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[Cancer Research 64, 6416-6423, September 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Potential Role of a Novel Transcriptional Coactivator PELP1 in Histone H1 Displacement in Cancer Cells

Sujit S. Nair, Sandip K. Mishra, Zhibo Yang, Seetharaman Balasenthil, Rakesh Kumar and Ratna K. Vadlamudi

Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The estrogen receptor plays an important role in breast cancer progression. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), also called modulator of nongenomic activity of estrogen receptor (MNAR), a novel coactivator of estrogen receptor, modulates estrogen receptor transactivation functions. The mechanisms by which PELP1 modulates estrogen receptor genomic functions is not known. Here, using biochemical and scanning confocal microscopic analysis, we have demonstrated nuclear localization and functional implications of PELP1. Subnuclear fractionation showed PELP1 association with chromatin and nuclear matrix fractions. Ligand stimulation promoted recruitment of PELP1 to 17ß-estradiol responsive promoters, its colocalization with acetylated H3, and increased PELP1-associated histone acetyltransferase enzymatic activity. Far Western analysis revealed that PELP1 interacts with histone 1 and 3, with more preference toward histone 1. Using deletion analysis, we have identified the PELP1 COOH-terminal region as the histone 1 binding site. The PELP1 mutant lacking histone 1-binding domain acts as a dominant-negative and blocks estrogen receptor {alpha}-mediated transcription. Chromatin immunoprecipitation analysis showed a cyclic association and dissociation of PELP1 with the promoter, with recruitment of histone 1 and PELP1 occurring in opposite phases. PELP1 overexpression increased the micrococcal nuclease sensitivity of estrogen response element-containing nucleosomes. Our results provide novel insights about the transcription regulation of PELP1 and suggest that PELP1 participates in chromatin remodeling activity via displacement of histone 1 in cancer cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breast cancer is the most frequent type of cancer among women. Steroid hormone 17ß-estradiol plays an important role in controlling the expression of genes involved in breast cancer progression (1 , 2) . The biological effects of estrogen are mediated by its binding to the structurally and functionally distinct estrogen receptors {alpha} and ß. Estrogen receptor is composed of a NH2-terminal AF1 domain, a DNA-binding domain, and a COOH-terminal ligand-binding region that contains an AF2 domain (3) . Upon the binding of 17ß-estradiol to estrogen receptor {alpha}, the ligand-activated estrogen receptor {alpha} translocates to the nucleus, binds to the responsive element in the target gene promoters, and stimulates gene transcription (genomic/nuclear signaling; refs. 4 , 5 ). Increasing evidence also suggests that estrogen receptor can also activate signaling at the membrane and cytosol via its interactions with other signaling kinases/adaptors (nongenomic/extranuclear signaling; refs. 6 , 7 ).

It is generally accepted that some of the diverse functions of estrogens depend on differential recruitment of coregulators to the ligand-bound estrogen receptor complex (8) . Coregulators that regulate the activity of estrogen receptor are thought to play a role in tumor progression (8, 9, 10) . Many breast tumors are hormone dependent, and 70% of primary breast tumors are estrogen receptor positive (8, 9, 10) . Very little is known about how these coregulators regulate the action of estrogen receptor and how these regulatory molecules control hormonal sensitivity. Recent work suggests that chromatin remodeling might constitute one possible mechanism (11) . Estrogen receptor transcriptional outcome is regulated by a dynamic interaction of histone acetyltransferases and histone deacetylases, which are generally associated with coactivators and corepressors, respectively (4 , 12 , 13) . Coactivators like steroid receptor coactivators (SRC1–3), and CBP/p300 have been shown to possess histone acetyltransferase activity (14 , 15) , whereas corepressors such as nuclear receptor corepressor and the metastasis-associated 1 protein are associated with histone deacetylases (14 , 16 , 17) . Accumulating evidence also suggests that nuclear receptors recruit multiprotein complexes that regulate higher-order chromatin domains into which nucleosomes are organized (18) . SW1/SNF, a complex that possesses ATPase activity, alters the nucleosomal structure and has been shown to be involved in the transcriptional regulation of nuclear receptors (19) .

In addition to nucleosomal constraints, histone 1, which binds to the DNA flanking the nucleosome core, is also implicated in the repression of transcription with a variety of DNA-binding activators, including nuclear receptors (20 , 21) . Accumulating evidence suggests that histone 1 in the promoter regions needs to be displaced by transcription factors to achieve productive transcription initiation (22) . Proteins that directly interact with histone 1, including prothymosin {alpha} (23) , nucleolin (24) , and high mobility group I (25) are thought to play a role in histone displacement (26) . Histone 1 was shown recently to be a potent repressor of estrogen receptor-mediated transcription by selectively inhibiting estrogen receptor-mediated transcriptional initiation (27) . Despite the identification of a number of estrogen receptor coregulators, very little is known about the mechanism by which estrogen receptor relieves histone 1 inhibition and whether estrogen receptor-associated novel regulators play a role in histone 1 displacement.

We recently cloned a novel estrogen receptor-regulatory protein named proline-, glutamic acid-, and leucine-rich protein-1 (PELP1; 28 ), also referred to as modulator of nongenomic activity of estrogen receptor (MNAR; ref. 29 ). PELP1 is abundantly expressed in a number of tissues including the mammary gland and the endometrium (28 , 30) . PELP1 contains 10 nuclear receptor interaction motifs (LXXLL) and a nuclear localization signal. PELP1 expression is up-regulated by estrogen (31) and functions as a coactivator of estrogen receptor {alpha} and estrogen receptor ß (28 , 29) . PELP1 plays a permissive role in 17ß-estradiol–mediated cell-cycle progression through its regulatory interactions with the retinoblastoma pathway (30) .

In the present study, we investigated the localization and the nuclear functions of PELP1 in human breast cancer cells. We found that PELP1 localizes in both cytosolic and nuclear compartments. In addition, endogenous PELP1 associates with active chromatin, nuclear matrix, and histone 1 and plays a mechanistic role in the enhanced ligand sensitivity of estrogen receptor by displacing histone 1 in breast cancer cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures and Reagents.
Breast cancer cell line MCF-7 and osteosarcoma cell line SAOS2 were obtained from the American Type Culture Collection (Manassas, VA). Dextran-coated, charcoal-treated fetal calf serum; antibodies against actin, vinculin, and 17-ß-estradiol; {alpha}-Amanitin, and a nuclei isolation kit (Nuclei EZ Prep) were purchased from Sigma Chemical Company (St. Louis, MO). Antibodies against paxillin were purchased from NeoMarkers (Fremont, CA). Antibodies against estrogen receptor {alpha}, histone 1, and acetylated histone 3 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The Lamin B antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The T7 monoclonal antibody was purchased from Novagen (Milwaukee, WI). Micrococcal nuclease was purchased from Roche Biochemical (Indianapolis, IN).

Plasmid Construction.
PELP1 wild-type and T7-tagged PELP1 constructs have been described previously (28) . For generation of PELP1 deletion constructs, indicated lengths of PELP1 cDNA were amplified by PCR, and the amplified products were cloned into either pcDNA 3.1 vector (Invitrogen, Carlsbad, CA) or pEBG vector (provided by Bruce Mayer, University of Connecticut Health Center, Farmington, CT).

Establishment of PELP1 Stable Cell Lines.
T7-PELP1 was subcloned into pcDNA4-TO vector and transfected into MCF-7 cells stably expressing pcDNA6-TR (Invitrogen). Stable clones were selected in the antibiotics blasticidin (for pcDNA-TR) and zeocin (for pcDNA4-TO-T7-PELP1). For generation of PELP1 mutant-expressing cells, MCF-7 cells were transfected with pcDNA3.1-PELP1H1 mutant, and pooled clones expressing PELP1-H1MT were selected in neomycin.

Cell Lysate Preparation, Northern Blotting, and Reporter Gene Assays.
Preparation of cell extracts, immunoblotting, immunoprecipitation, Northern blotting, and estrogen response element reporter gene assays were carried out as described previously (28) .

Chromatin Immunoprecipitation Analysis.
Approximately 106 cells were treated with 1% formaldehyde (final concentration, v/v) for 10 min at 37°C to cross-link histones to DNA. Chromatin immunoprecipitation was performed as described previously (32) .

Sequential Fractionation of the Nuclear Constituents.
Sequential fractionation of the nuclear constituents were carried out as described previously (33, 34, 35) .

Histone Binding and Histone Acetyltransferase Assays.
Total histones purified from MCF-7 or purified histone 1 and histone 3 (10 µg) purchased from Roche Diagnostics Corporation (Indianapolis, IN), were separated on a 20% SDS polyacrylamide gel and transferred to nitrocellulose, and the ability of PELP1 to bind to histones was analyzed using 35S-labeled PELP1 full-length or PELP1 mutants using the Far-Western analysis as described previously (36) . PELP1 wild-type or mutants were 35S-labeled using a transcription and translation kit (Promega). Washed blots were developed by autoradiography using phosphoimaging. For the histone acetyltransferase assay, MCF-7 cells expressing T7-PELP1 were either treated with estrogen (10–9 mol/L) or left untreated. Cells were lysed, and an equal amount of protein was immunoprecipitated with anti-T7 antibody. The immunoprecipitates were taken for histone acetyltransferase assay by the Histone Acetyltransferase-Check Activity Assay kit (Pierce Biotechnology Inc., Rockford, IL). Histone acetyltransferase assays with positive control in each assay was performed as per the manufacturer’s instructions.

Micrococcal Nuclease Digestion-Preparation of Mononucleosomes.
MCF-7 cells (5 x 105) were synchronized in G1/G2 phase by culturing them in DMEM/2% dextran-charcoal–treated fetal calf serum for 3 days. The cells were then treated with 2.5 µmol/L {alpha}-amanitin for 2 hours, followed by exposure to 10–8 mol/L 17ß-estradiol or EtOH for indicated time points, and micrococcal digestions were performed as described (37 , 38) .

Immunofluorescence and Confocal Studies of PELP1.
The cellular localization of PELP1 was determined by indirect immunofluorescence as described previously (32) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Localization of Endogenous PELP1.
To gain insight into the subcellular localization of PELP1, we have used a widely-used biochemical sequential extraction procedure (33, 34, 35) as described in Materials and Methods. MCF-7 cells were treated with 17ß-estradiol (10–9 mol/L) for 60 minutes, and cellular components were then sequentially extracted as soluble (NP40 buffer), cytoskeletal/nucleoplasm-associated (Triton X100), chromatin associated (DNase treated), and nuclear matrix-associated proteins (urea buffer). Localization of PELP1 was determined by Western blotting using an antibody that recognizes endogenous PELP1 (Fig. 1A)Citation . As seen in the gel, PELP1 was found tightly bound to different cellular compartments, and only a minor portion of PELP1 was seen soluble in NP40 buffer extraction. Significant amounts of PELP1 were extracted with Triton X100 buffer suggesting that some of the PELP1 is tightly associated with cytoskeleton and/or present in the nucleoplasm. In addition, a substantial amount of PELP1 was present in chromatin and nuclear matrix fractions. Estrogen receptor {alpha} was present in the cytoplasm/nucleoplasm, chromatin, and nuclear matrix fractions; however, the majority of the estrogen receptor was present in the chromatin fraction. These results suggest that PELP1 is localized in both cytoplasmic and nuclear compartments.



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  		Fig. 1. Cellular distribution of PELP1. A, MCF-7 cells treated with 17ß-estradiol (E2 10–9 mol/L) for 60 minutes were fractionated using a biochemical method as described in Materials and Methods. Cytosolic (NP 40), cytoskeletal/nucleoplasm (Triton X100), chromatin (DNAse1), high salt (2 mol/L NaCl), and nuclear matrix (Urea) fractions were analyzed by SDS-polyacrylamide gel followed by Western analysis using endogenous PELP1, estrogen receptor (ER), and CBP antibodies. Paxillin, poly(ADP-ribose) polymerase (PARP), and Lamin B1 were used as positive controls for cytosolic, chromatin, and nuclear matrix, respectively. B, Expression of T7-PELP1 was analyzed by treating PELP1-Teton model cells at various time points with 1 µg/ml of doxycycline, and PELP1 induction was analyzed by Western blotting using anti-T7 monoclonal antibody. The blot was reprobed with an antivinculin antibody as a loading control. C, Functionality of T7-PELP1 was analyzed by ERE-luc reporter gene assay. PELP-Teton cells were cultured in the presence or absence of doxycycline, transfected with 3x estrogen response element (ERE) reporter gene, and then treated with or without 17ß-estradiol (E2, 10–9 mol/L), and the reporter gene activity was measured after 24 hours. D, PELP1-Teton cells treated with 17ß-estradiol (E2 10–9 mol/L) were biochemically fractionated as described in A and Western blotted with antibodies that recognize T7 and estrogen receptor (ER). E, PELP1-Teton cells were cultured in the presence or absence of doxycycline, and the localization of PELP1 was analyzed by confocal microscopy using T7 monoclonal antibody; bars, ±SD.

 
Generation and Characterization of Epitope-Tagged MCF-7-PELP1 Model Cells.
Because estrogen receptor genomic functions are mediated in the nuclear compartment, we focused on understanding the role of PELP1 in estrogen receptor-mediated genomic transactivation functions. Because endogenous PELP1 antibody is not good for immunoprecipitation, we have generated a MCF-7 model system, which expresses T7-epitope–tagged PELP1 under the control of a Tetracycline-regulated promoter (PELP1-Teton cells). To minimize the clonal variation, we have generated a pooled clone expressing T7-epitope–tagged PELP1. Results show that tetracycline analogue doxycycline induces PELP1 expression in a time-dependent manner in PELP1-Teton cells (Fig. 1B)Citation . Functionality of the expressed protein was analyzed by estrogen response element luciferase assays (Fig. 1C)Citation . Doxycycline-mediated induction of PELP1 increased estrogen response element reporter activity in the presence of 17ß-estradiol compared with control uninduced PELP1-Teton cells treated with estrogen. Biochemical fractionation showed that T7-PELP1 is localized in cytoskeletal/nucleoplasm and nuclear compartments in a similar fashion as endogenous PELP1 (Fig. 1D)Citation . Confocal analysis using T7-epitope tag also showed that PELP1 is predominantly nuclear in localization; however, some diffused cytoplasm staining was also observed (Fig. 1E)Citation . In the nucleus PELP1 was present in punctuate/speckle like distributions. These results suggest that the T7-tagged PELP1 behaves similarly to endogenous PELP1, and a substantial amount of PELP1 localizes in the nuclear compartment.

PELP1 Is Recruited to 17ß-Estradiol–Responsive Promoters.
We then tested whether PELP1 is recruited to the proximal regions of estrogen response elements of 17ß-estradiol–responsive genes in vivo using the chromatin immunoprecipitation assay. Stable cells expressing Teton vector or PELP1-Teton were treated with 17ß-estradiol (10–9 mol/L) for 60 minutes, and T7-PELP1–bound chromatin was immunoprecipitated, eluted, and PCR amplified using primers that are specific to the estrogen receptor-responsive gene promoters such as trefoil factor 1 precursor (pS2), progesterone receptor, and insulin-like growth factor. Results showed increased recruitment of PELP1 to pS2, progesterone receptor, and insulin-like growth factor promoters (Fig. 2A)Citation . We then examined the kinetics of PELP1 recruitment to the pS2 promoter (Fig. 2B)Citation . The results show that PELP1 is specifically recruited to the pS2 promoter in a dynamic manner upon 17ß-estradiol stimulation. Increased recruitment of PELP1 was observed at 60 minutes of 17ß-estradiol treatment at the pS2 promoter; however, some basal association of PELP1 was observed when 17ß-estradiol was absent. Continuation of treatment for 3 hours resulted in complete loss of PELP1 from the pS2 promoter. Furthermore, treatment with Trichostatin A, an inhibitor of histone deacetylase, substantially increased the association of PELP1 with the pS2 promoter (Fig. 2B)Citation . These results suggest that PELP1 is recruited to the 17ß-estradiol–responsive promoters in a dynamic manner and that histone deacetylase complexes may have a role in PELP1 detainment on the pS2 promoter. These results suggest that 17ß-estradiol promotes PELP1 recruitment to 17ß-estradiol–responsive promoters.



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  		Fig. 2. PELP1 is recruited to 17ß-estradiol–responsive promoter regions. A, Teton-vector or PELP1-Teton cells were treated with or without 17ß-estradiol for 60 minutes, immunoprecipitated with anti-T7 monoclonal antibody to precipitate T7-PELP1, and chromatin immunoprecipitation analysis was performed using primers specific for pS2, insulin-like growth factor, progesterone receptor, and or actin promoters. As a positive control, chromatin immunoprecipitation analysis was done using estrogen receptor (ER) antibody, and as a negative control chromatin immunoprecipitation analysis was performed using control IgG, followed by PCR amplification using pS2 primers. B, PELP1-Teton cells were treated with 17ß-estradiol (E2, 10–9 mol/L) for indicated periods of time. When indicated, cells were pretreated with histone deacetylase inhibitor trichostatin A. T7 monoclonal antibody immunoprecipitated DNA was subjected to PCR analysis using primers specific for pS2 regions containing estrogen responsive element. C, PELP1-Teton cells grown on coverslips were treated with 17ß-estradiol (10–9 mol/L) for 60 minutes and fixed in methanol. Cells were double labeled with T7-PELP1 (green) and acetylated histone 3 (red). Cellular DNA was labeled with Topro3 (blue). Colocalization of PELP1 with acetylated histone 3 (H3) was analyzed by confocal microscopy. D, PELP1-Teton cells were treated with or with out 17ß-estradiol (E2), subjected to chromatin immunoprecipitation using acetylated histone 3 antibody. The immunoprecipitates were eluted using 0.1 mol/L DDT solutions and then subjected to secondary chromatin immunoprecipitation using T7 monoclonal antibody to precipitate T7-PELP1–associated chromatin. The elute from the second chromatin immunoprecipitation was then subjected to PCR using primers specific to pS2 and progesterone receptor regions, which contain estrogen response element. E, PELP1 associates with histone acetyltransferase activity. MCF-7 cells expressing T7-PELP1 or pcDNA were treated with or without 17ß-estradiol (E2, 10–9 mol/L) for 3 hours. Cell lysates were immunoprecipitated with antibodies against T7, and the associated histone acetyltransferase activity was determined as described in Materials and Method (n = 2). +ve control represents the activity of the positive control provided in the kit; bars, ±SD.

 
PELP1 Colocalizes with Active Chromatin.
We then examined whether PELP1 associates with active chromatin. PELP1-Teton cells were cultured in doxycycline-containing media for 2 days and then treated with 17ß-estradiol (10–9 mol/L) for 60 minutes. Confocal analysis using acetylated histone 3 antibody and T7 epitope antibody showed some areas of colocalization of PELP1 with acetylated histone 3 in the nuclear compartment (Fig. 2CCitation , white). To additionally confirm PELP1 association with active chromatin, chromatin was isolated and immunoprecipitated with an antibody that specifically recognizes acetylated histone 3. The immunoprecipitated chromatin was eluted and reimmunoprecipitated with T7 monoclonal antibody. Results showed that PELP1 is recruited to the active chromatin region of 17ß-estradiol responsive genes pS2 and progesterone receptor (Fig. 2D)Citation . Because PELP1 promoted transcription from the estrogen response element containing promoter and interacted effectively with the estrogen receptor target gene chromatin (Fig. 2, A and B)Citation , this raises the possibility that PELP1 influences the status of chromatin remodeling, presumably through histone acetyltransferase activity. Therefore, we next explored this possibility and examined whether such activity was either intrinsic or associated with T7-PELP1. Immunoprecipitation showed the presence of increased associated functional histone acetyltransferase activity with the PELP1 (Fig. 2E)Citation .

PELP1 Is a Histone Binding Protein.
Analysis of the primary structure of PELP1 suggests that it has an 80 amino acid glutamic acid-rich region, which has some homology with the putative histone-binding proteins including prothymosin {alpha}, nucleolin, and high mobility group I (Fig. 3A)Citation . Therefore, we examined whether PELP1 interacts with histones using Far Western method (36) . Native histones were purified from MCF-7 cells and resolved on a 20% SDS-polyacrylamide gel along with the purified histone 3 or histone 1 histones. 35S-labeled PELP1 was generated using an in vitro (transcription and translation) system as a probe. The PELP1 interacting bands were identified by autoradiography. Autoradiogram showed two positive bands with molecular weights corresponding to histone 1 and histone 3, respectively, in the total histone lane (Fig. 3B)Citation . Furthermore, purified histone 1 showed higher binding of PELP1 compared with purified histone 3. To confirm the interactions of PELP1 with histone 1 in vivo, we performed immunoprecipitation using nuclear lysates from PELP1-Teton cells. Immunoprecipitation of histone 1 showed the presence of PELP1, confirming that PELP1 interacts with histone 1 in vivo (Fig. 3C)Citation .



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  		Fig. 3. PELP1 interacts with histone. A, alignment of PELP1 glutamic acid-rich domain with known histone 1 binding proteins. B, PELP1 interacts with histones in far Western analysis. Total histones (isolated from MCF-7 cells), as well as purified histone 1 and histone 3 (10 µg each), were run on a SDS-PAGE, transferred to a nitrocellulose membrane, and probed with 35S-labeled PELP1. C, total cellular extracts from PELP1-Teton clones were immunoprecipitated with histone 1 (H1) antibody. Immunoprecipitates were Western blotted with antibodies that recognize histone H1 and T7-PELP1.

 
PELP1 COOH-Terminal Region Contains Histone-Binding Domain.
We then identified the histone-binding domain in PELP1 using a series of constructs that span the entire coding region of PELP1. Far Western analysis of 35S-labeled constructs revealed that COOH-terminal amino acid 246 is sufficient to interact with histone 1 and histone 3; however, affinity to histone 1 appears to be higher than histone 3. Additional deletion of the proline-rich region amino acid 800 to amino acid 884 resulted in the decreased affinity of PELP1 to histone 1; however, weak binding of these regions to histone 1 and histone 3 was observed. Deletion of COOH-terminal amino acid 884 to amino acid 960 that contained glutamic acid-rich region completely abolished the ability of PELP1 to interact with histones (Fig. 4A)Citation . These results suggest that the PELP1 glutamic acid region can interact with both histone 1 and histone 3, and the proximal proline-rich region contributes more to the affinity of PELP1 to histone 1.



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  		Fig. 4. The histone-binding domain is localized in the COOH-terminus of PELP1. A, PELP1 peptides of indicated lengths were in vitro translated using the TNT system (left), and their ability to bind histone 1 (H1) and histone 3 (H3) was analyzed by far Western analysis (right). B, PELP1 peptides of indicated lengths were expressed in MCF-7 cells as glutathione S-transferase fusion proteins using pEBG vector, and the ability of these peptides to activate 17ß-estradiol (E2)-mediated estrogen responsive element (ERE)-luc reporter activity was measured. C, schematic representation of PELP1 deletion constructs used in the assay; bars, ±SD.

 
We then examined whether the histone-binding region of PELP1 plays a role in estrogen receptor coactivation function. MCF-7 cells were cotransfected with the estrogen response element reporter gene alone with indicated regions of PELP1 expressed as glutathione S-transferase fusions. Results showed that PELP1 COOH-terminal region amino acids 800 to 1130 but not amino acids 960 to 1130 can positively augment estrogen receptor transactivation functions (Fig. 4, B and C)Citation . Furthermore, overexpression of NH2-terminal region, which contains LXXLL motifs and estrogen receptor-binding domain, repressed estrogen receptor transactivation functions in a dominant-negative manner (Fig. 4B)Citation . These results suggest that PELP1 COOH-terminal histone binding region plays an important role in PELP1-mediated transactivation functions.

PELP1 Lacking Glutamic Acid-Rich Region Functions as a Dominant-Negative Mutant.
Because the PELP1 COOH-terminal region interacted with histones, we hypothesized that the COOH-terminal region of PELP1-containing glutamic acid region plays an important role in PELP1 genomic functions. To test this possibility, we generated a PELP1 construct that lacks COOH-terminal 253 amino acids and that includes the histone-binding region (PELP1-H1MT). We tested this construct using transient estrogen response element luciferase reporter assay in MCF-7 cells (Fig. 5A)Citation . The results show that the PELP1-H1MT is effective in blocking 17ß-estradiol–mediated increase in luciferase activity. Results show that this mutant is a potent inhibitor of 17ß-estradiol-mediated transcription not only in breast cancer cells but also in endometrial Ishikawa and osteosarcoma SA0S2 cell lines.



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  		Fig. 5. PELP1 mutant lacking COOH-terminal histone-binding domain interferes with 17ß-estradiol (E2)-mediated transactivation functions. A, Breast cancer cell line MCF-7, endometrial cell line Ishikawa, and osteosarcoma SA0S2 cells were transiently transfected with the estrogen responsive element (ERE) reporter gene ER along with or without PELP1-H1MT. After 24 hours, cells were treated with E2 (10–9 mol/L), and luciferase activity was measured after 24 hours. B, expression of T7-PELP1 and T7-PELP1H1MT in MCF-7 pooled clones analyzed by Western blotting. C, pcDNA or PELP1-H1MT stably expressing cells were transfected with ERE-luc reporter gene, and treated with E2 (10–9 mol/L), and reporter activity was measured after 24 hours. D, pcDNA, PELP1-WT, and PELP-H1MT-expressing stable clones were treated with E2 (10–9 mol/L) for 8 hours, and the induction of E2 responsive gene pS2 was analyzed by Northern analysis. E, MCF-7 pooled clones stably expressing PELP-H1MT were treated with E2 for indicated periods of time, and recruitment of T7-PELP1-H1MT and ER{alpha} to the pS2 promoter was analyzed by chromatin immunoprecipitation analysis; bars, ±SD.

 
We then generated a pooled cell line that stably expresses T7-PELP1-H1MT. Stable cells expressed PELP1-H1MT at a level similar to wild-type T7-PELP1 (Fig. 5B)Citation . Estrogen response element reporter gene assays showed reduced estrogen response element reporter gene activity in the PELP1-H1MT mutant cells compared with the vector transfected cells (Fig. 5C)Citation . We then analyzed the expression of endogenous 17ß-estradiol–responsive gene pS2 using Northern analysis. PELP1-H1MT cells showed reduced induction (1.5-fold) compared with wild-type (6.0-fold) and vector-expressing control cells (3.0-fold). To check the possibility of whether the lack of 17ß-estradiol response in PELP1-H1MT is not caused by sequestration of estrogen receptor in the cytoplasm or due to the inability of PELP1-H1MT to localize to the nuclear compartment, we analyzed the recruitment of PELP1-H1MT and estrogen receptor to the pS2 promoter region using chromatin immunoprecipitation analysis. The results showed that both PELP1-H1MT and estrogen receptor were recruited efficiently to the pS2 promoter (Fig. 5E)Citation , indicating that the observed inhibitory effect of PELP1-H1MT is not due to estrogen receptor mislocalization.

PELP1 Modulates the Chromatin Structure.
To examine whether PELP1 alters the nucleosomal structure at the estrogen receptor target gene promoters, we performed micrococcal nuclease digestion. We limited our analysis to a single nucleosome, surrounding the area of estrogen response element present in the pS2 promoter as described by Sewack and Hansen (37) and used a PCR amplification procedure described previously (38) . In the absence of 17ß-estradiol, nucE, which encodes the complete nucleosome regions of the estrogen response element in the pS2 promoter, will be protected from micrococcal nuclease digestion; therefore, PCR amplifies this region. Alterations in the structure of the nucleosome expose this area to micrococcal nuclease digestion; therefore, these areas will not be amplified. For this experiment, pcDNA or PELP1-WT cells were synchronized by {alpha}-amanitin and then treated with 17ß-estradiol for various periods of time. DNA was isolated and treated with micrococcal nuclease, and the resulting chromatin fragments were amplified by PCR using primers specific to nucE nucleosomal regions. The results show that in pcDNA-expressing cells, 17ß-estradiol treatment decreased the amplification of nucE in a time-dependent manner, suggesting alterations in the nucleosome structure at the estrogen response element. After 80 minutes, the PCR band reappeared suggesting the completion of one productive transcriptional cycle and regaining of the compact nucleosomal structure (38) . In cells that overexpress PELP1, 17ß-estradiol treatment decreased the amplification of nucE in a similar manner as pcDNA-expressing cells; however, the decrease in amplification of nucE also occurred at 80 minutes of 17ß-estradiol treatment. These results suggest that PELP1 overexpression maintained the nucleosomal alterations for an extended period of time and that PELP1-mediated interactions with histone 1 may help PELP1 coactivation functions by allowing relaxation of estrogen response element-containing nucleosome.

Because PELP1-H1MT is recruited to the pS2 promoter but inhibits ligand-induced reporter gene activity, we next examined whether PELP1 has any effect on the status of histone 1 in the pS2 promoter, which is known to act as a repressor of estrogen receptor-mediated transcription initiation (27) . To test this, we used PELP1-WT and PELP1-H1MT–expressing cells, and the status of PELP1 and histone 1 in the pS2 promoter was analyzed by chromatin immunoprecipitation analysis. Results showed that PELP1-WT and PELP1-H1MT were recruited to the pS2 promoter in a similar fashion. However, during the period when PELP1-WT was recruited to the pS2 promoter, the occupancy of histone 1 in this region was decreased. However, no change in the status of histone 1 was observed in the PELP1-H1MT–expressing clones (Fig. 6B)Citation . These results suggest the possibility that PELP1 recruitment to the 17ß-estradiol target promoters may locally affect the status of histone 1, and the interaction of PELP1 with histone 1 may lead to displacement of histone 1 to maintain a locally active chromatin conformation so that transcriptional regulators can access 17ß-estradiol regulatory elements (Fig. 6C)Citation .



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  		Fig. 6. PELP1 recruitment alters the status of histone 1 (H1) at the pS2 promoter. A, Mononucleosomes were prepared from PELP1-WT and PELP-H1MT stable clones synchronized by {alpha}-amanitin treatment and treated with 17ß-estradiol (E2) for 60 minutes. Chromatin was subjected to micrococcal nuclease, and nucleosome-protected fragments were analyzed by PCR using primers spanning pS2 estrogen responsive element (ERE) nucleosome (NucE; PCR product a) and primers spanning pS2ERE-pS2 TATA nucleosomes (PCR product b). Schematic representation of nucleosomes around ERE and TATA boxes of pS2 promoter as identified by Sewack and Hansen (37) is shown at right. Lack of PCR product b indicates that micrococcal digestion has occurred. B, MCF-7 cells stably expressing PELP1-WT and PELP1-H1MT were treated with or without E2 (10–9 mol/L) for 60 minutes. Chromatin immunoprecipitation analysis was performed using antibodies against T7 (top) and H1 (bottom). C, schematic representation showing a possible model in which PELP1 promotes chromatin remodeling by displacing H1.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PELP1/MNAR is a novel estrogen receptor coregulatory protein that positively modulates estrogen receptor transcriptional functions. Some evidence suggests that PELP1 participates in estrogen receptor nongenomic signaling through the activation of the Src-mitogen-activated protein kinase pathway (29 , 39) . In this study, we examined whether PELP1 has any role in the estrogen receptor genomic functions. Using biochemical and confocal microscopy, we have established that substantial amounts of PELP1 localize to the nuclear compartment. Ligand stimulation promotes PELP1 localization to the estrogen receptor target gene promoters, and in some areas, colocalization of PELP1 with active chromatin and estrogen receptor was also observed. Collectively, these results suggest that PELP1 has nuclear functions, and such functions may have a role in estrogen receptor transactivation functions.

Cellular, molecular, biochemical, and in vivo genetic evidence support an obligatory relationship between nuclear microenvironments and fidelity of transcriptional control (40) . Several studies have shown that transcriptionally active DNA is tightly associated with the nuclear matrix (41) . The nuclear matrix is defined as the nuclear structure that remains after salt extraction of nuclease-treated chromatin (42) . A number of steroid receptors including estrogen receptor has been shown to be associated with nuclear matrix, and several studies have shown that actively transcribed genes are associated with the nuclear matrix (43 , 44) . Similarly, nuclear receptor-associated coregulatory proteins, including SW1/SNF and SRC, were reported to be enriched in active chromatin and are associated with the nuclear matrix (43 , 44) . In this study, we observed that PELP1 adopts a punctate subnuclear localization pattern in MCF-7 cells suggesting that PELP1 may be associated with structural components within the nucleus. Using endogenous as well as epitope-tagged PELP1, we have demonstrated that PELP1 localizes to the chromatin and nuclear matrix. PELP1 interaction with estrogen receptor and its tight association with the nuclear matrix strongly suggest that PELP1 may have a role in estrogen receptor genomic functions. Earlier studies suggest that PELP1 plays a permissive role in 17ß-estradiol–mediated cell cycle progression, presumably via its regulatory interaction with retinoblastoma protein (30) . The punctate subnuclear localization and nuclear matrix association of PELP1 might represent focal concentrations of the regulatory machinery for estrogen receptor-responsive integration of regulatory signals like growth and proliferation in breast cancer cells. It is also possible that PELP1 might function as a landing platform for several other chromatin remodeling complexes and thereby regulate gene expression.

Histone acetylation and deacetylation have been suggested to be mechanisms by which nuclear receptor coactivators modify chromatin structure. Several nuclear receptor coactivators, including SRC family members CBP, p300, and PCAF, have been shown to possess histone acetyltransferase activity (12 , 13) . Chromatin immunoprecipitation analysis suggested that PELP1 is recruited to the promoters of estrogen receptor target genes and colocalizes with acetylated histones. Immunoprecipitation and histone acetyltransferase activity assay showed increased associated histone acetyltransferase activity with PELP1 upon ligand treatment. Because PELP1 has no intrinsic histone acetyltransferase domain, its recruitment to the estrogen receptor target genes may also promote increased acetylation through its interactions with CBP and p300, which are known to interact with PELP1 (28) . Earlier studies have showed that PELP1 interacts with estrogen receptor via NH2-terminal region, which contains LXXLL motifs (28) . The ability of the PELP1 COOH-terminal region alone to promote estrogen receptor transcriptional activation also suggests a possibility that this region can associate and promote estrogen receptor-dependent transactivation functions via indirect interactions. Because PELP1 interacts with CBP and p300, it is possible that when COOH-terminal region is overexpressed, it might be recruited to the estrogen receptor target promoter region via its potential interactions with CBP/p300.

Acidic regions have been identified previously in a number of nuclear proteins, such as high mobility-group chromatin proteins-1 and -2, nucleoplasmin, prothymosin {alpha}, and nucleolin (23 , 45, 46, 47, 48, 49, 50) . Experimental in vitro and in vivo evidence suggests a role of glutamic acid-rich regions in modifying the chromatin structure via electrostatic interactions between acidic domains and histone 1 and, to some extent, with the core histones (45 , 50) . The presence of the 80 amino acid glutamic acid-rich region in PELP1, nuclear localization of PELP1, and its ability to interact with histone 1 suggest that PELP1-mediated estrogen receptor genomic functions involve PELP1–histone 1 interactions. The blockage of estrogen receptor-mediated transcriptional activation by the PELP1 mutant, which lacks histone 1 binding region, also supports this hypothesis.

The linker histone 1 is believed to be involved in chromatin organization by stabilizing higher-order chromatin structure by restricting the translational mobility of nucleosomes (50) . Histone 1 is generally viewed as a repressor of transcription, because it prevents the access of transcription factors and chromatin remodeling complexes to DNA (21) . Histone 1 is also shown as a potent repressor of ligand- and coactivator-regulated transcription by estrogen receptor {alpha} (27) . Our results suggest that during the time when PELP1 occupies the target gene promoter, less histone 1 residence was observed. This raises the possibility that PELP1 facilitates the histone 1 displacement at the estrogen receptor target promoter. In addition, alterations in the nucleosome position in PELP1-overexpressing cells (Fig. 6B)Citation also suggest that PELP1, due to its glutamic acid-rich domain may alter chromatin structure, probably by interacting with histone 1. Furthermore, PELP1 ability to associate with histone acetyltransferase enzymes could also indirectly contribute to the alteration of the local chromatin structure.

In summary, our findings suggest that PELP1 is distributed in different compartments including cytoplasm, active chromatin, and nuclear matrix. PELP1 interacts with histone 1, and such interactions play an essential role in estrogen receptor-mediated genomic functions by facilitating remodeling of estrogen receptor-bound nucleosomes.


   FOOTNOTES
 
Grant support: NIH Grant CA 095681 (R. Vadlamudi) and NIH Grants CA 90970 and CA 98823 (R. Kumar).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Ratna Vadlamudi or Rakesh Kumar, Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009. E-mail: rvadlamu{at}mdanderson.org or rkumar{at}mdanderson.org

Received 5/21/04. Revised 7/15/04. Accepted 7/26/04.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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