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• W2962952397 abstract "•Bcl6 ensures robust neurogenesis by repressing major extrinsic self-renewal pathways•Bcl6 inhibits the Notch, Wnt, SHH, and FGF signaling pathways at multiple levels•Bcl6 represses transcription through Sirt1 recruitment and histone deacetylation During neurogenesis, progenitors switch from self-renewal to differentiation through the interplay of intrinsic and extrinsic cues, but how these are integrated remains poorly understood. Here, we combine whole-genome transcriptional and epigenetic analyses with in vivo functional studies to demonstrate that Bcl6, a transcriptional repressor previously reported to promote cortical neurogenesis, acts as a driver of the neurogenic transition through direct silencing of a selective repertoire of genes belonging to multiple extrinsic pathways promoting self-renewal, most strikingly the Wnt pathway. At the molecular level, Bcl6 represses its targets through Sirt1 recruitment followed by histone deacetylation. Our data identify a molecular logic by which a single cell-intrinsic factor represses multiple extrinsic pathways that favor self-renewal, thereby ensuring robustness of neuronal fate transition. During neurogenesis, progenitors switch from self-renewal to differentiation through the interplay of intrinsic and extrinsic cues, but how these are integrated remains poorly understood. Here, we combine whole-genome transcriptional and epigenetic analyses with in vivo functional studies to demonstrate that Bcl6, a transcriptional repressor previously reported to promote cortical neurogenesis, acts as a driver of the neurogenic transition through direct silencing of a selective repertoire of genes belonging to multiple extrinsic pathways promoting self-renewal, most strikingly the Wnt pathway. At the molecular level, Bcl6 represses its targets through Sirt1 recruitment followed by histone deacetylation. Our data identify a molecular logic by which a single cell-intrinsic factor represses multiple extrinsic pathways that favor self-renewal, thereby ensuring robustness of neuronal fate transition. During neural development, the generation of the appropriate type and number of differentiated neurons and glial cells is controlled by a complex interplay between extrinsic and intrinsic cues acting on neural progenitors, thus regulating the balance between differentiation and self-renewal (Martynoga et al., 2012Martynoga B. Drechsel D. Guillemot F. Molecular control of neurogenesis: a view from the mammalian cerebral cortex.Cold Spring Harb. Perspect. Biol. 2012; 4: a008359Google Scholar, Rossi et al., 2017Rossi A.M. Fernandes V.M. Desplan C. Timing temporal transitions during brain development.Curr. Opin. Neurobiol. 2017; 42: 84-92Google Scholar, Tiberi et al., 2012bTiberi L. Vanderhaeghen P. van den Ameele J. Cortical neurogenesis and morphogens: diversity of cues, sources and functions.Curr. Opin. Cell Biol. 2012; 24: 269-276Google Scholar). In the developing cortex, radial glial cells are the main progenitors that will differentiate into specific postmitotic neuron populations, directly or through various classes of intermediate progenitors (Götz and Huttner, 2005Götz M. Huttner W.B. The cell biology of neurogenesis.Nat. Rev. Mol. Cell Biol. 2005; 6: 777-788Google Scholar, Kriegstein and Alvarez-Buylla, 2009Kriegstein A. Alvarez-Buylla A. The glial nature of embryonic and adult neural stem cells.Annu. Rev. Neurosci. 2009; 32: 149-184Google Scholar). Proneural factors act on these progenitors as the main intrinsic drivers of neurogenesis (Guillemot and Hassan, 2017Guillemot F. Hassan B.A. Beyond proneural: emerging functions and regulations of proneural proteins.Curr. Opin. Neurobiol. 2017; 42: 93-101Google Scholar, Guillemot et al., 2006Guillemot F. Molnár Z. Tarabykin V. Stoykova A. Molecular mechanisms of cortical differentiation.Eur. J. Neurosci. 2006; 23: 857-868Google Scholar), through cross-repression with the Notch pathway, which promotes self-renewal, and by directly inducing various classes of genes involved in neuronal differentiation. Key features of the Notch signaling pathway, such as lateral inhibition and oscillatory behavior, contribute in a major way to the irreversible commitment of differentiating cells toward neuronal fate (Kageyama et al., 2008Kageyama R. Ohtsuka T. Shimojo H. Imayoshi I. Dynamic Notch signaling in neural progenitor cells and a revised view of lateral inhibition.Nat. Neurosci. 2008; 11: 1247-1251Google Scholar). Moreover, many classes of extrinsic morphogen cues, including Wnt ligands, Sonic Hedgehog (SHH), and fibroblast growth factors (FGFs) can act on cortical progenitors to promote expansion and self-renewal and thereby effectively block neurogenesis (Chenn and Walsh, 2002Chenn A. Walsh C.A. Regulation of cerebral cortical size by control of cell cycle exit in neural precursors.Science. 2002; 297: 365-369Google Scholar, Kang et al., 2009Kang W. Wong L.C. Shi S.H. Hébert J.M. The transition from radial glial to intermediate progenitor cell is inhibited by FGF signaling during corticogenesis.J. Neurosci. 2009; 29: 14571-14580Google Scholar, Lien et al., 2006Lien W.H. Klezovitch O. Fernandez T.E. Delrow J. Vasioukhin V. alphaE-catenin controls cerebral cortical size by regulating the hedgehog signaling pathway.Science. 2006; 311: 1609-1612Google Scholar, Rash et al., 2011Rash B.G. Lim H.D. Breunig J.J. Vaccarino F.M. FGF signaling expands embryonic cortical surface area by regulating Notch-dependent neurogenesis.J. Neurosci. 2011; 31: 15604-15617Google Scholar, Wang et al., 2016Wang L. Hou S. Han Y.G. Hedgehog signaling promotes basal progenitor expansion and the growth and folding of the neocortex.Nat. Neurosci. 2016; 19: 888-896Google Scholar). Intriguingly, it has long been proposed that postmitotic cells undergoing neuronal differentiation become insulated from extrinsic signaling (Edlund and Jessell, 1999Edlund T. Jessell T.M. Progression from extrinsic to intrinsic signaling in cell fate specification: a view from the nervous system.Cell. 1999; 96: 211-224Google Scholar). Whether and how responsiveness to extrinsic cues is negatively modulated to allow neuronal commitment remains essentially unclear. Delamination of the progenitors away from the ventricular zone could contribute to this process, as some of these cues are secreted in the embryonic cerebrospinal fluid and are thought to act through the apical processes or cilia of the radial glial cells (Lehtinen et al., 2011Lehtinen M.K. Zappaterra M.W. Chen X. Yang Y.J. Hill A.D. Lun M. Maynard T. Gonzalez D. Kim S. Ye P. et al.The cerebrospinal fluid provides a proliferative niche for neural progenitor cells.Neuron. 2011; 69: 893-905Google Scholar). However, several cues, most strikingly Wnts, are also present in the cortical tissue (Harrison-Uy and Pleasure, 2012Harrison-Uy S.J. Pleasure S.J. Wnt signaling and forebrain development.Cold Spring Harb. Perspect. Biol. 2012; 4: a008094Google Scholar), where they can act on progenitors to block differentiation. Moreover, the signaling components of these various pathways, as well as some key downstream targets, are often partially overlapping. For instance, cross-talk of the Notch and Wnt pathways (Hayward et al., 2008Hayward P. Kalmar T. Arias A.M. Wnt/Notch signalling and information processing during development.Development. 2008; 135: 411-424Google Scholar), or Notch and FGFs (Rash et al., 2011Rash B.G. Lim H.D. Breunig J.J. Vaccarino F.M. FGF signaling expands embryonic cortical surface area by regulating Notch-dependent neurogenesis.J. Neurosci. 2011; 31: 15604-15617Google Scholar), has been documented during embryonic development and, despite the relatively simple intracellular regulation of the Wnt/β-catenin pathway, many of its components are used by other pathways or participate in distinct cellular activities. For instance, the deletion of Gsk3a/b, a major intracellular component of the β-catenin destruction complex, increases the proliferation of radial glial cells at the expense of their differentiation by altering not only Wnt but also Notch and FGF signaling activity (Kim et al., 2009Kim W.Y. Wang X. Wu Y. Doble B.W. Patel S. Woodgett J.R. Snider W.D. GSK-3 is a master regulator of neural progenitor homeostasis.Nat. Neurosci. 2009; 12: 1390-1397Google Scholar). Also, some key effectors genes, such as Cyclin d1/d2, are found as common targets of all morphogen pathways, depending on cellular context (Cohen et al., 2010Cohen B. Shimizu M. Izrailit J. Ng N.F. Buchman Y. Pan J.G. Dering J. Reedijk M. Cyclin D1 is a direct target of JAG1-mediated Notch signaling in breast cancer.Breast Cancer Res. Treat. 2010; 123: 113-124Google Scholar, Kalita et al., 2013Kalita A. Gupta S. Singh P. Surolia A. Banerjee K. IGF-1 stimulated upregulation of cyclin D1 is mediated via STAT5 signaling pathway in neuronal cells.IUBMB Life. 2013; 65: 462-471Google Scholar, Katoh and Katoh, 2009Katoh Y. Katoh M. Hedgehog target genes: mechanisms of carcinogenesis induced by aberrant hedgehog signaling activation.Curr. Mol. Med. 2009; 9: 873-886Google Scholar, Nilsson et al., 2012Nilsson E.M. Brokken L.J. Narvi E. Kallio M.J. Härkönen P.L. Identification of fibroblast growth factor-8b target genes associated with early and late cell cycle events in breast cancer cells.Mol. Cell. Endocrinol. 2012; 358: 104-115Google Scholar, Shtutman et al., 1999Shtutman M. Zhurinsky J. Simcha I. Albanese C. D’Amico M. Pestell R. Ben-Ze’ev A. The cyclin D1 gene is a target of the β-catenin/LEF-1 pathway.Proc. Natl. Acad. Sci. USA. 1999; 96: 5522-5527Google Scholar). How these intermingled pathways are effectively shut down during neurogenesis is therefore a complex issue and remains largely unresolved. We previously reported that the transcriptional repressor Bcl6 (Baron et al., 1993Baron B.W. Nucifora G. McCabe N. Espinosa 3rd, R. Le Beau M.M. McKeithan T.W. Identification of the gene associated with the recurring chromosomal translocations t(3;14)(q27;q32) and t(3;22)(q27;q11) in B-cell lymphomas.Proc. Natl. Acad. Sci. USA. 1993; 90: 5262-5266Google Scholar, Chang et al., 1996Chang C.C. Ye B.H. Chaganti R.S. Dalla-Favera R. BCL-6, a POZ/zinc-finger protein, is a sequence-specific transcriptional repressor.Proc. Natl. Acad. Sci. USA. 1996; 93: 6947-6952Google Scholar) is required for neuronal differentiation in the cerebral cortex and directly represses the Notch-dependent Hes5 target (Tiberi et al., 2012aTiberi L. van den Ameele J. Dimidschstein J. Piccirilli J. Gall D. Herpoel A. Bilheu A. Bonnefont J. Iacovino M. Kyba M. et al.BCL6 controls neurogenesis through Sirt1-dependent epigenetic repression of selective Notch targets.Nat. Neurosci. 2012; 15: 1627-1635Google Scholar), and in the cerebellum, Bcl6 promotes neurogenesis through repression of SHH pathway effectors Gli1/2 (Tiberi et al., 2014Tiberi L. Bonnefont J. van den Ameele J. Le Bon S.D. Herpoel A. Bilheu A. Baron B.W. Vanderhaeghen P. A BCL6/BCOR/SIRT1 complex triggers neurogenesis and suppresses medulloblastoma by repressing Sonic Hedgehog signaling.Cancer Cell. 2014; 26: 797-812Google Scholar). This raises the question whether Bcl6 promotes neurogenic conversion through the repression of distinct targets, depending on the cellular context, or through a more generic transcriptional repression program. Here, we combine transcriptome, epigenome, and in vivo functional analyses to determine the molecular logic of action of Bcl6 during neurogenesis, focusing on the cerebral cortex. We find that Bcl6 acts as a global repressor of a repertoire of signaling components of most signaling pathways known to promote self-renewal, including Notch, SHH, FGF, and most strikingly the Wnt pathway. These data define a molecular logic of neurogenesis whereby a single intrinsic factor downregulates the responsiveness to extrinsic cues, through transcriptional repression at multiple parallel and serial levels along these pathways, to ensure irreversible neurogenic fate transition. To determine the primary molecular mechanisms of Bcl6 action in cortical neurogenesis, we performed RNA sequencing (RNA-seq) transcriptome analysis on in vitro embryonic-stem-cell-derived cortical progenitors driving inducible Bcl6 expression in order to timely control the transgene induction (Figure 1A; Gaspard et al., 2008Gaspard N. Bouschet T. Hourez R. Dimidschstein J. Naeije G. van den Ameele J. Espuny-Camacho I. Herpoel A. Passante L. Schiffmann S.N. et al.An intrinsic mechanism of corticogenesis from embryonic stem cells.Nature. 2008; 455: 351-357Google Scholar, Tiberi et al., 2012aTiberi L. van den Ameele J. Dimidschstein J. Piccirilli J. Gall D. Herpoel A. Bilheu A. Bonnefont J. Iacovino M. Kyba M. et al.BCL6 controls neurogenesis through Sirt1-dependent epigenetic repression of selective Notch targets.Nat. Neurosci. 2012; 15: 1627-1635Google Scholar). We found that, 24 h following Bcl6 induction, 764 genes were significantly upregulated, with Bcl6 being the most increased, and 610 genes were significantly downregulated, with Hes5 being the most repressed (Table S1). Gene Ontology analysis of the upregulated genes revealed a significant enrichment in categories linked to development and cell or neuron differentiation (Figure 1B; Table S2). More specifically, most of the canonical markers of differentiation into intermediate progenitors and neurons were significantly upregulated (Figure 1C; Table S1). On the other hand, downregulated genes showed an enrichment in gene categories linked to regulation of translation, negative regulation of neurogenesis, and most strikingly to signaling pathways promoting the expansion and self-renewal of cortical progenitors (Figure 1B; Table S2). Among the significantly downregulated genes, we found, as expected, markers of radial glial cells and transcriptional targets of Notch but also many signaling components of FGF and SHH-dependent pathways and, most strikingly, a high number of genes belonging to the Wnt signaling cascade, from ligands to receptors to target genes (Figures 1C–1E; Tables S1 and S3). Given that Bcl6 has strong effects on neurogenesis, it could be that these global transcriptional changes reflect the consequence of changes in cell fate rather than direct regulation by Bcl6. However, the majority of the tested genes belonging to these proliferative pathways (8/11) were downregulated following Bcl6 induction several hours earlier than changes in cell fate markers (Figures 1F and S1A), indicating that their downregulation is not the mere result of Bcl6-mediated differentiation. Given the importance of the Wnt pathway in the regulation of self-renewal versus differentiation balance in the cortex (Chenn and Walsh, 2002Chenn A. Walsh C.A. Regulation of cerebral cortical size by control of cell cycle exit in neural precursors.Science. 2002; 297: 365-369Google Scholar, Fang et al., 2013Fang W.Q. Chen W.W. Fu A.K. Ip N.Y. Axin directs the amplification and differentiation of intermediate progenitors in the developing cerebral cortex.Neuron. 2013; 79: 665-679Google Scholar, Hirabayashi and Gotoh, 2005Hirabayashi Y. Gotoh Y. Stage-dependent fate determination of neural precursor cells in mouse forebrain.Neurosci. Res. 2005; 51: 331-336Google Scholar, Hirabayashi et al., 2004Hirabayashi Y. Itoh Y. Tabata H. Nakajima K. Akiyama T. Masuyama N. Gotoh Y. The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells.Development. 2004; 131: 2791-2801Google Scholar, Kuwahara et al., 2010Kuwahara A. Hirabayashi Y. Knoepfler P.S. Taketo M.M. Sakai J. Kodama T. Gotoh Y. Wnt signaling and its downstream target N-myc regulate basal progenitors in the developing neocortex.Development. 2010; 137: 1035-1044Google Scholar, Munji et al., 2011Munji R.N. Choe Y. Li G. Siegenthaler J.A. Pleasure S.J. Wnt signaling regulates neuronal differentiation of cortical intermediate progenitors.J. Neurosci. 2011; 31: 1676-1687Google Scholar, Mutch et al., 2010Mutch C.A. Schulte J.D. Olson E. Chenn A. Beta-catenin signaling negatively regulates intermediate progenitor population numbers in the developing cortex.PLoS ONE. 2010; 5: e12376Google Scholar, Wrobel et al., 2007Wrobel C.N. Mutch C.A. Swaminathan S. Taketo M.M. Chenn A. Persistent expression of stabilized beta-catenin delays maturation of radial glial cells into intermediate progenitors.Dev. Biol. 2007; 309: 285-297Google Scholar, Zhang et al., 2010Zhang J. Woodhead G.J. Swaminathan S.K. Noles S.R. McQuinn E.R. Pisarek A.J. Stocker A.M. Mutch C.A. Funatsu N. Chenn A. Cortical neural precursors inhibit their own differentiation via N-cadherin maintenance of beta-catenin signaling.Dev. Cell. 2010; 18: 472-479Google Scholar) and the number of downregulated genes belonging to this pathway, we tested the global impact of Bcl6 on the Wnt pathway in vivo. Axin 2, a classical Wnt/β-catenin-dependent target gene, was found to be upregulated in Bcl6−/− mouse embryonic cortex using in situ hybridization. Although Axin2 expression is normally detected in the medial pallium of the frontal cortex in wild-type animals, a higher signal was found throughout dorsolateral levels in Bcl6−/− mice (Figures 2A and 2B ), suggesting that β-catenin/Tcf activity is increased in the mutant cortex. Interestingly, this difference was not detectable at more posterior levels (Figures 2A and 2B), in accordance with the frontal high occipital low graded Bcl6 expression (Tiberi et al., 2012aTiberi L. van den Ameele J. Dimidschstein J. Piccirilli J. Gall D. Herpoel A. Bilheu A. Bonnefont J. Iacovino M. Kyba M. et al.BCL6 controls neurogenesis through Sirt1-dependent epigenetic repression of selective Notch targets.Nat. Neurosci. 2012; 15: 1627-1635Google Scholar). Given that these gene expression changes are specific to the frontal cortex, we tested whether they could affect areal patterning in the mutant mice. However, analysis of the pattern of expression of several area-specific markers did not detect any obvious changes in areal patterning in the Bcl6 mutant mice (Figures S2A–S2J), suggesting that Bcl6 effect on the Wnt pathways does not affect regional patterning of the cortex. On the other hand, these data suggest that Bcl6 neurogenic function could depend on the downregulation of the canonical Wnt pathway. We first tested this in vitro by examining potential genetic interactions between Bcl6 and β-catenin, the main signaling hub protein of the pathway. Neurogenic genes upregulated in vitro by Bcl6 were prevented by CHIR99021, a GSK3 inhibitor over-activating the canonical Wnt pathway. CHIR99021, which increased the levels of the Wnt reporter gene Lef1, also prevented Bcl6-mediated downregulation of Wnt target genes but did not prevent repression of Notch targets (Figure S2K). We then examined in vivo the impact of the gain of function of a stabilized β-catenin mutant on Bcl6 overexpression using in utero electroporation. Bcl6 gain of function alone led to increased neurogenesis, as assessed by increased number of cells in the cortical plate (CP) and an increase in Neurod2+ and Tuj1+ neurons, at the expense of ventricular zone (VZ)-located Pax6+ and Sox2+ progenitors, without detectable effect on neuronal migration or progenitor delamination (Figures 2C–2E and S3A–S3G). Importantly, these effects were suppressed by overexpression of stabilized β-catenin (Figures 2C–2E and S4C). Conversely, we combined Bcl6 and β-catenin (Ctnnb1) knockdown to assess potential epistasis. As expected (Tiberi et al., 2012aTiberi L. van den Ameele J. Dimidschstein J. Piccirilli J. Gall D. Herpoel A. Bilheu A. Bonnefont J. Iacovino M. Kyba M. et al.BCL6 controls neurogenesis through Sirt1-dependent epigenetic repression of selective Notch targets.Nat. Neurosci. 2012; 15: 1627-1635Google Scholar), Bcl6 knockdown led to decreased neurogenesis per se, as reflected by increased cell number in the VZ at the expense of the CP, associated with increased levels of radial glial cell progenitors and decreased neurons (Figures 2F–2H and S3H–S3N), and this phenotype was significantly rescued by the Ctnnb1 knockdown (Figures 2F–2H). These data collectively suggest that Bcl6 functionally acts on the Wnt pathway to promote neurogenesis, in addition to its effect on the Notch target Hes5 (Tiberi et al., 2012aTiberi L. van den Ameele J. Dimidschstein J. Piccirilli J. Gall D. Herpoel A. Bilheu A. Bonnefont J. Iacovino M. Kyba M. et al.BCL6 controls neurogenesis through Sirt1-dependent epigenetic repression of selective Notch targets.Nat. Neurosci. 2012; 15: 1627-1635Google Scholar). We next directly compared the impact of Bcl6 on Wnt and Notch pathways in vivo by combining Ctnnb1 and Hes5 short hairpin RNAs (shRNAs) to further assess whether Bcl6 represses them in parallel or sequentially. Hes5 knockdown rescued some of the Bcl6 loss-of-function-mediated phenotype to levels similar to the rescue obtained using the Ctnnb1 knockdown. However, the association of both Ctnnb1 and Hes5 shRNAs showed additive rescue of Bcl6 knockdown, reaching the corresponding control levels (Ctnnb1+Hes5 shRNA combination; Figures 2F–2H), suggesting that Bcl6 alters these two cascades at least in part in parallel. Altogether, these data indicate that Bcl6-mediated repression of the Wnt pathway, already at the level of β-catenin, is necessary to elicit neurogenic activity, in parallel to Notch signaling repression. Thus, Bcl6 acts by repression of multiple pathways promoting progenitor self-renewal and proliferation. As β-catenin is not only a key component of the Wnt pathway but also an important regulator of adherens junctions (Nelson and Nusse, 2004Nelson W.J. Nusse R. Convergence of Wnt, beta-catenin, and cadherin pathways.Science. 2004; 303: 1483-1487Google Scholar), we tested further the specific implication of the Wnt pathway by focusing on Tcf7l1, which was also found to be downregulated in response to Bcl6 in vitro (Figure S1A) and is the most heavily expressed Tcf/Lef transcription factor in cortical progenitors (Galceran et al., 2000Galceran J. Miyashita-Lin E.M. Devaney E. Rubenstein J.L. Grosschedl R. Hippocampus development and generation of dentate gyrus granule cells is regulated by LEF1.Development. 2000; 127: 469-482Crossref Google Scholar). Remarkably, we found that overexpression of Tcf7l1 blocked Bcl6-mediated neurogenesis in vivo (Figures S4A–S4D). Hence, these data strongly suggest that β-catenin downregulation by Bcl6 is mostly linked to its Wnt-related transcriptional activity rather than its activity in adherens junctions. This is in line with our RNA-seq analyses, which show that the expression of most genes related to adherens junctions tend to increase upon Bcl6 overexpression, especially Jup/γ-catenin (Figure S1B), thereby compensating β-catenin decrease, as previously reported (Wickline et al., 2013Wickline E.D. Du Y. Stolz D.B. Kahn M. Monga S.P. γ-catenin at adherens junctions: mechanism and biologic implications in hepatocellular cancer after β-catenin knockdown.Neoplasia. 2013; 15: 421-434Google Scholar). Although Bcl6 appears to repress multiple serial components of individual pathways, it could also act through common effectors of parallel signals. In line with this hypothesis, Cyclin d1/d2 genes were found to be downregulated by Bcl6 overexpression in vitro (Figure 1D; Table S1) and are known to be upregulated by pathways driving progenitor self-renewal, including Wnt (Shtutman et al., 1999Shtutman M. Zhurinsky J. Simcha I. Albanese C. D’Amico M. Pestell R. Ben-Ze’ev A. The cyclin D1 gene is a target of the β-catenin/LEF-1 pathway.Proc. Natl. Acad. Sci. USA. 1999; 96: 5522-5527Google Scholar) but also SHH (Kasper et al., 2006Kasper M. Schnidar H. Neill G.W. Hanneder M. Klingler S. Blaas L. Schmid C. Hauser-Kronberger C. Regl G. Philpott M.P. Aberger F. Selective modulation of Hedgehog/GLI target gene expression by epidermal growth factor signaling in human keratinocytes.Mol. Cell. Biol. 2006; 26: 6283-6298Google Scholar, Katoh and Katoh, 2009Katoh Y. Katoh M. Hedgehog target genes: mechanisms of carcinogenesis induced by aberrant hedgehog signaling activation.Curr. Mol. Med. 2009; 9: 873-886Google Scholar), FGF-insulin growth factor (IGF) (Kalita et al., 2013Kalita A. Gupta S. Singh P. Surolia A. Banerjee K. IGF-1 stimulated upregulation of cyclin D1 is mediated via STAT5 signaling pathway in neuronal cells.IUBMB Life. 2013; 65: 462-471Google Scholar, Nilsson et al., 2012Nilsson E.M. Brokken L.J. Narvi E. Kallio M.J. Härkönen P.L. Identification of fibroblast growth factor-8b target genes associated with early and late cell cycle events in breast cancer cells.Mol. Cell. Endocrinol. 2012; 358: 104-115Google Scholar), and Notch (Cohen et al., 2010Cohen B. Shimizu M. Izrailit J. Ng N.F. Buchman Y. Pan J.G. Dering J. Reedijk M. Cyclin D1 is a direct target of JAG1-mediated Notch signaling in breast cancer.Breast Cancer Res. Treat. 2010; 123: 113-124Google Scholar). Moreover, Cyclin d1/d2 are key promoters of cortical progenitor proliferation and consequently block neurogenesis (Lange et al., 2009Lange C. Huttner W.B. Calegari F. Cdk4/cyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors.Cell Stem Cell. 2009; 5: 320-331Google Scholar, Pilaz et al., 2009Pilaz L.J. Patti D. Marcy G. Ollier E. Pfister S. Douglas R.J. Betizeau M. Gautier E. Cortay V. Doerflinger N. et al.Forced G1-phase reduction alters mode of division, neuron number, and laminar phenotype in the cerebral cortex.Proc. Natl. Acad. Sci. USA. 2009; 106: 21924-21929Google Scholar, Tsunekawa et al., 2012Tsunekawa Y. Britto J.M. Takahashi M. Polleux F. Tan S.S. Osumi N. Cyclin D2 in the basal process of neural progenitors is linked to non-equivalent cell fates.EMBO J. 2012; 31: 1879-1892Google Scholar). We first examined their expression in the developing cortex in wild-type and Bcl6−/− brains. Ccnd1 was found in the VZ and SVZ in wild-type mice as previously reported (Glickstein et al., 2007Glickstein S.B. Alexander S. Ross M.E. Differences in cyclin D2 and D1 protein expression distinguish forebrain progenitor subsets.Cereb. Cortex. 2007; 17: 632-642Google Scholar), and its levels were significantly increased in Bcl6−/− animals (Figures 3A and 3B ). Ccnd2 mRNA was mostly detected in basal endfeet of radial glial cells and at lower intensity in the ventricular zone at embryonic day 12.5 (E12.5), as previously described (Tsunekawa et al., 2012Tsunekawa Y. Britto J.M. Takahashi M. Polleux F. Tan S.S. Osumi N. Cyclin D2 in the basal process of neural progenitors is linked to non-equivalent cell fates.EMBO J. 2012; 31: 1879-1892Google Scholar). Although the high density of labeling and limited resolution of in situ hybridization precluded detecting upregulation in the basal endfeet, Ccnd2 levels were significantly increased in the VZ in Bcl6−/− cortex (Figures 3C and 3D). Hence, Bcl6 negatively controls Ccnd1/2 expression in cortical progenitors in vivo. We next examined the functional impact of Bcl6 loss of function on Ccnd1/Ccnd2 double knockdown, as these two cyclins regulate cell cycle progression in a redundant manner (Ciemerych et al., 2002Ciemerych M.A. Kenney A.M. Sicinska E. Kalaszczynska I. Bronson R.T. Rowitch D.H. Gardner H. Sicinski P. Development of mice expressing a single D-type cyclin.Genes Dev. 2002; 16: 3277-3289Google Scholar, Glickstein et al., 2007Glickstein S.B. Alexander S. Ross M.E. Differences in cyclin D2 and D1 protein expression distinguish forebrain progenitor subsets.Cereb. Cortex. 2007; 17: 632-642Google Scholar, Tsunekawa et al., 2012Tsunekawa Y. Britto J.M. Takahashi M. Polleux F. Tan S.S. Osumi N. Cyclin D2 in the basal process of neural progenitors is linked to non-equivalent cell fates.EMBO J. 2012; 31: 1879-1892Google Scholar). Decreased neurogenesis observed following Bcl6 shRNA was completely rescued by the dual Ccnd1/Ccnd2 knockdown (Figures 3E–3G and S4E–S4G). Conversely, Ccnd1 overexpression blocked Bcl6-elicited neurogenesis in vivo (Figures S4A–S4D). This indicates that, in addition to repressing specific signaling pathway components, Bcl6 action also involves repression of common terminal effector targets, such as Ccnd1/2, driving progenitor proliferation and self-renewal. Overall, our data indicate that Bcl6 acts through inhibition of multiple pathways to promote neurogenesis, raising the question of which effects are directly related to Bcl6 transcriptional repression or reflect its indirect consequences. To address this point, we performed chromatin immunoprecipitation (ChIP)-seq to identify Bcl6 binding sites using in vitro cortical progenitors driving inducible Bcl6 expression in order to increase the efficiency of the immunoprecipitation (Figure S5). This revealed that Bcl6 binding was predominantly found on promoter regions, with a significant enrichment for Bcl6 matrix binding motif (Figures S5A–S5D). Further, 39% of the Bcl6-predicted targets (1,701/4,366 peak-associated genes) were similar to those previously reported by ChIP-seq in human B cells (Basso et al., 2010Basso K. Saito M. Sumazin P. Margolin A.A. Wang K. Lim W.K. Kitagawa Y. Schneider C. Alvarez M.J. Califano A. Dalla-Favera R. Integrated biochemical and computational approach identifies BCL6 direct target genes controlling multiple pathways in normal germinal center B cells.Blood. 2010; 115: 975-984Google Scholar). In line with our transcriptomics data, Bcl6 bound to numerous genes of the multiple cascades regulating the proliferation of cortical progenitors (Figure S5E), although it should be noted that only 19% of the downregulated genes from the RNA-seq dataset were found in the ChIP-seq screen (115/610 genes), indicating partial discrepancy between transcription factor binding and transcriptional effect, at least at the time points tested. Then we validated in vivo some of these targets using ChIP-qPCR on the identifie" @default.
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• W2962952397 title "Cortical Neurogenesis Requires Bcl6-Mediated Transcriptional Repression of Multiple Self-Renewal-Promoting Extrinsic Pathways" @default.
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