Interaction of estrogen receptor alpha with proliferating cell nuclear antigen

Abstract

The ability of estrogen receptor alpha (ERalpha) to modulate gene expression is influenced by the recruitment of a host of co-regulatory proteins to target genes. To further understand how estrogen-responsive genes are regulated, we have isolated and identified proteins associated with ERalpha when it is bound to DNA containing the consensus estrogen response element (ERE). One of the proteins identified in this complex, proliferating cell nuclear antigen (PCNA), is required for DNA replication and repair. We show that PCNA interacts with ERalpha in the absence and in the presence of DNA, enhances the interaction of ERalpha with ERE-containing DNA, and associates with endogenous estrogen-responsive genes. Interestingly, rather than altering hormone responsiveness of endogenous, estrogen-responsive genes, PCNA increases the basal expression of these genes. Our studies suggest that in addition to serving as a platform for the recruitment of DNA replication and repair proteins, PCNA may serve as a platform for transcription factors involved in regulating gene expression.

Figure 1.

Isolation of PCNA. (A) Radiolabeled ERE-containing oligos were incubated with purified ERα in the absence or presence of HeLa nuclear extracts as indicated. Protein–DNA complexes were resolved on an agarose gel, excised and analyzed by mass spectrometry. (B) The locations of peptides identified by mass spectrometry analysis in two independent experiments are indicated by gray bars.

Figure 2.

PCNA interacts with ERα. (A) Flag affinity resin without (lanes 2) or with (lanes 3 and 4) purified, flag-tagged ERα was combined with purified, untagged PCNA. (B) Nickel affinity resin without (lanes 2) or with (lanes 3 and 4) purified PCNA was combined with in vitro translated ³⁵S-labeled full-length or truncated ERα or unprogrammed lysate (UPL). (C) Nickel affinity resin without (lanes 2) or with (lanes 3 and 4) purified his-tagged ERα E domain was combined with purified, untagged PCNA. E₂ was added as indicated. Proteins were separated on a denaturing gel and proteins were detected by western analysis (A and C) or autoradiography (B). Ten percent of PCNA input was included for reference (lane 1). Results are representative of three independent experiments.

Figure 3.

ERα interacts with endogenously expressed PCNA. Flag-affinity resin without (lanes 3 and 4) or with flag-tagged ERα (lanes 5–10) was combined with 20 μg MCF-7 nuclear extract in the absence (lanes 3–6) or presence of oligos containing a non-specific DNA sequence (lanes 7 and 8) or an ERE (lanes 9 and 10). E₂ was added as indicated. ERα and its associated proteins were eluted and separated on a denaturing gel. PCNA was detected by western analysis. Ten percent input was included for reference (lanes 1 and 2). Results are representative of two independent experiments.

Figure 4.

PCNA enhances ERα–ERE complex formation. ³²P-labeled oligos containing the consensus ERE were incubated with 10 fmol of purified ERα in the presence of E₂.Purified his-labeled PCNA (lanes 2–6) and ERα- (lane 5) or PCNA- (lane 6) specific antibody (Ab) were added to the binding reactions as indicated. Bound and unbound ³²P-labeled oligos were fractionated on a non-denaturing polyacrylamide gel and visualized by autoradiography. Complexes containing ERα are indicated (ERα→). Results are representative of three independent experiments.

Figure 5.

PCNA associates with endogenous estrogen-responsive genes. (A) Sheared chromatin from MCF-7 cells, which had been treated with ethanol vehicle (white bars) or 10 nM E₂ for 2 h (gray bars), was immunoprecipitated with an ERα- or PCNA-specific antibody. DNA was isolated and real-time PCR was performed in triplicate to monitor the association of ERα and PCNA with the region of the pS2 gene containing an imperfect ERE or a region 2.8-kb upstream of the pS2 ERE (Control). Standard curves were derived for each primer set and the relative copy number for each sample was obtained based on the standard curve. Data from four independent experiments are expressed as the mean ± SEM. A significant change in the copies associated induction in the presence of E₂ was determined by Student's t-test and is indicated by an asterisk (*, P < 0.05). (B) Sheared chromatin from MCF-7 cells, which had been treated with ethanol vehicle or 10 nM E₂ for 15, 45 or 120 min, was immunoprecipitated with an ERα- or PCNA-specific antibody or non-specific IgG. DNA was isolated and subjected to PCR amplification to monitor the association of ERα and PCNA with the ERE-containing region of the oxytocin gene or the non-estrogen-responsive 36B4 gene. Ten percent of input DNA was included as a control. PCR products were run on 1.5% agarose gels, stained and visualized using UV light. Results are representative of three independent experiments.

Figure 6.

Knocking down endogenous PCNA expression alters gene expression. MCF-7 cells were transfected with 50 pmol of double-stranded control siRNA directed against renilla luciferase (Control) or PCNA-specific siRNA. (A) After 0–72 h, whole cell lysates were subjected to western analysis using a PCNA- or Sp1-specific antibody. Results are representative of two independent experiments. (B) After 72 h, cells were treated with ethanol vehicle (white bars) or 10 nM E₂ (gray bars) for 24 h, RNA was harvested and cDNA was synthesized. Real-time PCR was performed using primers specific to PCNA, PR, pS2, ERα or 36B4 mRNA sequences. Standard curves were derived for each primer set in each experiment. The relative amount of RNA obtained for each sample was calculated from the standard curve. Data are reported as the mean of three replicates ± SEM. One representative of four independent experiments is shown. Significant differences (P < 0.05) in E₂-treated cells compared to the corresponding ethanol vehicle (a) or control siRNA (b) were determined by ANOVA and are indicated. Some error bars are too small to be visible.