Transcriptional Regulatory Events Initiated by Ascl1 and Neurog2 During Neuronal Differentiation of P19 Embryonic Carcinoma Cells

Abstract

As members of the proneural basic-helix-loop-helix (bHLH) family of transcription factors, Ascl1 and Neurog2 direct the differentiation of specific populations of neurons at various times and locations within the developing nervous system. In order to characterize the mechanisms employed by these two bHLH factors, we generated stable, doxycycline-inducible lines of P19 embryonic carcinoma cells that express comparable levels of Ascl1 and Neurog2. Upon induction, both Ascl1 and Neurog2 directed morphological and immunocytochemical changes consistent with initiation of neuronal differentiation. Comparison of Ascl1- and Neurog2-regulated genes by microarray analyses showed both shared and distinct transcriptional changes for each bHLH protein. In both Ascl1- and Neurog2-differentiating cells, repression of Oct4 mRNA levels was accompanied by increased Oct4 promoter methylation. However, DNA demethylation was not detected for genes induced by either bHLH protein. Neurog2-induced genes included glutamatergic marker genes while Ascl1-induced genes included GABAergic marker genes. The Neurog2-specific induction of a gene encoding a protein phosphatase inhibitor, Ppp1r14a, was dependent on distinct, canonical E-box sequences within the Ppp1r14a promoter and the nucleotide sequences within these E-boxes were partially responsible for Neurog2-specific regulation. Our results illustrate multiple novel mechanisms by which Ascl1 and Neurog2 regulate gene repression during neuronal differentiation in P19 cells.

Reconstructed cell fate–regulatory programs in stem cells reveal hierarchies and key factors of neurogenesis

“…3E,F; Voronova et al 2011;Huang et al 2012Huang et al , 2015; Gata3 [Martinez-Monedero et al 2008]). Interestingly, however, the expression of some P19-specific TGs was already affected during the first hours of RA-treatment, among them, the TFs Gbx2 (Bouillet et al 1995;Inoue et al 2012;Nakayama et al 2013), and Tal2, which is essential for midbrain neurogenesis (Achim et al 2013) and contains an intronic RA response element (Kobayashi et al 2014(Kobayashi et al , 2015.…”

“…S13). Among them, several known neuronal TFs, like NEUROD1, NEUROG2, POU3F2, or MYT1L, reconstitute <20% of the P19 program, while other "early" factors, like ASCL1 (Huang et al 2012(Huang et al , 2015, NR2F2 (Zhou et al 2015), or NR4A2 (Park et al 2006) reconstitute >60% (Fig. 6B).…”

Regulation of downstream neuronal genes by proneural transcription factors during initial neurogenesis in the vertebrate brain

“…Cell cycle exit is controlled by a limited number of basic helix-loop-helix (bHLH) proneural genes that are both necessary and sufficient to activate neurogenesis [5, 28]. Loss of function studies indicate that proneural transcription factors direct not only general aspects of neuronal differentiation, but also specific aspects of neuronal identity within NPCs [23, 39, 60]. These proneural transcription factors include ASCL1 and members of the Neurogenin family.…”

Mechanisms Regulating Stemness and Differentiation in Embryonal Carcinoma Cells

“…Undifferentiated P19 cells maintain high levels of Oct4 expression by promoting a low methylation profile on the Oct4 locus [255, 256]. Taken together, these studies would indicate that the ratio of demethylation-to-methylation dictates the expression of pluripotency genes in ECCs, and thus the maintenance of stemness [255, 256]. If so, this role of methylation status could account for the heterogeneity in pluripotency as others and we have noted in ECCs populations.…”

“…This is recapitulated in human ESCs where high c-Myc levels maintain pluripotency in the absence of LIF/STAT3 [254]. Undifferentiated P19 cells maintain high levels of Oct4 expression by promoting a low methylation profile on the Oct4 locus [255, 256]. Taken together, these studies would indicate that the ratio of demethylation-to-methylation dictates the expression of pluripotency genes in ECCs, and thus the maintenance of stemness [255, 256].…”