The Deoxyribonucleic Acid Repair Protein Flap Endonuclease-1 Modulates Estrogen-Responsive Gene Expression

Abstract

The ligand-occupied estrogen receptor alpha (ERalpha) initiates changes in gene expression through its interaction with target DNA. The capacity of ERalpha to modulate gene expression is influenced by the association of the receptor with a variety of coregulatory proteins. To further understand the role of these coregulatory proteins in ERalpha-mediated transcription, we have isolated and identified proteins associated with ERalpha when it is bound to the consensus estrogen response element. One of the proteins identified in this complex, flap endonuclease-1 (FEN-1), is required for DNA replication and repair. We show that FEN-1 interacts directly with ERalpha and enhances the interaction of ERalpha with estrogen response element-containing DNA. More importantly, chromatin immunoprecipitation and RNA interference assays demonstrate that endogenously expressed FEN-1 associates with the native pS2 gene in MCF-7 cells and influences estrogen-responsive gene expression. Interestingly, estrogen differentially regulates expression of FEN-1 in mouse uterine epithelial, stromal, and myometrial cells. Together, our studies help to elucidate the functional consequence of the ERalpha-FEN-1 interaction and increase our understanding of the elaborate regulatory mechanisms that drive estrogen-responsive gene expression and DNA repair.

Rho GDP dissociation inhibitor α interacts with estrogen receptor α and influences estrogen responsiveness

“…A careful examination of RhoGDIa amino acid sequence failed to identify any nuclear localization signal, suggesting that another protein may assist in the shuttling of RhoGDIa between the cytoplasm and the nucleus. It seems plausible that cdc42 isoform1, a RhoGTPase family member with a polybasic region in its C-terminal end that can function as a nuclear localization signal (Lanning et al 2003, Williams 2003, might perform this function since we identified both isoforms 1 and 2 of cdc42 in our agarose gel purification experiments as ERa-associated proteins (Schultz-Norton et al 2007). Alternatively, RhoGDIa could be accompanied by a protein such as 14-3-3, which binds and helps to redistribute an array of signaling proteins (Fu et al 2000, Kino et al 2003, Diviani et al 2004.…”

“…Thus, we were surprised when we identified endogenously expressed RhoGDIa from HeLa nuclear extracts as a component of a large multiprotein complex associated with the DNA-bound ERa (Schultz-Norton et al 2007).…”

“…RhoGDIa was isolated with the ERE-bound ERa using HeLa nuclear extracts, purified ER, ERE-containing oligos, and agarose gel fractionation (Schultz-Norton et al 2007). Five unique peptides (SIQEQELDKDDESLR, VAV-SADPNVPNVVVTGLTLVCSSAPGPLELDLTGDLESFKK, IDKTDYMVGSYGPR, FTDDDKTDHLSWEWNLTIK, and AEEYEFLTPVEEAPK), which exclusively map to RhoGDIa, were identified by mass spectrometry analysis as described (Loven et al 2003).…”

“…Since RhoGDIa was originally identified in a complex with the ERE-bound ERa (Schultz-Norton et al 2007) and we had detected endogenously expressed RhoGDIa in MCF-7 nuclei, we determined whether RhoGDIa could influence ERa-mediated transactivation. Transient transfections were carried out in U2OS cells using an ERa expression vector, a luciferase reporter plasmid containing two copies of the consensus ERE, and a renilla reporter plasmid, which was used as an internal control.…”

“…Using agarose gel mobility shift assays and mass spectrometry analysis, we identified novel HeLa nuclear proteins associated with the DNA-bound estrogen receptor a (ERa; Schultz-Norton et al 2007). One of these ERa-associated proteins was the 28 kDa protein Rho GDP dissociation inhibitor a (RhoGDIa), which was originally characterized as a negative regulator of the RhoGTPase family members RhoA, Rac1, and Cdc42 (Fukumoto et al 1990, Leonard et al 1992, Masuda et al 1994, Koch et al 1997, Olofsson 1999.…”