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Molecular Endocrinology, doi:10.1210/me.2006-0519
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Molecular Endocrinology 21 (7): 1569-1580
Copyright © 2007 by The Endocrine Society

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

Jennifer R. Schultz-Norton, Kjirsten A. Walt, Yvonne S. Ziegler, Ian X. McLeod, John R. Yates, Lori T. Raetzman and Ann M. Nardulli

Department of Molecular and Integrative Physiology (J.R.S.-N., K.A.W., Y.S.Z., L.T.R., A.M.N.), University of Illinois, Urbana, Illinois 61801; and Department of Cell Biology (I.X.M., J.R.Y.), The Scripps Institute, La Jolla, California 92037

Address all correspondence and requests for reprints to: Ann M. Nardulli, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801. E-mail: anardull{at}life.uiuc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ligand-occupied estrogen receptor {alpha} (ER{alpha}) initiates changes in gene expression through its interaction with target DNA. The capacity of ER{alpha} 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 ER{alpha}-mediated transcription, we have isolated and identified proteins associated with ER{alpha} 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 ER{alpha} and enhances the interaction of ER{alpha} 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 ER{alpha}-FEN-1 interaction and increase our understanding of the elaborate regulatory mechanisms that drive estrogen-responsive gene expression and DNA repair.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE HORMONE ESTROGEN activates transcription of a wide variety of genes (1, 2). The genomic effects of this hormone are initiated by interaction of the ligand-occupied estrogen receptor {alpha} (ER{alpha}), with estrogen response elements (EREs) residing in target genes. The DNA-bound receptor recruits a diverse array of coregulatory proteins involved in numerous cellular processes to exert fine-tuned control of estrogen-responsive genes. Many of these coregulatory proteins, such as kinases (3, 4, 5, 6, 7), histone acetyl transferases (8, 9, 10, 11, 12), and inhibitors of acetylation (13, 14), influence ER{alpha} function through specific posttranslational modifications.

In addition to proteins that modify receptor activity through chemical adducts, other coregulatory proteins recruited to ER{alpha} participate in DNA repair and are required to maintain DNA integrity. Our laboratory previously demonstrated that the DNA repair protein 3-methyladenine DNA glycosylase (MPG) interacts with ER{alpha} and modulates ER{alpha}-mediated transactivation (15). Other DNA repair proteins, including thymidine DNA glycosylase, O(6)-methylguanine-DNA methyltransferase, and poly(ADP-ribose) polymerase 1 (PARP-1), have also demonstrated the capacity to associate with estrogen-responsive genes and influence estrogen-mediated transactivation (16, 17, 18). Thus, there is accumulating evidence to link ER{alpha}-mediated transcription and DNA repair.

Using a modified gel mobility shift assay (19), we isolated a large complex of proteins that were associated with ER{alpha} when it was bound to ERE-containing DNA and then identified these ER{alpha}-associated proteins using mass spectrometry analysis. In addition to the previously described MPG (15), we identified another protein that plays a critical role in DNA replication and repair, flap endonuclease-1 (FEN-1). FEN-1 is required for processing of Okazaki fragments during DNA replication and for long patch base excision repair (BER;20, 21, 22, 23). In this study, we demonstrate that FEN-1 interacts with ER{alpha}, increases the ER{alpha}-ERE interaction, is recruited to the endogenous pS2 gene in MCF-7 cells, and influences endogenous, estrogen-responsive gene expression. The identification of FEN-1 as a regulator of ER{alpha} function helps to further establish a functional link between estrogen-responsive gene expression and DNA repair.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FEN-1 Is Expressed in a Variety of Cultured Cell Lines
To isolate proteins associated with the ERE-bound ER{alpha}, HeLa nuclear extracts were combined with purified ER{alpha} and 32P-labeled ERE-containing oligos and then fractionated on an agarose gel (19). The receptor-DNA complex was isolated, and mass spectrometry was used to identify FEN-1, a protein that is required for processing of Okazaki fragments during DNA replication and removal of nucleotide flaps during long patch base excision repair (24, 25, 26, 27, 28, 29).

Because HeLa nuclear extracts had been used for the initial isolation and identification of FEN-1, we monitored the FEN-1 expression in a number of cultured cell lines that have been used to study estrogen responsiveness (14, 15, 19, 30, 31, 32, 33, 34). FEN-1 was expressed in U2 osteosarcoma (U2-OS) and MDA-MB-231 and MCF-7 breast cancer cells (Fig. 1AGo). As expected, ER{alpha} was only present in MCF-7 nuclear extracts, but Sp1, which was used as a loading control, was expressed to a similar extent in all three cell lines.


Figure 1
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  		Fig. 1. FEN-1 Is Expressed in Cultured Cell Lines and Interacts with ER{alpha}

A, Nuclear extracts from U2-OS or MDA-MB-231 (231) or MCF-7 breast cancer cells were fractionated on a denaturing acrylamide gel and subjected to Western blot analysis using antibodies directed against ER{alpha}, FEN-1, or Sp1 as indicated. B, FLAG-affinity resin without (lanes 3 and 4) or with (lanes 5 and 6) FLAG-tagged ER{alpha} was combined with 20 µg MCF-7 nuclear extracts. E2 was added as indicated. ER{alpha} and its associated proteins were eluted and separated on a denaturing gel. FEN-1 was detected by Western blot analysis. "10% input" was included for reference (lanes 1 and 2). Results are representative of three independent experiments.

 
FEN-1 Interacts with ER{alpha}
Although FEN-1 was isolated in a complex with ER{alpha}, we wanted to confirm that endogenously expressed FEN-1 could interact with ER{alpha} using another method. Therefore, baculovirus-expressed, FLAG-tagged ER{alpha} was immobilized and incubated with MCF-7 nuclear extracts. As shown in Fig. 1BGo, FEN-1 was pulled down in the presence (lanes 5 and 6) but not in the absence (lanes 3 and 4) of ER{alpha}. Thus, ER{alpha} was able to interact with endogenously expressed FEN-1.

To determine the region(s) of ER{alpha} required for interaction with FEN-1, bacterially expressed, His-tagged FEN-1 was immobilized and incubated with in vitro transcribed and translated 35S-labeled full-length or truncated ER{alpha}. As shown in Fig. 2Go, FEN-1 interacted with the full-length ER{alpha} and with ABC, which contained the amino terminus and DNA binding domain, but failed to interact with AB, which lacked the DNA binding domain. FEN-1 also interacted with CD and the carboxy-terminal DEF. Because the ligand binding domain (E) interacted with the nickel resin used for protein immobilization, we were unable to further define those regions of the receptor required for interaction with FEN-1 (data not shown). However, we were able to use incremental deletions of the hinge domain (D) to further define the region of CD required for interaction. Partial deletion of the hinge domain to amino acid 292 ({Delta}CD1) maintained the interaction of this truncated ER{alpha} with FEN-1. However, removal of either a larger portion of the hinge domain to amino acid 272 ({Delta}CD2) or the entire hinge domain so that only the DNA binding domain (C) remained abrogated the ability of these truncated ER{alpha} proteins to interact with FEN-1. Interestingly, the region of the hinge domain required for FEN-1 interaction was defined previously as the C-terminal extension (amino acids 252–288), which is essential for stabilizing receptor-DNA interactions (35, 36, 37). Thus, FEN-1 interacts with multiple ER{alpha} domains, including the DNA binding domain, C-terminal extension, and carboxy-terminal domains.


Figure 2
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  		Fig. 2. FEN-1 Interacts with Multiple ER{alpha} Domains

Nickel affinity resin without (lane 2) or with (lanes 3 and 4) purified FEN-1 was combined with in vitro-translated 35S-labeled full-length or truncated ER{alpha} or unprogrammed lysate (UPL). E2 was added as indicated. Proteins were separated on a denaturing gel and detected by autoradiography. "10% input" was included for reference (lane 1). The results are representative of at least three independent experiments.

 
FEN-1 Influences the ER{alpha}-ERE Interaction
Given that FEN-1 interacted with ER{alpha}, it seemed possible that it might influence the ER{alpha}-ERE interaction. Gel mobility shift assays were used to determine whether this was the case. When 10 fmol purified, FLAG-tagged ER{alpha} were incubated with radiolabeled ERE-containing oligos in the absence of hormone, a receptor-DNA complex was formed (Fig. 3AGo, lane 1). When increasing amounts of purified, His-tagged FEN-1 were added to the binding reaction, there was a dramatic increase in the ER{alpha}-ERE interaction. Similar results were obtained when increasing amounts of FEN-1 were added to the binding assay in the presence of 17ß-estradiol (E2) (Fig. 3BGo).


Figure 3
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  		Fig. 3. FEN-1 Enhances ER{alpha}-ERE Complex Formation

32P-labeled oligos containing the consensus ERE were incubated with 10 fmol purified ER{alpha}. Purified His-FEN-1 was added to the binding reactions as indicated in the absence (A) or presence (B and C) of E2. Antibodies (Ab) specific to ER{alpha} (E1 and E2), FEN-1 (F1 and F2), or His tag (His) were added as indicated. Bound and unbound 32P-labeled oligos were fractionated on a nondenaturing polyacrylamide gel and visualized by autoradiography. Results are representative of three independent experiments.

 
To determine whether FEN-1 was present in the complexes formed, gel mobility shift assays were performed in the absence and presence of ER{alpha}- or FEN-1-specific antibody. In the absence of FEN-1, an ER{alpha}-specific antibody was able to supershift the ER{alpha}-ERE complex (Fig. 3CGo, compare lanes 1 and 2). In the presence of FEN-1, the complex increased in intensity, and two ER{alpha}-specific antibodies were able to supershift the complex, indicating that ER{alpha} was present (lanes 4 and 5). However, two different FEN-1-specific antibodies were unable to alter the complex migration (lanes 6 and 7). Because the ER{alpha}-FEN-1 interaction might mask the epitopes required for FEN-1 antibody recognition, an antibody that recognizes the amino-terminal His tag of the purified FEN-1 was used. Again, the migration of the complex was unaltered (lane 8). Simultaneous addition of ER{alpha}- and FEN-1-specific antibodies did not change the migration when compared with an ER{alpha}-specific antibody alone (compare lanes 4 and 9). Thus, although FEN-1 enhanced ER{alpha}-ERE interaction, it did not remain associated with the complex in a manner that could be resolved in this assay. We and others have reported a similar capacity of other purified regulatory proteins to enhance the interaction of nuclear receptors with their cognate binding sites but fail to form stable trimeric complexes (13, 14, 15, 19, 38, 39, 40). Furthermore, Tom et al. (41) reported that proliferating cell nuclear antigen (PCNA) enhances binding of FEN-1 to DNA but that PCNA is not present in the FEN-1-DNA complex. Thus, the ability of an individual protein to enhance interaction of a DNA-binding protein to its recognition sequence, but not form a stable trimeric complex, is not uncommon and could indicate that FEN-1 induces a structural change in ER{alpha} that increases the stability of the receptor-DNA complex. The failure of purified FEN-1 to remain in a stable complex with the DNA-bound receptor suggests that the interaction of FEN-1 with the ERE-bound ER{alpha} is transient or that other nuclear proteins are needed to stabilize complex formation.

To determine whether FEN-1 was associated with a naturally occurring ERE in its native environment, chromatin immunoprecipitation assays were performed in MCF-7 cells to examine the ERE-containing region of the pS2 gene. Using this method, we could monitor the association of ER{alpha} and FEN-1 with an endogenous, estrogen-responsive gene in its normal environment. The association of ER{alpha} with the ERE-containing region of the pS2 gene was increased after 2 and 24 h of E2 treatment (Fig. 4Go) as we and others have reported previously (19, 31, 42). When E2 was present, there was also a substantial increase in the association of FEN-1 with this gene region in a pattern that mimicked the association of ER{alpha}. In contrast, no changes were detected in the association of ER{alpha} or FEN-1 with a region 2.8 kb upstream of the pS2 ERE, except for a slight increase in FEN-1 association after 24 h of E2 treatment. Thus, although we were unable to form a trimeric complex with purified ER{alpha} and FEN-1 in our gel shift experiments, FEN-1 did functionally associate with the ERE-containing region of the endogenous pS2 gene in MCF-7 cells.


Figure 4
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  		Fig. 4. FEN-1 Is Associated with the ERE-Containing Region of the pS2 Gene

Sheared chromatin from MCF-7 cells, which had been treated with ethanol vehicle (white bars) or 10 nM E2 for 2 or 24 h (light and dark gray bars, respectively), was immunoprecipitated with an ER{alpha}- or FEN-1-specific antibody. DNA was isolated, and real-time PCR was performed in triplicate to monitor the association of ER{alpha} and FEN-1 with the region of the pS2 gene containing an imperfect ERE or a region 2.8 kb upstream of the pS2 ERE. Standard curves were derived for each primer set, and the relative copy number for each sample was obtained based on the standard curve. The average ± SEM fold induction for three independent experiments is shown. A significant increase in the fold induction in the presence of E2 was determined by Student’s t test and is indicated (*, P < 0.05).

 
FEN-1 Influences Transcription of an Estrogen-Responsive Reporter Plasmid
Because FEN-1 interacted with ER{alpha} and was associated with the endogenous, estrogen-responsive pS2 gene in MCF-7 cells, it seemed likely that FEN-1 might also affect ER{alpha}-mediated transactivation. MCF-7 cells were transfected with a luciferase reporter plasmid containing a single consensus ERE upstream of a minimal thymidine kinase promoter. When increasing amounts of a FEN-1 expression vector were included, a dose-dependent decrease in ER{alpha}-mediated transcription was observed in the presence, but not in the absence, of hormone (Fig. 5AGo), supporting the idea that FEN-1 modulates ER{alpha}-mediated transcription. Similar results were observed when MDA-MB-231 cells were transfected with increasing amounts of a FEN-1 expression vector (Fig. 5BGo). However, a slight decline in ER{alpha}-mediated transcription was also observed in these cells in the absence of E2. Together, these transfection experiments demonstrate that FEN-1 limited ER{alpha}-mediated transcription of an ERE-containing reporter plasmid in two different breast cancer cell lines.


Figure 5
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  		Fig. 5. FEN-1 Decreases ER{alpha}-Mediated Transcription

Transient transfections were performed in MCF-7 (A) or MDA-MB-231 (B) cells with a luciferase reporter plasmid containing a single consensus ERE and increasing amounts of a FEN-1 expression vector. A parental expression vector lacking the FEN-1 sequence was included as needed to maintain constant DNA levels, and an ER{alpha} expression vector was included in MDA-MB-231 cell transfections. Ethanol vehicle (white bars) or 10 nM E2 (dark gray bars) was included as indicated. Data from four independent experiments, which were performed in duplicate, were combined and are presented as the mean ± SEM relative luciferase units (RLU). The magnitude of E2-induced transactivation was compared in the absence and presence of the FEN-1 expression vector. Significant differences in transcription in the presence of FEN-1 in the presence (a) or absence (b) of E2 were determined by ANOVA and are indicated. No significant differences were detected in the absence of hormone.

 
FEN-1 Influences Transcription of the Endogenous, Estrogen-Responsive Progesterone Receptor (PR) Gene
Although FEN-1 was able to influence ER{alpha}-mediated transcription in our transient transfection experiments, we felt that it was important to examine the effect of FEN-1 on the expression of endogenous, estrogen-responsive genes in their native cellular context. RNA interference assays were performed to assess the effect of knocking down endogenously expressed FEN-1 in MCF-7 cells. In addition, a small interfering RNA (siRNA) directed at renilla luciferase mRNA was used as a control.

In the presence of control (Con) siRNA, increased FEN-1, pS2, and PR mRNA levels were observed after hormone treatment (Fig. 6AGo). In contrast, 36B4 mRNA levels were not altered by exposure of MCF-7 cells to hormone. These findings, which demonstrate the estrogen responsiveness of the pS2 and PR genes but not the 36B4 gene, are in agreement with previous studies (1, 19, 43).


Figure 6
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  		Fig. 6. Knocking Down FEN-1 Influences Estrogen Responsiveness

MCF-7 cells were transfected with 50 pmol double-stranded control siRNA directed against renilla luciferase (Con) or FEN-1-specific siRNA. A, Cells were treated with ethanol vehicle (white bars) or 10 nM E2 (dark gray bars) for 24 h. RNA was harvested and cDNA was synthesized. Real-time PCR was performed using primers specific to FEN-1, pS2, PR, or 36B4 mRNA sequences. Standard curves were derived for each primer set in each experiment. The relative amount of RNA was obtained for each sample based on the standard curve. Data are reported as the average ± SEM of three replicates. Some error bars are too small to be visible. One representative of four independent experiments is shown. Significant differences (P < 0.05) in E2-treated cells compared with the corresponding ethanol vehicle (a) or FEN-1-specific siRNA compared with 36B4 control siRNA under the corresponding hormone treatment (b) were determined by ANOVA and are indicated. B, Cells were treated with ethanol vehicle or 10 nM E2 for 24 h as indicated. Cells were lysed, and proteins were subjected to Western blot analysis using antibodies (Ab) directed against FEN-1, ER{alpha}, PR-A, PR-B, APE-1, or Sp1. Results are representative of three independent experiments.

 
When siRNA directed against the 3' region of the FEN-1 mRNA sequence was used, FEN-1 mRNA levels were reduced. Although we were unable to decrease the levels of FEN-1 mRNA by more than 40%, distinct changes were observed in the mRNA levels of two ER{alpha}-regulated genes. The basal levels of pS2 and PR mRNA declined, and a decrease was observed in the E2-induced expression of pS2 mRNA. In contrast, a striking 18-fold, E2-induced increase in PR mRNA levels was observed in the presence of FEN-1 siRNA, which was substantially greater than the 4.5-fold increase in PR mRNA levels observed in the presence of E2 and control siRNA. These gene-specific effects of FEN-1 on the pS2 and PR genes most likely reflect the distinct nature of the promoters regulating the expression of these genes (43, 44, 45, 46, 47, 48, 49, 50, 51, 52).

The effect of knocking down FEN-1 on estrogen-mediated gene expression was also examined using Western blot analysis. When MCF-7 cells were transfected with control siRNA, the expected E2-mediated increase in both forms of the progesterone receptor (PR-A and PR-B) and decrease in ER{alpha} protein levels were observed (Fig. 6BGo, compare lanes 1 and 2). Knocking down FEN-1 augmented both PR-A and PR-B protein levels in the presence of E2 (lanes 3 and 4), which was consistent with the changes we observed at the mRNA level. Interestingly, knocking down FEN-1 inhibited the E2-mediated decrease in ER{alpha} protein normally observed after 24 h of E2 treatment but did not affect the level of Sp1 or apurinic endonuclease-1 (APE-1).

To ensure that the effects we observed could be attributed to the decrease in FEN-1 expression, we used siRNA directed against another FEN-1 region located in the central portion of exon 2. This second set of FEN-1-specific siRNA also enhanced PR mRNA and protein levels and diminished the E2-induced expression of pS2 mRNA levels (data not shown), as was observed with the first set of siRNA directed against a more 3' region of the FEN-1 mRNA sequence. Thus, two separate sets of siRNA directed against different FEN-1 regions produced similar results. Furthermore, knocking down FEN-1 did not affect the expression of another endonuclease, APE-1. Together with our transient transfection experiments, these studies reinforce the idea that FEN-1 is an effective modulator of ER{alpha}-mediated transcription.

FEN-1 Expression Is Regulated by Estrogen in the Uterus
Because FEN-1 is essential for in utero survival of mouse embryos (53), it has not been possible to use FEN-1 knockout mice to examine the effect of FEN-1 on estrogen-mediated physiological processes. Interestingly, Reese et al. (54) reported that FEN-1 mRNA is more highly expressed at the site of implantation than in the surrounding uterine tissue, suggesting that FEN-1 may play an integral role in the uterus during implantation. To determine whether E2 priming might alter FEN-1 expression in the uterus, the level of FEN-1 protein was monitored in the uteri of ovariectomized mice that had been treated with vehicle or E2 for 24 h. As seen in Fig. 7AGo, FEN-1 expression was mainly restricted to the uterine luminal (L) and glandular (G) epithelial cells in the absence of hormone. When mice were treated with E2, FEN-1 expression was dramatically increased in the stroma (panel B, S) and myometrium (M). On closer examination, a somewhat diffuse staining of FEN-1 was observed in the luminal and glandular epithelial cells in the absence of E2 (C), but, after E2 treatment, these epithelial cells exhibited more discrete nuclear staining, indicating the increased localization of FEN-1 in the nuclei (panel D). FEN-1 expression was dramatically increased in the uterine stromal and myometrial cells after hormone exposure such that that the majority of these cells expressed FEN-1. Thus, E2 has a dramatic effect on FEN-1 expression in the uterus.


Figure 7
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  		Fig. 7. E2 Treatment of Ovariectomized Mice Increases FEN-1 Expression in the Uterus

Ovariectomized mice were treated with vehicle (A and C) or E2 (B and D) for 24 h. A representative slide stained with a FEN-1-specific antibody from one of three E2-treated and two vehicle-treated mice is shown at x5 magnification (A and B). The boxed areas in A and B are shown at x40 magnification in C and D, respectively. Control slides, which were not exposed to FEN-1 antibody, are shown in the insets in C and D. Stromal (S), luminal epithelial (L), myometrial (M), and glandular epithelial (G) cells are indicated. Scale bars, 100 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The ability of ER{alpha} to initiate changes in gene expression requires the recruitment of a vast array of coregulatory proteins with a variety of enzymatic functions (55, 56, 57). We discovered a novel interaction between ER{alpha} and FEN-1, a protein required for DNA replication and repair, and uncovered a new role for FEN-1 in regulating ER{alpha}-mediated transactivation. FEN-1 interacts with ER{alpha}, fosters the interaction of ER{alpha} with ERE-containing DNA, associates with the endogenous pS2 gene in MCF-7 cells, and alters endogenous, estrogen-responsive gene expression.

FEN-1 and Estrogen-Responsive Gene Expression
The ability of FEN-1 to interact with ER{alpha} and foster the receptor-DNA interaction may influence gene expression by assisting in the formation of a stable transcription complex. This would not, however, explain the paradoxical ability of FEN-1 to differentially alter expression of the estrogen-responsive pS2 and PR genes. In contrast to many of the coregulatory proteins identified to date, which either enhance or inhibit hormone responsiveness (55, 56, 57), FEN-1 appears play a role in enhancing and inhibiting estrogen responsiveness. The ability of FEN-1 to inhibit estrogen responsiveness was observed in our transient transfection experiments in which increased expression of FEN-1 decreased the activity of an ERE-containing reporter plasmid but was even more evident in our siRNA experiments in which knocking down FEN-1 expression significantly increased expression of the endogenous PR gene and resulted in increased PR mRNA and protein levels. However, FEN-1 also has the capacity to increase transcription of the pS2 gene as illustrated by the decreased estrogen responsiveness of the pS2 gene when FEN-1 expression was knocked down. Thus, FEN-1 alters estrogen responsiveness in a gene-specific manner. The dual effects of FEN-1 on ER{alpha}-mediated transcription most likely result from the contributions of multiple cis elements residing in target genes and the recruitment of their associated trans acting factors to foster the assembly of a multiprotein transcription complex, which in turn determines the transcriptional response. It is important to note that varying the level of FEN-1 influences this transcriptional response.

Previous studies have demonstrated that the DNA repair proteins O(6)-methylguanine-DNA methyltransferase and MPG decrease ER{alpha}-mediated transactivation (15, 16) and suggest that the stalling of DNA polymerase II at sites of DNA damage may account for this decreased transcription (58). However, DNA repair proteins do not always limit ER{alpha}-mediated gene expression as illustrated by the DNA repair protein thymine-DNA glycosylase, which enhances ER{alpha}-mediated transactivation (17). Regardless of the transcriptional outcome, these reports combined with the studies described here add to the growing body of evidence supporting a functional link between ER{alpha}-mediated transcription and DNA repair.

FEN-1 and DNA Repair
Cells are continually exposed to metabolic byproducts that can damage DNA. These DNA lesions must be repaired to maintain DNA integrity and cell viability. To accomplish this, chromatin must be remodeled so that DNA repair proteins can access the damaged DNA (59). Our studies and previous reports (for review, see Ref. 29) suggest that FEN-1 may contribute to DNA repair in two ways. First, when target cells are exposed to a mitogen such as E2, FEN-1 is recruited to DNA to process Okazaki fragments formed during DNA replication (29). To initiate DNA replication, chromatin must be reorganized so that FEN-1 and other proteins can access the DNA. At this time, when DNA is accessible, FEN-1 can also recognize and repair damaged DNA. Thus, FEN-1 has global effects on replication and repair of bulk DNA in response to hormone. Second, the recruitment of FEN-1 to estrogen-responsive genes by the ERE-bound receptor could provide another mechanism to target this repair protein to DNA. However, in this case, FEN-1 is targeted to actively transcribing, estrogen-responsive genes. In support of this idea, we have shown that more FEN-1 is recruited to the ERE-containing region of the pS2 gene than to an upstream region, which lacks any known ER{alpha} binding site. By recruiting FEN-1 to transcriptionally active genes, the DNA can be surveyed for lesions at the time of transcription when the DNA is accessible.

Ju et al. (18) recently reported a transient, topoisomerase IIß-mediated double-stranded DNA break in the proximity of the pS2 ERE during E2 exposure. During cleavage of the pS2 gene, increases in PARP-1 association with the pS2 gene were observed. Interestingly, both PARP-1 and FEN-1 are required for long patch BER (60). Using a variety of methods, we have isolated four of the proteins involved in BER, including MPG, APE-1, PCNA, and FEN-1 (Ref. 15 and our unpublished data). Hence, in addition to its more global effects on DNA repair that occur during E2-induced replication, FEN-1 may have more localized effects on DNA repair at specific estrogen-responsive genes as they are transcribed. The enhanced repair of transcriptionally active genes compared with nontranscribed DNA regions and the preferential repair of the DNA coding strand provide evidence for the coupling of transcription and DNA repair (58, 61, 62). Meijer and Smerdon (59) have suggested that DNA replication, repair, and transcription all depend on the ability of proteins to access DNA and that, when the DNA is accessible, multiple processes can proceed simultaneously.

We demonstrated previously that ER{alpha} can influence MPG-initiated DNA repair (15). Recently, Crowe and Lee (63) reported that E2 treatment of MCF-7 cells decreases etoposide-induced DNA damage and that transient expression of the receptor in ER{alpha}-negative MDA-MB-231 breast cancer cells helps to protect these cells from etoposide-induced DNA damage. At the time this study was reported, it was unclear how hormone or ER{alpha} might decrease DNA damage. Our studies suggest that the recruitment of FEN-1 and other DNA repair proteins to the ERE-bound receptor may play a role in enhanced DNA repair (15, 16, 17, 18). Thus, the recruitment of FEN-1 to DNA may influence gene expression and help ensure that the integrity of the genome is maintained.

Biological Effects of FEN-1
Because of its increased expression at the site of implantation (54), it has been suggested that FEN-1 may serve as a marker of uterine receptivity at the time of implantation and may be instrumental in maintaining fertility and normal reproductive function. The expression of FEN-1 is required for viability as indicated by the early embryonic lethality in FEN-1 null mice (53). We have now demonstrated that E2 has a profound effect on the expression of FEN-1 in specific uterine cell types. E2 dramatically increased the expression of FEN-1 in uterine stromal and myometrial cells and modestly affected FEN-1 expression in luminal and glandular epithelial cells. Although it has been suggested that FEN-1 is constitutively expressed in all cells (29), we have shown that the majority of uterine stromal and myometrial cells are nearly devoid of FEN-1 in the absence of hormone and that the luminal and glandular epithelial cells expressed significantly higher levels of FEN-1. Our combined studies suggest that FEN-1 would influence ER{alpha}-mediated gene expression in epithelial cells in the absence of hormone and that, in the presence of E2, estrogen-responsive gene expression would be differentially altered by FEN-1 expression in all uterine cell types.

Decreased expression of FEN-1 has been linked to rapid tumor progression (64), but overexpression of FEN-1 has also been associated with tumorigenesis in reproductive tissues, including the endometrium, ovary, and prostate gland (65, 66, 67). These studies, combined with the work presented here, provide evidence for an inextricable link between FEN-1 and effective DNA repair, suggest that FEN-1 levels must be carefully titrated to maintain normal cell biology, and implicate a role of E2 in helping to maintain appropriate FEN-1 expression in hormone-responsive tissues.

Although it has been clear for some time that FEN-1 plays a critical role in DNA replication and repair, we identified yet another role for this multifunctional protein in regulating estrogen-responsive gene expression. Together, our studies suggest that FEN-1 serves as a critical integrator of cellular functions and helps to coordinate the processes of replication, repair, and transcription in estrogen-responsive cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Isolation and Identification of FEN-1
Nuclear extracts from HeLa cervical cancer cells were incubated with purified, baculovirus-expressed ER{alpha} in the absence or presence of annealed, 32P-labeled oligos containing the Xenopus laevis vitellogenin A2 ERE (5'-GAT TAA CTG TCC AAA GTC AGG TCA CAG TGA CCT GAT CAA AGT TAA TGT AA-3' and 5'-TTA CAT TAA CTT TGA TCA GGT CAC TGT GAC CTG ACT TTG GAC AGT TAA TC-3'). Proteins associated with the ERE-bound ER{alpha} were isolated using agarose gel mobility shift assays and identified using mass spectrometry analysis essentially as described previously (19). Parallel experiments were performed using 32P-labeled oligos containing a nonspecific DNA sequence (5'-CTA GAT TAC TTC TCA TGT TAG ACA TAC TCA GAT CTA GAC ATA CTC AGA TC-3' and 5'-GAT CTG AGT ATG TCT AGA TCT GAG TAT GTC TAA CAT GAG AAG TAA TCT AG-3') instead of the ERE to ensure that isolated proteins were associated with the DNA-bound ER{alpha} and did not simply comigrate with the receptor-DNA complex. Four peptide fragments that had amino acid sequence identical to FEN-1 and covered 18% of the protein (MMENGIKPVYVFDGKPPQLK, KLPIQEFHLSR, RLDPNKYPVPENWLHK, EAHQLFLEPEVLDPESVELK) were isolated in a complex with the ERE-bound ER{alpha}.

Western Blots
Nuclear extracts were prepared from U2-OS and MDA-MB-231 and MCF-7 breast cancer cells as described previously (68). Ten micrograms of nuclear extracts were fractionated on a 10% SDS polyacrylamide gel, transferred to nitrocellulose (69), and detected with antibodies directed against ER{alpha}, FEN-1, and Sp1 (sc-8002, sc-28355, and sc-59, respectively; Santa Cruz Biotechnology, Santa Cruz, CA). The blots were probed with a horseradish peroxidase-conjugated secondary antibody and developed using a chemiluminescent detection system as described previously (70).

Expression and Purification of His-Tagged FEN-1
A bacterial expression vector encoding His-tagged FEN-1 (phis-FEN-1), which had been synthesized as described by Stols et al. (71), was graciously provided by K. Nettles (The Scripps Institute, Jupiter, FL). Expression and purification of His-tagged FEN-1 was performed as described previously (19). Protein purity was assessed on Coomassie blue-stained gels, and protein concentrations were determined using the Bio-Rad (Hercules, CA) protein assay with BSA as a standard.

Pull-Down Assays
Pull-down assays using in vitro-transcribed and -translated 35S-labeled full-length ER{alpha} or truncated ER{alpha} proteins ABC, AB, CD, and DEF (72, 73, 74) were performed essentially as described previously (19). Expression vectors for {Delta}CD1 (amino acids 180–292), {Delta}CD2 (amino acids 180–272), and C (amino acids 180–262) were provided by K. Nettles and synthesized as described by Stols et al. (71). Full-length and truncated ER{alpha} proteins were synthesized using the TNT T7 Quick Coupled Transcription/Translation system (Promega, Madison, WI) and incubated with immobilized, His-tagged FEN-1 at 4 C for 45 min in binding buffer [15 mM Tris (pH 7.9), 20 mM KCl, 0.2 mM EDTA, and 4 mM dithiothreitol] with or without 10 µM E2. Bound proteins were washed once with binding buffer, once with wash buffer [15 mM Tris (pH 7.9), 100 mM KCl, 0.2 mM EDTA, and 4 mM dithiothreitol], and were eluted with 2x loading buffer [125 mM Tris (pH 6.8), 4% SDS, 20% glycerol, and 1.44 M ß-mercaptoethanol]. Eluted proteins were separated by SDS-PAGE and subjected to autoradiography.

For ER{alpha} pull-down assays, FLAG-tagged full-length ER{alpha} was expressed in Sf9 cells as described previously (43, 75), immobilized on M2-agarose (Sigma, St. Louis, MO), and washed with purification buffer [20 mM Tris (pH 7.5), 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 10% vol/vol glycerol]. Twenty micrograms of MCF-7 nuclear extracts were incubated with immobilized ER{alpha} in binding buffer, followed by washing and elution as described. Eluted proteins were separated by SDS-PAGE and subjected to Western blot analysis with an antibody specific for FEN-1 (sc-28355; Santa Cruz Biotechnology). The blots were probed with a horseradish peroxidase-conjugated secondary antibody and developed using a chemiluminescent detection system as described previously (70).

Gel Mobility Shift Assays
ER{alpha} was expressed and purified to near homogeneity as we described previously (43). Ten to 50 fmol purified ER{alpha} were incubated without or with 0.5–2.0 µg of purified His-FEN-1 in binding buffer with 10% glycerol and 100 ng poly-dI/dC with or without 50 nM E2 in a final volume of 20 µl for 10 min on ice. BSA and His elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole) were included as needed to maintain constant protein and salt concentrations. For antibody supershift experiments, an ER{alpha}-, FEN-1-, or His tag-specific antibody (ER{alpha}, sc-8002 or sc-8005; FEN-1, sc-28355 or sc-13051; or His, sc-803; Santa Cruz Biotechnology) was added to the binding reaction and incubated for 10 min on ice before addition of DNA. Radiolabeled ERE-containing oligos were added to the binding reactions and incubated for 10 min at room temperature before fractionation on low-ionic-strength polyacrylamide gels (76) at 4 C with buffer recirculation. Radioactive bands were visualized by autoradiography or were quantitated by phosphor imager analysis with Image Quant software (GE Healthcare, Piscataway, NJ).

Transient Transfections
MCF-7 cells were maintained in phenol red-containing MEM supplemented with 5% calf serum, transferred to phenol red-free MEM with 5% charcoal dextran-treated calf serum (CDCS) 40 h before transfection, and seeded in 24-well plates in phenol red-free MEM with 5% CDCS 16 h before transfection. Cells were transfected with 5 ng TK-renilla (Promega) and 1 µg of ERE-TK-Luc3 reporter plasmid (generously provided by Mark Nichols, University of Pittsburgh Cancer Institute, Pittsburgh, PA) (77) without or with 0.1–2.5 µg of an FEN-1 mammalian expression vector (a gift from C. McMurray, Mayo Clinic, Rochester, MN) (78) using Lipofectin (Invitrogen, Carlsbad, CA).

MDA-MB-231 cells were maintained in L-15 medium supplemented with 10% fetal bovine serum and placed on improved MEM with 5% CDCS 24 h before transfection. Cells were seeded in 24-well plates and transfected with 25 ng CMV5-hER{alpha} (79), 10 ng TK-renilla, and 1 µg ERE-TK-Luc3 without or with 0.1–2.5 µg of an FEN-1 mammalian expression vector.

For all transfection experiments, the transfection media was removed after 8 h, and the cells were treated with ethanol vehicle or 10 nM E2 for 24 h. The parental expression vector pCDNA3.1 (Invitrogen) was included to maintain a constant DNA concentration in each well. Luciferase activity was quantitated using the Dual Luciferase Assay kit (Promega).

Chromatin Immunoprecipitation Assays
MCF-7 cells were maintained in phenol red-containing MEM supplemented with 5% calf serum, placed on phenol red-free MEM with 5% CDCS for 72–96 h, and exposed to ethanol vehicle or 10 nM E2 for 2 or 24 h. Chromatin immunoprecipitation assays were performed essentially as recommended by Millipore (Charlottesville, VA) except that pelleted cells were washed three times in lysis buffer [10 mM Tris (pH 7.5), 10 mM NaCl, and 3 mM MgCl2] with 0.5% Nonidet P-40, resuspended in lysis buffer with 10 mM CaCl2 and 4% Nonidet P-40, and treated with 75 U micrococcal nuclease (USB, Cleveland, OH) for 10 min before sonication. The ER{alpha}- and FEN-1-specific antibodies sc-8002 and sc-28355, respectively (Santa Cruz Biotechnology) were used for immunoprecipitation of protein-DNA complexes. PCR primers flanking the pS2 ERE (forward, 5'-CCC GTG AGC CAC TGT TGT C-3'; and reverse, 5'-CCT CCC GCC AGG GTA AAT AC-3') or a region 2.8 kb upstream of this site, which contains no known ER{alpha} binding sequence (forward, 5'-GTA TGG TGT GGT CTT GGG TTC C-3'; and reverse, 5'-GGG TTG GAG CGG CTG GAG-3'), were used for semiquantitative PCR analysis with iQ SyBr Green Supermix and the iCycler PCR thermocycler according to directions of the manufacturer (Bio-Rad). One thousand, 5000, 10,000, and 25,000 genomic copies were run in parallel with each primer set during each experiment to derive a standard curve. Samples were run in triplicate, and the relative copy number was determined from the standard curve. Data from three independent experiments are reported as the fold induction in the presence of E2 compared with ethanol control. Significant changes in induction were calculated using the Student’s t test.

RNA Interference Assays
For siRNA experiments, MCF-7 cells were maintained as stated above and seeded in 12-well plates 24 h before transfection. Cells were transfected with 50 pmol control (renilla luciferase, 4630; Ambion, Austin, TX) or one of two sets of FEN-1-specific siRNA oligos [sc-37795, containing three discrete oligos (Santa Cruz Biotechnology) or a single oligo sequence, GUUCUCUGAGGAGCGAAUC (Dharmacon, Lafayette, CO)] in the absence of antibiotics using siLentFect (Bio-Rad) for 48 h. The second sequence did not align to any mRNA other than FEN-1 in a BLAST (basic local alignment search tool) search and was shown previously to knock down FEN-1 protein expression in FLAH25 cells (80). Medium was replaced with phenol red-free MEM containing 5% CDCS for an additional 0–24 h, followed by treatment with 10 nM E2 or ethanol vehicle for 24 h. Preliminary time course experiments demonstrated that 72 h of siRNA exposure was required to reduce FEN-1 protein levels (data not shown). RNA was harvested using Trizol (Invitrogen) and processed according to the directions of the manufacturer. cDNA was synthesized using the Reverse Transcription System (Promega). Real-time PCR was performed using iQ SYBR Green Supermix and the iCycler PCR thermocycler (Bio-Rad) according to directions of the manufacturer with primer sequences specific for FEN-1 (forward, 5'-AAG GTC ACT AAG CAG CAC AAT G-3'; and reverse, 5'-GTA GCC GCA GCA TAG ACT TTG-3'), pS2 (forward, 5'-GCT GTT TCG ACG ACA CCG TT-3'; and reverse, 5'-TTC TGG AGG GAC GTC GAT G-3'), or PR (forward, 5'-GTG CCT ATC CTG CCT CTC AAT C-3'; and reverse, 5'-CCC GCC GTC GTA ACT TTC G-3') mRNA. Primers to 36B4 mRNA (forward, 5'-GTG TTC GAC AAT GGC AGC AT-3'; and reverse, 5'-GAC ACC CTC CAG GAA GCG A-3') were used as a control. The 36B4 gene is not regulated by E2. Standard curves were derived using serial dilutions of cDNA equivalent to 0.02, 0.2, 2, and 20 ng of input RNA and were run in duplicate for each primer set during each experiment. The relative nanograms of RNA was determined from the standard curve. The average of three replicates from one experiment is shown and is representative of four independent experiments. Significant changes in RNA levels attributable to specific siRNA or hormone exposure were calculated by ANOVA with ezANOVA (C. Rorden, University of South Carolina, Columbia, SC; www.mricro.com). Protein knockdown was monitored by Western blot analysis of whole-cell lysates as described above using antibodies to FEN-1, ER{alpha}, APE-1, Sp1 (sc-28355, sc-8002, sc-334, sc-59, respectively; Santa Cruz Biotechnology), or PR (RM-9102; LabVision, Fremont, CA).

Immunohistochemistry
All animal procedures were done under a protocol approved by the University of Illinois Institutional Animal Care and Use Committee. Ovariectomized C57BL/6 mice were injected sc with oil or 125 ng E2 and killed after 24 h. Uteri were removed, fixed in formalin, embedded in paraffin, and sectioned. The 6-µm sections were deparaffinized in xylene and rehydrated with a series of graded ethanols. PBS washes were used between each of the following steps. Slides were boiled in 0.01 M citric acid for 10 min and cooled for 15 min at room temperature for antigen retrieval. Endogenous peroxidase activity was quenched with 1.5% hydrogen peroxide for 20 min at room temperature. Slides were incubated in blocking solution (PBS with 3% BSA and 0.1% Triton X-100) with 5% normal donkey serum for 10 min at room temperature. Control and experimental slides were incubated overnight at 4 C in blocking solution with 5% normal donkey serum or with FEN-1 antibody (1:100, sc-13051; Santa Cruz Biotechnology), respectively. All slides were incubated at room temperature for 30 min with a biotin-conjugated secondary antibody (1:200; Jackson ImmunoResearch, West Grove, PA). The Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) was used with a 1:100 dilution each of reagents A and B in PBS. FEN-1 was visualized with peroxidase-sensitive 3,3'-diaminobenzidine (SigmaFast tablets; Sigma). Samples were counterstained with 0.1% methyl green (Sigma), dehydrated in ethanol, cleared in xylene, and mounted with permount (Fisher, Pittsburgh, PA). Images were obtained using a Leica (Nussloch, Germany) DM2500 microscope fitted with a Qimaging Retiga 2000R camera (Qimaging, Burnaby, British Columbia, Canada) and white-balanced using Adobe Photoshop (Adobe Systems, San Jose, CA).


   ACKNOWLEDGMENTS
 
We thank W. L. Kraus and J. Kadonaga for ER{alpha} viral stock; M. Nichols, K. Nettles, B. Katzenellenbogen, and C. McMurray for plasmids; and P. Cooke for mouse uteri.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grants R01 DK 53884 (to A.M.N.) and P41 RR11823-10 (to J.R.Y.), Reproductive Biology Training Grant T32 HD07028 (to J.R.S.-N.), and Cell and Molecular Biology Training Grant T32 GM007283 (to K.A.W.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 8, 2007

Abbreviations: APE-1, Apurinic endonuclease-1; BER, base excision repair; CDCS, charcoal dextran-treated calf serum; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; FEN-1, flap endonuclease-1; MPG, 3-methyladenine DNA glycosylase; PARP-1, poly(ADP-ribose) polymerase 1; PCNA, proliferating cell nuclear antigen; PR, progesterone receptor; OS, osteosarcoma; siRNA, small interfering RNA.

Received for publication December 4, 2006. Accepted for publication April 30, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  PR
Ligands:   17β-Estradiol



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