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

Eva Porlan1,2,5, José Manuel Morante-Redolat1,2,5, María Ángeles Marqués-Torrejón1,2,5, Celia Andreu-Agulló1,2,4, Carmen Carneiro3, Esther Gómez-Ibarlucea3, Atenea Soto3, Anxo Vidal3,

Sacri R. Ferrón2,6 and Isabel Fariñas1,2,6

1Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 2Departamento de Biología Celular, Universidad de Valencia, Spain. 3Departamento 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,

Spain.

4Present address: Sloan-Kettering Institute, Department of Developmental Biology, New York, USA.

5These authors contributed equally to this work

6To whom all correspondence should be addressed, at:

Isabel Fariñas or Sacri R. FerrónDepartamento de Biología CelularUniversidad de Valencia46100 Burjassot, SpainPhones: +34-963 543784/3246FAX: +34-963 543404isabel.farinas@uv.es or sacramento.rodriguez@uv.es

Supplementary Figures & Figure Legends

Nature Neuroscience: doi:10.1038/nn.3545

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Figure S1. 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 repre-senting 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.

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Figure S2. Analysis of B-cell derivatives in the SEZ of 2-month old p21-null mice. (a) Immunohistochemis-try 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; Scale bar in a: 10 µm; in b: 10 µm.

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Figure S4. 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.

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Nature Neuroscience: doi:10.1038/nn.3545

Figure S5. 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 proxi-mal, (-643/+165)-luciferase, purple bar. Neurospheres were transfected with either of these reporter constructs. F/DFn/Dfd: 4.187/2/2. P=0.3856. (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. Interfe-red shRNAp21-c17.2 cells were transfected in parallel with the proximal Bmp2 promoter in the presence or in the absence of the inhibitor. F/DFn/Dfd: 1.653/5/5; 10.12/5/3; 8.761/2/2. P= 0.5947; 0.0854; 0.2049. (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. (d) Immu-noblot 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 3T3 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.

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Figure S6. Uncropped protein immunoblots and DNA agarose gels. (a) Immunoblots in Fig. 2d. (b) Immunob-lot 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. C: cytosolic fraction, N: nuclear fraction, MWM: molecular weight marker, IB: immunoblot, IP: immunoprecipitation, e.v.: empty vector, NTC: non template control.

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