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

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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.

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Abbreviations

bHLH:

Basic helix-loop-helix

CamKII:

Ca2+/calmodulin-dependent protein kinase

CNS:

Central nervous system

Dner:

Delta/Notch-like EGF-related receptor

Dnmts:

DNA methyltransferases

Dox:

Doxycycline

EC:

Embryonic carcinoma

EGFP:

Enhanced green fluorescent protein

ERas:

ES cell-expressed Ras

ES:

Embryonic stem

Gadd45γ:

Growth arrest and DNA damage-inducible gamma

ILK:

Integrin-linked kinase

IRES:

Internal ribosome entry site

MAPK:

Mitogen-activated protein kinase

PAK:

p21-activated protein kinase

PKA:

cAMP-dependent protein kinase

PKC:

Protein kinase C

PKN:

Protein kinase N

PP1:

Protein phosphatase 1

qRT-PCR:

Quantitative real-time PCR

ROCK:

Rho-associated coiled-coil kinase

rtTA:

Reverse transcriptional activator protein

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Acknowledgments

The authors would like to thank Dr. Fan Meng for helpful discussions of the microarray data. This work was supported by NIH/NINDSR01NS051472 (MDU), the Medical School of the University of Michigan, and the Pritzker Neuropsychiatric Disorders Research Fund.

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Authors and Affiliations

  1. Molecular and Behavioral Neuroscience Institute, University of Michigan, 109 Zina Pitcher Pl, Ann Arbor, MI, 48109-2200, USA

    Holly S. Huang, Tanya M. Redmond, Shweta Gupta, Robert C. Thompson, David L. Turner & Michael D. Uhler

  2. Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, USA

    Tanya M. Redmond, David L. Turner & Michael D. Uhler

  3. Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA

    Ginger M. Kubish

  4. Department of Psychiatry, University of Michigan, Ann Arbor, MI, 48109, USA

    Robert C. Thompson

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Correspondence to Michael D. Uhler.

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Supplementary Fig. 1

Gene expression of Ascl1, Neurog2, EGFP, and Mtap2 in A6 and N3 cells following Dox treatment. a qRT-PCR analysis of gene expression changes over the course of 8 days in response to 0.5 μg/ml Dox. Treating A6 and N3 cells with 0.5 μg/ml Dox resulted in a transient increase in Ascl1 or Neurog2 expression, respectively. Bicistronic Egfp expression is also transiently induced, with kinetics mirroring bHLH expression for each cell line. Mtap2 expression significantly, but transiently, increases in both cell lines, albeit to a greater extent in response to Neurog2. b qRT-PCR analysis of gene expression in response to varying concentrations of Dox at 48 h. A6 and N3 cells express Ascl1, Neurog2, and Egfp in a dose-dependent manner, with significant increases in gene expression in as low as 100 ng/ml of Dox. While Ascl1 and Neurog2 both induce expression of general neuronal differentiation marker, Mtap2, N3 cells appear to be more sensitive to lower concentrations of Dox (GIF 62 kb)

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Supplementary Fig. 2

Characterization of Isl1 gene regulation by Ascl1 and Neurog2. a qRT-PCR analysis of Isl1 gene expression changes over the course of 8 days in response to 0.5 μg/ml Dox. Isl1 mRNA transiently increases in response to both Ascl1 and Neurog2, with expression peaking 2 days after treatment with Dox. The induction of Isl1 appears to be 2.8-fold higher in response to Ascl1 after 2 days of treatment with Dox. b Western blot for Isl1 protein expression changes over the course of 8 days shows a significant increase in Isl1 protein expression after 2 days of Dox treatment, with elevated levels in response to Ascl1. c qRT-PCR analysis of Isl1 gene expression in response to varying concentrations of Dox again shows higher induction by Ascl1 (GIF 42 kb)

High-Resolution Image (TIFF 3,652 kb)

Supplementary Fig. 3

Analysis of Cldn5 gene expression changes. a qRT-PCR analysis of Cldn5 gene expression changes over the course of 8 days in response to 0.5 μg/ml Dox. Cldn5 exhibits induction by only Ascl1, and the expression is undetectable in response to Neurog2. b Western blot for Cldn5 protein expression changes over the course of 8 days shows a substantial increase in Cldn5 protein expression in response to Ascl1, but not to Neurog2. c qRT-PCR analysis of Cldn5 gene expression in response to varying concentrations of Dox again shows induction by only Ascl1 and not Neurog2 (GIF 42 kb)

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Supplementary Table 2

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Supplementary Table 3

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Supplementary Table 4

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Huang, H.S., Redmond, T.M., Kubish, G.M. et al. Transcriptional Regulatory Events Initiated by Ascl1 and Neurog2 During Neuronal Differentiation of P19 Embryonic Carcinoma Cells. J Mol Neurosci 55, 684–705 (2015). https://doi.org/10.1007/s12031-014-0408-2

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  • DOI: https://doi.org/10.1007/s12031-014-0408-2

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