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Endocrinology Vol. 142, No. 8 3380-3388
Copyright © 2001 by The Endocrine Society

ARTICLES

Inhibition of ER{alpha}-Mediated Trans-Activation of Human Coagulation Factor XII Gene by Heteromeric Transcription Factor NF-Y

Antonella Farsetti1, Michela Narducci1, Fabiola Moretti, Simona Nanni, Roberto Mantovani, Ada Sacchi and Alfredo Pontecorvi

Istituto di Neurobiologia e Medicina Molecolare, Consiglio Nazionale delle Ricerche (A.F., F.M.); Cattedra di Endocrinologia, Università di Roma La Sapienza (M.N.); Dipartimento Biologia Animale, Università di Modena e Reggio (R.M.); Istituto di Patologia Medica, Università Cattolica del Sacro Cuore (A.P.); and Laboratorio di Oncogenesi Molecolare, Istituto Tumori Regina Elena (A.F., M.N., F.M., S.N., A.S., A.P.), 00158 Rome, Italy

Address all correspondence and requests for reprints to: Dr. Alfredo Pontecorvi, Laboratorio di Oncogenesi Molecolare, Istituto Tumori Regina Elena, via delle Messi d’Oro No. 156, 00158 Rome, Italy. E-mail: pontecorvi{at}ifo.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human coagulation factor XII promoter contains an estrogen response element that mediates ligand-activated ER{alpha} induction of coagulation factor XII gene expression. The 3'-half of coagulation factor XII-estrogen response element overlaps a putative CCAAT box, the widespread regulatory element specifically recognized by the heteromeric transcription factor NF-Y. Transient cotransfection of NF-Y and ER{alpha} results in strong inhibition of estrogen stimulation of coagulation factor XII promoter activity. NF-Y antagonism is primarily exerted by the NF-YA subunit and does not require binding to the CCAAT element, as NF-YA mutants with impaired DNA binding capacity retain the ability to inhibit ER{alpha} trans-activation. EMSAs with increasing concentrations of recombinant NF-Y do not detect the formation of NF-Y-DNA complexes or show impairment of ER{alpha} binding to estrogen response element. Immunoprecipitation of whole cell extracts with anti-ER{alpha} antibody reveals an in vivo association between the two transcription factors, which is abolished by deletion of the NF-YA carboxyl-terminus. In functional experiments with sequential NF-YA deletion mutants the HAP2-homology region appears essential in eliciting NF-YA antagonistic activity. In conclusion, our results demonstrate that heteromeric transcription factor NF-Y inhibits estrogen induction of coagulation factor XII promoter in a DNA binding-independent fashion and suggest a novel role for NF-Y as a partner for the ER{alpha} transcription complex.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FACTOR XII (FXII), also known as Hageman factor, is a major component of the contact activation system, a multienzymatic cascade of events that controls blood coagulation, fibrinolysis, and complement activation (1). It is synthesized in the liver as an 80-kDa protein and secreted into the circulation as an inert zymogen. After contacting negatively charged surfaces, released after local trauma or inflammation, FXII is converted to its active form (FXIIa) by limited proteolysis, forming a two-polypeptide chain molecule bridged by a disulfide bond that exhibits serine protease activity (2). Although originally discovered as a clotting factor, FXII appears to play a major role in the kallikrein-kinin and fibrinolytic systems, because 1) its molecular structure more resembles that of other fibrinolytic proteins (i.e. tissue plasminogen activator) (3); 2) it releases the potent vasodilator bradykinin from high mol wt kininogen (4) and stimulates consumption of the plasminogen activator inhibitor-1 (5); and 3) inherited deficiency of FXII produces a thrombophilic state, rather than an hemophilic state as for most coagulation factors (6). Modification of circulating FXII levels and/or function in vivo may therefore alter the fine balance existing between pro- and anticoagulant activities.

Indeed, circulating FXII levels may change under different pathological conditions, such as tissue injury, infection, inflammation, or in the presence of artificial surfaces (i.e. prosthetic valves, dialysis membranes, etc.) as well as in response to physiological stimuli (1, 3). In particular, FXII gene expression is tightly regulated by circulating estrogens (7, 8). We have previously demonstrated that ligand-activated ER{alpha} is able to induce transcription of the FXII gene, both in vitro and in vivo. This effect of estrogens follows direct binding of ER{alpha} to the estrogen response element (ERE), identified in the context of FXII gene promoter (9). Ligand-induced ER conformational changes direct the assembly of a preinitiation complex at the promoter of target genes by allowing direct or indirect interactions with a large array of molecules, such as factors contained in the transcription initiation complex, other DNA-bound transcription factors, as well as a set of recently identified proteins, defined as coactivators, that enhance receptor trans-activation properties (10, 11, 12). In addition, posttranslational modifications (i.e. phosphorylation) may contribute to activate ER{alpha} in a ligand-independent manner (13). Several coactivators possess intrinsic histone acetyl transferase activity and may therefore be acting in concert to remodel chromatin and create a transcriptionally permissive environment (14, 15). Most of these ER-interacting proteins do not require DNA binding, but exert their action directly modulating ER{alpha} trans-activation properties.

We have also demonstrated that the effects of estrogen on FXII gene promoter activity may be modulated by additional factors that may act in a general or tissue-specific manner. ER{alpha}-mediated transcriptional induction of FXII promoter, in fact, is specifically antagonized, in a liver cell environment but not in a nonliver cell context, by the former orphan receptor hepatocyte nuclear factor-4 (HNF-4) (16, 17). Distinct cis-acting elements, which are bound by HNF-4 and mediate its transcriptional interference on ER{alpha} trans-activation, have been identified and characterized in the 5'-flanking region of the FXII gene (18). Therefore, the combination of positive and negative regulatory factors is in play to ensure precise and fine modulation of FXII gene expression under different conditions.

The present study give further insight into ER{alpha}-mediated transcriptional regulation of FXII gene promoter by demonstrating an additional level of control of ER{alpha} activity. This mechanism involves the heteromeric transcription factor NF-Y that appears to exert an inhibitory action on ER{alpha} trans-activation properties through a DNA binding-independent mechanism. This novel mode of action of NF-Y introduces it as a new partner of ER{alpha} multiprotein transcription complex.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfections
Mouse fibroblast NIH-3T3 and human hepatoma HepG2 cells were cultured in DMEM containing 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Two days before transfection, cells were seeded at a density of 1.2 x 106/175-cm2 flask in phenol red-free DMEM containing 10% hormone-stripped FCS (19). Plasmid transfections, chloramphenicol acetyltransferase (CAT) assays, and quantitation of CAT activity were performed as previously described (20). The results of transfections were expressed as the mean ± SE of the number of experiments indicated in corresponding figure legends.

Plasmids
Reporter plasmid PT-CAT-181 (9) contains a fragment (-181/+49) of human genomic DNA at the 5'-end of the FXII transcription unit, cloned upstream of the CAT reporter gene, myelin basic protein (MBP)-CAT (21) contains a fragment (-256/+1) of the 5'-flanking region of mouse MBP gene, and p240B1CAT (22) contains a (-57/+182) promoter fragment of the human cyclin B1 gene, both cloned in pBLCAT3 vector; vitellogenin B1 (VIT)-CAT (23) contains Xenopus laevis VIT gene promoter (nucleotides -596/+8) fused to the CAT reporter gene. Expression vectors for human TRß (24); human wild-type NF-YA, NF-YB, and NF-YC (25, 26); and mutant NF-YA constructs (27, 28, 29) were previously described. Plasmid pSG5-HEO (30), expressing the human ER{alpha} with a point mutation at amino acid position 400 that renders the receptor ligand dependent for dimerization and DNA binding was a gift from Prof. P. Chambon (Institute de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France). pCMV-ß-gal, containing the lacZ gene under the control of the immediate early gene promoter of cytomegalovirus (CMV) was used as internal control to monitor transfection efficiency.

Western blot analysis
NIH-3T3 cells (1.5 x 105) were lysed directly in 15 µl protein sample buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol (DTT), and 1.1% bromophenol blue]. Whole cell extracts were sonicated to reduce solution viscosity and boiled for 5 min. Equal amounts of protein were loaded onto a 12% SDS-polyacrylamide gel, electrophoresed, and electroblotted onto nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA). As a loading control, proteins were stained with Ponceau S (data not shown). Filters were blocked by incubation in 5% nonfat dry milk for 1 h at room temperature, then probed with anti-ER{alpha} HC-20 polyclonal antibody (Santa-Cruz, CA), polyclonal antiserum pR{alpha}YA/C or pR{alpha}YB (31), or anti-70-kDa heat shock protein (anti-Hsp70) monoclonal antibodies (StressGen, Biotechnologies Corp., Victoria, Canada), as indicated. Immunoreactivity was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL).

Immunoprecipitation
Transfected cells were washed twice and harvested in ice-cold PBS, pelleted by centrifugation at 3,000 rpm at 4 C for 5 min, and dissolved in 100 µl extraction buffer containing 20 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.5 mM NaCl, 10% glycerol, 0.1 mM EDTA, 50 µg/ml leupeptin, 5 µg/ml phenylmethysulfonylfluoride, 1 µg/ml pepstatin, and 5 µg/ml aprotinin. Whole cell extracts were prepared by two freeze-thaw cycles, followed by centrifugation at 14,000 rpm at 4 C for 30 min. Cell extracts containing 400 µg total protein were preincubated at 4 C for 1 h with immobilized protein G (Pierce Chemical Co., Rockford, IL) in buffer containing 20 mM HEPES (pH 7.8), 50 mM KCl, 10% glycerol, 1 mM DTT, as well as protease inhibitors (50 µg/ml leupeptin, 5 µg/ml phenylmethysulfonylfluoride, 1 µg/ml pepstatin, and 5 µg/ml aprotinin). Nonspecific protein complexes were removed by centrifugation, and the supernatant was incubated with 2 µg anti-ER{alpha} monoclonal antibody C-311 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 C overnight, followed by incubation with immobilized protein G at 4 C for 3 h. The immunoprecipitates were pelleted by centrifugation at 14,000 rpm for 5 min at 4 C, boiled for 5 min into 20 µl 1 x SDS-PAGE buffer, electrophoresed on a 12% polyacrylamide gel, and blotted onto nitrocellulose membranes, and immunoblots were decorated with anti-NF-YA (pR{alpha}YA/C), anti-ER{alpha} (HC-20, Santa Cruz Biotechnology, Inc.), or anti-NF-YB (pR{alpha}YB) polyclonal antibodies.

EMSAs
A 32P-labeled double stranded oligonucleotide spanning ERE-CCAAT sequences of FXII promoter (from -49 to -18) was assayed by native gel electrophoresis for binding to the NF-Y complex. As controls, double stranded oligonucleotides containing a canonical (E{alpha} Y-box) or mutated (E{alpha} Y-box mut) CCAAT box were used. Binding reactions with NIH-3T3 or HepG2 nuclear extracts were performed as previously described (21). Anti-NF-YB polyclonal antibody was added to some samples after incubation with the 32P-labeled probe. Samples were loaded onto a 5% polyacrylamide gel and electrophoresed for 3 h at 150 V using 0.5 x TBE (45 mM Tris-borate, 45 mM boric acid, and 2 mM EDTA, pH 7.6) as running buffer. For competition binding experiments, increasing concentrations of recombinant NF-Y trimeric complex, produced with the Escherichia coli expression vector pET3b and the BL21(DE3)LysE bacterial strain according to the method described by Mantovani et al. (26), were added to the binding mixture containing Sf9 (Spodoptera frugiperda) extracts from cells infected with a recombinant baculovirus expressing human ER{alpha} (from Dr. Myles Brown, Dana Farber Cancer Institute, Boston, MA).

Statistical analysis
Data are expressed as the mean ± SE. Results were analyzed by one-way ANOVA, followed by t test for significance between unpaired mean values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NF-Y inhibits ER{alpha}-dependent estrogen induction of FXII gene promoter
Estrogens are potent regulators of FXII gene expression, both in vitro and in vivo (9). This effect is modulated by functional interaction of hormone-bound ER{alpha} with other transcription factors, which, by acting in a tissue-specific manner, may contribute to regulate FXII gene transcription (18). A computer-assisted analysis of transcription factor signal sequences was therefore performed in search of additional factors that could modulate FXII promoter activity. A putative CCAAT box was identified at nucleotide position -32/-28 from the major transcription start site of FXII promoter, partially overlapping the previously described ERE, at its 3'-half-site (Fig. 1Go). The CCAAT box has been shown to bind NF-Y, a heteromeric transcription complex, composed of three different subunits, NF-YA, NF-YB, and NF-YC (30), which appears to reinforce the transcriptional activity of several transcription factors.



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  		Figure 1. Sequence overlap between ER{alpha} and NF-Y-binding elements in FXII promoter. The arrow indicates the position of the major transcription start site (+1).

 
To investigate whether NF-Y could modulate the estrogen inducibility of human FXII gene promoter, NF-Y subunits were transiently transfected into different cell types, either alone or in combination, in the presence or absence of ligand-activated human ER{alpha}. As previously described, transfection of ER{alpha} in NIH-3T3 fibroblasts in the presence of the natural ligand 17ß-estradiol (E2), resulted in transcriptional stimulation of cotransfected FXII promoter-CAT reporter plasmid (PT-CAT 181) of about 30-fold above basal levels (Fig. 2AGo). ER{alpha}-mediated trans-activation was strongly inhibited after cotransfection of the whole NF-Y complex. When each NF-Y subunit was independently transfected, NF-YA elicited a similar degree of repression as the whole trimer (P < 0.001). On the contrary, expression of NF-YB alone slightly impaired (P < 0.05), and NF-YC was totally ineffective in modifying ER{alpha}-dependent transcriptional activity, even after transfection of a greater amount (5 µg) of the corresponding plasmids (data not shown). Similar results were obtained in the more physiological context of liver-derived human HepG2 cells, although inhibition of ER{alpha} trans-activation by NF-YA alone was less pronounced (P < 0.01) than that carried out by the whole NF-Y complex (P < 0.001). (Fig. 2BGo). In HepG2 cells overexpression of NF-YB and NF-YC did not affect ER{alpha}-mediated trans-activation, even at a higher concentration of transfected plasmids (5 µg/dish). The NF-YA inhibitory effect was elicited in the presence of an NF-YA:ER{alpha} 1:4 plasmid ratio, resulting in approximately 80% repression of E2 induction of FXII promoter activity (Fig. 2CGo). To investigate whether NF-Y inhibition was specific to the FXII gene promoter or was also exerted on other estrogen-responsive genes, transient transfection experiments were performed in NIH-3T3 cells using a genomic fragment (-596/+8) of X. Laevis vitellogenin B1 gene promoter (VIT-CAT). This promoter is known to contain a canonical ERE not flanked by nearby CCAAT elements. Interestingly, NF-Y also inhibited (P < 0.001) ER{alpha}-mediated trans-activation of VIT-CAT, suggesting that the NF-Y inhibitory action was independent of the presence of its consensus DNA-binding sequence (Fig. 2AGo).



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  		Figure 2. NF-Y inhibits ER{alpha}-dependent trans-activation of the FXII promoter. A, NIH-3T3 cells were cultured in the presence or absence of 10-7 M E2 or 10-7 M T3 and cotransfected with FXII promoter PT-CAT181, VIT-CAT, or MBP-CAT reporter vectors (5 µg each) and, as indicated, with expression vectors for ER{alpha} (5 µg); TRß (5 µg); NF-YA, NF-YB, or NF-YC (1.25 µg each); and the CMV-ß gal construct (250 ng) for determination of transfection efficiency. B, HepG2 cells were similarly cultured and cotransfected in the presence or absence of E2. Transfected cells were assayed for CAT and ß-galactosidase activities after 72 h. Values are expressed as the fold hormone induction, calculated as the ratio of normalized CAT activity (counts per min/U ß-galactosidase) in the presence or absence of hormones (+/- hormone or +/- E2). Results represent the mean ± SE of six (A) or three (B) independent experiments, each performed in duplicate. C, NIH-3T3 cells, cultured under the same conditions as those described in A, were transfected with increasing amount (micrograms) of the NF-YA expression vector. Data are expressed as counts per min/U ß-galactosidase x 10-3 in the absence (-E2) or presence (+E2) of E2. Results represent the average ± SE of three independent experiments, each performed in duplicate. D, Western blot analysis of cell extracts from NIH-3T3 cells transiently transfected with ER{alpha} (lanes 1–8), NF-YA (lanes 1, 2, 7, and 8), NF-YB (lanes 3, 4, 7, and 8), NF-YC (lanes 5–8), or empty (lane 9) vectors. Extracts were probed with polyclonal antisera raised against a C-terminal peptide of NF-YA, the recombinant NF-YB protein, ER{alpha} as well as Hsp70 as a control (see Materials and Methods).

 
To verify whether NF-Y inhibitory activity was specifically exerted on ER{alpha}-mediated transcription, the whole NF-Y trimer was cotransfected in NIH-3T3 cells with TRß in the presence of the thyroid hormone-responsive MBP promoter-CAT reporter (MBP-CAT) (21). However, NF-Y trimer failed to show any inhibition of T3-mediated stimulation of MBP promoter activity (Fig. 2AGo). As controls, ER{alpha} and the NF-Y trimer were cotransfected in NIH-3T3 cells together with the cell cycle-related cyclin B1 gene promoter p240B1CAT reporter plasmid (22), which contains two canonical CCAAT boxes and is transcriptionally regulated by NF-Y in a cell cycle-dependent fashion. No modification of cyclin B1 promoter activity was observed by ER{alpha} over NF-Y (data not shown).

These results indicate that the NF-Y heteromeric complex significantly reduces ER{alpha}-mediated trans-activation of FXII promoter and that the inhibitory effect is mainly ascribable to the NF-YA subunit. In addition, NF-Y antagonism is specifically exerted against ER{alpha} trans-activation properties and not toward another member of the nuclear receptor superfamily, TRß.

To ascertain whether different intracellular levels of transfected NF-Y subunits could account for the different inhibitory effects observed, Western blot analysis of transfected NIH-3T3 cell extracts was performed (1.25 µg plasmid/dish), and results were quantitated by densitometry and normalized for Hsp70 protein levels, as a control. When transfected alone, NF-YA and NF-YB protein levels were 2- to 4-fold and 5- to 6-fold higher than endogenous levels, respectively, (see Fig. 2DGo, lanes 1–2 vs. lanes 3–4 for NF-YA and vice versa for NF-YB). Therefore, the stronger inhibition of NF-YA on ER{alpha}-mediated trans-activation on FXII promoter vs. that observed with the other NF-Y subunits cannot be attributed to higher intracellular protein levels. Cotransfection of all three NF-Y subunits caused a significant increase in their intracellular levels, particularly NF-YA (Fig. 2DGo). This effect can be accounted for by posttranslational stabilization of the NF-Y complex when present throughout (Piaggio, G., personal communication). Similar results were obtained in the HepG2 cell line (data not shown). Cotransfection of NF-Y subunits did not modify the ER{alpha} protein concentration, thus ruling out the possibility that the weaker ER{alpha}-dependent trans-activation of FXII promoter could be due to a decrease in intracellular receptor levels (Fig. 2DGo).

These findings indicate that NF-Y overexpression specifically impairs ER{alpha}-mediated trans-activation of FXII promoter in both a liver and nonliver cell environment, and that the NF-YA subunit is sufficient to exert the inhibitory effect.

NF-YA inhibits ER{alpha}-mediated induction of FXII promoter activity by a DNA binding-independent mechanism
To investigate the mechanism by which NF-YA represses ER{alpha}-mediated trans-activation of FXII promoter, transient transfection experiments were performed using two different NF-YA mutants unable to bind DNA (m29) or defective in the interaction with the other NF-Y subunits (m24). In the presence of E2, both NF-YA mutants inhibited ER{alpha}-dependent trans-activation of FXII promoter activity to a similar extent (P < 0.001) as wild-type NF-YA (Fig. 3AGo).



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  		Figure 3. NF-YA inhibits ER{alpha} trans-activation of FXII promoter via a DNA binding-independent mechanism. A, NIH-3T3 cells were cotransfected with PT-CAT181 and expression vectors for ER{alpha}, and either wild-type NF-YA or NF-YA mutants (NF-Ym29 and NF-Ym24; see schematic diagram) under the conditions described in Fig. 2Go. Data represent the mean of three independent experiments, each performed in duplicate. B and C, EMSAs were performed with nuclear extracts (NE) from HepG2 (B) or NIH-3T3 (C) cells and with 32P-labeled oligonucleotides spanning FXII-CCAAT element (Y box), and wild-type (E{alpha}) or mutated (E{alpha} mut) Y boxes from the major histocompatibility complex class II promoter E{alpha}, as probes. B, Lanes 1, 4, and 7, Probe alone; lanes 2, 3, 5, 6, 8, and 9, plus HepG2 nuclear extracts; lanes 3, 6, and 9, plus a specific antiserum raised against the NF-YB subunit. C, Lanes 1 and 3, Probe alone; lanes 2 and 4, plus NIH-3T3 nuclear extracts.

 
This result indicates that DNA binding and formation of the NF-Y heteromeric complex are not strictly required to interfere with the trans-activation properties of ligand-activated ER{alpha} and suggest that NF-YA could negatively modulate estrogen induction of FXII promoter by a novel DNA binding-independent mechanism. These results are also supported by the observation that NF-Y was able to inhibit ER{alpha}-dependent trans-activation of vitellogenin B1 promoter despite the absence of a CCAAT element surrounding the canonical ERE (Fig. 2AGo).

To verify whether NF-Y was able to bind the CCAAT box of FXII promoter, EMSAs were performed (Fig. 3Go, B and C). Incubation of nuclear extracts from HepG2 or NIH-3T3 cells with a 32P-labeled oligonucleotide containing the putative CCAAT box of the NF-Y-responsive E{alpha} promoter showed a retarded band (Fig. 3BGo, lane 5, and Fig. 3CGo, lane 4) that was competed out by addition of anti-NF-YB antibody (Fig. 3BGo, lane 6). These results indicate that nuclear extracts from both cell lines contain NF-Y complex. As control, a mutated E{alpha} oligonucleotide, containing a disrupted CCAAT box (32), was unable to bind the NF-Y complex (Fig. 3BGo, lanes 7–9). Incubation of a labeled oligonucleotide spanning FXII ERE-CCAAT sequences with nuclear extracts from both cell lines did not show NF-Y binding (Fig. 3BGo, lanes 2 and 3, and Fig. 3CGo, lane 2).

These observations confirm the finding that the NF-Y complex, endogenously present in nuclear extracts from different cell lines, is unable to bind FXII-CCAAT box. This strengthens the idea that NF-Y trans-repression of ER{alpha}-mediated transcription of the FXII promoter does not require binding to DNA, thus ruling out the possibility of a competition between the two transcription factors in interacting with DNA.

The inability of NF-Y to interact with FXII promoter could be due to a lower affinity of the FXII-ERE-CCAAT box for binding the heteromeric complex. Indeed, the FXII-CCAAT box exhibits noncanonical nucleotides flanking the core CCAAT sequence at its 3'-end. Nevertheless, to rule out the possibility that a weak interaction between NF-Y and its putative DNA-binding sequence on FXII promoter could indeed occur, EMSAs were performed using excess recombinant NF-Y proteins (Fig. 4Go). Although the recombinant NF-Y complex was able to bind DNA at the canonical E{alpha}-Y box (Fig. 4Go, lane 2), it completely failed to interact with the FXII-ERE-CCAAT sequence (Fig. 4Go, lane 5).



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  		Figure 4. NF-YA does not bind the Y box of FXII. A 32P-labeled double stranded oligonucleotide containing the E{alpha}-Y box was incubated alone (lane 1) or with purified NF-Y complex in the absence or presence of anti-NF-YB polyclonal antibody (lanes 2 and 3, respectively). A 32P-labeled double stranded oligonucleotide spanning the FXII promoter Y box and ERE sequences was incubated in the presence of E2 alone (lane 4) or with the recombinant NF-Y complex (lane 5), recombinant human ER{alpha} (lane 6), or a fixed amount of recombinant human ER{alpha} and increasing concentrations of the purified NF-Y complex as indicated (lanes 7–10). Lane 10, Anti NF-YB polyclonal antibody was also added.

 
To definitively dismiss the possibility that NF-Y could interfere with ER{alpha} binding to FXII-ERE, increasing concentrations of recombinant NF-Y were incubated with a fixed amount of baculovirus-expressed human ER{alpha} in the presence of labeled FXII-ERE-CCAAT oligonucleotide. Neither modification of ER{alpha} binding to FXII-ERE-CCAAT sequence (Fig. 4Go, lanes 6–9) nor the appearance of additional bands after incubation with anti-NF-YB antibody (Fig. 4Go, lane 10) were observed.

These experiments confirm that NF-Y does not bind FXII-CCAAT box or interfere with ER{alpha} binding to the overlapping ERE. Therefore, NF-Y trans-repression of ER{alpha}-mediated trans-activation of the FXII gene promoter does not occur through competition with ER{alpha} for binding to its responsive element.

NF-Y interacts with the ER{alpha} transcription complex
NF-Y is known to interact with components of the intracellular transcription machinery (33). However, it is currently unknown whether NF-Y may contact members of the nuclear hormone receptor family. To investigate the possibility that NF-Y could interact with the ER{alpha} transcriptional complex, immunoprecipitation and Western blot analysis were performed on whole extracts of NIH-3T3 cells after forced coexpression of NF-YA, -B, and -C subunits and ER{alpha} in the presence or absence of E2 (Fig. 5Go). Anti-ER{alpha} antibody immunoprecipitated a protein immunologically related to NF-YA, as indicated by subsequent Western blot analysis using anti-NF-YA antibody. The association of NF-Y with ER{alpha} was evident after transfection of all three NF-Y subunits as well as NF-YA alone. In the presence of E2, immunoreactivity of ER{alpha} was enhanced in immunoprecipitates (Fig. 5Go, lanes 1, 4, and 6), probably due to a greater affinity of the monoclonal antibody used for immunoprecipitation toward the liganded receptor. The specificity of the ER{alpha}-NFY interaction was also confirmed by lack of NF-YA immunoprecipitation in cells not cotransfected with ER{alpha} (lanes 9 and 10) or transfected with ER{alpha} alone (lanes 11 and 12). Interestingly, after cotransfection of the sole NF-YB expression plasmid, a slight amount of NF-YB protein immunoprecipitated together with ER{alpha} (data not shown). Whether this association is mediated through the endogenous NF-YA or is due to a different interaction between NF-YB and ER{alpha} remains to be elucidated.



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  		Figure 5. ER{alpha} and NF-YA interacts in vivo. NIH-3T3 cells cultured in the presence or absence of E2 were cotransfected with expression vectors for human ER{alpha}, NF-YA, NF-YB, NF-YC, or empty vectors, as indicated. Whole cell extracts were immunoprecipitated with anti ER{alpha} monoclonal antibody. Whole cell extracts (lanes 1–3) and immunoprecipitated proteins (lanes 4–12) were visualized by Western blotting using anti-ER{alpha} or anti NF-YA, as indicated.

 
These data indicate that the NF-Y trimer is able to associate with the ER{alpha} transcription complex and that this interaction occurs mainly through the NF-YA subunit. These results provide a putative molecular mechanism for the NF-Y antagonistic effect on ER{alpha}-mediated trans-activation of the FXII gene promoter.

Mapping of NF-YA domains required for trans-repression of ER{alpha}-mediated trans-activation of FXII promoter
The results obtained from transient transfection experiments, EMSA, and immunoprecipitation show a repressive action of NF-Y on ER{alpha}-mediated trans-activation of the FXII promoter, which appears to occur in a DNA binding-independent fashion and probably through interaction of NF-YA with the ER{alpha} transcription complex.

To characterize which portion of the NF-YA molecule was responsible for the association with ER{alpha}, different NF-YA deletion mutants fused to the Gal4 DNA-binding domain (see schematic diagram in Fig. 6BGo) were used in transient cotransfection experiments in the presence or absence of the ER{alpha} expression vector (Fig. 6AGo). Immunoprecipitation of whole cell extracts with anti-ER{alpha} antibody and subsequent staining with anti-Gal4 antibody showed that the carboxyl-terminus of NF-YA (YA-2) was responsible for the association with ER{alpha}, whereas the subunit NH2 portion (YA-6) was unable to interact with the receptor complex. In particular, the serine/threonine-rich domain of NF-YA (YA-5) appeared sufficient to elicit the protein-protein interaction.



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  		Figure 6. NF-YA domains involved in physical and functional interaction with ER{alpha}. A, HeLa cells were cultured as described in Fig. 2Go and cotransfected with ER{alpha} and NF-YA deletion mutants YA2, YA5, and YA6 (see diagram in B) fused to the DNA-binding domain of the yeast Gal4 protein (0.125 µg/each). Whole cell extracts were either visualized by Western blotting using anti-Gal4 antibodies (left panel) or immunoprecipitated with anti-ER{alpha} antibodies and subsequently electrophoresed, blotted, and stained with anti-Gal4 antibodies (right panel). B, HeLa cells were cultured and cotransfected as described in Fig. 2Go, except that NF-YA deletion mutants YA2, YA5, and YA6 (1.25 µg each) were added to transfection mix, as indicated. Data are expressed as counts per min/U ß-galactosidase x 10-3 in the absence (-E2) or presence (+E2) of E2. The figure shows results from a representative experiment of three independent experiments, each performed in duplicate. Left, Schematic diagram of immunoprecipitation results, NF-YA deletion mutants, and protein domains (Q, glutamine-rich; S/T, serine/threonine-rich; HAP2, yeast HAP-2 homology domain).

 
Functional experiments were also performed by using the same deletion constructs in the presence or absence of the ER{alpha} expression vector and the PT-CAT181 reporter (Fig. 6BGo). The integrity of the carboxyl-terminal HAP-2 domain, a region that shares high degree homology with HAP-2, the yeast homologue of NF-YA, as in construct YA-2, was strictly required to elicit repression of activated ER{alpha} trans-activation of the FXII promoter (P < 0.01 vs. control). All NF-YA mutants deleted in this region were, in fact, ineffective in inhibiting E2-mediated induction of FXII promoter activity.

These results map the NF-YA domain required for interaction with ER{alpha} to the carboxyl region of the NF-YA molecule, whereas NF-YA trans-repression of ER{alpha}-mediated induction of FXII promoter appears confined to the COOH-terminal HAP-2 domain.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The FXII gene may be considered a sensitive target of estrogen action, because it is one of the few members of the clotting factor family whose plasma levels are induced even with a low estrogen therapeutic regimen (34). This FXII property may be of relevance in view of its major fibrinolytic role and the antithrombotic, cardiovascular and cerebrovascular protective effects exerted by estrogen replacement therapy in postmenopausal women (35). In this study we demonstrate that ER{alpha}-mediated trans-activation of the FXII promoter is strongly inhibited by the ubiquitous heteromeric transcription factor NF-Y. To our knowledge this is the first evidence of an antagonistic role played by NF-Y toward the activity of another transcription factor.

The NF-Y complex, also known as CCAAT-binding factor, is composed of three highly conserved subunits, NF-YA, NF-YB, and NF-YC (25, 36), that must be associated to bind DNA (37, 38, 39). The activity of NF-Y is mediated by interaction of the trimeric complex with the CCAAT box, a DNA element present in about 30% of eukaryotic promoters (40), mostly among members of large gene families such as {alpha}-collagens, globins, histones, cell cycle-regulated genes, coagulation factors, etc. (41). NF-Y appears to play an important role both in basal transcription (26), by promoting reinitiation of transcription, and in activated transcription, by cooperating with other factors in eliciting their specific transcriptional activities. Indeed, the increase in basal transcription activity of FXII promoter observed in our experiments after forced NF-Y expression may be attributed to this cooperating action of NF-Y with components of the basal transcription machinery (26). The CCAAT box, in fact, is usually flanked by at least one functionally important promoter element, and the distance between the two elements is crucial for transcriptional interaction between the two factors. This is also the case for human FXII promoter, in which a computerized homology search showed an overlap between the the 3'-half-site of the previously identified ERE and a putative CCAAT element.

Several mechanisms may underlie the reciprocal interplay between NF-Y and other transcription factors. In some cases NF-Y considerably increases the affinity of the neighboring transcription factors for its cognate DNA-binding site, and the increased stability of the two complexes on DNA partly explains the transcriptional synergism (42). In the farnesyl diphosphate synthase promoter, a gene involved in cholesterol metabolism, functional cooperation between sterol regulatory element-binding protein 1a and NF-Y on adjacent sterol regulatory element and CAAT binding sites occur in a sterol-regulated manner. In vitro, sterol regulatory element-binding protein 1a binds the sterol regulatory element very weakly unless recombinant NF-Y is added (43).

The sequence overlap between ERE and the CCAAT box led us to speculate that the observed NF-Y repression of ER{alpha}-mediated trans-activation of the FXII promoter could be due to mutual competition between the two transcription factors in binding their cognate DNA-binding sites. However, our results clearly demonstrated that this is not the case, as both functional and binding studies showed that the NF-Y inhibitory effect occurred through a DNA binding-independent mechanism. This hypothesis is further confirmed by the observation of a similar NF-Y-mediated inhibition of activated ER{alpha}-dependent trans-activation in the VIT promoter that does not contain any CCAAT box surrounding the canonical ERE. Lack of DNA binding by NF-Y could be due to a lower affinity for the FXII-ERE-CCAAT box. However, competition-binding experiments in the presence of increasing concentrations of recombinant NF-Y still did not show any DNA binding, nor did they produce any interference with binding of ER{alpha} to FXII-ERE. A careful analysis of the sequences surrounding the core CCAAT pentanucleotide revealed the presence of noncanonical nucleotides at the 3'-end, diverging from the extended consensus C,G/A,G/A,C,C,A,A,T,C/G,A/G,C,A/C sequence (41).

A second mechanism through which NF-Y interacts with other transcription factors is that described by Milos and Zaret in the albumin promoter (44). This system resembles that of the FXII promoter, in that the albumin promoter contains a CCAAT box included in a CCAAT/enhancer-binding protein-binding site. The sequence overlap results in mutually exclusive DNA binding between NF-Y and CCAAT/enhancer-binding protein. However, despite the lack of cooperation in DNA binding, the two factors functionally synergize in promoting transcription, suggesting a precise mechanism requiring protein-protein contacts with portions of the partner transcription factor other than the DNA-binding domain. Moving the CCAAT box 10 bp away, in fact, allowed simultaneous binding of both factors, but caused the loss of transcriptional synergism (44).

Several factors have been reported to bind NF-Y in the absence of DNA. Among others, the liver-enriched nuclear receptor HNF-4 (16) has been demonstrated to interact with NF-YA both in solution and in a yeast two-hybrid system (45). The Tax1 protein, the activator of the oncogenic human T cell lymphotropic virus type 1 virus, interacts with NF-YB and is immunoprecipitated by anti-NF-YA antibodies, suggesting in vivo association with the trimer (46). A direct, DNA-independent cooperative interaction between the general transcription factor stimulating protein 1 and the NF-YA subunit on the fatty acid synthase gene promoter has recently been described (47). Therefore, at least in some transcriptional units, protein-protein interactions, that are independent from cooperative DNA binding may be adopted by NF-Y to cross-talk with other transcription factors. Our in vivo immunoprecipitation experiments show that NF-Y is coimmunoprecipitated with ER{alpha}, enlightening a novel role for NF-Y as a partner of the ER{alpha} multiprotein transcription complex. As experiments were performed using crude whole cell extracts, ER{alpha}/NF-Y coimmunoprecipitation could well be mediated by third interacting partners.

The major role in eliciting NF-Y inhibition of ER{alpha}-mediated induction of FXII promoter is played by NF-YA, which carries most of the inhibitory potency expressed by the whole NF-Y complex. In fact, NF-YA mutants, unable to bind DNA or to participate in trimer formation, still retain estrogen inhibitory action. In vivo immunoprecipitation experiments with NF-YA deletion mutants showed that the serine/threonine and the Heme activated protein 2 homology domains were important for interaction with the ER{alpha} transcription complex. In functional experiments the Heme activated protein 2 domain, located at the COOH-terminal, appeared necessary to elicit inhibition of ER{alpha}-mediated trans-activation of the FXII promoter, as all mutants devoid of this region failed to exhibit trans-repression.

A possible mechanism for NF-Y antagonistic action could be by interfering with the interaction of ER{alpha} with coactivators. Recent reports have demonstrated the association of NF-Y with histone acetyltransferase (HAT) activities, which could be immunoprecipitated by anti-NF-Y antibodies. Interaction of HAT proteins hGCN5, P/CAF, and p300 with NF-YA and NF-YB subunits has been recently documented (48, 49, 50), and coexpression of NF-Y and the HAT domain of P/CAF increases in vivo multidrug resistance 1 promoter activity (48). However, attempts to rescue full ER{alpha}-mediated transcriptional induction of FXII promoter by overexpressing coactivators P/CAF or p300, which also interacts, directly or indirectly, with ER (51, 52), have been unsuccessful (data not shown). Therefore, squelching of these cofactors by NF-Y could be ruled out. A second hypothesis, which remains to be elucidated, is that NF-Y may prevent ER{alpha} interaction with components of the basal transcription machinery such as TATA binding protein and/or TATA-associated factor (TAFIIs). NF-Y, in fact, has been shown to interact with TBP, via the conserved domains of NF-YB/NF-YC (37), and with Drosophila TAFII110, via the Q-rich activating regions of NF-YA (53).

In conclusion, this study provides the first evidence of an antagonistic role for the heteromeric transcription factor NF-Y toward a member of the steroid/thyroid nuclear hormone receptor superfamily and introduces NF-Y as a novel functional partner of the ER{alpha} transcription complex.


   Acknowledgments
 
We are grateful to Giulia Piaggio (Regina Elena Cancer Institute, Rome, Italy) for suggestions and helpful discussion.


   Footnotes
 
This work was supported by research grants from Ministero dell’Università e della Ricerca Scientifica e Tecnologica, Ministero della Sanità, and Associazione Italiana per la Ricerca sul Cancro.

1 A.F. and M.N. equally contributed to this study. Back

Abbreviations: CAT, Chloramphenicol acetyltransferase; CMV, cytomegalovirus; DTT, dithiothreitol; E2, 17ß-estradiol; ERE, estrogen response element; FXII, coagulation factor XII; HAT, histone acetyltransferase; HNF-4, hepatocyte nuclear factor-4; Hsp, heat shock protein; MBP, myelin basic protein; VIT, vitellogenin B1.

Received January 9, 2001.

Accepted for publication April 23, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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