The Chromatin Remodeler CHD7 Regulates Adult Neurogenesis via Activation of SoxC Transcription Factors

•CHD7 is associated with the lineage commitment period in adult neurogenesis•Selective ablation of CHD7 in adult NSCs impairs neuronal differentiation•Physical exercise rescues the adult hippocampal neurogenesis defect in CHD7 mutant•CHD7 is required for the activation of Sox4 and Sox11 in adult neurogenesis Chromatin factors that regulate neurogenesis in the central nervous system remain to be explored. Here, we demonstrate that the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7), a protein frequently mutated in human CHARGE syndrome, is a master regulator of neurogenesis in mammalian brain. CHD7 is selectively expressed in actively dividing neural stem cells (NSCs) and progenitors. Genetic inactivation of CHD7 in NSCs leads to a reduction of neuronal differentiation and aberrant dendritic development of newborn neurons. Strikingly, physical exercise can rescue the CHD7 mutant phenotype in the adult hippocampal dentate gyrus. We further show that in NSCs, CHD7 stimulates the expression of Sox4 and Sox11 genes via remodeling their promoters to an open chromatin state. Our study demonstrates an essential role of CHD7 in activation of the neuronal differentiation program in NSCs, thus providing insights into epigenetic regulation of stem cell differentiation and molecular mechanism of human CHARGE syndrome. Chromatin factors that regulate neurogenesis in the central nervous system remain to be explored. Here, we demonstrate that the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7), a protein frequently mutated in human CHARGE syndrome, is a master regulator of neurogenesis in mammalian brain. CHD7 is selectively expressed in actively dividing neural stem cells (NSCs) and progenitors. Genetic inactivation of CHD7 in NSCs leads to a reduction of neuronal differentiation and aberrant dendritic development of newborn neurons. Strikingly, physical exercise can rescue the CHD7 mutant phenotype in the adult hippocampal dentate gyrus. We further show that in NSCs, CHD7 stimulates the expression of Sox4 and Sox11 genes via remodeling their promoters to an open chromatin state. Our study demonstrates an essential role of CHD7 in activation of the neuronal differentiation program in NSCs, thus providing insights into epigenetic regulation of stem cell differentiation and molecular mechanism of human CHARGE syndrome. Epigenetic regulations are essential for the maintenance of cell identity and the guidance of stepwise cell differentiation. Mutations in epigenetic regulators are linked to many human diseases, including cancer and mental retardation (Berdasco and Esteller, 2010Berdasco M. Esteller M. Aberrant epigenetic landscape in cancer: how cellular identity goes awry.Dev. Cell. 2010; 19: 698-711Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, Jakovcevski and Akbarian, 2012Jakovcevski M. Akbarian S. Epigenetic mechanisms in neurological disease.Nat. Med. 2012; 18: 1194-1204Crossref PubMed Scopus (321) Google Scholar). As one family of chromatin regulators, ATP-dependent chromatin remodelers utilize the energy from ATP hydrolysis to slide nucleosomes, dissociate core histones, or relocate the entire histone octamers (Li et al., 2007Li B. Carey M. Workman J.L. The role of chromatin during transcription.Cell. 2007; 128: 707-719Abstract Full Text Full Text PDF PubMed Scopus (2677) Google Scholar). The dynamic change of nucleosome occupancy at gene promoters provides temporal control of transcription. Chromodomain-helicase-DNA-binding protein 7 (CHD7) belongs to the CHD family of chromatin remodelers. CHD proteins are involved in the regulation of multiple biological processes, including chromatin structure reorganization and gene expression (Hall and Georgel, 2007Hall J.A. Georgel P.T. CHD proteins: a diverse family with strong ties.Biochem. Cell Biol. 2007; 85: 463-476Crossref PubMed Scopus (157) Google Scholar). Importantly, de novo heterozygous mutations of the CHD7 gene are the major cause of the human CHARGE syndrome, a genetic disease characterized by a complex constellation of birth defects (coloboma of the eye, heart defects, atresia of the choanae, severe retardation of growth and development, genital abnormalities, and ear abnormalities) (Vissers et al., 2004Vissers L.E. van Ravenswaaij C.M. Admiraal R. Hurst J.A. de Vries B.B. Janssen I.M. van der Vliet W.A. Huys E.H. de Jong P.J. Hamel B.C. et al.Mutations in a new member of the chromodomain gene family cause CHARGE syndrome.Nat. Genet. 2004; 36: 955-957Crossref PubMed Scopus (949) Google Scholar). It has been shown that CHD7 cooperates with another chromatin remodeling complex PBAF (polybromo- and BRG1-associated factor-containing complex) to regulate neural crest migration, implicating its role in the peripheral system (Bajpai et al., 2010Bajpai R. Chen D.A. Rada-Iglesias A. Zhang J. Xiong Y. Helms J. Chang C.P. Zhao Y. Swigut T. Wysocka J. CHD7 cooperates with PBAF to control multipotent neural crest formation.Nature. 2010; 463: 958-962Crossref PubMed Scopus (426) Google Scholar). Interestingly, most of the CHARGE patients have mental retardation and olfactory anomalies ranging from absence to hypoplasia of the olfactory bulbs (OBs) (Blustajn et al., 2008Blustajn J. Kirsch C.F. Panigrahy A. Netchine I. Olfactory anomalies in CHARGE syndrome: imaging findings of a potential major diagnostic criterion.AJNR Am. J. Neuroradiol. 2008; 29: 1266-1269Crossref PubMed Scopus (42) Google Scholar, Vissers et al., 2004Vissers L.E. van Ravenswaaij C.M. Admiraal R. Hurst J.A. de Vries B.B. Janssen I.M. van der Vliet W.A. Huys E.H. de Jong P.J. Hamel B.C. et al.Mutations in a new member of the chromodomain gene family cause CHARGE syndrome.Nat. Genet. 2004; 36: 955-957Crossref PubMed Scopus (949) Google Scholar), suggesting that the corresponding neurogenic systems are impaired in those patients. A recent study identified CHD7 as a transcriptional cofactor of the essential NSC regulator Sox2 in NSCs using both proteomic and genomic approaches (Engelen et al., 2011Engelen E. Akinci U. Bryne J.C. Hou J. Gontan C. Moen M. Szumska D. Kockx C. van Ijcken W. Dekkers D.H. et al.Sox2 cooperates with Chd7 to regulate genes that are mutated in human syndromes.Nat. Genet. 2011; 43: 607-611Crossref PubMed Scopus (192) Google Scholar), suggesting a role of CHD7 in neurogenesis. However, the function of CHD7 in mammalian neurogenesis and the molecular mechanism underlying its role in the human CHARGE syndrome remains largely unknown. The subventricular zone (SVZ) of the lateral ventricle (LV) and the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus are the major germinal zones of active neurogenesis during adulthood in the mammalian central nervous system (CNS) (Alvarez-Buylla and Garcia-Verdugo, 2002Alvarez-Buylla A. Garcia-Verdugo J.M. Neurogenesis in adult subventricular zone.J. Neurosci. 2002; 22: 629-634Crossref PubMed Google Scholar, Gage, 2000Gage F.H. Mammalian neural stem cells.Science. 2000; 287: 1433-1438Crossref PubMed Scopus (4077) Google Scholar). Astrocyte-like type B cells in the adult SVZ are multipotent neural stem cells (NSCs) (Doetsch et al., 1999Doetsch F. Caillé I. Lim D.A. García-Verdugo J.M. Alvarez-Buylla A. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain.Cell. 1999; 97: 703-716Abstract Full Text Full Text PDF PubMed Scopus (3226) Google Scholar). These cells give rise to transit amplifying type C cells, which in turn differentiate into type A cells (neuroblasts) that migrate to the OB through the rostral migratory stream (RMS) (Alvarez-Buylla and Garcia-Verdugo, 2002Alvarez-Buylla A. Garcia-Verdugo J.M. Neurogenesis in adult subventricular zone.J. Neurosci. 2002; 22: 629-634Crossref PubMed Google Scholar). NSCs in the SGZ are also astrocyte-like cells with their cell bodies residing in the SGZ and their radial processes extending into the granular layer (GL) of the DG (Ming and Song, 2005Ming G.L. Song H. Adult neurogenesis in the mammalian central nervous system.Annu. Rev. Neurosci. 2005; 28: 223-250Crossref PubMed Scopus (1501) Google Scholar). The SGZ NSCs mainly give rise to newborn granule cells that are integrated into GL. The adult neurogenic system has been used to study many human neurological-disease-related genes, in particular their roles in regulating NSC self-renewal, differentiation, and maturation of newborn neurons (Zhao et al., 2008Zhao C. Deng W. Gage F.H. Mechanisms and functional implications of adult neurogenesis.Cell. 2008; 132: 645-660Abstract Full Text Full Text PDF PubMed Scopus (2425) Google Scholar). Molecularly, adult neurogenesis represents a robust process involving sequential regulation of crucial transcription factors important for cell-fate commitment (Suh et al., 2009Suh H. Deng W. Gage F.H. Signaling in adult neurogenesis.Annu. Rev. Cell Dev. Biol. 2009; 25: 253-275Crossref PubMed Scopus (283) Google Scholar). Epigenetic regulation is proposed to be actively involved in regulation of this process (Ma et al., 2010Ma D.K. Marchetto M.C. Guo J.U. Ming G.L. Gage F.H. Song H. Epigenetic choreographers of neurogenesis in the adult mammalian brain.Nat. Neurosci. 2010; 13: 1338-1344Crossref PubMed Scopus (259) Google Scholar). As a group of important epigenetic regulators, the roles of chromatin remodelers in adult neurogenesis remain largely unknown. To gain insights into the mechanism of chromatin remodelers in regulating adult NSCs, we investigated the function of CHD7 using mouse genetic approaches. Here, we demonstrate that CHD7 expression is specifically enriched in active NSCs and progenitors in the SVZ and SGZ. A NSC-specific inactivation of CHD7 in adult mice leads to a dramatic decrease of neurogenesis. Strikingly, physical exercise can rescue the hippocampal neurogenesis defect in CHD7 mutants. We further show that loss of CHD7 in NSCs represses the expression of Sox4 and Sox11 genes by inducing chromatin condensation of their promoters. And, forced expression of Sox4 and Sox11 in CHD7 mutant NSCs can rescue the neuronal differentiation defect. In summary, our study reveals that the CHARGE syndrome protein CHD7 is a master regulator of governing the neurogenic potential of NSCs. Rescue of the CHD7 mutant phenotype via physical exercise indicates an alternative pathway to overcome CHD7 deficiency, demonstrating the advantage of using adult NSCs to study human brain disease causing genes. These results provide significant insights into the molecular mechanism of chromatin regulation of NSCs fate commitment, which also provide implication to molecular pathogenesis of CHARGE syndrome. We first examined the expression of CHD7 in adult mouse brain, particularly in adult NSCs, which to our knowledge has not been reported yet. Data from the Allen Brain Atlas show that the messenger RNA (mRNA) of the CHD7 gene is highly expressed in adult neurogenic regions of the mouse brain, i.e., in the SVZ, RMS, and SGZ (Figure S1A available online). Consistent with the expression of the mRNA, immunostaining assays show that CHD7 protein is present in both the SVZ and SGZ cells (Figures S1B and S1C). To identify the CHD7-expressing cell population in the above-mentioned regions, we performed coimmunostainings of CHD7 with various cellular markers. Glial fibrillary acidic protein (GFAP) is a marker for astrocyte-like type B cells, namely, the NSCs in the SVZ (Doetsch et al., 1997Doetsch F. García-Verdugo J.M. Alvarez-Buylla A. Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain.J. Neurosci. 1997; 17: 5046-5061Crossref PubMed Google Scholar), whereas Mash1 and DCX (doublecortin), respectively, mark type C cells, i.e., the transit amplifying cells (Parras et al., 2004Parras C.M. Galli R. Britz O. Soares S. Galichet C. Battiste J. Johnson J.E. Nakafuku M. Vescovi A. Guillemot F. Mash1 specifies neurons and oligodendrocytes in the postnatal brain.EMBO J. 2004; 23: 4495-4505Crossref PubMed Scopus (310) Google Scholar), and type A cells, the neuroblasts. We found that some of the GFAP-positive cells express CHD7 (Figure 1A), indicating that CHD7 is expressed in a subpopulation of SVZ NSCs. Most of Mash1-positive or DCX-positive cells express CHD7 (Figures 1B and 1C), demonstrating that CHD7 is expressed in most of type C and A cells. We next analyzed the proliferation state of CHD7-expressing cells by costaining of CHD7 with a proliferation marker MCM2 (minichromosome maintenance complex component 2). In both the SVZ and SGZ, CHD7 colabels with MCM2 in most of the cells (Figures 1D and 1E), demonstrating that CHD7 is expressed in most fast-dividing cells. To further determine the expression of CHD7 in the adult NSCs, we made use of a transgenic mouse in which GFP expression is under the control of the Tlx BAC-based promoter. Tlx has been shown to be specifically expressed in NSCs, i.e., type B cells in the SVZ and type 1 cells in the SGZ (Liu et al., 2008Liu H.K. Belz T. Bock D. Takacs A. Wu H. Lichter P. Chai M. Schütz G. The nuclear receptor tailless is required for neurogenesis in the adult subventricular zone.Genes Dev. 2008; 22: 2473-2478Crossref PubMed Scopus (126) Google Scholar, Niu et al., 2011Niu W. Zou Y. Shen C. Zhang C.L. Activation of postnatal neural stem cells requires nuclear receptor TLX.J. Neurosci. 2011; 31: 13816-13828Crossref PubMed Scopus (71) Google Scholar). Our analyses demonstrate that the expression of Tlx-GFP recapitulates endogenous Tlx expression (Figures S1D–S1F). Adult Tlx-GFP mice were injected with bromodeoxyuridine (BrdU) 30 min before sacrifice in order to visualize dividing cells in neurogenic regions. Interestingly, most of Tlx-GFPhigh cells in both the SVZ and SGZ were negative for BrdU (Figures 1F and 1G, white arrows), suggesting that they are in a quiescent state. In support of this finding, we observed that Tlx-GFPhigh cells express NSC markers Nestin and GFAP, but not proliferation markers Ki67 and MCM2 (Figures S1G–S1I, arrows). Costaining of CHD7 with GFP and BrdU in these animals shows that some Tlx-GFP-positive cells express CHD7, and many of these colabeled cells were BrdU positive (Figures 1F and 1G, yellow arrows; Figure 1H). In contrast, most Tlx-GFPhigh cells did not express CHD7 (Figures 1F and 1G, white arrows). Moreover, CHD7 is coexpressed with Nestin and Sox2 in both cultured neurospheres and monolayer NSCs (Figures 1I and 1J). These data reveal that NSCs start to express CHD7 upon exiting the quiescent state, and the expression of CHD7 persists in neural progenitors and neuroblasts (Figure 1K). This intriguingly selective expression pattern of this chromatin remodeler suggests that CHD7 is probably involved in a temporal regulatory program during NSC activation, lineage commitment, and progression. To investigate the function of CHD7 in adult neurogenesis, a CHD7 conditional knockout (KO) mouse line (CHD7fl/fl) was established in which the exon 3 is flanked by two loxP sites (Figure S2A). We have previously generated a Tlx-CreERT2 mouse line using a BAC-mediated transgenic approach, where the Cre recombinase is only expressed in type B cells of the adult SVZ (Liu et al., 2008Liu H.K. Belz T. Bock D. Takacs A. Wu H. Lichter P. Chai M. Schütz G. The nuclear receptor tailless is required for neurogenesis in the adult subventricular zone.Genes Dev. 2008; 22: 2473-2478Crossref PubMed Scopus (126) Google Scholar). The CHD7fl/fl mice were crossed with Tlx-CreERT2 mice to achieve a tamoxifen (TMX)-inducible mutation of CHD7 in adult SVZ NSCs and their derivatives. Two weeks post-TMX induction (2 wpi), CHD7 was completely removed from the adult mouse SVZ as shown by immunohistochemistry (IHC) assay using a CHD7 antibody (Figure S2B). NSCs in the SVZ mainly give rise to neuroblasts that migrate to the OB and differentiate into mature neurons. To investigate the role of CHD7 in these processes, TMX-treated mice (Tlx-CreERT2; CHD7fl/fl and the littermate control) were injected with BrdU and traced for 4 weeks. As shown in Figure 2A, there was a dramatic decrease of BrdU-positive cells in the OB of CHD7 mutants. The number of newborn neurons, as shown by costaining of BrdU and a mature neuronal marker NeuN, was significantly decreased (Figure 2B). Consistently, there were less DCX-positive neuroblasts in the OB of CHD7 mutants compared to the control (Figure 2C). Moreover, the CHD7fl/fl mice were mated to another NSC-specific cre line, Nestin-CreERT2. As shown in Figure S2C, CHD7 is efficiently depleted in Nestin-CreERT2; CHD7fl/fl mice upon TMX treatment. By applying the same BrdU tracing approach as illustrated in Figure 2A, we observed a similar mutant phenotype in these animals as in the Tlx-CreERT2; CHD7fl/fl mice (data not shown). It has been proposed that SVZ NSCs are already predetermined to generate a certain subtype of neurons in the OB (Merkle et al., 2007Merkle F.T. Mirzadeh Z. Alvarez-Buylla A. Mosaic organization of neural stem cells in the adult brain.Science. 2007; 317: 381-384Crossref PubMed Scopus (652) Google Scholar). To analyze whether the decrease of neurogenesis is restricted to certain types of neurons, we performed BrdU costaining with markers (calretinin [CR], tyrosine hydroxylase [TH], and calbindin [CB]) representing different subtypes of interneurons in the OB. As shown in Figure 2D, no preferential loss of certain types of neurons was found in CHD7 mutants, indicating that there is a panneuronal phenotype. We thus assessed whether the self-renewal of NSCs is affected upon CHD7 inactivation. Immunostaining assays did not show any major change in expression of two NSC markers Nestin and Sox2 in the SVZ of CHD7 mutants (Figure S2D), suggesting that the number of NSCs was not altered. We then analyzed the cell proliferation in the SVZ using a 2 hr BrdU-pulse labeling assay. As shown in Figure 2E, there was no significant difference of BrdU incorporation in the control and CHD7 mutants. Analysis with the proliferation marker Ki67 showed similar results (Figure S2E). We next monitored the self-renewal of NSCs using neurosphere formation assay. The number of primary, secondary, and long-term passaged neurospheres formed from the SVZ-derived cells did not significantly differ between the control and CHD7 mutants (Figures 2F and 2G). Thus, loss of CHD7 impairs neurogenesis in the SVZ-OB without affecting the self-renewal of NSCs, which indicates that CHD7 is specifically required for the neuronal differentiation of NSCs. Indeed, the number of transit amplifying cells marked by Mash1 was slightly increased in CHD7 mutants (Figure S2E), indicating a possible blockage of these cells for further neuronal differentiation. Moreover, DCX immunostaining showed that the chain-like structure of migrating neuroblasts was not affected (Figure S2F), ruling out a major defect in the migration of neuroblasts. To further confirm the phenotype of CHD7 mutated NSCs observed in vivo, we established a SVZ NSC culture and tested the neuronal differentiation capacity of control and CHD7 mutant cells. In agreement with the in vivo finding, CHD7 mutant cells, both adult and fetal NSCs, generated significantly less Tuj1-positive neurons upon induction of differentiation (Figure S2G), which demonstrates an indispensable role of CHD7 during neuronal differentiation. In the SVZ, the majority of NSCs differentiate into neuronal lineage. The selective expression pattern of CHD7 in adult SVZ and the neuronal differentiation defect in CHD7 mutants suggest that CHD7, as a chromatin remodeler, plays a specific role for initiating the neuronal differentiation program on the chromatin level. These results also help to understand the olfaction defect that is frequently associated in the human CHARGE patient. The adult hippocampal neurogenic system has often been used to study functions of neurological disease genes linked to learning disability (Zhao et al., 2008Zhao C. Deng W. Gage F.H. Mechanisms and functional implications of adult neurogenesis.Cell. 2008; 132: 645-660Abstract Full Text Full Text PDF PubMed Scopus (2425) Google Scholar). Many CHARGE patients have a learning disability (Bergman et al., 2011Bergman J.E. Janssen N. Hoefsloot L.H. Jongmans M.C. Hofstra R.M. van Ravenswaaij-Arts C.M. CHD7 mutations and CHARGE syndrome: the clinical implications of an expanding phenotype.J. Med. Genet. 2011; 48: 334-342Crossref PubMed Scopus (210) Google Scholar). The specific expression pattern of CHD7 in the adult SGZ suggests a potential role in regulating the hippocampal neurogenesis. We generated a Nestin-CreERT2; CHD7fl/fl mouse line, which allows us to ablate CHD7 in the SGZ NSCs upon TMX induction (Figure S3A). NSCs in the SGZ generate neuroblasts that give rise to granule cells in the GL. Using the same BrdU tracing approach as used for the SVZ-OB system, we observed that loss of CHD7 in the SGZ NSCs results in a significant reduction of the number of BrdU-positive newborn neurons in the GL (Figure 3A). Moreover, DCX-positive neuroblasts in CHD7 mutants have shorter and less branched dendrites compared to the control (Figure 3B), suggesting a possible defect of dendritic development of newborn neurons. Both Sox2 and CCND2 have been shown to be essential for the maintenance of the SGZ NSCs (Favaro et al., 2009Favaro R. Valotta M. Ferri A.L. Latorre E. Mariani J. Giachino C. Lancini C. Tosetti V. Ottolenghi S. Taylor V. Nicolis S.K. Hippocampal development and neural stem cell maintenance require Sox2-dependent regulation of Shh.Nat. Neurosci. 2009; 12: 1248-1256Crossref PubMed Scopus (371) Google Scholar, Ferri et al., 2004Ferri A.L. Cavallaro M. Braida D. Di Cristofano A. Canta A. Vezzani A. Ottolenghi S. Pandolfi P.P. Sala M. DeBiasi S. Nicolis S.K. Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain.Development. 2004; 131: 3805-3819Crossref PubMed Scopus (529) Google Scholar, Suh et al., 2007Suh H. Consiglio A. Ray J. Sawai T. D’Amour K.A. Gage F.H. In vivo fate analysis reveals the multipotent and self-renewal capacities of Sox2+ neural stem cells in the adult hippocampus.Cell Stem Cell. 2007; 1: 515-528Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). By analyzing the RNA extracted from the microdissected hippocampus, we did not observe significant alteration in the expression Sox2 and CCND2 genes in CHD7 mutants as compared to the control (Figure S3B). Intriguingly, we observed a slight increase of Ki67-positive cells and Tbr2-positive transit amplifying cells in the SGZ of CHD7 mutants (Figures 3C and S3C). The accumulation of these cells could be due to a possible blockage of further neuronal differentiation. Thus, like in the SVZ-OB system, depletion of CHD7 in the SGZ NSCs leads to reduction of adult hippocampal neurogenesis. Ablating CHD7 in NSCs of the two adult neurogenic regions results in similar phenotype of neuronal differentiation defect, suggesting a general role of CHD7 in regulating neurogenesis. In support of this, we observed a similar phenotype by ablating CHD7 in radial glial cells during embryonic brain development (Figures S3D and S3E). Moreover, we analyzed whether loss of CHD7 in NSCs leads to an alternative fate of their progeny. There was no significant change of glial differentiation in all systems analyzed (SVZ, SGZ, and embryonic brain development) (Figures S3F–S3H). Interestingly, we observed an increase of cell death of newborn CHD7 mutant cells (Figures S3I and S3J), suggesting that CHD7 mutant cells may undergo apoptosis if they cannot be properly differentiated. Epigenetic regulators are considered to be essential players of regulating cell responses to environmental stimuli (Ma et al., 2010Ma D.K. Marchetto M.C. Guo J.U. Ming G.L. Gage F.H. Song H. Epigenetic choreographers of neurogenesis in the adult mammalian brain.Nat. Neurosci. 2010; 13: 1338-1344Crossref PubMed Scopus (259) Google Scholar). Interestingly, environmental stimuli can greatly affect the proliferation and survival of newborn cells in the adult CNS. For example, exposure of rodents to an enriched environment increases the survival of newborn neurons in the SGZ without affecting SVZ neurogenesis (Kempermann et al., 1997Kempermann G. Kuhn H.G. Gage F.H. More hippocampal neurons in adult mice living in an enriched environment.Nature. 1997; 386: 493-495Crossref PubMed Scopus (2894) Google Scholar). Physical exercise, such as voluntary running, promotes SGZ neurogenesis by increasing cell proliferation and survival of the newborn granule neurons (van Praag et al., 1999van Praag H. Kempermann G. Gage F.H. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus.Nat. Neurosci. 1999; 2: 266-270Crossref PubMed Scopus (3069) Google Scholar). Given that CHD7 is expressed in active, but not quiescent, NSCs, and the chromatin remodeler identity of CHD7, we anticipated that CHD7 is maybe involved in regulating exercise-induced hippocampal neurogenesis. To test this, we divided the control and CHD7 mutant mice into nonrunner and runner groups. Running was performed as a voluntary exercise in a running wheel and newborn neurons were labeled with BrdU as illustrated in Figure 4A. First, we confirmed the reduction of BrdU-positive cells in the GL of CHD7 mutant nonrunners as compared to the control nonrunners (Figure 4A). As expected, one month of running significantly increased the number of BrdU-positive cells in the GL of the control runners as compared to the control nonrunners (Figure 4A). To our surprise, after running, numbers of BrdU-positive cells in CHD7 mutants were not significantly different from the control runners (Figure 4A). Similar results were obtained when older mice were used for this assay (Figure S4A). Moreover, there was no significant difference in the number of Ki67-positive cells between the control and mutant runners (Figure S4B), indicating that running attenuates the accumulation of proliferating cells in CHD7 mutants (compared to Figure 3C). The observation that CHD7-mutated neuroblasts have shorter and less branched dendrites indicates a defect of dendritic development (Figure 3B). We therefore monitored the dendritic development of newborn neurons in the control and CHD7 mutants. For this, retroviruses expressing GFP were stereotactically injected into the DG to label newborn cells. The animals were analyzed 4 weeks postretroviral injection, and GFP-positive newborn neurons were traced. Intriguingly, the total dendritic length of GFP-positive newborn neurons in the GL of CHD7 mutants was significantly shorter compared to the control (Figures 4B, 4C, and S4C). Although not statistically significant, GFP-positive neurons in CHD7 mutants have less total dendrite branch numbers as compared to the control (Figures 4B, 4C, and S4C). Sholl analysis further demonstrated a decrease in the dendritic complexity of CHD7 mutant neurons (Figure 4D). Thus, loss of CHD7 in the SGZ NSCs not only reduces the number of newborn neurons but also leads to the dendritic abnormality in newborn neurons. To test whether running can have any effect on the dendritic abnormality in CHD7 mutant neurons, we applied the same retroviral-labeling approach to control and CHD7 mutant running mice. Strikingly, all of these above-mentioned defects of dendritic development in newborn neurons of CHD7 mutants were completely rescued after running (Figures 4B–4D; Figure S4C). Together, these results suggest that physical exercise is capable of overcoming the neurogenic defect in the DG of CHD7 mutants, including both the number and the dendritic development of newborn neurons. Our data demonstrate that exercise-induced neurogenesis does not depend on CHD7, which suggests an alternative pathway can guide the neuronal differentiation in the absence of CHD7. All of our above-mentioned results suggest that CHD7 is important for activation of a neuronal differentiation program in NSCs. Next, we aimed to identify the direct target genes of CHD7 in NSCs that are responsible for its function in neurogenesis. For this, we took a computational approach by analyzing the expression profiling data from the Cancer Genome Atlas Project (TCGA) (Network and Cancer Genome Atlas Research Network, 2008Network, T.C.G.A.R.; Cancer Genome Atlas Research NetworkComprehensive genomic characterization defines human glioblastoma genes and core pathways.Nature. 2008; 455: 1061-1068Crossref PubMed Scopus (5794) Google Scholar) because we observed a heterogeneous expression of CHD7 in human brain tumors (data not shown). Because CHD7 occupancy at genes is correlated with active gene expression (Engelen et al., 2011Engelen E. Akinci U. Bryne J.C. Hou J. Gontan C. Moen M. Szumska D. Kockx C. van Ijcken W. Dekkers D.H. et al.Sox2 cooperates with Chd7 to regulate genes that are mutated in human syndromes.Nat. Genet. 2011; 43: 607-611Crossref PubMed Scopus (192) Google Scholar, Schnetz et al., 2009Schnetz M.P. Bartels C.F. Shastri K. Balasubramanian D. Zentner G.E. Balaji R. Zhang X. Song L. Wang Z. Laframboise T. et al.Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns.Genome Res. 2009; 19: 590-601Crossref PubMed Scopus (189) Google Scholar), we focused on genes that are most positively correlated with CHD7 expression in the database. Interestingly, Sox4 and Sox11, two group C genes of the Sox gene family (Kuhlbrodt et al., 1998Kuhlbrodt K. Herbarth B. Sock E. Enderich J. Hermans-Borgmeyer I. Wegner M. Cooperative function of POU proteins and SOX proteins in glial cells.J. Biol. Chem. 1998; 273: 16050-16057Crossref PubMed Scopus (194) Google Scholar), are on top of the list of genes correlated most with CHD7 expression (Figure 5A). Several studies have shown that Sox4 and Sox11 are essential for neuronal property determination (Bergsland et al., 2006Bergsland