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Transcriptional repression of Bmp2 by p21Waf1/Cip1 links quiescence to neural stem cell maintenance

Nature Neuroscience volume 16, pages 1567–1575 (2013)Cite this article

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Abstract

Relative quiescence and self renewal are defining features of adult stem cells, but their potential coordination remains unclear. Subependymal neural stem cells (NSCs) lacking cyclin-dependent kinase (CDK) inhibitor (CKI) 1a (p21) exhibit rapid expansion that is followed by their permanent loss later in life. Here we demonstrate that transcription of the gene encoding bone morphogenetic protein 2 (Bmp2) in NSCs is under the direct negative control of p21 through actions that are independent of CDK. Loss of p21 in NSCs results in increased levels of secreted BMP2, which induce premature terminal differentiation of multipotent NSCs into mature non-neurogenic astrocytes in an autocrine and/or paracrine manner. We also show that the cell-nonautonomous p21-null phenotype is modulated by the Noggin-rich environment of the subependymal niche. The dual function that we describe here provides a physiological example of combined cell-autonomous and cell-nonautonomous functions of p21 with implications in self renewal, linking the relative quiescence of adult stem cells to their longevity and potentiality.

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Figure 1: The CKI p21 is expressed in stem cell–like astrocytes and promotes multipotency.
Figure 2: BMP2 production is increased in the absence of p21.
Figure 3: The CKI p21 prevents B cell terminal differentiation.
Figure 4: Enhanced BMP secretion is responsible for the deficit in self renewal of p21-null neurospheres.
Figure 5: p21 acts as a Bmp2 transcriptional repressor.

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Acknowledgements

We thank C. Gallego (Institut de Biologia Molecular de Barcelona), M.R. Campanero (Instituto de Investigaciones Biomédicas Alberto Sols, Madrid), S. Harris (University of Texas Health Science Center at San Antonio), S. Hitoshi (Shiga University of Medical Science), D. van der Kooy (University of Toronto) and W. Zhang (The Johns Hopkins University School of Medicine) for generously providing constructs. We also thank the Servicios Centrales de Soporte a la Investigación Experimental of the Universidad de Valencia for technical support. This work was supported by grants from the Ministerio de Economía y Competitividad (MINECO) (SAF program) to I.F., S.R.F. and A.V. and the Ministerio de Sanidad (Red TerCel and CIBERNED) and Generalitat Valenciana (Programas Prometeo, ISIC and ACOMP) to I.F. M.A.M.-T. was a recipient of a predoctoral fellowship from the Ministerio de Educación y Ciencia (FPI program), C.A-A. was a recipient of a predoctoral fellowship from the Ministerio de Educación y Ciencia (FPU program), E.G.-I. is supported by a predoctoral fellowship from Xunta de Galicia, and C.C. was a 'Parga Pondal' investigator supported by Xunta de Galicia.

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  1. Celia Andreu-Agulló

    Present address: Present address: Sloan-Kettering Institute, Department of Developmental Biology, New York, New York, USA.,

  2. Eva Porlan, José Manuel Morante-Redolat and María Ángeles Marqués-Torrejón: These authors contributed equally to this work.

Authors and Affiliations

  1. Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain

    Eva Porlan, José Manuel Morante-Redolat, María Ángeles Marqués-Torrejón, Celia Andreu-Agulló & Isabel Fariñas

  2. Departamento de Biología Celular, Universidad de Valencia, Valencia, Spain

    Eva Porlan, José Manuel Morante-Redolat, María Ángeles Marqués-Torrejón, Celia Andreu-Agulló, Sacri R Ferrón & Isabel Fariñas

  3. Departamento de Fisiología and Centro de Investigación en Medicina Molecular (CIMUS), Facultad de Medicina, Instituto de Investigación Sanitaria de Santiago (IDIS), Universidad de Santiago de Compostela, Santiago de Compostela, Spain

    Carmen Carneiro, Esther Gómez-Ibarlucea, Atenea Soto & Anxo Vidal

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Contributions

E.P. performed blots and ChIP in neurospheres and contributed to transcription assays. J.M.M.-R. performed transcription assays and contributed to the expression analyses. E.P. and J.M.M.-R. performed ELISA, intracerebro-ventricular infusion of Noggin and rescue experiments in vitro. M.A.M.-T. contributed to the analysis of the mouse phenotype in vivo and in vitro and to the multipotency analyses. C.A.-A. contributed to the coculture experiments and expression analysis. A.V. designed the generation and validation of p21 mutant constructs and the interference of p21 with shRNA on c17.2 cells. C.C., E.G.-I., A.S. and A.V. generated and validated p21 mutant constructs and the shRNA to p21 in c17.2 and 3T3 cells and performed ChIP in c17.2 cells. S.R.F. performed the analysis of the phenotype in vivo and in vitro, performed terminal astrocyte differentiation experiments and contributed to expression analyses and rescue experiments. I.F. designed and supervised the study, secured funding and analyzed the data. All authors contributed to experimental design, discussions and data analysis. E.P., J.M.M.-R., A.V., S.R.F. and I.F. wrote the manuscript.

Corresponding authors

Correspondence to Sacri R Ferrón or Isabel Fariñas.

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Integrated supplementary information

Supplementary Figure 1 Primary neurosphere-forming cells in p21-null mice.

(a) Phase contrast micrographs of primary neurospheres formed by cells directly obtained from 2-m Cdkn1a wild-type and mutant mice. (b) Graph representing the numbers of primary neurospheres obtained from individual SEZs of wild-type and Cdkn1a mutant mice at different adult ages, from 1-m to 12-m. Notice that between 1-m and 5-m of age more neurospheres are recovered from mutant tissue but that, after 5-m, the mutant yield decreases dramatically (2-m: n = 5; 4-m: n = 4; 5-m: n = 3; 6-m: n = 6; 12-m: n = 4 animals per genotype). (c) Senescence-associated (SA) β-galactosidase staining in early and late passages of wild-type and Cdkn1a-null cultures. (d) Self-renewal assays in early and late-passage secondary neurospheres derived from wild-type and Cdkn1a-null animals. F/DFn/ Dfd: 1.833/7/8; 4.770/6/6. P=0.4143, 0.0789. (e) γH2AX and 53BP1 simultaneous immunofluorescent detection in early and late-passage secondary neurospheres cells derived from wild-type and Cdkn1a-null animals. (f) Percentage of Sox2-high expressing cells and (g) percentage of γH2AX-53BP1 double-positive cells in early and late passages of wild-type and Cdkn1a-null neurosphere-derived cells. F/DFn/Dfd: 3.829/2/1; P=0.0229 (f); F/DFn/Dfd: 5.051/2/1; P=0.6003 (g). Data are shown as mean values ± s.e.m.; *p<0.05. Scale bar in a, c: 50 μm; in e: 25 μm; insert in f: 10 μm.

Supplementary Figure 2 Analysis of B-cell derivatives in the SEZ of p21-null 2-month old mice.

(a) Immunohistochemistry for Mash1 (red) and Ki67 (green) in coronal sections through the SEZ of 2-m wild-type and Cdkn1a mutant mice, as seen by confocal microscopy. White arrows indicate double-positive cells. (b) Immunohistochemistry for βIII-tubulin (green) and Ki67 (red) in the SEZ of 2-m Cdkn1a wild-type and mutant mice. White arrows indicate double-positive cells. (c) Percentage of Mash1+ and βIII-tubulin+ cells and (d) Mash1-Ki67 and βIIItubulin-Ki67 double positive cells found in the SEZ of young animals of the two genotypes. Data are shown as mean values ± s.e.m. (n = 3); *p<0.05, **p<0.01. Scale bar in a: 10 μm; in b: 10 μm.

Supplementary Figure 3 Enhanced astroglial differentiation in p21-null mice.

(a) Coronal sections of the SEZ of 12-m wild-type and Cdkn1a mutant mice stained for S100β (blue) and p-SMAD1 (red). Notice increased numbers of double positive cells, indicated by white arrows, in the mutant sample. (b) Percentage of pSMAD1+ and pSMAD1+S100β+ cells in the SEZ of 12-m wild-type and Cdkn1a mutant mice. Data are shown as mean values ± s.e.m. (n = 3); *p<0.05. Scale bars: in a, 20 μm.

Supplementary Figure 4 BMP2-induced astroglial differentiation in p21 null mice is independent of Sox2-induced replicative stress.

(a) Percentage of GFAP+ cells expressing high levels of Sox2 (Sox2high) amongst GFAP-Sox2 double positive cells, in saline (−) vs. Noggin (+) infused p21-null mice. F/DFn/Dfd: 17.30/2/2; P=0.1093. (b) Percentage of S100β/γH2AX double positive cells in wild-type and saline and noggin-infused p21-null animals. F/DFn/Dfd: 2.046/4/2; 12.63/2/1 P=0.7085; 0.3902. (c) Examples of the simultaneous detection of S100β and γH2AX in the SEZ of wild-type and saline and Noggin-infused p21-null animals. (d) Percentage of cells expressing high levels of Sox2 and (e) percentages of γH2AX and 53BP1 doubly positive cells in early and late-passage, wild-type and p21-null Noggin treated cells. (f) Fold change in neurosphere numbers produced by FACS-sorted cells from neurospheres infected with a retroviral construct for the expression of Sox2 (pMY-sox2) or empty vector (pMY), treated or not with 100 ng/ml Noggin. Data are shown as mean values ± s.e.m; *p<0.05. Scale bar in c: 35 μm.

Supplementary Figure 5 Bmp2 proximal promoter is preferentially used in neurospheres.

(a) Schematic representation of the murine Bmp2 promoter (grey bar) showing two alternative transcription initiation sites (TSS) at +1 and −736, and the two fragments used for reporter experiments: distal, (−2712/−700)-luciferase, red bar, and proximal, (−643/+165)-luciferase, purple bar. Neurospheres were transfected with either of these reporter constructs and luciferase activity was measured 24 h later. (b) Interfered shRNAp21-c17.2 cells were transfected with an E2F-luciferase reporter bearing 3 tandem repeats of the hydropholate reductase E2F-binding site (E2F-luc) and treated with 2.5 μM CDK inhibitor 2-bromo-12,13-dihydro-5H-indolo [2,3-a] pyrrolo [3,4-c] carbazole-5,7(6H)-dione. Interfered shRNAp21-c17.2 cells were transfected in parallel with the proximal Bmp2 promoter in the presence or in the absence of the inhibitor. (c) p21-null neurospheres were transfected with the E2F-luciferase reporter (E2F-luc), or co-transfected with pCDNA-p21 and the proximal Bmp2-promoter, and cells were treated or not with 40 μM E2F inhibitor HLM006474. Luciferase activity was measured 24h after transfection. (d) Immunoblot for Flag epitope showing the expression of the p21-mutants used (upper panel) and co-immunoprecipitation of p21-Flag and CDK2 or Cyclin A in mouse fibroblasts transfected with the different p21-mutants used (bottom panels). (e) Percentage of cells transfected with the different p21-mutant constructs that are retained in G1 phase of the cell cycle shown as increase relative to empty vector values. Data are shown as mean values ± s.e.m. (n = 4, panels a–c; n =2, panel e); **p<0.05,**p<0.01, **p<0.001.

Supplementary Figure 6 Uncropped protein immunoblots and DNA agarose gels.

(a) Immunoblots in Fig. 2d. (b) Immunoblot in Fig.4d. (c) ChIP agarose gels in Fig. 5d. (d) Immunoblots in Fig. S5d. Dashed squares outline the cropped area presented in the corresponding figure. MWM: molecular weight marker, IB: immunoblot, IP: immunoprecipitation, e.v.: empty vector, NTC: non template control.

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Porlan, E., Morante-Redolat, J., Marqués-Torrejón, M. et al. Transcriptional repression of Bmp2 by p21Waf1/Cip1 links quiescence to neural stem cell maintenance. Nat Neurosci 16, 1567–1575 (2013). https://doi.org/10.1038/nn.3545

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