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SummaryDuring mitosis, chromatin condensation is accompanied by a global arrest of transcription. Recent studies suggest transcriptional reactivation upon mitotic exit occurs in temporally coordinated waves, but the underlying regulatory principles have yet to be elucidated. In particular, the contribution of sequence-specific transcription factors (TFs) remains poorly understood. Here we report that Brn2, an important regulator of neural stem cell identity, associates with condensed chromatin throughout cell division, as assessed by live-cell imaging of proliferating neural stem cells. By contrast, the neuronal fate determinant Ascl1 dissociates from mitotic chromosomes. ChIP-seq analysis reveals that Brn2 mitotic-chromosome binding does not result in sequence-specific interactions prior to mitotic exit, relying mostly on electrostatic forces. Nevertheless, surveying active transcription using single-molecule RNA-FISH against immature transcripts, indicates the differential presence of TF near chromatin when exiting mitosis is associated with early (anaphase) versus late (early G1) reactivation of key targets of Brn2 and Ascl1, respectively. Moreover, by using a mitotic-specific dominant negative approach, we show that competing with Brn2 binding during mitotic exit reduces the transcription of its target gene Nestin. Our study shows an important role for differential binding of TFs to mitotic chromosomes, governed by their electrostatic properties, in defining the temporal order of transcriptional reactivation during mitosis-to-G1 transition.

During mitosis, chromatin condensation is accompanied by a global arrest of transcription. Recent studies suggest transcriptional reactivation upon mitotic exit occurs in temporally coordinated waves, but the underlying regulatory principles have yet to be elucidated. In particular, the contribution of sequence-specific transcription factors (TFs) remains poorly understood. Here we report that Brn2, an important regulator of neural stem cell identity, associates with condensed chromatin throughout cell division, as assessed by live-cell imaging of proliferating neural stem cells. In contrast, the neuronal fate determinant Ascl1 dissociates from mitotic chromosomes. ChIP-seq analysis reveals that Brn2 mitotic chromosome binding does not result in sequence-specific interactions prior to mitotic exit, relying mostly on electrostatic forces. Nevertheless, surveying active transcription using single-molecule RNA-FISH against immature transcripts reveals differential reactivation kinetics for key targets of Brn2 and Ascl1, with transcription onset detected in early (anaphase) versus late (early G1) phases, respectively. Moreover, by using a mitotic-specific dominant-negative approach, we show that competing with Brn2 binding during mitotic exit reduces the transcription of its target geneNestin. Our study shows an important role for differential binding of TFs to mitotic chromosomes, governed by their electrostatic properties, in defining the temporal order of transcriptional reactivation during mitosis-to-G1 transition.

SUMMARYTransition from smooth, lissencephalic brains to highly-folded, gyrencephalic structures is associated with neuronal expansion and breaks in neurogenic symmetry. Here we show thatNeurog2andAscl1proneural genes regulate cortical progenitor cell differentiation through cross-repressive interactions to sustain neurogenic continuity in a lissencephalic rodent brain. Usingin vivolineage tracing, we found thatNeurog2andAscl1expression defines a lineage continuum of four progenitor pools, with ‘double+progenitors’ displaying several unique features (least lineage-restricted, complex gene regulatory network, G2pausing). Strikingly, selective killing of double+progenitors using split-Cre;Rosa-DTAtransgenics breaks neurogenic symmetry by locally disrupting Notch signaling, leading to cortical folding. Finally, consistent withNEUROG2andASCL1driving discontinuous neurogenesis and folding in gyrencephalic species, their transcripts are modular in folded macaque cortices and pseudo-folded human cerebral organoids.Neurog2/Ascl1double+progenitors are thus Notch-ligand expressing ‘niche’ cells that control neurogenic periodicity to determine cortical gyrification.HIGHLIGHTSNeurog2 and Ascl1 expression defines four distinct transitional progenitor statesDouble+NPCs are transcriptionally complex and mark a lineage branch pointDouble+NPCs control neurogenic patterning and cortical folding via Notch signalingNeurog2 and Ascl1 expression is modular in folded and not lissencephalic corticeseTOC BLURBEmergence of a gyrencephalic cortex is associated with a break in neurogenic continuity across the cortical germinal zone. Han et al. identify a pool of unbiased neural progenitors at a lineage bifurcation point that co-express Neurog2 and Ascl1 and produce Notch ligands to control neurogenic periodicity and cortical folding.

Transition from smooth, lissencephalic brains to highly-folded, gyrencephalic structures is associated with neuronal expansion and breaks in neurogenic symmetry. Here we show that Neurog2 and Ascl1 proneural genes regulate cortical progenitor cell differentiation through cross-repressive interactions to sustain neurogenic continuity in a lissencephalic rodent brain. Using in vivo lineage tracing, we found that Neurog2 and Ascl1 expression defines a lineage continuum of four progenitor pools, with ‘double+ progenitors’ having several unique features (least lineage-restricted, complex gene regulatory network, G 2 pausing). Strikingly, selective killing of double+ progenitors using split-Cre;Rosa-DTA transgenics breaks neurogenic symmetry by locally disrupting Notch signaling, leading to cortical folding. Finally, consistent with NEUROG2 and ASCL1 driving discontinuous neurogenesis and folding in gyrencephalic species, their transcripts are modular in folded macaque cortices and pseudo-folded human cerebral organoids. Neurog2/Ascl1 double + progenitors are thus Notch-ligand expressing ‘niche’ cells that control neurogenic periodicity to determine cortical gyrification.

Cadherins are calcium-binding proteins with a pivotal role in cell adhesion and tissue homeostasis. The cadherin-dependent mechanisms of cell adhesion and migration are exploited by cancer cells, contributing to tumor invasiveness and dissemination. In particular, cadherin switch is a hallmark of epithelial to mesenchymal transition, a complex development process vastly described in the progression of most epithelial cancers. This is characterized by drastic changes in cell polarity, adhesion, and motility, which lead from an E-cadherin positive differentiated epithelial state into a dedifferentiated mesenchymal-like state, prone to metastization and defined by N-cadherin expression. Although vastly explored in epithelial cancers, how these mechanisms contribute to the pathogenesis of other non-epithelial tumor types is poorly understood. Herein, the current knowledge on cadherin expression in normal development in parallel to tumor pathogenesis is reviewed, focusing on epithelial to mesenchymal transition. Emphasis is taken in the unascertained cadherin expression in CNS tumors, particularly in gliomas, where the potential contribution of an epithelial-to-mesenchymal-like process to glioma genesis and how this may be associated with changes in cadherin expression is discussed.

International audience; Asymmetric neuronal expansion is thought to drive evolutionary transitions between lissencephalic and gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes together sustain neurogenic continuity and lissencephaly in rodent cortices. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage restricted due to Neurog2-Ascl1 cross-repression and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selectively eliminating double+ NPCs by crossing Neurog2-Ascl1 split-Cre mice with diphtheria toxin-dependent "deleter" strains locally disrupts Notch signaling, perturbs neurogenic symmetry, and triggers cortical folding. In support of our discovery that double+ NPCs are Notch-ligand-expressing "niche" cells that control neurogenic periodicity and cortical folding, NEUROG2, ASCL1, and HES1 transcript distribution is modular (adjacent high/low zones) in gyrencephalic macaque cortices, prefiguring future folds.

The continuous proliferation of distributed energy resources (DER), mainly from renewable energy sources (RES) is changing the operational planning of distribution grids. Microgrids (MGs) as a small part of distribution grids are characterized by their ability to partially/fully self-producing their energy needs, and for the ability to trade different energy products (e.g. energy and reserve). This paper, addresses the energy and reserve market problem within the MG environment considering the RES uncertain production. Thus, a two-stage stochastic programming was modelled, minimizing the energy and reserve costs of the MG operator. A DC Optimal Power Flow (OPF) was incorporated to mitigate potential congestion that may occur in the MG. The assessment of the model is carried out through a test case based on actual generation data, considering a 37-bus distribution grid. The performance and accuracy of the model is determined based on the expected value of perfect information (EVPI) and value of stochastic solution (VSS).

Citrullination, the deimination of arginine residues into citrulline, has been implicated in the aetiology of several diseases. In multiple sclerosis (MS), citrullination is thought to be a major driver of pathology, through hypercitrullination and destabilization of myelin. As such, inhibition of citrullination has been suggested as a therapeutic strategy for MS. Here, in contrast, we show citrullination by peptidylarginine deiminase 2 (PADI2) is required for normal oligodendrocyte differentiation, myelination and motor function. We identify several targets for PADI2, including myelin-related proteins and chromatin-associated proteins, implicating PADI2 in epigenetic regulation. Accordingly, we observe that PADI2 inhibition and its knockdown affect chromatin accessibility and prevent the upregulation of oligodendrocyte differentiation genes. Moreover, mice lacking PADI2, display motor dysfunction and decreased number of myelinated axons in the corpus callosum. We conclude that citrullination is required for oligodendrocyte lineage progression and myelination and suggest its targeted activation in the oligodendrocyte lineage might be beneficial in the context of remyelination.

Zeb2 is a homeodomain transcription factor that plays pleiotropic functions during embryogenesis, but its role for midbrain dopaminergic (mDA) neuron development is unknown. Here we report that Zeb2 is highly expressed in progenitor cells in the ventricular zone of the midbrain floor plate and downregulated in postmitotic neuroblasts. Functional experiments show that Zeb2 expression in the embryonic ventral midbrain is dynamically regulated by a negative feedback loop that involves miR-200c. We also find that Zeb2 overexpression reduces the levels of CXCR4, NR4A2, and PITX3 in the developing ventral midbrain in vivo, resulting in migration and mDA differentiation defects. This phenotype was recapitulated by miR-200c knockdown, suggesting that the Zeb2-miR-200c loop prevents the premature differentiation of mDA progenitors into postmitotic cells and their migration. Together, our study establishes Zeb2 and miR-200c as critical regulators that maintain the balance between mDA progenitor proliferation and neurogenesis.

The gene regulatory network (GRN) that supports neural stem cell (NS cell) self-renewal has so far been poorly characterized. Knowledge of the central transcription factors (TFs), the noncoding gene regulatory regions that they bind to, and the genes whose expression they modulate will be crucial in unlocking the full therapeutic potential of these cells. Here, we use DNase-seq in combination with analysis of histone modifications to identify multiple classes of epigenetically and functionally distinct cis-regulatory elements (CREs). Through motif analysis and ChIP-seq, we identify several of the crucial TF regulators of NS cells. At the core of the network are TFs of the basic helix-loop-helix (bHLH), nuclear factor I (NFI), SOX, and FOX families, with CREs often densely bound by several of these different TFs. We use machine learning to highlight several crucial regulatory features of the network that underpin NS cell self-renewal and multipotency. We validate our predictions by functional analysis of the bHLH TF OLIG2. This TF makes an important contribution to NS cell self-renewal by concurrently activating pro-proliferation genes and preventing the untimely activation of genes promoting neuronal differentiation and stem cell quiescence.

Chromatin immunoprecipitation (ChIP) is considered the method of choice for characterizing interactions between a protein of interest and specific genomic regions. It is of paramount importance in gene-regulation studies, as it can be used to map the target regions of sequence-specific transcription factors and cofactors, or histone marks that characterize distinct chromatin states. ChIP can be used directly to probe interactions with candidate regions (ChIP-PCR), or coupled to Next-Generation Sequencing (ChIP-seq) to generate genome-wide information. This chapter describes a protocol for performing ChIP and ChIP-seq of transcription factors, starting either from mouse embryonic tissue or adherent cells in culture.

Chromatin immunoprecipitation (ChIP) is considered the method of choice for characterizing interactions between a protein of interest and specific genomic regions. It is of paramount importance in gene-regulation studies, as it can be used to map the target regions of sequence-specific transcription factors and cofactors, or histone marks that characterize distinct chromatin states. ChIP can be used directly to probe interactions with candidate regions (ChIP-PCR), or coupled to Next-Generation Sequencing (ChIP-seq) to generate genome-wide information. This chapter describes a protocol for performing ChIP and ChIP-seq of transcription factors, starting either from mouse embryonic tissue or adherent cells in culture.

Midbrain dopaminergic neurons have been the subject of intense research, because of their importance in pathologies such as Parkinson9s disease. In this issue of The EMBO Journal , Villaescusa et al (2016) show the transcription factor Pbx1 is required for the differentiation and survival of midbrain dopaminergic neurons. Notably, Pbx1 protects from oxidative stress by inducing Nfe2l1, and the expression of both genes is reduced in dopaminergic neurons from Parkinson9s patients. This is an important study, with possible implications in regenerative medicine and drug design.

Glioblastoma is the most common and aggressive brain tumor, with a subpopulation of stem‐like cells thought to mediate its recurring behavior and therapeutic resistance. The epithelial–mesenchymal transition (EMT) inducing factor Zeb1 was linked to tumor initiation, invasion, and resistance to therapy in glioblastoma, but how Zeb1 functions at molecular level and what genes it regulates remain poorly understood. Contrary to the common view that EMT factors act as transcriptional repressors, here we show that genome‐wide binding of Zeb1 associates with both activation and repression of gene expression in glioblastoma stem‐like cells. Transcriptional repression requires direct DNA binding of Zeb1, while indirect recruitment to regulatory regions by the Wnt pathway effector Lef1 results in gene activation, independently of Wnt signaling. Amongst glioblastoma genes activated by Zeb1 are predicted mediators of tumor cell migration and invasion, including the guanine nucleotide exchange factor Prex1, whose elevated expression is predictive of shorter glioblastoma patient survival. Prex1 promotes invasiveness of glioblastoma cells in vivo highlighting the importance of Zeb1/Lef1 gene regulatory mechanisms in gliomagenesis.

Citrullination, the deimination of peptidylarginine residues into peptidylcitrulline, has been implicated in the etiology of several diseases. In multiple sclerosis, citrullination is thought to be a major driver of pathology through hypercitrullination and destabilization of myelin. As such, inhibition of citrullination has been suggested as a therapeutic strategy for MS. Here, in contrast, we show that citrullination by peptidylarginine deiminase 2 (PAD2) contributes to normal oligodendrocyte differentiation, myelination, and motor function. We identify several targets for PAD2, including myelin and chromatin-related proteins, implicating PAD2 in epigenomic regulation. Accordingly, we observe that PAD2 inhibition and its knockdown affect chromatin accessibility and prevent the upregulation of oligodendrocyte differentiation genes. Moreover, mice lacking PAD2 display motor dysfunction and a decreased number of myelinated axons in the corpus callosum. We conclude that citrullination contributes to proper oligodendrocyte lineage progression and myelination.

SummaryDirect lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an “on-target” pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types.

In the developing mammalian brain, differentiating neurons mature morphologically via neuronal polarity programs. Despite discovery of polarity pathways acting concurrently with differentiation, it's unclear how neurons traverse complex polarity transitions or how neuronal progenitors delay polarization during development. We report that zinc finger and homeobox transcription factor-1 (Zeb1), a master regulator of epithelial polarity, controls neuronal differentiation by transcriptionally repressing polarity genes in neuronal progenitors. Necessity-sufficiency testing and functional target screening in cerebellar granule neuron progenitors (GNPs) reveal that Zeb1 inhibits polarization and retains progenitors in their germinal zone (GZ). Zeb1 expression is elevated in the Sonic Hedgehog (SHH) medulloblastoma subgroup originating from GNPs with persistent SHH activation. Restored polarity signaling promotes differentiation and rescues GZ exit, suggesting a model for future differentiative therapies. These results reveal unexpected parallels between neuronal differentiation and mesenchymal-to-epithelial transition and suggest that active polarity inhibition contributes to altered GZ exit in pediatric brain cancers. DOI: http://dx.doi.org/10.7554/eLife.12717.001, eLife digest During the formation of the brain, developing neurons are faced with a logistical problem. After newborn neurons form they must change in shape and move to their final location in the brain. Despite much speculation, little is known about these processes. Neurons mature via the activity of several pathways that control the activity, or expression, of the neuron’s genes. One way of controlling such gene expression is through proteins called transcription factors. At the same time, the developing neurons go through a process called polarization, where different regions of the cell develop different characteristics. However, it was not known how the maturation and polarization processes are linked, or how the developing neurons actively regulate polarization. By studying the developing mouse brain, Singh et al. found that a transcription factor called Zeb1 keeps neurons in a immature state, stopping them from becoming polarized. Further investigation revealed that Zeb1 does this by preventing the production of a group of proteins that helps to polarize the cells. The most common type of malignant brain tumour in children is called a medulloblastoma. Singh et al. analyzed the genes expressed in mice that have a type of medulloblastoma that results from the constant activity of a gene called Sonic Hedgehog in developing neurons. This revealed that these tumour cells contain abnormally high levels of Zeb1, and so do not take on a polarized form. However, artificially restoring other factors that encourage the cells to polarize caused the neurons to mature normally. Further investigation is now needed to find out whether the activity of the Sonic Hedgehog gene regulates Zeb1 activity, and to discover whether inhibiting Zeb1 could prevent brain tumours from developing. DOI: http://dx.doi.org/10.7554/eLife.12717.002

Motility is a universal property of newly generated neurons. How cell migration is coordinately regulated with other aspects of neuron production is not well understood. Here we show that the proneural protein neurogenin 2 (Neurog2), which controls neurogenesis in the embryonic cerebral cortex1,2, directly induces the expression of the small GTP-binding protein Rnd2 (ref. 3) in newly generated mouse cortical neurons before they initiate migration. Rnd2 silencing leads to a defect in radial migration of cortical neurons similar to that observed when the Neurog2 gene is deleted. Remarkably, restoring Rnd2 expression in Neurog2-mutant neurons is sufficient to rescue their ability to migrate. Our results identify Rnd2 as a novel essential regulator of neuronal migration in the cerebral cortex and demonstrate that Rnd2 is a major effector of Neurog2 function in the promotion of migration. Thus, a proneural protein controls the complex cellular behaviour of cell migration through a remarkably direct pathway involving the transcriptional activation of a small GTP-binding protein.

The proneural factor Ascl1 controls multiple steps of neurogenesis in the embryonic brain, including progenitor division and neuronal migration. Here we show that Cenpj, also known as CPAP, a microcephaly gene, is a transcriptional target of Ascl1 in the embryonic cerebral cortex. We have characterized the role of Cenpj during cortical development by in utero electroporation knockdown and found that silencing Cenpj in the ventricular zone disrupts centrosome biogenesis and randomizes the cleavage plane orientation of radial glia progenitors. Moreover, we show that downregulation of Cenpj in post-mitotic neurons increases stable microtubules and leads to slower neuronal migration, abnormal centrosome position and aberrant neuronal morphology. Moreover, rescue experiments shows that Cenpj mediates the role of Ascl1 in centrosome biogenesis in progenitor cells and in microtubule dynamics in migrating neurons. These data provide insights into genetic pathways controlling cortical development and primary microcephaly observed in humans with mutations in Cenpj., The proneural factor Ascl1/Mash1 is an important regulator of embryonic neurogenesis. Here the authors identify that the microcephaly protein Cenpj/CPAP is essential for several microtubule-dependent steps in the neurogenic program driven by Ascl1 in the developing cerebral cortex.

Summary The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program., Graphical Abstract, Highlights • Genome-wide binding of Ascl1 correlates with transcription activation • Ascl1 can bind to both open and closed chromatin in proliferating cells • Ascl1 promotes local chromatin accessibility at its target sites • Chromatin dynamics at Ascl1 sites regulates temporal progression of its program, The proneural transcription factor Ascl1 sequentially activates target genes in proliferating and differentiating progenitors during neurogenesis. Here Raposo et al. show that Ascl1 binds closed and open chromatin in proliferating cells. Binding to closed chromatin promotes accessibility and activation of differentiation specific genes. Thus, dynamics of chromatin landscape at Ascl1 target regions regulate the temporal onset of Ascl1 targets.

The gene regulatory network (GRN) that supports neural stem cell (NS cell) self-renewal has so far been poorly characterized. Knowledge of the central transcription factors (TFs), the noncoding gene regulatory regions that they bind to, and the genes whose expression they modulate will be crucial in unlocking the full therapeutic potential of these cells. Here, we use DNase-seq in combination with analysis of histone modifications to identify multiple classes of epigenetically and functionally distinct cis-regulatory elements (CREs). Through motif analysis and ChIP-seq, we identify several of the crucial TF regulators of NS cells. At the core of the network are TFs of the basic helix-loop-helix (bHLH), nuclear factor I (NFI), SOX, and FOX families, with CREs often densely bound by several of these different TFs. We use machine learning to highlight several crucial regulatory features of the network that underpin NS cell self-renewal and multipotency. We validate our predictions by functional analysis of the bHLH TF OLIG2. This TF makes an important contribution to NS cell self-renewal by concurrently activating pro-proliferation genes and preventing the untimely activation of genes promoting neuronal differentiation and stem cell quiescence. Welcome Trust grants: (WT095908, WT098051), FEBS Long-Term Fellowship, Medical Research Council Grant-in-Aid (U117570528).

Proneural transcription factors (TFs) such as Ascl1 function as master regulators of neurogenesis in vertebrates, being both necessary and sufficient for the activation of a full program of neuronal differentiation. Novel insights into the dynamics of Ascl1 expression at the cellular level, combined with the progressive characterization of its transcriptional program, have expanded the classical view of Ascl1 as a differentiation factor in neurogenesis. These advances resulted in a new model, whereby Ascl1 promotes sequentially the proliferation and differentiation of neural/stem progenitor cells. The multiple activities of Ascl1 are associated with the activation of distinct direct targets at progressive stages along the neuronal lineage. How this temporal pattern is established is poorly understood. Two modes of Ascl1 expression recently described (oscillatory vs. sustained) are likely to be of importance, together with additional mechanistic determinants such as the chromatin landscape and other transcriptional pathways. Here we revise these latest findings, and discuss their implications to the gene regulatory functions of Ascl1 during neurogenesis. FCT grants: (PTDC/SAU-BID/117418/2010, PTDC/NEU-NMC/0315/2012), Marie Curie CIG, FCT fellowships: (SFRH/BD/51178/2010, IF/00413/2012).

During development of the CNS, neurons and glia are generated in a sequential manner. The mechanism underlying the later onset of gliogenesis is poorly understood, although the cytokine-induced Jak-STAT pathway has been postulated to regulate astrogliogenesis. Here, we report that the overall activity of Jak-STAT signaling is dynamically regulated in mouse cortical germinal zone during development. As such, activated STAT1/3 and STAT-mediated transcription are negligible at early, neurogenic stages, when neurogenic factors are highly expressed. At later, gliogenic periods, decreased expression of neurogenic factors causes robust elevation of STAT activity. Our data demonstrate a positive autoregulatory loop whereby STAT1/3 directly induces the expression of various components of the Jak-STAT pathway to strengthen STAT signaling and trigger astrogliogenesis. Forced activation of Jak-STAT signaling leads to precocious astrogliogenesis, and inhibition of this pathway blocks astrocyte differentiation. These observations suggest that autoregulation of the Jak-STAT pathway controls the onset of astrogliogenesis.

Nurr1 is an orphan nuclear receptor essential for the development of midbrain dopaminergic neurons. Activation of Nurr1 depends on two so-called activation functions (AFs) situated in the N- and C-terminal regions, respectively. The region important for activation within the C-terminal domain has been shown to promote activation in a highly cell-type specific fashion in the absence of added exogenous ligands. In contrast, the region in the N-terminal domain (AF1) has been much less characterized. Here we mutagenized the N-terminal domain of Nurr1 to define essential activation regions. The results identified a short core activation region localized close to the N-terminus of Nurr1. In addition, cell-type specific influences by other signaling pathways were analyzed by mutagenesis of specific conserved phosphorylation sites. The results indicate that mitogen-activated protein kinase activity (MAPK) positively influences Nurr1 AF1-dependent transcriptional activation via a conserved phosphorylation site outside the core activation region.

The proper balance of excitatory and inhibitory neurons is crucial for normal processing of somatosensory information in the dorsal spinal cord. Two neural basic helix-loop-helix transcription factors (TFs), Ascl1 and Ptf1a, have contrasting functions in specifying these neurons. To understand how Ascl1 and Ptf1a function in this process, we identified their direct transcriptional targets genome-wide in the embryonic mouse neural tube using ChIP-Seq and RNA-Seq. We show that Ascl1 and Ptf1a directly regulate distinct homeodomain TFs that specify excitatory or inhibitory neuronal fates. In addition, Ascl1 directly regulates genes with roles in several steps of the neurogenic program, including Notch signaling, neuronal differentiation, axon guidance and synapse formation. By contrast, Ptf1a directly regulates genes encoding components of the neurotransmitter machinery in inhibitory neurons, and other later aspects of neural development distinct from those regulated by Ascl1. Moreover, Ptf1a represses the excitatory neuronal fate by directly repressing several targets of Ascl1. Ascl1 and Ptf1a bind sequences primarily enriched for a specific E-Box motif (CAGCTG) and for secondary motifs used by Sox, Rfx, Pou and homeodomain factors. Ptf1a also binds sequences uniquely enriched in the CAGATG E-box and in the binding motif for its co-factor Rbpj, providing two factors that influence the specificity of Ptf1a binding. The direct transcriptional targets identified for Ascl1 and Ptf1a provide a molecular understanding of how these DNA-binding proteins function in neuronal development, particularly as key regulators of homeodomain TFs required for neuronal subtype specification.

The generation of neurons at the correct time and location in the developing nervous system requires a fine balance between gene expression programs that regulate differentiation and maintenance of neural stem cells. During vertebrate neurogenesis, cell fate commitment and differentiation of neural stem cells toward the neuronal lineage are regulated by the opposing activities of the proneural and Notch pathways. Neuronal differentiation is inhibited by high Notch signaling characteristic of neural stem/progenitor cells, and requires the repression of the Notch transcriptional program by mechanisms that are still poorly understood. In a recent study1, we showed the zinc-finger transcription factor MyT1 promotes neurogenesis downstream the proneural factor Ascl1. MyT1 functions as a repressor of many Notch transcriptional target genes, linking the activation of a differentiation program by Ascl1 with the repression of the neural progenitor identity. Here we analyze our findings in light of the curr...

The proper balance of excitatory and inhibitory neurons is crucial for normal processing of somatosensory information in the dorsal spinal cord. Two neural basic helix-loop-helix transcription factors (TFs), Ascl1 and Ptf1a, have contrasting functions in specifying these neurons. To understand how Ascl1 and Ptf1a function in this process, we identified their direct transcriptional targets genome-wide in the embryonic mouse neural tube using ChIP-Seq and RNA-Seq. We show that Ascl1 and Ptf1a directly regulate distinct homeodomain TFs that specify excitatory or inhibitory neuronal fates. In addition, Ascl1 directly regulates genes with roles in several steps of the neurogenic program, including Notch signaling, neuronal differentiation, axon guidance and synapse formation. By contrast, Ptf1a directly regulates genes encoding components of the neurotransmitter machinery in inhibitory neurons, and other later aspects of neural development distinct from those regulated by Ascl1. Moreover, Ptf1a represses the excitatory neuronal fate by directly repressing several targets of Ascl1. Ascl1 and Ptf1a bind sequences primarily enriched for a specific E-Box motif (CAGCTG) and for secondary motifs used by Sox, Rfx, Pou and homeodomain factors. Ptf1a also binds sequences uniquely enriched in the CAGATG E-box and in the binding motif for its co-factor Rbpj, providing two factors that influence the specificity of Ptf1a binding. The direct transcriptional targets identified for Ascl1 and Ptf1a provide a molecular understanding of how these DNA-binding proteins function in neuronal development, particularly as key regulators of homeodomain TFs required for neuronal subtype specification. National Institutes of Health grants: ([F31NS705592, R01 HD037932, R01 NS032817, F31 NS061440

Dopamine cells are generated in the ventral midbrain during embryonic development. The progressive degeneration of these cells in patients with Parkinson's disease, and the potential therapeutic benefit by transplantation of in vitro generated dopamine cells, has triggered intense interest in understanding the process whereby these cells develop. Nurr1 is an orphan nuclear receptor essential for the development of midbrain dopaminergic neurons. However, the mechanism by which Nurr1 promotes dopamine cell differentiation has remained unknown. In this study we have used a dopamine-synthesizing cell line (MN9D) with immature characteristics to analyze the function of Nurr1 in dopamine cell development. The results demonstrate that Nurr1 can induce cell cycle arrest and a highly differentiated cell morphology in these cells. These two functions were both mediated through a DNA binding-dependent mechanism that did not require Nurr1 interaction with the heterodimerization partner retinoid X receptor. However, retinoids can promote the differentiation of MN9D cells independently of Nurr1. Importantly, the closely related orphan receptors NGFI-B and Nor1 were also able to induce cell cycle arrest and differentiation. Thus, the growth inhibitory activities of the NGFI-B/Nurr1/Nor1 orphan receptors, along with their widespread expression patterns both during development and in the adult, suggest a more general role in control of cell proliferation in the developing embryo and in adult tissues.

Accurately characterizing transcription factor (TF)-DNA affinity is a central goal of regulatory genomics. Although thermodynamics provides the most natural language for describing the continuous range of TF-DNA affinity, traditional motif discovery algorithms focus instead on classification paradigms that aim to discriminate ‘bound’ and ‘unbound’ sequences. Moreover, these algorithms do not directly model the distribution of tags in ChIP-seq data. Here, we present a new algorithm named Thermodynamic Modeling of ChIP-seq (TherMos), which directly estimates a position-specific binding energy matrix (PSEM) from ChIP-seq/exo tag profiles. In cross-validation tests on seven genome-wide TF-DNA binding profiles, one of which we generated via ChIP-seq on a complex developing tissue, TherMos predicted quantitative TF-DNA binding with greater accuracy than five well-known algorithms. We experimentally validated TherMos binding energy models for Klf4 and Esrrb, using a novel protocol to measure PSEMs in vitro. Strikingly, our measurements revealed strong non-additivity at multiple positions within the two PSEMs. Among the algorithms tested, only TherMos was able to model the entire binding energy landscape of Klf4 and Esrrb. Our study reveals new insights into the energetics of TF-DNA binding in vivo and provides an accurate first-principles approach to binding energy inference from ChIP-seq and ChIP-exo data. Published version

In the developing vertebrate nervous system, bHLH proneural factors, such as Ascl1, are known to play important regulatory roles at different stages of the neurogenic differentiation process. In spite of the wealth of information gathered on the cellular functions of proneural factors, little was known of the molecular basis for their activities and, in particular, of the identity of their target genes. The development of genomic approaches is making possible the characterization of transcriptional programs at an unprecedented scale. Recently, we have used a combination of genomic location analysis by ChIP-on-chip and expression profiling in order to characterize the proneural transcription program regulated by Ascl1 in the ventral telencephalon of the mouse embryonic brain. Our results demonstrate that Ascl1 directly controls successive steps of neurogenesis and provide a molecular frame for previously described Ascl1 functions. In addition, we uncovered an important but previously unrecognized role for Ascl1 in promoting the proliferation of neural progenitors. Here we discuss our recent findings and review them in light of efforts from other laboratories to characterize the transcriptional programs downstream various proneural factors.

Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. The mechanisms by which proneural genes activate neurogenesis—and, in particular, the genes that they regulate—however, are mostly unknown. We performed a genome-wide characterization of the transcriptional targets of Ascl1 in the embryonic brain and in neural stem cell cultures by location analysis and expression profiling of embryos overexpressing or mutant for Ascl1. The wide range of molecular and cellular functions represented among these targets suggests that Ascl1 directly controls the specification of neural progenitors as well as the later steps of neuronal differentiation and neurite outgrowth. Surprisingly, Ascl1 also regulates the expression of a large number of genes involved in cell cycle progression, including canonical cell cycle regulators and oncogenic transcription factors. Mutational analysis in the embryonic brain and manipulation of Ascl1 activity in neural stem cell cultures revealed that Ascl1 is indeed required for normal proliferation of neural progenitors. This study identified a novel and unexpected activity of the proneural gene Ascl1, and revealed a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation.

Summary Little is known of the intracellular machinery that controls the motility of newborn neurons. We have previously shown that the proneural protein Neurog2 promotes the migration of nascent cortical neurons by inducing the expression of the atypical Rho GTPase Rnd2. Here, we show that another proneural factor, Ascl1, promotes neuronal migration in the cortex through direct regulation of a second Rnd family member, Rnd3. Both Rnd2 and Rnd3 promote neuronal migration by inhibiting RhoA signaling, but they control distinct steps of the migratory process, multipolar to bipolar transition in the intermediate zone and locomotion in the cortical plate, respectively. Interestingly, these divergent functions directly result from the distinct subcellular distributions of the two Rnd proteins. Because Rnd proteins also regulate progenitor divisions and neurite outgrowth, we propose that proneural factors, through spatiotemporal regulation of Rnd proteins, integrate the process of neuronal migration with other events in the neurogenic program., Highlights ► The small GTPase Rnd3 is a direct target of the proneural transcription factor Ascl1 ► Rnd3 promotes cortical neuron migration by inhibiting RhoA signaling ► Rnd3 and the related protein Rnd2 have distinct roles in neuronal migration ► Rnd3 and Rnd2 have distinct subcellular distributions in cortical neurons

Monoaminergic neurons include the physiologically important central serotonergic and noradrenergic subtypes. Here, we identify the zinc-finger transcription factor, Insm1, as a crucial mediator of the differentiation of both subtypes, and in particular the acquisition of their neurotransmitter phenotype. Insm1 is expressed in hindbrain progenitors of monoaminergic neurons as they exit the cell cycle, in a pattern that partially overlaps with the expression of the proneural factor Ascl1. Consistent with this, a conserved cis-regulatory sequence associated with Insm1 is bound by Ascl1 in the hindbrain, and Ascl1 is essential for the expression of Insm1 in the ventral hindbrain. In Insm1-null mutant mice, the expression of the serotonergic fate determinants Pet1, Lmx1b and Gata2 is markedly downregulated. Nevertheless, serotonergic precursors begin to differentiate in Insm1 mutants, but fail to produce serotonin because of a failure to activate expression of tryptophan hydroxylase 2 (Tph2), the key enzyme of serotonin biosynthesis. We find that both Insm1 and Ascl1 coordinately specify Tph2 expression. In brainstem noradrenergic centres of Insm1 mutants, expression of tyrosine hydroxylase is delayed in the locus coeruleus and is markedly deficient in the medullary noradrenergic nuclei. However, Insm1 is dispensable for the expression of a second key noradrenergic biosynthetic enzyme, dopamine β-hydroxylase, which is instead regulated by Ascl1. Thus, Insm1 regulates the synthesis of distinct monoaminergic neurotransmitters by acting combinatorially with, or independently of, Ascl1 in specific monoaminergic populations.

Neural precursor cells (NPCs) are present in most regions of the adult central nervous system (CNS). Using NPCs in a therapeutical perspective, that is, to regenerate CNS tissue after injury or in neurodegenerative diseases, will require the efficient manipulation of their fate. Proneural gene overexpression in NPCs represents a promising strategy to promote neuronal differentiation. The activity of the proneural proteins is, however, context-dependent and can be inhibited/modulated by binding with other bHLH (basic helix-loop-helix) or HLH transcription factors. In this study, we show that the two proneural proteins, Ngn2 and Mash1, are differentially sensitive to negative regulation by gliogenic factors or a gliogenic substrate (i.e., postnatal spinal cord slices). Coexpressing E-proteins with proneural proteins was efficient to rescue proneural proteins neurogenic activity, suggesting a central role for E-protein sequestration in mediating postnatal CNS gliogenic inhibition. Tethering of proneural proteins with E47 further insulated Mash1 from negative environmental influences whereas this strategy was not successful with Ngn2, suggesting that mechanisms of inhibition differ in between these two proneural proteins. Our results demonstrate that a better understanding of proneural protein modulation by environmental cues is a prerequisite to develop innovative approaches that will permit the manipulation of the fate of NPCs in the adult CNS after trauma or disease. Disclosure of potential conflicts of interest is found at the end of this article.

After cell birth, almost all neurons in the mammalian central nervous system migrate. It is unclear whether and how cell migration is coupled with neurogenesis. Here we report that proneural basic helix-loop-helix (bHLH) transcription factors not only initiate neuronal differentiation but also potentiate cell migration. Mechanistically, proneural bHLH factors regulate the expression of genes critically involved in migration, including down-regulation of RhoA small GTPase and up-regulation of doublecortin and p35, which, in turn, modulate the actin and microtubule cytoskeleton assembly and enable newly generated neurons to migrate. In addition, we report that several DNA-binding-deficient proneural genes that fail to initiate neuronal differentiation still activate migration, whereas a different mutation of a proneural gene that causes a failure in initiating cell migration still leads to robust neuronal differentiation. Collectively, these data suggest that transcription programs for neurogenesis and migration are regulated by bHLH factors through partially distinct mechanisms.

Nurr1, a transcription factor belonging to the nuclear receptor family, is essential for the generation of midbrain dopamine (DA) cells during embryonic development. Nurr1 continues to be expressed in adult DA neurons but the role for Nurr1 in inducing and regulating basic dopaminergic functions such as dopamine synthesis and storage has remained unknown. We have previously used MN9D dopamine cells to analyze the role of Nurr1 and retinoids in DA cell maturation. These studies demonstrated that both Nurr1 and retinoids induce cell cycle arrest and a mature morphology. Here we used MN9D cells to investigate how Nurr1 regulates dopaminergic functions. Our results demonstrate that Nurr1, but not retinoids, increases DA content and the expression of aromatic L-amino acid decarboxylase (AADC) and vesicular monoamine transporter-2 (VMAT2) in MN9D cells. In a Nurr1-inducible cell line upregulation of VMAT2 is dependent on continuous Nurr1 expression. Moreover, AADC and VMAT2 are deregulated in midbrain DA cells of Nurr1 knockout embryos as revealed by in situ hybridization. Together, the results provide evidence indicating an instructive role for Nurr1 in controlling DA synthesis and storage.

Nurr1, a member of the nuclear hormone receptor superfamily, was recently demonstrated to be of critical importance in the developing central nervous system, where it is required for the generation of midbrain dopamine cells. Nuclear receptors encompass a transcriptional activation function (activation function 2; AF2) within their carboxyl-terminal domains important for ligand-induced transcriptional activation. Since a Nurr1 ligand remains to be identified, the role of the Nurr1 AF2 region in transcriptional activation is unclear. However, here we show that the Nurr1 AF2 contributes to constitutive activation independent of exogenously added ligands in human embryo kidney 293 cells and in neural cell lines. Extensive mutagenesis indicated a crucial role of the AF2 core region for transactivation but also identified unique features differing from previously characterized receptors. In addition, Nurr1 did not appear to interact with, and was not stimulated by, several previously identified coactivators such as the steroid receptor coactivator 1. In contrast, adenovirus protein E1A, stably expressed in 293 cells, was shown to contribute to AF2-dependent activation. Finally, while the AF2 core of RXR is required for ligand-induced transcriptional activation by Nurr1-RXR heterodimers, the functional integrity of Nurr1 AF2 core is not critical. These results establish that the ligand binding domain of Nurr1 has intrinsic capacity for transcriptional activation depending on cell type and mode of DNA binding. Furthermore, these results are consistent with the possibility that gene expression in the central nervous system can be modulated by an as yet unidentified ligand interacting with the ligand binding domain of Nurr1.

The implementation of neural stem cell lines as a source material for brain tissue transplants is currently limited by the ability to induce specific neurochemical phenotypes in these cells. Here, we show that coordinated induction of a ventral mesencephalic dopaminergic phenotype in an immortalized multipotent neural stem cell line can be achieved in vitro. This process requires both the overexpression of the nuclear receptor Nurr1 and factors derived from local type 1 astrocytes. Over 80% of cells obtained by this method demonstrate a phenotype indistinguishable from that of endogenous dopaminergic neurons. Moreover, this procedure yields an unlimited number of cells that can engraft in vivo and that may constitute a useful source material for neuronal replacement in Parkinson's disease.