpaired Student’s t-test. (f) Number of myelinated axons per area in sciatic nerves of Zeb2loxP/loxP and Zeb2loxP/+;Dhh-cre. Data are mean ± S.E.M., n = 3; Mann-Whitney test; P = 0.9000. (g) (Left) Number of unmyelinated axons per Remak bundle in sciatic nerves of 3-week-old Zeb2loxP/loxP and Zeb2loxP/+;Dhh-cre. Data are mean ± S.E.M., n = 3 animals/genotype; Mann-Whitney test; P = 0.4000. (Right) Percentage frequency distribution of the number of axons per Remak bundles in Zeb2loxP/loxP and Zeb2loxP/+;Dhh-cre sciatic nerves. (h) Zeb2loxP/loxP, Zeb2loxP/+;Dhh-cre and Zeb2loxP/loxP;Dhh-cre mice were tested for heat hypersensitivity to 50oC water 24h following carrageenan injection into the hairy hindpaw skin. Data are mean ± S.E.M., Zeb2loxP/loxP, n = 6; Zeb2loxP/+;Dhh-cre, n = 7; and Zeb2loxP/loxP;Dhh-cre, n = 4; *P < 0.05; **P < 0.01; Two-way repeated measures ANOVA with Sidak’s multiple comparisons test; Baseline: P > 0.05 Zeb2loxP/loxP vs Zeb2loxP/+;Dhh-cre; P > 0.05 Zeb2loxP/+;Dhh-cre vs Zeb2loxP/loxP;Dhh-cre, P > 0.05 Zeb2loxP/loxP vs Zeb2loxP/loxP;Dhh-cre; Carrageenan: P > 0.05 Zeb2loxP/loxP vs Zeb2loxP/+;Dhh-cre; P > 0.05 Zeb2loxP/+;Dhh-cre vs Zeb2loxP/loxP;Dhh-cre, P > 0.05 Zeb2loxP/loxP vs Zeb2loxP/loxP;Dhh-cre. Within animal group-baseline vs Carrageenan: Zeb2loxP/loxP P < 0.05; Zeb2loxP/+;Dhh-cre P < 0.01; Zeb2loxP/loxP;Dhh-cre P < 0.01. Interaction F (2, 14) = 0.7859; Time F (1, 14) = 44.03; Column Factor F (2, 14) = 0.1186; Subjects (matching) F (14, 14) = 2.49.

Supplementary Figure 2 Zeb2 is not required for myelin maintenance in SCs

(a) Survival curves of control (red) and Zeb2 cKO (black) mice (n = 40 animals/genotype). (b) Electron micrographs of cross sections of 5-month-old sciatic nerves from control (Zeb2loxP/+) and Zeb2 iKO (Zeb2loxP/loxP;Plp-creERT) induced by tamoxifen from 4 weeks old. n = 4. Scale bar: 2 µm.

Supplementary Figure 3 siRNA-mediated Zeb2 knockdown causes a defect in SC differentiation

(a) qRT-PCR analysis showing expression of Zeb2, Oct6, Krox20, Mbp and Mpz in rat SCs transfected with scrambled and Zeb2 siRNA cultured in neuregulin-containing proliferation medium. (*P < 0.05, **P < 0.01; ***P < 0.001; n = 3 independent experiments; Data are presented as mean ± S.E.M., P(Zeb2) = 0.0001 t = 14.14 df = 4; P(Sox10) = 0.0416 t = 2.959 df = 4; P(Oct6) = 0.0003 t = 11.58 df = 4; P(Krox20) = 0.0072 t = 5.052 df = 4; P(Mbp) = 0.0077 t = 4.953 df = 4; P(Mpz) = 0.0037 t = 6.089 df = 4; Two-tailed unpaired Student’s t-test). (b) qRT-PCR analysis showing Oct6, Krox20, Mbp and Mpz expression in rat SCs transfected with scrambled and Zeb2 siRNA and induced to differentiate for 18 hours. (*P < 0.05, **P < 0.01; n = 3 independent experiments; Data are presented as mean ± S.E.M., P(Zeb2) = 0.0054 t = 5.489 df = 4; P(Oct6) = 0.0098 t = 5.891 df = 4; P(Krox20) = 0.0063 t = 5.254 df = 4; P(Mbp) = 0.0175 t = 3.905 df = 4; P(Mpz) = 0.0101 t = 4.586 df = 4; Two-tailed unpaired Student’s t-test). (c) ImmunoloxPuorescence labeling for differentiation markers, Oct6 and Krox20 in rat SCs transfected with scrambled and Zeb2 siRNA and induced to differentiate for 18 hours; n = 3 independent experiments. Scale bar: 50 µm. (d) Quantification of Oct6+ and Krox20+ cells relative to DAPI+ nuclei in scrambled and Zeb2-siRNA knockdown SCs induced to differentiate. Data are presented as mean ± S.E.M (***P < 0.001, n = 3 independent experiments; P(Oct6) < 0.0001 t = 40.4 df = 4; P(Krox20) = 0.0005 t = 10.31 df = 4; Two-tailed unpaired Student’s t-test). (e) ChIP assays using a Zeb2 antibody for its recruitment to the Zeb2 binding sites at -0.8kb and -3.9 kb from TSS in the Oct6 promoter were performed on chromatin from primary SCs exposed in proliferation or differentiation (9 h) media. IgG IP was used as control. Data are mean ± S.E.M; ***P < 0.001; n = 3 independent experiments; P(-0.8 kb) = 0.0061 t = 5.294 df = 4; P(-3.9 kb) = 0.0047 t = 4.367 df = 4; Two-tailed unpaired Student’s t-test.

Supplementary Figure 4 ChIP assays show that Zeb2 does not bind c-Jun promoter or to distal control regions of Sox2, Hey2 or Oct6 gene loci

(a) Zeb2 occupancy by ChIP–PCR in differentiating rat SCs on the promoter of c-Jun (-6 kb from TSS) shows no binding of Zeb2 to the putative Zeb2 binding site in the c-Jun promoter. n = 3 independent experiments; P = 0.9682 t = 0.04134 df = 7; Two-tailed unpaired Student’s t-test. (b) Distal control region of Sox2 is located at 25 kb upstream of TSS, that of Hey2 at 20 kb upstream of TSS and that of Oct6 at 18 kb. Input chromatin from rat SCs differentiated in the presence of cAMP for 9 hours shows amplification of the PCR fragment, Zeb2 binding in these regions was not detected in ChIP samples using anti-Zeb2 antibody. Full-length images are presented in Supplementary Figure 10d.

Supplementary Figure 5 Zeb2 regulates genes involved in the regulation of radial sorting

(a) Differential expression of selected genes involved in radial sorting regulation was detected based on transcriptome profiling of P7 control and Zeb2 cKO sciatic nerves. The table lists selected genes reported to inloxPuence radial sorting, their fold change in Zeb2 cKO sciatic nerves relative to control nerves and the sorting defects in corresponding mutants. Downregulated and upregulated genes are highlighted in red and blue, respectively. (b) qRT-PCR analysis of independently prepared P7 control and Zeb2 cKO sciatic nerves confirms the differential gene expression. Data are presented as mean ± S.E.M (**p < 0.01; ***p < 0.001; n = 3 independent experiments, P(Cdc42) = 0.0057 t = 5.402 df = 4; P(Dag1) = 0.0631 t = 2.553 df = 4; P(Itga6) = 0.001 t = 8.511 df = 4; P(Itga7) = 0.0004 t = 11.28 df = 4; P(Lama2) = 0.0007 t = 9.289 df = 4; Two-tailed unpaired Student’s t-test).

Supplementary Figure 6 Zeb2 deletion does not lead to activation of BMP signaling in sciatic nerves

Immunoblot analysis of p-Smad (1/5/8) and Smad7 in sciatic nerves of control and Zeb2 cKO mutants at P14. No increase in p-Smad (1/5/8) or alteration of Smad7 was detected in in Zeb2 cKO sciatic nerves. The p-Smad level appears to be even reduced in Zeb2 cKO sciatic nerves. GAPDH was used as a loading control. n = 3 independent experiments. Full-length blots are presented in Supplementary Figure 10c.

Supplementary Figure 7 Model schematics

(a) Strategy to detect Notch-mediated activation of eGFP expression in the transgenic Notch/RBP-J-RE-EGFP reporter line. (b) A schematic representation of the NuRD complex, composed of multiple subunits including the histone deacetylase core proteins HDAC1 and HDAC2, the histone-binding proteins RbAp46 and RbAp48, the chromatin remodeling ATPase Mi-2, the metastasis-associated proteins MTA1 (or MTA2/MTA3), the methyl-CpG-binding domain protein MBD3. (c) Schematic representation of SC lineage development and the dynamic expression of the transcription regulators as indicated. (d) A model depicts a dual mode of action by Zeb2 to promote SC differentiation by forming a transcriptional co-repressor complex with Hadc1/2-NuRD to inhibit differentiation inhibitory genes, e.g. Notch-Hey2 and Sox2 in pre-myelinating SCs, while activating pro-myelinating genes e.g. Oct6 and Krox20.

Supplementary Figure 8 Hey2 or Sox2 gene expression is not altered in Zeb2-deficient nerves after injury compared with controls

qRT-PCR analysis of Hey2 or Sox2 in transected distal sciatic nerves from adult control and Zeb2 iKO mice 7 days post-lesion showing no significant changes in mRNA levels. n = 3 animals/genotypes; data are presented as mean ± S.E.M; P(Zeb2) = 0.046 t = 2.422 df = 7; P(Hey2) = 0.3864 t = 0.9713 df = 4; P(Sox2) = 0.4095 t = 0.9203 df = 4; Two-tailed unpaired Student’s t-test.

Supplementary Figure 9 Uncropped western blots for Figures 5c, 6g and 8b,d

Supplementary Figure 10 Uncropped western blots for Figure 8g,f and Supplementary Figures 4 and 6

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Wu, L., Wang, J., Conidi, A. et al. Zeb2 recruits HDAC–NuRD to inhibit Notch and controls Schwann cell differentiation and remyelination. Nat Neurosci 19, 1060–1072 (2016). https://doi.org/10.1038/nn.4322

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