SUPPLEMENTAL INFORMATION

Immunohistochemistry

Mice were perfused intracardially with 1× PBS followed by 4% paraformaldehyde. Brains were dissected and fixed overnight in 4% paraformaldehyde, rinsed, cryoprotected, and frozen in liquid N2. Cryosections (16 or 30 µm) were sliced on a cryostat. Standard immunostaining procedures were used for most antibodies, and appropriate conjugated secondary antibodies were used. For BrdU immunostaining, sections were pretreated in 2N HCL for 30 min. For in vitro immunofluorescence staining, cells were fixed with 4% paraformaldehyde, and immunofluorescence staining was performed. Labeled cells were visualized using a Olympus IX71 inverted microscope and a high resolution digital camera (Hamamatsu). Quantification of cell phenotype was done by sampling 5-10 random fields in each well and counting a total of 250-500 cells at 20X magnification. Throughout the study, definitive neuronal or glial cells were scored on the basis of morphological criteria (elaboration of processes), as well as immunoreactivity with various markers (e.g., Tuj1 and Map2ab staining). In some cases, Hoechst or GFP was used to identify individual cells as counterstaining.

Stereological Cell Quantification and Digital Image Analysis

Cells positive for BrdU, CFPnuc, GFAP, CldU, IdU, NeuN, PCNA, AC3, TUNEL, NGN2, NEUROD1, PROX1, TUJ1, MAP2ab, DCX, GFP, and mCherry were analyzed in serial sections through the DG of SOX2cKO and wild-type. We counted positive cells under a ×63 objective using Slidebook software (Olympus). The total number of cells was counted using an optical dissector technique. Pictures were taken with the same exposure time and contrast/brightness parameters. The average intensity for a particular marker was determined using ImageJ software (http://rsb.info.nih.gov/ij/) and normalized to the average intensity of dentate gyrus granule neurons. A minimum of 6 pictures containing at least 40 cells was analyzed for each marker.

Western blotting

Adult hipNPCs were washed in cold PBS and lysed with ice-cold Trizol buffer. Lysates were run on SDS-Tris glycine gels and transferred to PVDF membranes, which were blocked in 5% w/v skim milk in TBST and stained in 5% w/v BSA in TBST. Detection was performed using ECL detection kit (Advansta) and the ChemiDoc XRS system (Bio- Rad).

Electrophysiological recordings

Animals were anaesthetized with isoflurane, and brains removed and placed into ice- cold sucrose ACSF (83mM NaCl, 2.5mM KCl, 1mM NaH2PO4, 26.2mM NaHCO3, 22mM Glucose, 72mM Sucrose, 0.5mM CaCl2, 3.3mM MgSO4), with 95% O2, 5% CO2. 300µm sagittal slices were cut using a Leica VT 1000S vibratome, then allowed to recover for 30-40 min at 32°C, followed by at least 30 min at room temperature prior to recording. Slices were transferred to an electrophysiology rig, and maintained in circulating bicarb ACSF (119mM NaCl, 2.5mM KCl, 26mM NaHCO3, 1mM NaH2PO4, 11mM Glucose, 2.5mM CaCl2, 1.3mM MgSO4), plus 100µM PTX (Tocris). GFP-positive dentate granule cells were identified using epifluorescence (Olympus BX51WI). Whole cell recordings were made using an Axopatch 200B amplifier. Pipettes (resistance 3-5 MΩ) were pulled on a horizontal puller (Sutter P-97) and filled with a CsGluc internal solution (130mM D-Gluconic Acid, 130mM CsOH, 5mM NaCl, 12mM Phosphocreatine, 10mM HEPES, 10mM EGTA, 3mM adenosine triphosphate, 0.2mM guanosine triphosphate [pH 7.3]). Signals were low-pass filtered at 2kHz and digitized at 5kHz. Access resistance (Ra) was assessed throughout recordings by monitoring the capacitative transient response to a 5 mV test pulse. Mean Ra was 23.9 ± 1.2 MΩ. Experiments in which Ra varied by more than 20% over the course of the recordings were discarded. For recording spontaneous EPSCs, cells were held at -70mV under voltage-clamp conditions. Events were identified by fitting to an event template using pClamp 10 software. For evoked action-potentials, cells were switched to current-clamp and subject to a series of 100 pA current steps, ranging from -200 to + 700 pA, each step lasting 300ms. Recordings were analyzed using pClamp 10 software.

Retroviral and lentiviral vectors and virus preparation pTRIPZ-based plasmids from Open Biosystems (now ThermoFisher Scientific) were used for knockdown experiments, as previously described (1). Briefly, the Sox2-specific shRNA was generated using 5’-TCCATTGTTTATAAGCTGAGAA-3’ as target sequence obtained from RNAi Codex (Cold Spring Harbor Laboratory) and cloned into doxycycline inducible pTRIPZ lentivector expressing red fluorececent protein (RFP). Vectors were packaged into lentiviral particles and used to generate stable hipNPCs lines. To eliminate nontransduced hipNPCs, the puromycin resistance gene present in the pTRIPZ vector was exploited. Cells were cultured in the presence of 1 µg/mL puromycin for at least 10 d before being used in experiments. A hipNPC cell line expressing inducible scramble shRNA was used as a control (shCTRL). TaqMan qPCR and western blotting was used to confirm doxycycline-dependent Sox2 expression in these lines. The lentiviral PGK-GFP-IRES-GFP (phosphoglicokinase hybrid-green fluorescent protein), and the retroviral CAG-GFP-IRES-GFP (cytomegalovirus immediate early enhancer- chicken β-actin hybrid-green fluorescent protein) and CAG-mCherry) have been previously described (2, 3). Lentiviral PGK-NeuroD1-IRES-GFP and retroviral CAG- NeuroD1-IRES-GFP were generated from the original PGK-GFP-IRES-GFP and CAG- GFP-IRES-GFP vectors by replacing the first GFP coding sequence. Functionality of the vectors with regard to NeuroD1 expression was confirmed via transfection in HEK293T cells. In addition, immunohistochemical analysis revealed that NeuroD1-transduced cells in the adult subgranular zone (SGZ) were positive for nuclear NeuroD1. All Lenti- and retro-vectors were packaged into viral particles by the Viral Vector Core at the Sanford-Burnham Medical Research Institute (La Jolla, CA).

Genome-wide chromatin analysis All analyses were done using the mm9 release of the mouse genome (NCBI37/mm9 assemby from July 2007). SOX2 binding sites were searched using publicly available data (ENCODE, GSE35496) (4), which report chromatin occupancy in mouse NPCs. Genomic coordinates were remapped to mm9 by the LiftOver tool (UCSC Genome Browser). H3K27m3 and H3K4me3 histone mark densities in mouse NPCs were obtained by publicly available data (ENCODE, GSE38118) (5). The Cistrome/Galaxy “integrative analysis – Heatmap” was used to draw the scores of H3K27me3 and H3K4me3 near SOX2 binding sites (±5kb) and cluster the intervals using k-means. The Cistrome/Galaxy “SitePro” was used to obtain the signal profile of these histone marks near SOX2 binding sites.

REFERENCES SI 1. Cimadamore F, Amador-Arjona A, Chen C, Huang CT, & Terskikh AV (2013) SOX2-LIN28/let-7 pathway regulates proliferation and neurogenesis in neural precursors. Proc Natl Acad Sci U S A 110(32):E3017-3026. 2. Cimadamore F, et al. (2011) Human ESC-derived neural crest model reveals a key role for SOX2 in sensory neurogenesis. Cell Stem Cell 8(5):538-551. 3. Zhao C, Teng EM, Summers RG, Jr., Ming GL, & Gage FH (2006) Distinct morphological stages of dentate granule neuron maturation in the adult mouse hippocampus. J Neurosci 26(1):3-11. 4. Lodato MA, et al. (2013) SOX2 co-occupies distal enhancer elements with distinct POU factors in ESCs and NPCs to specify cell state. PLoS genetics 9(2):e1003288. 5. Hahn MA, et al. (2013) Dynamics of 5-hydroxymethylcytosine and chromatin marks in Mammalian neurogenesis. Cell Rep 3(2):291-300

SUPPLEMENTARY FIGURE LEGENDS Figure S1. SOX2 Binds to a Subset of Bivalent Promoters in Adult NPCs (Related to Figure 1) (A) Venn diagram showing the overlap of bivalent chromatin (genes) bound by SOX2 (± 2.5 kB of SOX2 binding site) and bivalent promoters (± 2.5 kB of transcription start sites [TSS]). SOX2 binds in the proximity of the TSS in a subset (368) of bivalent promoters in mouse NPCs. (B) GO analysis of genes with bivalent promoters lacking SOX2 binding.

Figure S2. SOX2 Silencing in Adult Hippocampal NPCs (Related to Figure 1) (A) Bright field imaging of cultured adult hippocampal neural progenitor cells (hNPCs, left panel). Adult wild-type hNPCs transduced with a dox-inducible SOX2-targeting shRNA labeled with red fluorescent protein (RFP) reporter is shown in the right panel. Scale bars: 20 µm. (B) Western blot and quantification of SOX2 protein levels in cultured adult hNPCs after 4 days of doxycycline induction. Results are the mean ± SEM (**p < 0.01).

Figure S3. SOX2 is Ablated in GFAP-Expressing NPCs of SOX2cKO Mice (Related to Figure 1) (A) Immunofluorescence staining of SOX2 and CFPnuc in dentate gyrus (DG) section of 2-month-old mice demonstrates efficient SOX2 deletion in NPCs in the subgranular zone (SGZ) of SOX2cKO mice. The section was counterstained with Hoechst (grey). Scale bar: 50 µm. (B) Immunofluorescence staining of SOX2 and CFPnuc in lateral ventricle (LV) section of 2-month-old mice demonstrates efficient SOX2 deletion in the subventricular zone (SVZ) of SOX2cKO mice. The section was counterstained with Hoechst (grey). Scale bar: 50 µm.

Figure S4. Reduced Expression of Ngn2 and NeuroD1 in SOX2cKO hNPCs Responding to Neurogenic Cues (Related to Figure 1) (A) Schematic of a 7 day cell culture protocol used to differentiate SOX2-deficient hNPCs under neurogenic signals (Wnt3a, left panel). In the right panel, qRT-PCR analysis of Ngn2 and NeuroD1 gene expression changes in SOX2cKO and wild-type hNPCs exposed to Wnt3a. (B) qRT-PCR analysis of Wnt/beta-catenin target genes Axin2, cMyc, cJun, Btrc, Lef-1, and Ccnd1 mRNA expression in adult hNPCs from SOX2cKO and wild-type mice. (C) Schematic of a 36 h cell culture protocol used with adult hNPCs. Cells were exposed to EGF and bFGF for 12 h and then treated for 24 h with the histone deacetylase inhibitor valproic acid (VPA). In the right panel, qRT-PCR analysis of Ngn2 and NeuroD1 gene expression changes in NPCs from SOX2cKO and wild-type mice. For all quantifications dot lines show relative mRNA expression in wild- type hNPCs under self-renewal conditions. Data are plotted as the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure S5. Repressive Chromatin State in SOX2-Deficient hNPCs Responding to Neurogenic Cues (Related to Figure 2) (A) Schematic of a 7 day cell culture protocol used to differentiate SOX2-deficient hNPCs under neurogenic signals (Wnt3a). (B) H3K27me3 levels at regulatory regions of Ngn2, NeuroD1, Sox21, Bdnf, and Gadd45b promoters in mouse adult hNPCs induced by neurogenic signals. (C) ChIP-qPCR analyses show reduced H3K9ac levels on regulatory regions of Ngn2, NeuroD1, Sox21, Bdnf, and Gadd45b in SOX2-deficient hNPCs exposed to Wnt3a. (D) ChIP-qPCR analyses show reduced occupancy of GCN5 protein on regulatory regions of Ngn2, NeuroD1, Sox21, Bdnf, and Gadd45b in SOX2- deficient hNPCs exposed to Wnt3a. For all quantifications, data are plotted as the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure S6. Expression of PROX1 in Neuroblasts is Not Affected by SOX2cKO Ablation (Related to Figure 3) (A) Sections from 2-month-old mice were immunostained for PROX1. Scale bar: 20 µm. Quantification results shown in the right panel are expressed as the average intensity of PROX1 staining in neuroblasts (DCX+) cells relative to mature granule neurons. (B) Quantification of the relative mRNA levels of Prox1 in adult hippocampal progenitors. For all quantifications, data are plotted as the mean ± SEM.

Figure S7. Reduction in Neural Stem/Progenitor Cell Density and Proliferation in the SGZ of SOX2cKO Mice (Related to Figure 4) (A) Hippocampal DG sections from 1- and 6-month-old mice (SOX2 and the littermate wild-type) were stained for CFPnuc. Scale bar: 50 µm. The number and percentage of NESTIN+ stem/neural progenitors (CFPnuc+ cells) in the DG was analyzed in SOX2cKO and wild-type mice at 1, 2, and 6 months (m) of age. (B) Hippocampal DG sections from 1- and 6-month-old mice (SOX2 and the littermate wild-type) were stained for the proliferative marker PCNA. Scale bar: 50 µm. The number and percentage of proliferative stem/progenitor cells (PCNA+ cells) in the DG was analyzed in SOX2cKO and wild-type mice at 1, 2, and 6 months of age. (C) Hippocampal DG sections from 1- and 6-month-old mice (SOX2 and the littermate wild-type) were stained for the neuroblast marker DCX. Scale bar: 50 µm. The number and percentage of neuroblasts (DCX+ cells) in the DG was analyzed in SOX2cKO and wild-type mice at 1, 2, and 6 months of age. For all quantifications, data are plotted as the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure S8. Increased Cell Cycle Exit in Hippocampal Amplifying Progenitors from SOX2cKO Mice (Related to Figure 4) (A) Schematic of the double-injection protocol of CldU and IdU (28 h apart at 50 mg kg-1 body weight) used in 2-month-old SOX2cKO and wild-type mice. CldU- and IdU-labeled cells were examined 2 h after the last injection. (B) Sections from CldU- and IdU-injected mice were immunostained for CldU, IdU, GFAP, and CFPnuc. Scale bar: 25 µm. Results are expressed as the percentage of CldU+ IdU+ among radial NPCs (rNPCs) and amplifying progenitors (ANPs) in the right panel. For all quantifications, data are plotted as the mean ± SEM (*p < 0.05).

Figure S9. SOX2cKO Hippocampal NPCs Show Reduced Ability to Generate Neurons In Vitro (Related to Figure 4) (A) Bright field imaging of neurospheres generated from hippocampal DG of 2-month-old SOX2cKO and wild-type mice. Scale bar: 200 µm. Quantification of Sox2 gene expression in adult neurospheres is shown in the right panel. (B) Immunofluorescence staining of the neuroblast marker TUJ1 in adult stem/progenitor cells responding to differentiation cues (Wnt3a). Scale bar: 50 µm. Quantification of the percentage of TUJ1+ cells in cultures from SOX2cKO and wild-type mice is shown in the right panel. For all quantifications, data are plotted as the mean ± SEM (***p < 0.001).

Figure S10. Transduction of NeuroD1 in Adult NPCs from SOX2cKO Recovers Neurogenesis In Vitro (Related to Figure 6) (A) Immunofluorescence staining of adult NPCs with the NPC markers SOX2 and NESTIN. Scale bar: 100 µm. (B) SOX2cKO adult NPCs were transduced with GFP- or NeuroD1-expressing lentiviruses and then induced to differentiate for 10 days. The cells were stained with antibody against MAP2ab. Scale bar: 50 µm. In the right panel, quantification of MAP2ab+ cells. For all quantifications, data are plotted as the mean ± SEM (**p < 0.01, ###p < 0.001).

Figure S11. Electrophysiological Properties of Hippocampal Newborn Neurons in SOX2cKO Mice (Related to Figure 7) (A) Electrophysiological analysis showing resting membrane potentials of newborn neurons in the DG at 42 dpi of a retrovirus expressing GFP in brain slices of SOX2cKO and wild-type mice. (B) Quantification of the number of action-potential spikes generated in response to positive current injections in current-clamp conditions of newborn neurons in the DG at 42 dpi of a retrovirus expressing GFP in brain slices of SOX2cKO and wild- type mice. For all quantifications, data are plotted as the mean ± SEM.

Figure S12. Epigenetic Regulation of SOX2 at the Promoters of Poised Genes in Adult Hippocampal NPCs Adult hippocampal NPCs (purple cells) in wild-type mice generate neuroblasts and newborn neurons (green cells) that integrate in the DG. In NPCs SOX2 binds to the promoters of poised genes and limits the activity of the PRC2 complex (H3K27me3), enabling proper gene expression (active chromatin) in the neuroblasts. In SOX2 mutants the binding of EZH2 and PRC2 activity (repressive chromatin mark, H3K27me3) are increased at the promoters of poised proneural and neurogenic genes. Consequently, transcriptional activation of poised genes (e.g. Ngn2, NeuroD1, Sox21, Bdnf, Gadd45b) is reduced in SOX2 mutants resulting in increased neuroblasts apoptosis and altered morphology and function of newborn DG neurons.

TABLES SI Table S1. List of antibodies used

Table S2. List of primers used in qPCR

Table S3. List of primers used in ChIP-qPCR