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Regulation of Oct-4 gene expression during differentiation of EC cells

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Abstract

The stem cell-specific factor Oct-4 is expressed in undifferentiated embryonal carcinoma and embryonic stem cells and is quickly downregulated upon RA-induced differentiation. Irrespective of the direction of differentiation, Oct-4 repression in P19 EC cells requires treatment with high doses of either all-trans or 9-cis RA. Unlike in P19 cells, no RA-induced downregulation of Oct-4 expression is observed in the P19-derived RA-resistant RAC65 cells. However, in these cells Oct-4 promoter repression can be rescued in a RA-dependent manner by cotransfection of RARα2 or RARβ2 but not RARrγ1, matching previously reported transactivation properties of these receptor types. In the vicinity of the transcription initiation site of the Oct-4 gene, three Hormone Response Element (HRE) half sites are present which are arranged as direct repeats with different spacing.In vitro translated RAR and RXR proteins bind to this HRE as heterodimers with low affinity, in such a way that all three HRE half sites contribute to complex formation. Although P19 EC cells contain weak binding activity interacting with the Oct-4 promoter HRE, strong binding activity is observed in nuclear extracts from RA-treated P19 cells. This binding activity was shown to correspond to COUP-TFs but not nuclear RA receptors. Moreover, the presence of these binding factors in nuclear extracts corresponds to silencing of Oct-4 expression. These results implicate RA and the action of its nuclear receptors in silencing Oct-4 expression upon differentiation of EC cells. The observed silencing is most likely not exerted by direct binding of RARs to the Oct-4 proximal promoter HRE. Our results support models in which different nuclear receptor complexes sequentially occupy different sites in the Oct-4 promoter HRE to silence Oct-4 expression during RA-induced differentiation.

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Abbreviations

CAT:

chloramphenicol transferase

COUP-TF:

chicken ovalbumin upstream promoter — transcription factor

DCC:

dextran coated charcoal

DMSO:

dimethylsulfoxide

DR:

direct repeat

EAR-2:

erbA related protein 2

ELP:

embryonal LTR-binding protein

EC:

embryonal carcinoma

ES:

embryonic stem cells

FGF:

fibroblast growth factor

GAPDH:

glyceraldehyde phosphate dehydrogenase

HRE:

hormone responsive element

ICM:

inner cell mass

IL-6:

interleukin 6

LacZ:

β-galactosidase

LIF:

leukemia inhibitory factor

Luc:

luciferase

Oct:

octamer binding

PAA:

polyacrylamide

POU:

Pit/GHF1, Oct, Unc83 transcription regulators

RA:

retinoic acid

RAR:

retinoic acid receptor

RAR:

Retinoic acid responsive element

RXR:

retinoid X receptor

SV:

simian virus

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Author notes

  1. Jon Schoorlemmer

    Present address: Centro de Investigaciones Biologicas, Velázquez 144, 28006, Madrid, Spain

  2. Luigi Jonk & Wiebe Kruijer

    Present address: Biologisch Centrum, RU Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands

  3. Sanbing Shen

    Present address: MRC Human Genetics Unit, Western general Hospital Crewe road, Edinburgh, UK

Authors and Affiliations

  1. Hubrecht Laboratorium, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands

    Jon Schoorlemmer, Luigi Jonk, Sanbing Shen, André van Puijenbroek, Alie Feijen & Wiebe Kruijer

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Schoorlemmer, J., Jonk, L., Shen, S. et al. Regulation of Oct-4 gene expression during differentiation of EC cells. Mol Biol Rep 21, 129–140 (1995). https://doi.org/10.1007/BF00997235

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