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Molecular Endocrinology 21 (7): 1617-1629
Copyright © 2007 by The Endocrine Society

Estrogen-Enhanced Peptidylarginine Deiminase Type IV Gene (PADI4) Expression in MCF-7 Cells Is Mediated by Estrogen Receptor-{alpha}-Promoted Transfactors Activator Protein-1, Nuclear Factor-Y, and Sp1

Sijun Dong, Zilian Zhang and Hidenari Takahara

Department of Applied Biological Resource Sciences (S.D., H.T.), School of Agriculture, Ibaraki University, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan; and Research Institute for Cell Engineering (Z.Z.), National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan

Address all correspondence and requests for reprints to: Hidenari Takahara, Ph.D., Department of Applied Biological Resource Sciences, School of Agriculture, Ibaraki University, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan. E-mail: takahara{at}mx.ibaraki.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human peptidylarginine deiminase type IV (PAD4), a Ca2+-dependent enzyme known to convert arginine residues to citrulline residues in histones, has been shown to be associated with the development of rheumatoid arthritis. Recently, it was noted that the human peptidylarginine deiminase type IV gene (PADI4) regulates the expression of estrogen-responsive genes by modifying the methylated arginine sites in histones H3 and H4. In this study, we demonstrated that PADI4 was expressed in MCF-7 cells and was responsive to estrogen at the transcriptional level. Using the luciferase reporter gene fused to wild-type or mutated 5'-flanking region of PADI4, we characterized that as few as 348 bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Chromatin immunoprecipitation and small interfering RNA assays revealed that activator protein–1, Sp1/Sp3, and nuclear factor–Y were cis-acting factors bound to the minimal promoter of PADI4 and that they regulated gene expression in a cooperative manner. Moreover, it was indicated that estrogen stimulated PADI4 expression through binding of estrogen receptor (ER)-{alpha} to the upstream of the PADI4 gene and ER{alpha}-mediated enhancement of activator protein-1, Sp1, and nuclear factor-Y levels. These findings indicated that estrogen stimulated PADI4 expression through both of the classical and nonclassical ER-mediated pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PEPTIDYLARGININE DEIMINASE (PAD; EC 3.5.3.15) is an enzyme involved in the posttranslational deimination of protein-bound arginine to citrulline (1, 2). Five different types of human PADs encoded by the genes PADI1–PADI4 and PADI6 are described (3, 4). Among the human PAD genes, PAD type IV gene (PADI4) is expressed in bloodstream granulocytes and is localized in the cell nucleus via a nuclear localization signal (5, 6). PADI4 is also expressed in CD34 (+) cells of bone marrow and is speculated to be associated with abnormal proliferation of CD34 (+) stem cells (7). Furthermore, overexpression of PADI4 induces apoptosis mainly through cell cycle arrest in G1 phase and a mitochondria-mediated pathway, suggesting an important role for PADI4 in immune cell differentiation and cell death (8).

The presence of citrulline-modified target epitopes for autoantibody production is a well-known phenomenon in rheumatoid arthritis (RA) (9, 10). PADIs were recently implicated in the generation of anticyclic citrullinated peptide antibodies detectable in early stages of the disease. This process resulting in anticyclic citrullinated peptide formation is thought to play a pivotal role in early stages of RA evolvement (11, 12). Replication studies from the North American RA Consortium cohort strongly support an association of PADI4 with the development of RA (13), whereas variability of the PADI4 gene may influence susceptibility to RA in the German population (14). PADI4 has at least five main haplotypes, which differ by four exonic single nucleotide polymorphisms, and three of them involved amino acid substitutions (15, 16). Whereas the so-called susceptibility haplotypes 2, 3, and 4 were found to be significantly more frequent in Japanese individuals suffering from RA, the nonsusceptibility haplotype 1 predominated in healthy individuals (15). These reports indicated that PADI4 was actually responsible for the association with RA.

Estrogen seems to play an important role as a modulator and perpetuator of rheumatic disorders with autoimmune involvement, such as RA or systemic lupus erythematosus, because of the significant excess of affected females (17). Indeed, the cells in immune system, such as human CD8 (+)-T cells, B cells, monocytes, and synovial macrophages, have been found to possess functional sex hormone receptors (17). PADI4 is recruited to an estrogen-responsive gene, pS2, in MCF-7 cells coincident with the subsequent appearance of citrullinated histones and the disengagement of RNA polymerase II (18, 19). Multiple arginine sites in histones H3 (Arg2, Arg8, Arg17, and Arg26) and H4 (Arg3), including those sites methylated by coactivator-associated arginine methyltransferase 1 (H3/Arg17) and protein arginine methyltransferase 1 (H4/Arg3), are targets of PADI4 (18, 19, 20, 21). Furthermore, transcription of estrogen-regulated genes is activated by dimethylations of H3/Arg17 and H4/Arg3, but suppressed by citrullination (18, 19). In addition to these histone modifications, Arg2142 of coactivator p300 is methylated by coactivator-associated arginine methyltransferase 1 and citrullinated by PADI4 (22). These modifications result in a significant change of the assembly structure of the coactivator complex (22). Thus, Ca2+-dependent deimination of histone arginine residues by PADI4 has come into focus as a novel posttranslational modification linked to transcriptional regulation in eukaryotes (18, 19).

The tissue-specific expression and Ca2+-induced activation of PADI4 have been reported (5, 6, 23). The transcription and estrogen-induced activation mechanisms of PADI4 are still unclear, although the three-dimensional structure, enzyme activity, and tissue and cellular localization of PADI4 (5, 6, 23, 24) have been studied so far. Here we report that PADI4 is expressed in MCF-7 cells and is responsive in an estrogen receptor (ER){alpha}-dependent manner to 17ß-estradiol (E2) treatment. We cloned the 5'-flanking region of the PADI4 gene and identified the minimal promoter sequence required to efficiently drive the transcription of PADI4 in MCF-7 cells. We also report that activator protein-1 (AP-1), nuclear factor-Y (NF-Y), and Sp1/Sp3 bind to the proximal promoter to regulate the expression of PADI4, which is E2 responsive through ER{alpha}-mediated binding activities.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PADI4 Expression in Cultured Human MCF-7 Cells
We initially investigated the regulation of PADI4 expression in human MCF-7 cells using real-time PCR and Western blotting analyses. PAD4 mRNA expression was low in unstimulated cells and gradually increased with time after stimulation with 10 nM E2 (Fig. 1AGo). PAD4 protein levels mimicked mRNA expression (Fig. 1BGo). Furthermore, ICI 182,780 (ICI), a specific antagonist to ER{alpha} (25), completely abrogated the E2-induced PADI4 expression both at the mRNA and protein level (Fig. 1Go, A and B, last lanes). These findings suggest that E2 stimulates expression of PADI4 via ER{alpha}.


Figure 1
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  		Fig. 1. Estrogen Induces PADI4 Expression at Both the mRNA and Protein Levels

MCF-7 cells were exposed to 10 nM E2 without or with 1 µM ICI and incubated for the indicated length of time. A, Total RNA was isolated and analyzed by real-time RT-PCR. Data were normalized to the GAPDH gene. Results are means ± SD for four separate experiments. **, P < 0.01 (Student’s t test). B, Protein was extracted with sodium dodecyl sulfate buffer and analyzed by Western blotting for PAD4 with the monoclonal antibody (D7h35). Western blotting for actin was also carried out for input protein control.

 
Identification of the Minimal Promoter and Potential cis-Elements Involved in PADI4 Transcription
To determine which sequences were sufficient to direct transcription of PADI4, a 2004-bp fragment (at nucleotides –1977 to +27) containing the proximal 5'-flanking region, the transcription start site, and a portion of exon 1 was cloned and fused to the upstream of a promoterless luciferase reporter gene. In addition, progressive 5'-deletions in the putative promoter region were also examined for their effect on reporter gene activity. The resulting constructs were transiently transfected into MCF-7 cells, and luciferase activities were measured after 48 h. Significant transcriptional activity was observed with three constructs: pG4–1977/+27, pG4–661/+27, and pG4–348/+27 (Fig. 2AGo). However, further truncations of the regions at nucleotides –348 to –315, –199 to –82, and –82 to –39 (constructs pG4–315/+27, pG4–82/+27 and pG4–39/+27, respectively) gradually decreased the transcriptional activity, indicating that nucleotide –348 to +27 contained positive regulatory elements that were essential for basal promoter function. The sequence search with the GenomeNet MOTIF program (Motif library: TRANSFAC; Classification: vertebrates; Cut off score: 85, Kyoto University Bioinformatics Center, Kyoto, Japan) revealed several predicted cis-elements for binding of the transcription factors NF-Y, MZF1, Sp1, and AP-1 as well as a TATA-box (Fig. 2BGo). The presence of multiple putative transcription factor-binding sites near the transcription initiation site suggests that nucleotides –348 to +27 functions as a minimal promoter of PADI4.


Figure 2
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  		Fig. 2. Identification and Characterization of the Minimal Promoter Region of the PADI4 Gene

A, Identification of the minimal promoter of PADI4. MCF-7 cells were transfected with the indicated constructs and assayed for luciferase activity after 48 h. The numbers given to the constructs indicate the 5'- and 3'-ends of the 5'-flanking region of PADI4, position numbered +1 corresponding to the first base of the transcription initiation nucleotide. The transcription initiation site was predicted by cDNA sequence (GenBank accession no. AB017919). Luciferase activity is expressed as fold increase over promoterless vector, pGBasic (set as 1). Values were normalized for transfection efficiency by cotransfection with the Renilla expression plasmid and are expressed as means ± SD for four separate experiments. *, P < 0.05; **, P < 0.01 (Student’s t test). B, Sequence and putative transcription factor-binding sites of the minimal promoter of PADI4. Numbering of the nucleotides begins with the transcription initiation nucleotide as +1. Potential transcription factor-binding sites predicted by the GenomeNet MOTIF program (motif library: TRANSFAC; classification: vertebrates; cut-off score: 85, Kyoto University Bioinformatics Center, Kyoto, Japan) are underlined. C, Characterization of the transcription factor-binding sites in the PADI4 promoter. The numbers given to the constructs indicate the 5'- and 3'-ends of the PADI4 promoter region. The putative transcription factor-binding sites, which are targeted, are shown on the constructs. Site-directed mutagenesis was carried out with the construct spanning the –348 to +27 region. The relative positions of each mutation, designated as a cross, are shown. See text for additional details. Values were corrected for transfection efficiency by cotransfection with the Renilla expression plasmid and are expressed as means ± SD for four separate experiments. **, P < 0.01 vs. the difference between luciferase activity in pG4–348/+27 and mutant (Student’s t test).

 
To further determine potential cis-elements of the minimal promoter involved in the transcriptional control of PADI4 expression, we constructed a new series of motif mutants. They were transiently transfected into MCF-7 cells in addition to the parental construct pG4–348/+27. As shown in Fig. 2CGo, luciferase activities were significantly reduced by mutation of the NF-Y-, Sp1-, or AP-1-binding site compared with that of the original plasmid. Mutation of the TATA-box completely abolished PADI4 promoter activity (Fig. 2CGo). However, no significant difference was observed after mutation of the MZF1-binding site. These results strongly suggest that the NF-Y-, AP-1-, and Sp1-binding sites and the TATA-box found between nucleotides –348 to +27 are cooperative cis-elements critical for PADI4 promoter activity.

Binding of Transfactors to the PADI4 Minimal Promoter
To test whether the predicted transfactors actually bind to the PADI4 promoter in vivo, we performed a chromatin immunoprecipitation (ChIP) assay using specific antibodies, specific primers for the PADI4 promoter region, and formaldehyde-fixed chromatin isolated from cultured cells. As expected, AP-1, Sp1, and NF-YA (A subunit of NF-Y) bound to the 5'-flanking region of PADI4, whereas no binding of MZF1 to the cis-element was observed (Fig. 3AGo, panel 1). Sp3, which is part of the Sp1 family, is coexpressed in several tissues/cell types and recognizes the same DNA element (26). Therefore, we also performed the ChIP assay with Sp3-specific antibody and confirmed the binding of Sp3 to the proximal promoter region of PADI4 (Fig. 3AGo, panel 1). When PCR was performed on the chromatin samples immunoprecipitated with nonimmune IgG using the same primers, no PCR signal was observed, showing the specificity of binding. The input levels of chromatin for each cell line were shown by using nonimmunoprecipitated DNA as templates for PCR. Further specificity of the ChIP analysis was demonstrated by the inability to detect binding of AP-1, Sp1/Sp3, or NF-Y to the first intron region of PADI4 gene using the primers to amplify a segment from the first intron (Fig. 3AGo, panel 2). To quantify E2-induced effects of transcription factor binding, we carried out quantitative PCR with the immunoprecipitated chromatin. As shown in Fig. 3BGo, higher levels of AP-1, Sp1, and NF-Y recruitment were observed with E2 treatments than without. Interestingly, Sp3 binding decreased when cells were cultured with 10 nM E2, as compared with no E2 addition. However, in MCF-7 cells no MZF1 binding to the PADI4 promoter was observed under either condition (Fig. 3Go, A and B). Further analyses using the total cell lysates for Western blotting indicate that MZF1 was not expressed in MCF-7 cells when treated with or without E2 (Fig. 3CGo). These findings suggest that the association of AP-1, NF-Y, and Sp1/Sp3 on the promoter region of PADI4 may play a prominent role in the transcription of the gene.


Figure 3
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  		Fig. 3. AP-1, Sp1, Sp3, and NF-Y Binding to the PADI4 Promoter in Vivo

ChIP assays using anti-AP-1, anti-Sp1, anti-Sp3, anti-MZF1, anti-NF-YA antibodies, or IgG were performed using chromatin from MCF-7 cells cultured in medium with or without 10 nM E2 as described in Materials and Methods. A, AP-1, Sp1, Sp3, or NF-Y binding to the PADI4 promoter was detected by gel staining after PCR amplification using primers corresponding to the promoter region (panel 1). Results of control PCR, using the primers for the intron 1 region are shown in panel 2. The numbers shown on the right are given as nucleotide sizes in base pairs. B, For quantitative analyses of AP-1, Sp1, Sp3, or NF-Y binding to the PADI4 promoter, the samples from MCF-7 cells cultured with (black bar) or without (white bar) E2 were analyzed using real-time PCR. The relative DNA levels were calculated as described in Materials and Methods. Results are means ± SD for four separate experiments. **, P < 0.01 (Student’s t test). C, MZF1 was not expressed in MCF-7 cells cultured in medium with or without E2. The protein extracts from the immortalized keratinocyte line HaCaT cells were used as positive control (panel 1). Actin was used as loading control (panel 2).

 
Expression of AP-1, Sp1, and NF-Y Are Essential for PADI4 Expression
We carried out small interfering RNA (siRNA) experiments to investigate the role of AP-1, Sp1, Sp3, and NF-Y in vivo for PADI4 gene regulation. As shown in Fig. 4AGo by Western blot, the siRNAs transferred into MCF-7 cells hindered the synthesis of each transcription factor in a specific manner. When Sp1 expression was reduced by specific siRNA transfection, the level of PADI4 transcription in the cells was diminished by more than 90% (Fig. 4BGo). When AP-1 or NF-YA expression was reduced by specific siRNA, the level of PADI4 transcription was also diminished by more than 60% (Fig. 4BGo). Western blotting indicated that PAD4 protein levels significantly decreased upon individual transfection of AP-1, Sp1, and NF-YA siRNA (Fig. 4CGo). However, when Sp3 was effectively silenced by Sp3-specific siRNA (Fig. 4AGo), PADI4 expression was not significantly affected at either the mRNA or the protein level (Fig. 4Go, B and C). Taken together with the ChIP analysis, the binding of AP-1, Sp1, and NF-Y is essential for PADI4 transcription, and the binding of Sp3 might be competitive with that of Sp1, resulting in suppression of the transcription.


Figure 4
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  		Fig. 4. Binding of AP-1, Sp1, and NF-Y to the PADI4 Promoter Is Essential for PADI4 Expression

MCF-7 cells were transfected with 100 nM of the indicated siRNA and cultured in medium with 10 nM E2. After being cultured for 36 h, total proteins and RNA were prepared for Western blotting and quantitative real-time PCR analyses, respectively. A, Western blotting for each transfactor. Actin was used as a loading control. Inhibition of expression of AP-1, Sp1, Sp3, and NF-YA with their respective specific siRNA was confirmed by comparing with control siRNA treatment. B, Effects of siRNA AP-1, Sp1, Sp3, or NF-YA on the transcriptional level of PADI4. The transcriptional level of PADI4 in MCF-7 cells was normalized to the level of expression of GAPDH. The data were expressed as percentage relative to the control siRNA-treated MCF-7 cells. Results are means ± SD for four experiments. **, P < 0.01 vs. the difference between specific and control siRNA treatment (Student’s t test). C, Effects of siRNA AP-1, Sp1, Sp3, or NF-YA on the translational level of PAD4 by Western blotting. Actin was used as loading control.

 
E2 Induced the Expressions of AP-1, Sp1, and NF-Y
To investigate the effects of E2 on the nuclear localization of the transfactors, AP-1, Sp1/Sp3, and NF-Y, we carried out Western blot analyses of the nuclear extracts derived from E2-treated MCF-7 cells. As shown in Fig. 5Go, nuclear accumulation of AP-1, Sp1, and NF-Y gradually increased after stimulation with 10 nM E2, corresponding to the PADI4 expression pattern (Fig. 1Go). E2-induced expression of AP-1, Sp1, and NF-Y was abrogated by addition of ICI, suggesting an important role for ER{alpha} (Fig. 5Go, last lane). Furthermore, no significant changes were detected for Sp3 with E2 stimulation or ICI inhibition (Fig. 5Go). Thus, these results indicate that E2-enhanced PADI4 expression in MCF-7 cells is mediated by increasing the nuclear levels of AP-1, NF-Y, and Sp1 transfactors.


Figure 5
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  		Fig. 5. Estrogen Promoted the Expression of AP-1, Sp1, and NF-Y in MCF-7 Cells

MCF-7 cells were exposed to 10 nM E2 without or with 1 µM ICI and incubated for the indicated length of time. The nuclear proteins obtained from the cultured cells were prepared and analyzed by Western blotting using anti-AP-1, Sp1, or NF-Y antibodies. Actin was used as a loading control.

 
E2 Enhances PADI4 Transcription via ER{alpha}
To further demonstrate the involvement of ER in the E2-induced expression of PADI4 in vivo, we performed transient transfection of the expression plasmid for ER{alpha} or ERß into HeLa cells accompanied with the reporter gene of pG4–348/+27. We then measured luciferase activities in cells incubated with increasing E2 concentrations. As shown in Fig. 6Go, E2 activated transcription of pG4–348/+27 in a dose-dependent manner (≤10 nM) in the presence of ER{alpha}. In contrast, the transcription of pG4–348/+27 was not significantly stimulated by E2 with mock vector or ERß. Furthermore, the stimulating effect of E2 mediated by ER{alpha} was completely abrogated by ICI (Fig. 6Go). These results strongly support the idea that E2 enhances the transcription of PADI4 only via ER{alpha}.


Figure 6
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  		Fig. 6. Enhancement of PADI4 Transcription by E2 Is Dependent on ER{alpha}

HeLa cells were cotransfected with pG4–348/+27 plasmid with ER{alpha} (black bar) or with ERß (gray bar) plasmid. After 16 h of transfection, cells were incubated with varying concentrations of E2, as indicated. Cells were harvested 48 h after transfection, and luciferase activity was measured. White bars show the activity using mock plasmid. Values were corrected for transfection efficiency by cotransfection with the Renilla expression plasmid. Representative data of four experiments are shown. *, P < 0.05; **, P < 0.01 (Student’s t test).

 
To identify the transcription factor-binding elements responsible for the E2-induced PADI4 transcription via ER{alpha}, we performed a mutagenic analysis of the PADI4 promoter region. We evaluated the expression of the reporter gene pG4–348/+27 harboring various mutagenic nucleotides or further 5'-deletion in HeLa cells that were cotransfected with ER{alpha} plasmid in the presence or absence of E2. As shown in Fig. 7Go, the mutation of NF-Y- (at nucleotides –325 and –326) or Sp1/Sp3-binding cis-elements (at nucleotides –186 and –187) did not show remarkable loss of E2-induced activity compared with that of the native reporter gene. On the other hand, mutants of the Sp1/Sp3- (at nucleotides –90, –91 and –92) and AP-1-binding site (at nucleotides –52 and –53) abrogated more than 60% of E2-induced activity. In addition, two mutants that successively deleted the above two cis-elements (pG4–82/+27, pG4–39/+27) lost almost all responsiveness to E2 (Fig. 7Go). Neither basal nor E2-induced activity was detected upon mutation of the TATA-box. These results suggest that the enhancement of PADI4 transcription by E2 is mainly due to the association of transcription factors Sp1/Sp3 bound at nucleotides –84 to –93 and AP-1 bound at nucleotides –46 to –56.


Figure 7
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  		Fig. 7. Determination of Crucial Motif Site for the E2-Induced Activation of PADI4 Expression

Each PADI4 reporter construct was transfected into HeLa cells with ER{alpha} plasmid. After 16 h of transfection, cells were further incubated with (black bar) or without (white bar) 10 nM E2 for 36 h. Cells were harvested and then luciferase activity was measured. The numbers given to the constructs indicate the 5'- and 3'-ends of the 5'-flanking region of PADI4, position numbered +1 corresponding to the first base of the transcription initiation nucleotide. The putative factor-binding sites, NF-Y, Sp1/Sp3, AP-1, and TATA-box, that are targeted, are shown on the constructs. The positions of each mutation designated as a cross are shown. Values were corrected for transfection efficiency by cotransfection with the Renilla expression plasmid and are expressed as means ± SD for four separate experiments. *, P < 0.05; **, P < 0.01 vs. the difference between with and without 10 nM E2 (Student’s t test).

 
Rapid Regulation Mechanism Involved in E2-Enhanced PADI4 Transcription
According to the above experiments, several transfactors binding to the cis-elements at the proximal promoter appeared to contribute to the enhancement of PADI4 transcription by E2, but there is also a possibility that a more direct and rapid mechanism induces some of the PADI4 transcription. In addition, the ability of ER{alpha} to associate indirectly with the promoter through binding to the transcription factors expands the set of PADI4 capable of responding to E2 induction (27). We, therefore, performed a cycloheximide-blocking experiment to demonstrate whether the direct and rapid pathway was involved in the estrogen-induced PADI4 transcription. As shown in Fig. 8Go, E2 rapidly increased the PADI4 transcription, and pretreatment of MCF-7 cells with the translation inhibitor cycloheximide for 1 h before estrogen stimulation did not block early induction of this gene, indicating that PADI4 is a direct target of ER action. Furthermore, after incubation of MCF-7 cells with E2 alone or E2 together with cycloheximide for 24–72 h, the estrogen-induced expression of PADI4 was significantly blocked by cycloheximide (Fig. 8Go), suggesting that an indirect regulation mechanism is involved in the late induction of PADI4 by estrogen.


Figure 8
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  		Fig. 8. Assessment of Mechanisms of Early and Late PADI4 Expression

MCF-7 cells were pretreated with control (white bar) or 10 µg/ml cycloheximide (black bar) for 1 h and then stimulated with 10 nM E2 for the indicated length of time. The non-E2-treated cells were also used as relative expression control. Total RNA was collected, and transcript level of PADI4 was assessed by quantitative RT-PCR. PADI4 expression level was normalized to the level of expression of GAPDH and expressed relative to the non-E2-treated control cells (set as 1). The data are the average of triplicate experiments. *, P < 0.05; **, P < 0.01 vs. the difference between with and without cycloheximide.

 
Association of ER{alpha} with the Upstream PADI4 Gene in Vivo
Recent genome-wide analysis of ER-binding sites demonstrated the proximal promoter region, although important for some genes, does not constitute the majority of ER target sites and a full definition of ER binding to cis-elements [estrogen response elements (EREs)] distinct from promoters is required to fully understand the estrogen response (28). Indeed, we found that two ER-binding sites, assigned to ERE-125 and ERE-126 (28) (http://research.dfci.harvard.edu/brownlab/datasets/index.php?dir=ER_whole human/genome/), are located upstream of the PADI4 gene (Fig. 9AGo), whereas the proximal promoter of the gene lacks any ERE-like sequences. Therefore, we performed ChIP assay with ER{alpha}-specific antibody to demonstrate receptor occupancy for PADI4 transcriptional regulation. The product of PCR, using nonimmunoprecipitated DNA as template, was used to show the input level of chromatin. As shown in Fig. 9BGo, ChIP assays indicated that ER{alpha} bound to ERE-125 and ERE-126 sites and increased the binding level with estrogen stimulation. The specificity of the ChIP analysis was demonstrated by the inability to detect binding of ER{alpha} to the first intron region of PADI4 gene using the primers to amplify a segment from the first intron (Fig. 9BGo). When PCR was performed on the chromatin samples immunoprecipitated with nonimmune IgG using the same primers, no PCR signal was observed, showing the specificity of binding (data not shown). Figure 9BGo also demonstrated that ER{alpha} bound to the minimal promoter region and increased the binding level with estrogen stimulation. It is described that indirect binding of ER{alpha} to DNA and association with AP-1 and Sp1 can result in estrogen regulation (28). Therefore, we deduced that ER{alpha} bound to the PADI4 promoter via protein-protein interactions at AP-1, Sp1, and NF-Y sites. To quantify E2-induced effects of transcription factor binding, we carried out quantitative PCR with the immunoprecipitated chromatin. As shown in Fig. 9CGo, higher levels of ER{alpha} recruitment on ERE-125, -126, and the minimal promoter regions were observed with E2 treatments than without.


Figure 9
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  		Fig. 9. Association of ER{alpha} with Upstream Regions of PADI4 in Vivo

ChIP assays using anti-ER{alpha} antibody were performed using chromatin from MCF-7 cells cultured in medium with or without 10 nM E2 for 45 min as described in Materials and Methods. A, Genomic segments of PADI4, which were tested in this research. Two ER-binding regions (ERE-125 and ERE-126) as described by Carroll et al. (28 ) are located upstream of PADI4. The number given to the nucleotide position of each region, numbered +1 corresponding to the first base of the transcription initiation nucleotide of PADI4 gene. B, Association of ER{alpha} with ERE-125, ERE-126, or the minimal promoter was detected by gel staining after PCR amplification using primers corresponding to two ER-binding and the minimal promoter regions (panel 1). Results of control PCR, using the primers for the intron 1 region are also shown in panel 1. In panel 2, PCR were performed on the input chromatin samples as control. The numbers shown on the right are given as nucleotide sizes in base pairs. C, For quantitative analyses of association of ER{alpha} with ERE-125, ERE-126, or the minimal promoter, the samples from MCF-7 cells cultured with (black bar) or without (white bar) 10 nM E2 were analyzed using real-time PCR. Data were normalized to the input chromatin levels, and relative DNA levels were calculated as described in Materials and Methods. Results are means ± SD for four independent experiments. **, P < 0.01 (Student’s t test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We demonstrated that PADI4 was expressed in MCF-7 cells and responsive to estrogen at transcriptional and translational levels (Fig. 1Go). Using a reporter gene and site-deletion or site-mutation assays in combination with ChIP and siRNA assays, we mapped the minimal promoter region of PADI4 gene to –348/+27 in MCF-7 cells. Further, we identified AP-1, Sp1, and NF-Y as the cis-acting factors responsible for its basal and E2-induced transcription (Figs. 2Go and 3Go). AP-1 consists of a large variety of dimers composed of members of Fos (Fos, FosB, Fra1, and Fra2), Jun (Jun, JunB, and JunD), and activating transcription factor proteins (29). AP-1 activity is implicated in a wide range of biological processes including cell proliferation, differentiation, apoptosis, and oncogenesis (30). Many genes are regulated by AP-1, such as cell life and death control genes, Cyclin D1, p53, p21, p16, p19, granulocyte macrophage colony-stimulating factor, KGF (keratinocyte growth factor), FL1, FasL, Fas, and Bc13 (30). Here, we reveal that PADI4 is also an AP-1 target gene. E2-induced PADI4 expression was consistent with E2-induced AP-1 activity, and the expression of both PAD4 and AP-1 was inhibited by ICI, suggesting an ER-dependent regulation pathway (Figs. 1Go and 5Go). It was reported that E2 activated MAPK-signaling pathways to enhance the proliferation of MCF-7 cells (31, 32). In fact, MAPK cascades enhance AP-1 activity through the phosphorylation of distinct substrates and are responsive to stimuli from growth factors, cytokines, neurotransmitters, polypeptide hormones, cell-matrix interactions, bacterial and viral infections, and a variety of physical and chemical stresses (33). Therefore, we hypothesize that AP-1 not only plays a role in PADI4 expression in response to E2, but also for other extracellular signals.

The Sp1 family has been shown to be involved in inducible gene transcription, such as responses to glucose, serum, epidermal growth factor, platelet-derived growth factor, TGF, and IL-21 receptor (26, 34). We now show that Sp1 plays an essential role in regulating basal and E2-induced PADI4 expression. Mutation of the Sp1 binding sites in the PADI4 proximal promoter region diminished reporter activity. Strikingly, Sp1 expression was significantly increased in MCF-7 cells after E2 stimulation. The E2-induced expression of Sp1 correlated with induction of PAD4 mRNA, and transfection of Sp1 siRNAs into MCF-7 cells significantly decreased E2-induced PADI4 expression at the levels of both mRNA and protein. It has been reported that Sp3 influences Sp1 to exert its transcriptional activation (26, 35). The DNA-binding activity of the complex containing Sp3 was decreased by E2 stimulation, in contrast to Sp1, the binding activity of which was markedly induced (Fig. 3Go). Because Sp1 and Sp3 recognize the same DNA element (26), we hypothesize that decreased Sp3-binding activity was due to the increased Sp1-binding activity. In addition, siSp3 had little effect on E2-induced PADI4 expression, and the expression of Sp3 was not influenced by E2 stimulation. Thus, unlike Sp1, Sp3 plays only a minor role in the regulation of PADI4 expression.

We demonstrated that the NF-Y binding cis-element (CCAAT-box) is required for PADI4 transcription and showed that the NF-Y transcription factor, present in MCF-7 cell extracts, actually binds to the PADI4 proximal promoter region in vivo (Figs. 2Go, 3Go, and 4Go). Furthermore, when NF-YA expression was specifically reduced by siRNA, the level of PADI4 expression was significantly diminished (Fig. 4Go). The levels of NF-Y vary in different cell types and growth conditions (36), and its DNA-binding activities are required for E2-induced gene expression (37, 38). In addition, its expression level and DNA-binding activities were significantly induced by E2 (Figs. 3Go and 5Go). These findings strongly indicated that NF-Y plays an important role in the regulation of PADI4 expression.

Although two MZF1 motifs are present in PADI4 proximal promoter region, their DNA-binding activities could not be detected (Figs. 2BGo and 3Go). Moreover, the transcriptional activity of PADI4 was little influenced by MZF1-mutant or siRNA transfection (data not shown). We further demonstrated that MZF1 was not expressed in cultured MCF-7 cells (Fig. 3CGo). It has been reported that MZF1 is expressed in cell lines representing early stages of myeloid differentiation (39) to regulate hematopoiesis either positively or negatively (40, 41, 42). Therefore, whether MZF1 is involved in PADI4 regulation in myeloid cells would be an area for future investigation.

We reported that estrogen enhanced PADI4 expression through both of the classical and nonclassical transcription factor pathways. Three distinct pathways are involved in ER-mediated estrogen signaling. In the classical pathway of ER action, ligand-activated ER binds specifically to DNA at EREs of promoters through its DNA-binding domain and brings coactivators and corepressors to the transcription site via its activator function 1 and 2 domains (43). In this study, we confirmed that estrogen directly and rapidly induced the up-expression of PADI4, and ER{alpha} bound to two upstream regions of PADI4 gene (Figs. 8Go and 9Go). Therefore, we believe that the classical transcription factor pathway was involved in E2-induced PADI4 expression.

The second mechanism involved in the estrogen-induced gene expression, in which ER interacts with other transcription factors, is through a process referred to as transcription factor cross talk. In this case, ER modulates the activities of other transcription factors such as AP-1, Sp1, and NF-Y by stabilizing their DNA binding and/or recruiting coactivators to the complex (37, 38, 44, 45, 46). We demonstrate that AP-1, Sp1, and NF-Y are required for PADI4 basal and E2-induced transcription, and their expression levels and DNA-binding activities are up-regulated by E2 treatment. The cycloheximide-blocking experiment indicated that the ER interaction with estrogen-induced other transcription factors was involved in the regulation of late PADI4 expression (Fig. 8Go). These facts suggest that the nonclassical transcription factor pathway was also involved in E2-enhanced PADI4 expression, through ER modulating the DNA-binding activities of AP-1, Sp1, and NF-Y.

The nongenomic pathway is another mechanism estrogen uses to affect gene expression. Through this mechanism, signal transduction pathways are rapidly induced by activated MAPK, leading to protein kinase C and protein kinase A activation (31, 32, 47, 48). The estrogen-induced activation of these cytoplasmic protein kinases leads to induction of genes that are downstream of these kinase cascades. Furthermore, ERK-induced phosphorylation of the c-Jun transactivation domain activates the AP-1 transcription factor (49). Together, these reports and our findings that E2-stimulated expression of PADI4 depended on the ER{alpha}-mediated DNA-binding activities of AP-1, Sp1, and NF-Y (Figs. 1Go, 5Go, and 6Go), we believe that the nongenomic pathway is also involved in the regulation of PADI4 expression.

A coregulation mechanism was suggested to be involved in the E2 responses of PADIs. In previous studies, we demonstrated that the transcription factors binding to the minimal promoter regions of PADIs (Sp1/Sp3 for PADI2, and Sp1/Sp3 and NF-Y for PADI3) play a very important role for the expression of both genes in the epidermis (50, 51). The putative Sp1 binding sites are also present in the 5'-flanking sequences of PADI1 (4). Here, our data support the idea that AP-1, Sp1, and NF-Y are the cis-acting factors responsible for basal and E2-induced transcription of PADI4. The up-regulation of the four PAD genes (PADI1–4) by E2 stimulation in MCF-7 cells (our unpublished work) might be due to the same Sp1 regulation pathway. In addition, PADI family members are encoded by five genes (PADI1–4 and 6), which are clustered on a single chromosomic locus, i.e. 1p35–36 in human and likely 4E1 in mouse (4). These facts suggest that Sp1, NF-Y, or AP-1 coregulation of PADI family genes might be a genome-wide strategy for gene regulation. Furthermore, PADI4 regulates the expression of estrogen-responsive genes through modifying the arginine sites in histones H3 or H4 (18, 19, 20, 21, 22). Thus, cross talk between PADI4 and transcription factors might be an important mechanism by which hormones modulate genome-wide gene expression.

Multiple arginine sites in histones H3 and H4, including those sites methylated by coactivator-associated arginine methyltransferase 1 and protein arginine methyltransferase 1, are targets of PADI4 (18, 19, 20, 21). In addition, the methylation of a nonhistone coactivator (p300) can be similarly reversed by PADI4 and contribute to the transcriptional activation process (22). Moreover, PADI4 is recruited to the estrogen-activated gene’s promoter, and plays a role in suppression of transcriptional activation by ER (18, 19). In this study, we demonstrated that estrogen enhances PADI4 expression through ER-mediated pathway. Based on these facts, we deduce that PADI4 is involved in feedback regulation of estrogen-responsive genes. In this model, the transcription of some genes may be modulated by estrogen-enhanced expression of PADI4, and finally achieve a new dynamic balance of genes expression. On the other hand, overexpression of PADI4 induces cells apoptosis (8), suggesting that regulation of the expression of this gene may be a key issue for anticancer, such as human breast cancer. Therefore, in order to further understand the molecular mechanism of the feedback and cell death that is induced by PADI4, it is necessary to investigate genome-wide the genes that are responsive to the activation of PADI4.

Focus has been placed upon PADI4 due to its important role for autoimmune involvement in RA (9, 10, 11, 12, 13, 14, 15). Indeed, estrogen and androgen receptors are present in human synovial macrophages (52). Moreover, increased estrogen concentrations were observed in synovial fluids of RA patients (53, 54). In this study, we demonstrated that estrogen stimulated PADI4 expression in an ER{alpha}-dependent manner. Thus, we hypothesize that the activity of PADI4 may be up-regulated by the increase in estrogen accumulating in RA synovial fluids, at least at the level of expression. Upon stimulation of PADI4 activity, the level of citrullinated peptides may increase, enhancing the autoimmune responses. Due to the significant role of estrogen in this process, this hypothesis may well explain why the majority of RA patients are female.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Chemicals and Antibodies
E2 was purchased from Sigma-Aldrich Corp. (St. Louis, MO) and ICI was from Tocris Cookson, Inc. (Ellisville, MO). A 10 mM stock solution for each compound was dissolved in dimethylsulfoxide (DMSO) (vehicle). The final concentration of the solvent in the culture medium did not exceed 0.1%, which did not affect the yield of cells. Fetal bovine serum (FBS) was purchased from HyClone Laboratories, Inc. (Logan, UT). Penicillin-streptomycin solution and trypsin/EDTA solution were purchased from Life Technologies (Gaithersburg, MD). Monoclonal antihuman PAD4 antibody (D4h35), which was defined to be nonreactive against other human PAD, was supplied from MBL Inc. (Nagoya, Japan). The other antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cell Culture and RNA Extraction
Human breast cancer MCF-7 cells were obtained from the JCRB Cell Bank (National Institute of Health Sciences; Tokyo, Japan) and cultured in RPMI 1640 medium (Invitrogen; Carlsbad, CA) supplemented with 10% FBS at 37 C in a humidified atmosphere with 5% CO2. Cells were cultured in phenol red-free RPMI 1640 medium containing 10% dextran-coated charcoal-treated FBS (DCC-FBS) for 3 d and treated with 10 nM E2 with or without 1 µM ICI, or with 0.1% DMSO as control for 1–3 d. Total RNA was purified from the cells using the RNeasy Protect kit (QIAGEN, Hilden, Germany). The reagents for tissue culture were from Life Technologies.

HeLa cells were obtained from the Health Sciences Research Resources Bank (Osaka, Japan) and maintained in DMEM (Life Technologies) with 10% (vol/vol) FBS. For transfection, HeLa cells were cultured in phenol red-free DMEM containing 10% DCC-FBS. The immortalized keratinocyte line HaCaT, a generous gift of Prof. N. Fusenig (Heidelberg, Germany), was grown in DMEM supplemented with 5% FBS.

Real-Time RT-PCR
The relative degrees of PADI4 expression in response to E2 were analyzed by real-time RT-PCR as described previously (50). The PCR was performed four times using the forward primer, 5'-CGAAGACCCCCAAGGACT-3' (nucleotides 586–603 of human PAD4 cDNA, GenBank accession no. AB017919) and reverse primer, 5'-AGGACAGTTTGCCCCGTG-3' (nucleotides 676–693). The amplification program consisted of denaturation at 95 C for 3 min, followed by 50 cycles at 95 C for 30 sec and at 59 C for 30 sec using a two-step protocol on LightCycler (Roche; Mannheim, Germany). The level of expression was standardized to that of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) using the following primer set, forward primer: 5'-CATGTTCCAATATGATTCCAC-3' (nucleotides 187–207 of human GAPDH cDNA, GenBank accession no. M33197); reverse primer: 5'-CCTGGAAGATGGTGATG-3' (nucleotides 271–287).

Western Blotting
Total protein was extracted with SDS buffer (0.125 M Tris-HCl, 10% 2-mercaptoethanol, 4% SDS, 10% sucrose) and sonicated on ice for 30 sec. Nuclear extracts were prepared from cultured cells as described by Andrews and Faller (55). After incubation at 95 C for 5 min with loading buffer, total protein or nuclear extract (30 µg) was resolved by SDS-PAGE with 5–20% gradient gel, and then electrotransferred onto nitrocellulose membranes (Millipore Corp., Billerica, MA) using a semidry transfer cell (Bio-Rad Laboratories, Inc., Hercules, CA) at 1 mA/cm2 for 2 h. Proteins were analyzed by probing membranes, which were preblocked in Tris-buffered saline containing 0.1% Tween 20 and 5% BSA (TBST-BSA), with specific antibodies diluted 1:1000 in TBST-BSA overnight at 4 C. The complexes of rabbit, mouse, or goat antibodies-antigens were detected with horseradish peroxidase-coupled antibodies against rabbit, mouse, or goat IgG after 1:5000-dilution with TBST-BSA. Complexes were visualized using the ECL plus Western Blotting Detection System (Amersham Pharmacia Biotech; Arlington Heights, IL) using Cool Saver AE-6955 (ATTO; Tokyo, Japan).

Construction of Promoter Reporter Plasmids
Based on the nucleotide sequences of human PADI genes (GenBank accession no. AJ549502), the 5'-flanking region of PADI4 was amplified by PCR using human genomic DNA (BD Biosciences, Palo Alto, CA) as a template and a pair of specific primers: forward, 5'-GGCTCTATGAAGGCAGGATTTTTGTC-3' (nucleotides –1977 to –1952; position number +1 corresponding to the transcription initiation site); reverse, 5'-AATTTATCTGTCGTGTGTTCCTGGGG-3' (nucleotides +331 to +356). The PCR condition was an initial denaturation for 2 min at 95 C, 30 cycles (95 C for 30 sec, 56 C for 30 sec, and 72 C for 2 min) and a final extension at 72 C for 5 min, with Ex Taq DNA polymerase (Takara, Shiga, Japan) according to the manufacturer’s instructions. The PCR products were subcloned into the pGEM-T vector (Promega Corp., Madison, WI). The obtained plasmid was designated as pTAhPAD4.

To construct the reporter plasmid pG4–1977/+27, PCR was carried out using pTAhPAD4 as a template and a pair of specific PADI4 primers, P4–1977 and P4-RV (Table 1Go), under the following conditions: initial denaturation at 95 C for 1 min, 30 cycles (95 C for 30 sec, 50 C for 30 sec, and 72 C for 4 min) and a final extension at 72 C for 8 min. The PCR product was cloned into the MluI and HindIII sites of pGBasic vector 2 (Nippon Gene, Toyama, Japan). Sequential 5'-deletion constructs of the 5'-flanking region of PADI4 were generated by PCR using pTAhPAD4 as a template and the primers listed in Table 1Go. The thermocycler settings consisted of 2 min incubation at 95 C, followed by 30 cycles at 95 C for 30 sec, 30 sec at the predicted melting temperature for each forward primer and 1 min at 72 C, and a final extension for 10 min at 72 C. The resulting amplification products were cloned into the XhoI and HindIII sites of pGBasic vector 2. The –661/+27 DNA segment was cut out from plasmid pG4–1977/+27 by XhoI and HindIII digestion and then cloned into the XhoI and HindIII sites of pGBasic vector 2. Site-mutation of construct pG4–348/+27 was done using the mutation oligonucleotides (Table 1Go) and QuickChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. All of the constructs were prepared using the QIAfilter Plasmid Midi Kit (QIAGEN), and their nucleotide sequences were confirmed by double-stranded DNA sequencing.


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  		Table 1. Primers Used for Preparing the Deletion Mutants and Oligonucleotides for Motif Mutants

 
Transfection of Plasmid and Measurement of Promoter Activity
Transfection and measurement of promoter activities were carried out with MCF-7 cells following the procedures described previously (50). Expression plasmids of ER{alpha} and ERß were generous gifts from Professor S. Kato (The University of Tokyo, Japan). HeLa cells were cotransfected with 75 ng of ER{alpha} or ERß plasmid, 720 ng of luciferase reporter plasmid, and 30 ng of SV-40 Renilla expression plasmid. After 16 h of transfection, cells were incubated with varying concentrations of E2. Cells were harvested 48 h after transfection, and luciferase activity was measured. Four transfections were carried out independently for each construct, and the results were expressed as means ± SD.

ChIP Assays
For putative transcription factor-minimal promoter binding analysis, ChIP assays were performed as described previously (51). Immunoprecipitation was performed overnight with agitation at 4 C with 5 µg of normal IgG, anti-AP-1, anti-MZF1, anti-Sp1, anti-Sp3, or anti-NF-YA antibody. Immunoprecipitated chromatin was collected and analyzed by conventional PCR and quantitative PCR. The following primers were used for ChIP PCR analysis: 5'-CCAGCATTGACACCCATCT -3' (forward; nucleotides –175 to –157), and 5'-TTATATCGCCCAGCAGAGG-3' (reverse; nucleotides –44 to –26) to amplify the promoter region from nucleotides –175 to –26; 5'-TGGGATTACAGGCGGGAG-3' (forward; nucleotides +10880 to +10897) and 5'-GATGGCCGAGGTCTGGAA-3' (reverse; nucleotides +11020 to +11038) to amplify a segment in the first intron region of the human PAD4 gene. For ER{alpha}-binding analysis, ChIP assay using anti-ER{alpha} antibody was performed using chromatin from MCF-7 cells cultured in medium with or without 10 nM E2 for 45 min (28). The primers 5'-CACACAGCAAAGGACAGGGT-3' (forward; nucleotides –23023 to –23004) and 5'-GTCAGTAGAGGTCCAGCATGA-3' (reverse; nucleotides –22892 to –22872) were used to amplify a segment in ERE-125 region; 5'-AGGTGGAGGTTGCAGTGAG-3' (forward; nucleotides –6728 to –6710) and 5'-CTGGGATTATAGGCGCTTGC-3' (reverse; nucleotides –6626 to –6607) to amplify a segment in ERE-126 region. For ChIP PCR analysis of the minimal promoter or the first intron with ER{alpha}-specific antibody, we used the same primer sets as described above. The amplification program consisted of denaturation at 95 C for 3 min, followed by 50 cycles of 95 C for 15 sec, 56 C for 10 sec, and 60 C for 20 sec.

siRNA-Based Inhibition
MCF-7 cells were transfected using the siRNA transfection reagent (Santa Cruz Biotechnology) with 100 nM siAP-1, siSp1, siSp3, siNF-YA, or siControl (Santa Cruz Biotechnology) according to the manufacturer’s instructions. After culturing in antibiotic-free medium with 10 nM E2 for 36 h, total RNA was extracted and analyzed by quantitative real-time RT-PCR. To confirm the specific inhibitory activity of each siRNA, we carried out Western blotting analyses with the antibodies against each transcription factor. Nuclear extracts were prepared from untreated or siRNA-transfected cells as described previously (55) and used for Western blotting analyses.

Cycloheximide Blocking Experiment
MCF-7 cells were cultured in phenol red-free RPMI 1640 containing 10% DCC-FBS for 3 d, and then cells were pretreated with control (DMSO) or 10 µg/ml cycloheximide for 1 h and then stimulated with 10 nM of E2 for the 3–72 h (28). Total RNA was collected and transcript level of PADI4 gene was assessed by quantitative RT-PCR as described above.


   ACKNOWLEDGMENTS
 
We thank Professor Shigeuki Kato, The University of Tokyo, for providing the human ER{alpha} and ERß expression plasmids.


   FOOTNOTES
 
This work was supported by Grants-in-Aid for Scientific Research of the Ministry of Education, Science, Sports and Culture (16580071, 18591265).

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 24, 2007

Abbreviations: AP-1, Activator protein-1; ChIP, chromatin immunoprecipitation; DCC-FBS, dextran-coated charcoal-treated FBS; DMSO, dimethylsulfoxide; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICI, ICI 182,780; NF-Y, nuclear factor-Y; PAD, peptidylarginine deiminase; PAD4, PAD type IV; PADI4, human PAD4 gene; RA, rheumatoid arthritis; siRNA, small interfering RNA; TBST-BSA, Tris-buffered saline containing 0.1% Tween 20 and 5% BSA.

Received for publication December 26, 2006. Accepted for publication April 18, 2007.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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