Proteomics

Dataset Information

0

Affinity purification of DNA binding proteins from HeLa extracts LC-MS/MS


ABSTRACT: The enhancer/promoter of the vitellogenin II (VTG) gene 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 methyltranferase Eco72IM was sufficient to attenuate USF1/2 binding in vitro and abolish the hormone-induced transcription of the VTG gene in the reporter system.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo sapiens  

TISSUE(S): Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Lia Kallenberger  

LAB HEAD: Josef Jiricny

PROVIDER: PXD015594 | Pride | 2020-05-27

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
20171106lk_10173_NE1_C.mgf Mgf
20171106lk_10173_NE1_C.raw Raw
20171106lk_10173_NE1_dG.mgf Mgf
20171106lk_10173_NE1_dG.raw Raw
20171106lk_10173_NE1_mC.mgf Mgf
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Publications

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.

Kallenberger Lia L   Erb Rachel R   Kralickova Lucie L   Patrignani Andrea A   Stöckli Esther E   Jiricny Josef J  

Molecular and cellular biology 20191112 23


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-responsiv  ...[more]

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