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Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis

View Article: PubMed Central - PubMed

ABSTRACT

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.

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A Cellular Model of Neurogenesis Driven by Ascl1(A) Cellular localization of Ascl1-ERT2 in NS5 cells before and 30 min after the addition of tamoxifen, as assessed by immunostaining against HA-tag (green). White arrowheads mark cytoplasmic expression in the absence of tamoxifen. Cell nuclei are labeled with DAPI (blue). Scale bar represents 30 μm.(B) Quantification of total Ascl1 protein levels in E14.4 ventral telencephalic progenitors (left) or NS5 Ascl1-ERT2 cells (right). Histograms show absolute fluorescence intensity after normalization (see Experimental Procedures for details).(C) State of differentiation of NS5 Ascl1-ERT2 cells in the presence or absence of tamoxifen 3 days after induction of differentiation, as assessed by the expression of the neuronal marker TUJ1 (red). Cell nuclei are labeled with DAPI (blue). Scale bar represents 200 μm.(D) Expression of Gad65/67 (red) in NS5 Ascl1-ERT2 cells 4 days after induction of differentiation. Cell nuclei are labeled with DAPI (blue). Scale bar represents 200 μm.(E) Electrophysiological properties of GFP-labeled neurons generated from NS5 Ascl1-ERT2 cells 14 days upon induction of differentiation. Representative responses of two neurons to step-current injection at a holding potential of −70mV in current-clamp mode. Note the brief burst of action potentials on top of a prolonged calcium spike following depolarization (red traces) and the rebound burst following release from hyperpolarization (black trace). Scale bar represents 200 μm.(F) Experiment design for the transcriptome analysis and the resulting number of deregulated genes determined at different time points after induction of differentiation (fold change > 1.2, p < 10−3).(G) Enrichment of Gene Ontology biological process terms among genes deregulated during neuronal differentiation. Total number of genes associated with each term is in brackets.See also Figure S1.
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fig1: A Cellular Model of Neurogenesis Driven by Ascl1(A) Cellular localization of Ascl1-ERT2 in NS5 cells before and 30 min after the addition of tamoxifen, as assessed by immunostaining against HA-tag (green). White arrowheads mark cytoplasmic expression in the absence of tamoxifen. Cell nuclei are labeled with DAPI (blue). Scale bar represents 30 μm.(B) Quantification of total Ascl1 protein levels in E14.4 ventral telencephalic progenitors (left) or NS5 Ascl1-ERT2 cells (right). Histograms show absolute fluorescence intensity after normalization (see Experimental Procedures for details).(C) State of differentiation of NS5 Ascl1-ERT2 cells in the presence or absence of tamoxifen 3 days after induction of differentiation, as assessed by the expression of the neuronal marker TUJ1 (red). Cell nuclei are labeled with DAPI (blue). Scale bar represents 200 μm.(D) Expression of Gad65/67 (red) in NS5 Ascl1-ERT2 cells 4 days after induction of differentiation. Cell nuclei are labeled with DAPI (blue). Scale bar represents 200 μm.(E) Electrophysiological properties of GFP-labeled neurons generated from NS5 Ascl1-ERT2 cells 14 days upon induction of differentiation. Representative responses of two neurons to step-current injection at a holding potential of −70mV in current-clamp mode. Note the brief burst of action potentials on top of a prolonged calcium spike following depolarization (red traces) and the rebound burst following release from hyperpolarization (black trace). Scale bar represents 200 μm.(F) Experiment design for the transcriptome analysis and the resulting number of deregulated genes determined at different time points after induction of differentiation (fold change > 1.2, p < 10−3).(G) Enrichment of Gene Ontology biological process terms among genes deregulated during neuronal differentiation. Total number of genes associated with each term is in brackets.See also Figure S1.

Mentions: Overexpression of Ascl1 promotes cell cycle exit and neuronal differentiation of neural progenitor cells (Castro et al., 2006). To study this process in controlled conditions, we established a cellular model of Ascl1-driven neurogenesis by expressing an inducible version of Ascl1 in NS cells in culture (NS5 cell line) (Pollard et al., 2006). Fusion of full-length Ascl1 to the modified ligand binding domain of the estrogen receptor (ERT2) renders Ascl1 activity dependent on the presence of 4-hydroxytamoxifen (herein referred to as tamoxifen) (Bergstrom et al., 2002, Burk and Klempnauer, 1991, Littlewood et al., 1995). In a transcriptional assay in transfected NS cells, Ascl1-ERT2 induces the transcriptional activation of an enhancer of the Ascl1 target gene Dll1 (Castro et al., 2006) in an inducible manner to levels that are similar to its WT counterpart (Figure S1A). In order to test for the ability of the inducible Ascl1 protein to promote neuronal differentiation, we transduced NS cells in culture with a retrovirus vector co-expressing Ascl1-ERT2 and green fluorescent protein (GFP). In the vast majority of transduced cells, drastic morphological changes characterized by the extension of long processes and associated with the expression of the neuronal marker Tuj1 were observed only upon the addition of tamoxifen, confirming the ability of Ascl1-ERT2 to activate the neurogenic differentiation program in an inducible manner (Figure S1B). Although activation is associated with the nuclear translocation of a small fraction of Ascl1-ERT2, most of the protein is already nuclear prior to addition of tamoxifen (as previously reported with other cases of TFs fused to ERT2) (Burk and Klempnauer, 1991, Roemer and Friedmann, 1993), and additional mechanisms must therefore contribute to the inducibility of Ascl1-ERT2 (Figure 1A).


Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis
A Cellular Model of Neurogenesis Driven by Ascl1(A) Cellular localization of Ascl1-ERT2 in NS5 cells before and 30 min after the addition of tamoxifen, as assessed by immunostaining against HA-tag (green). White arrowheads mark cytoplasmic expression in the absence of tamoxifen. Cell nuclei are labeled with DAPI (blue). Scale bar represents 30 μm.(B) Quantification of total Ascl1 protein levels in E14.4 ventral telencephalic progenitors (left) or NS5 Ascl1-ERT2 cells (right). Histograms show absolute fluorescence intensity after normalization (see Experimental Procedures for details).(C) State of differentiation of NS5 Ascl1-ERT2 cells in the presence or absence of tamoxifen 3 days after induction of differentiation, as assessed by the expression of the neuronal marker TUJ1 (red). Cell nuclei are labeled with DAPI (blue). Scale bar represents 200 μm.(D) Expression of Gad65/67 (red) in NS5 Ascl1-ERT2 cells 4 days after induction of differentiation. Cell nuclei are labeled with DAPI (blue). Scale bar represents 200 μm.(E) Electrophysiological properties of GFP-labeled neurons generated from NS5 Ascl1-ERT2 cells 14 days upon induction of differentiation. Representative responses of two neurons to step-current injection at a holding potential of −70mV in current-clamp mode. Note the brief burst of action potentials on top of a prolonged calcium spike following depolarization (red traces) and the rebound burst following release from hyperpolarization (black trace). Scale bar represents 200 μm.(F) Experiment design for the transcriptome analysis and the resulting number of deregulated genes determined at different time points after induction of differentiation (fold change > 1.2, p < 10−3).(G) Enrichment of Gene Ontology biological process terms among genes deregulated during neuronal differentiation. Total number of genes associated with each term is in brackets.See also Figure S1.
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fig1: A Cellular Model of Neurogenesis Driven by Ascl1(A) Cellular localization of Ascl1-ERT2 in NS5 cells before and 30 min after the addition of tamoxifen, as assessed by immunostaining against HA-tag (green). White arrowheads mark cytoplasmic expression in the absence of tamoxifen. Cell nuclei are labeled with DAPI (blue). Scale bar represents 30 μm.(B) Quantification of total Ascl1 protein levels in E14.4 ventral telencephalic progenitors (left) or NS5 Ascl1-ERT2 cells (right). Histograms show absolute fluorescence intensity after normalization (see Experimental Procedures for details).(C) State of differentiation of NS5 Ascl1-ERT2 cells in the presence or absence of tamoxifen 3 days after induction of differentiation, as assessed by the expression of the neuronal marker TUJ1 (red). Cell nuclei are labeled with DAPI (blue). Scale bar represents 200 μm.(D) Expression of Gad65/67 (red) in NS5 Ascl1-ERT2 cells 4 days after induction of differentiation. Cell nuclei are labeled with DAPI (blue). Scale bar represents 200 μm.(E) Electrophysiological properties of GFP-labeled neurons generated from NS5 Ascl1-ERT2 cells 14 days upon induction of differentiation. Representative responses of two neurons to step-current injection at a holding potential of −70mV in current-clamp mode. Note the brief burst of action potentials on top of a prolonged calcium spike following depolarization (red traces) and the rebound burst following release from hyperpolarization (black trace). Scale bar represents 200 μm.(F) Experiment design for the transcriptome analysis and the resulting number of deregulated genes determined at different time points after induction of differentiation (fold change > 1.2, p < 10−3).(G) Enrichment of Gene Ontology biological process terms among genes deregulated during neuronal differentiation. Total number of genes associated with each term is in brackets.See also Figure S1.
Mentions: Overexpression of Ascl1 promotes cell cycle exit and neuronal differentiation of neural progenitor cells (Castro et al., 2006). To study this process in controlled conditions, we established a cellular model of Ascl1-driven neurogenesis by expressing an inducible version of Ascl1 in NS cells in culture (NS5 cell line) (Pollard et al., 2006). Fusion of full-length Ascl1 to the modified ligand binding domain of the estrogen receptor (ERT2) renders Ascl1 activity dependent on the presence of 4-hydroxytamoxifen (herein referred to as tamoxifen) (Bergstrom et al., 2002, Burk and Klempnauer, 1991, Littlewood et al., 1995). In a transcriptional assay in transfected NS cells, Ascl1-ERT2 induces the transcriptional activation of an enhancer of the Ascl1 target gene Dll1 (Castro et al., 2006) in an inducible manner to levels that are similar to its WT counterpart (Figure S1A). In order to test for the ability of the inducible Ascl1 protein to promote neuronal differentiation, we transduced NS cells in culture with a retrovirus vector co-expressing Ascl1-ERT2 and green fluorescent protein (GFP). In the vast majority of transduced cells, drastic morphological changes characterized by the extension of long processes and associated with the expression of the neuronal marker Tuj1 were observed only upon the addition of tamoxifen, confirming the ability of Ascl1-ERT2 to activate the neurogenic differentiation program in an inducible manner (Figure S1B). Although activation is associated with the nuclear translocation of a small fraction of Ascl1-ERT2, most of the protein is already nuclear prior to addition of tamoxifen (as previously reported with other cases of TFs fused to ERT2) (Burk and Klempnauer, 1991, Roemer and Friedmann, 1993), and additional mechanisms must therefore contribute to the inducibility of Ascl1-ERT2 (Figure 1A).

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus