Journal of Molecular Neuroscience

, Volume 55, Issue 3, pp 684–705 | Cite as

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

  • Holly S. Huang
  • Tanya M. Redmond
  • Ginger M. Kubish
  • Shweta Gupta
  • Robert C. Thompson
  • David L. Turner
  • Michael D. Uhler
Article

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.

Keywords

Ascl1 Differentiation Neurog2 Neuron Transcription 

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

Notes

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.

Supplementary material

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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)

12031_2014_408_MOESM1_ESM.tif (15.2 mb)
High-Resolution Image (TIFF 15,577 kb)
12031_2014_408_Fig10_ESM.gif (42 kb)
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)

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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 1 (DOCX 15 kb)
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Supplementary Table 2 (DOCX 34 kb)
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Supplementary Table 3 (XLSX 12 kb)
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Supplementary Table 4 (XLSX 28 kb)

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© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Holly S. Huang
    • 1
  • Tanya M. Redmond
    • 1
    • 2
  • Ginger M. Kubish
    • 3
  • Shweta Gupta
    • 1
  • Robert C. Thompson
    • 1
    • 4
  • David L. Turner
    • 1
    • 2
  • Michael D. Uhler
    • 1
    • 2
  1. 1.Molecular and Behavioral Neuroscience InstituteUniversity of MichiganAnn ArborUSA
  2. 2.Department of Biological ChemistryUniversity of MichiganAnn ArborUSA
  3. 3.Life Sciences InstituteUniversity of MichiganAnn ArborUSA
  4. 4.Department of PsychiatryUniversity of MichiganAnn ArborUSA

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