Two Estrogen Response Element Sequences Near the PCNA Gene Are Not Responsible for Its Estrogen-Enhanced Expression in MCF7 Cells

Abstract

BackgroundThe proliferating cell nuclear antigen (PCNA) is an essential component of DNA replication, cell cycle regulation, and epigenetic inheritance. High expression of PCNA is associated with poor prognosis in patients with breast cancer. The 5′-region of the PCNA gene contains two computationally-detected estrogen response element (ERE) sequences, one of which is evolutionarily conserved. Both of these sequences are of undocumented cis-regulatory function. We recently demonstrated that estradiol (E2) enhances PCNA mRNA expression in MCF7 breast cancer cells. MCF7 cells proliferate in response to E2.Methodology/Principal FindingsHere, we demonstrate that E2 rapidly enhanced PCNA mRNA and protein expression in a process that requires ERα as well as de novo protein synthesis. One of the two upstream ERE sequences was specifically bound by ERα-containing protein complexes, in vitro, in gel shift analysis. Yet, each ERE sequence, when cloned as a single copy, or when engineered as two tandem copies of the ERE-containing sequence, was not capable of activating a luciferase reporter construct in response to E2. In MCF7 cells, neither ERE-containing genomic region demonstrated E2-dependent recruitment of ERα by sensitive ChIP-PCR assays.Conclusion/SignificanceWe conclude that E2 enhances PCNA gene expression by an indirect process and that computational detection of EREs, even when evolutionarily conserved and when near E2-responsive genes, requires biochemical validation.