Ectopic Methylation of a Single Persistently Unmethylated CpG in the Promoter of the Vitellogenin Gene Abolishes Its Inducibility by Estrogen through Attenuation of Upstream Stimulating Factor Binding

ABSTRACT

The enhancer/promoter of the vitellogenin II gene (VTG) has been extensively studied as a model system of vertebrate transcriptional control. While deletion mutagenesis and in vivo footprinting identified the transcription factor (TF) binding sites governing its tissue specificity, DNase hypersensitivity and DNA methylation studies revealed the epigenetic changes accompanying its hormone-dependent activation. Moreover, upon induction with estrogen (E₂), the region flanking the estrogen-responsive element (ERE) was reported to undergo active DNA demethylation. We now show that although the VTG ERE is methylated in embryonic chicken liver and in LMH/2A hepatocytes, its induction by E₂ was not accompanied by extensive demethylation. In contrast, E₂ failed to activate a VTG enhancer/promoter-controlled luciferase reporter gene methylated by SssI. Surprisingly, this inducibility difference could be traced not to the ERE but rather to a single CpG in an E-box (CACGTG) sequence upstream of the VTG TATA box, which is unmethylated in vivo but methylated by SssI. We demonstrate that this E-box binds the upstream stimulating factor USF1/2. Selective methylation of the CpG within this binding site with an E-box-specific DNA methyltransferase, Eco72IM, was sufficient to attenuate USF1/2 binding in vitro and abolish the hormone-induced transcription of the VTG gene in the reporter system.

KEYWORDS:

• DNA demethylation
• DNA methylation
• E-box
• USF1
• USF2
• estrogen inducibility
• gene expression
• vitellogenin

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/MCB.00436-19.

ACKNOWLEDGMENTS

We thank Hanspeter Saluz for providing hen and rooster DNA samples, Peter Hunziker for the proteomic analysis, Giancarlo Russo for assistance with the bioinformatics, Thermo Fischer for the generous gift of Eco72I DNA, Beat Kunz for assistance with the egg manipulations, and Maite Olivera Harris for constructive discussions.

This work was funded by the Swiss National Science Foundation (grant no. 31003A-149989 and 31003B-170267 to J.J.). We gratefully acknowledge the financial support of the Giuliana and Giorgio Stefanini Foundation.