©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Vitamin D Receptor Interacts with General Transcription Factor IIB (*)

(Received for publication, September 16, 1994; and in revised form, November 10, 1994)

Paul N. MacDonald (1)(§) David R. Sherman (3) Diane R. Dowd (2) Stephen C. Jefcoat Jr. (1) Robert K. DeLisle (1)

From the  (1)Department of Pharmacological and Physiological Science and the (2)E. A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University Health Sciences Center, St. Louis, Missouri 63104 and the (3)PathoGenesis Corporation, Seattle, Washington 98119

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The vitamin D receptor (VDR) heterodimerizes with retinoid X receptors (RXR) on many vitamin D-responsive promoter elements, suggesting that this complex is the active factor in vitamin D-mediated transcription. However, the mechanism of transcriptional regulation following VDRbulletRXR binding to DNA is not well characterized. Using a yeast two-hybrid protein interaction assay, we demonstrate that VDR forms specific protein:protein contacts with the basal transcription factor TFIIB. Deletion analysis indicated that the carboxyl-terminal ligand binding domain of VDR interacted with a 43-residue amino-terminal domain in TFIIB. The interaction with TFIIB showed selectivity for the ligand binding domain of VDR as similar regions of RXRalpha or of retinoic acid receptor alpha did not couple with TFIIB. Binding assays with purified proteins showed a direct interaction between VDR and TFIIB in vitro. These data suggest a mechanism for VDR-dependent transcription in which protein contacts between VDR and TFIIB may impart regulatory information to the transcription preinitiation complex.


INTRODUCTION

The biological effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) (^1)are mediated through a soluble protein termed the vitamin D receptor, or VDR. The VDR is a member of the superfamily of nuclear receptors for steroid hormones, thyroid hormone, and retinoids. As such a member, the VDR acts as a ligand-induced transcription factor that binds to specific DNA elements in vitamin D-responsive genes and ultimately influences the rate of transcription(1) . Purified VDR does not bind to authentic vitamin D-responsive elements with high affinity unless an additional nuclear protein, designated NAF or RAF (for nuclear or receptor auxiliary factor) is present(2, 3, 4) . RAF may be related to retinoid X receptors (RXRs) since RXRs mimic RAF activity (5, 6, 7) and highly purified RAF contains RXR immunoreactivity(7) . Involvement of RXR in vitamin D function is supported by the observations that vitamin D-dependent transcription is augmented by exogenous RXR in transient expression systems (5, 7) and that numerous VDR mutants that do not interact with RXR also fail to activate transcription in vivo(8) . Thus, in one current hypothesis, 1,25(OH)(2)D(3)-dependent gene expression is mediated by heterodimeric binding of VDR and RXR to the vitamin D-responsive element with the heterodimer serving as the functional transcription factor.

The mechanisms that link receptor:response element binding to alterations in RNA polymerase II transcription are not well understood but are likely to involve protein:protein interactions between the receptor and the transcription preinitiation complex. Transcription factors IIB and IID (TFIIB and TFIID) are key targets for transactivator interactions, and, in many cases, additional adapter proteins function to mediate contacts between the transactivator and these basal factors. In this regard, TAF110 links transcription factor Sp1 to TFIID(9, 10) , TAF40 is involved in VP16/TFIIB interactions(11) , and a putative coactivator also links phosphorylated cAMP response element binding protein to TFIIB(12) . Alternatively, transactivators may contact components of the basal transcription machinery directly. Both the acidic activation domain of VP16 and the glutamine-rich activation domain of the Ftz protein interact directly with TFIIB(13, 14, 15) . Furthermore, progesterone receptor, estrogen receptor, thyroid hormone receptor, and chicken ovalbumin upstream promoter transcription factor interact with TFIIB, suggesting that steroid receptor function is mediated through TFIIB interactions(16, 17) . Presently, the mechanisms linking VDRbulletRXR heterodimers to the transcription preinitiation complex are not known. In the current study, we demonstrate that VDR forms a highly specific, direct protein:protein contact with TFIIB. The carboxyl-terminal portion of VDR contacts TFIIB, whereas similar regions of RXR or of retinoic acid receptor (RAR) do not associate with TFIIB.


MATERIALS AND METHODS

Preparation of Two-hybrid Expression Vectors

All two-hybrid plasmid constructs used the pAS1 and pGAD.GH yeast expression vectors(18, 19) . The HincII/EcoRI fragment of human RXRalpha cDNA was subcloned into SmaI-EcoRI-digested pGAD.GH. This vector generates a fusion between the GAL4 activation domain and RXRalpha (Thr-Thr). AS1-RARalpha contains the ScaI-EcoRI fragment from murine RARalpha cDNA subcloned in frame with the Gal4 DNA binding domain in pAS1. The AS1-VDR constructs were generated by inserting restriction enzyme fragments of the human VDR cDNA into the multiple cloning site of pAS1. All amino-terminal deletion constructs were sequenced through the fusion point to confirm that the reading frame was maintained. To produce AS1-VDR(93-427), human VDR cDNA was amplified by polymerase chain reaction using the following primer pairs: 5`-tcctgaaTTCATTCTGACAGATGAGGA-3` and 5`-acttggaTCCTAGTCAGGAGATCTCAT-3`. The amplified product was digested with EcoRI and BamHI and inserted into the pAS1 plasmid. AS1-SNF1 and GAD-SNF4 (pSE1112 and pSE1111, respectively) were previously described(18) .

Yeast Transformation and Library Screening

The HeLa cell cDNA library in the pGAD.GH vector was previously described (19) . The library was cotransformed with pAS1-VDR(93-427) into the yeast strain HF7c (ura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3,112 gal80-538 gal4-542 LYS2::GAL1-GAL1-HIS3 URA3:: GAL4-CYC1-lacZ), which was made competent with lithium acetate(20) . Transformants were plated on media lacking leucine, tryptophan, and histidine and containing 5 mM 3-amino-1,2,4-triazole. 3-Amino-1,2,4-triazole is a chemical inhibitor of imidazole glycerol phosphate dehydratase, the product of the HIS3 gene, thereby overcoming residual HIS3 expression in the HF7c strain. Colonies were assayed for beta-galactosidase expression using a colony lift filter assay(19) .

Liquid beta-Galactosidase Assays

The indicated plasmid pairs were transformed into HF7c yeast, plated on media lacking leucine and tryptophan, and incubated for 4 days at 30 °C. Triplicate colonies were grown overnight in LeuTrp liquid media, diluted in nutrient-rich media (A = 0.2), and grown to late log phase (A = 0.8). Cells were assayed for beta-galactosidase activity as described(21) .

Production of GST Fusion Proteins and Precipitation of VDR with Glutathione-Agarose

The NcoI/EcoRI fragment of human RXRalpha was converted to a blunt-ended fragment and inserted into the SmaI site of pGEX-KT(22) . The resulting plasmid produces an in frame fusion between GST and RXRalpha from Met-Thr. Full-length TFIIB and various deletion mutants were subcloned into pGEX-KT as well. All fusion proteins were expressed in the DH5alpha strain of bacteria and purified with glutathione-agarose(22) . Full-length VDR and RXRbeta were expressed in a baculovirus expression system(7) . Individual GST fusion proteins (7.5 µg) were incubated with 1.3 µg of VDR in 100 µl of GBB buffer (20 mM Tris-Cl, pH 7.6, 100 mM NaCl, 0.1% Nonidet P-40, 1 mM dithiothreitol, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 50 µg/ml pepstatin A, and 0.1 mM phenylmethylsulfonyl fluoride). VDR was incubated with and without 1,25(OH)(2)D(3) at a final concentration of 0.8 times 10M. After 1 h at 4 °C, 20 µl of a 50% slurry of glutathione-agarose was added, and, after 30 min at 4 °C, associated proteins were pelleted by centrifugation. The pellet was washed 5 times in 100 volumes of GBB buffer, solubilized in SDS sample buffer, and analyzed for VDR content by Western analysis. Immunoanalysis was as previously described (3) except a peroxidase-conjugated goat anti-rat IgG was used as secondary antibody and development was with a chemiluminescent substrate.


RESULTS

Two-hybrid Protein Interaction Screening System for VDR

Based on the discovery that VDR forms heterodimers with RXR and RAF, a search was initiated to identify VDR-specific interactive proteins with a two-hybrid system described by Hannon et al.(19) . The initial constructs are illustrated in Fig. 1A. One hybrid construct contained the Gal4 DNA binding domain (amino acids 1-147) fused to the carboxyl-terminal region of VDR (Phe-Ser) and was termed AS1-VDR(93-427). A second hybrid construct contained the Gal4 activation domain (amino acids 786-881) fused to the carboxyl-terminal region of human RXRalpha (Thr-Thr), termed GAD-RXR(223-462). Interaction between the VDR and RXR domains will bring the Gal4 activation and DNA binding domains in close proximity and lead to transcriptional activation of Gal4-responsive reporter genes. To aid in the elimination of false positives, two Gal4-regulated sequences are present in HF7c yeast: 1) the lacZ gene controlled by the CYC1 basal promoter and multiple GAL4-responsive elements and 2) the HIS3 reporter gene driven by the GAL1 promoter (Fig. 1A). Consequently, interaction between AS1-VDR(93-427) and GAD-RXR(223-462) fusion proteins is characterized by monitoring the expression of beta-galactosidase activity and by monitoring the growth of HF7c on selective media lacking histidine.


Figure 1: Specific interactions of VDR with RXRalpha and with TFIIB in a two-hybrid system. A, the two-hybrid protein interaction assay. The Gal4 DNA binding domain (DBD)-VDR hybrid protein and the Gal4 activation domain (ACT)-RXRalpha hybrid protein are illustrated schematically. The interaction of the VDR and RXR domains results in Gal4-dependent transcription of the lacZ and HIS3 reporter sequences that are integrated into the HF7c yeast genome. B, growth characteristics of various two-hybrid combinations on His-deficient media. Individual colonies were streaked on media containing histidine (leftplate) or lacking histidine (rightplate) to identify plasmid combinations that resulted in expression of the HIS3 gene. Area1, AS1-VDR(93-427) + GAD-SNF4; area2, AS1-SNF1 + GAD-RXRalpha(223-462); area3, AS1-SNF1 + GAD-SNF4; area4, AS1-VDR(93-427) + GAD-RXRalpha(223-462); area5, AS1-SNF1 + GAD-TFIIB; area6, AS1-VDR(93-427) + GAD-TFIIB. Specific interaction is observed only with the following hybrid pairs: yeast SNF1 and SNF4 (area3), VDR and RXR (area4), and VDR and TFIIB (area6).



Initial characterization of the two-hybrid system is presented in Fig. 1B. The specificity of the VDR/RXR interaction was examined using SNF1 and SNF4, authentic interacting yeast proteins. The AS1-SNF1 and GAD-SNF4 constructs expressed functional proteins since yeast transformed with this combination grew well on His-deficient media (right plate, area 3). In contrast, yeast expressing either the VDR and SNF4 combination (area 1) or the SNF1 and RXR combination (area 2) did not grow, indicating that neither the VDR nor the RXR fusion proteins interacted with the SNF proteins in this system. Importantly, specific interactions between VDR and RXR were evident as yeast expressing AS1-VDR(93-427) and GAD-RXR(223-462) grew well on histidine-deficient media (area 4).

Isolation of TFIIB from a HeLa Cell cDNA Library Using AS1-VDR(93-427)

Having demonstrated specific interactions between VDR and a known partner (RXR), the AS1-VDR(93-427) plasmid was then used to screen a HeLa cell library constructed in the GAD.GH plasmid. Positive clones were selected for growth on His media, expression of beta-galactosidase activity, and specificity for interaction with the AS1-VDR(93-427) bait plasmid. The specificity controls included testing each positive clone against pAS1, pAS1-SNF1, and pAS1-RXRalpha. Out of about 250,000 individual clones examined in the initial screen, 12 positives conformed to these stringent criteria. DNA sequence analysis identified one of the clones as full-length human TFIIB. The remaining 11 clones are the subject of further study and will not be described here. Fig. 1B illustrates the growth properties of TFIIB in the two-hybrid system demonstrating a specific interaction with the VDR hybrid protein (area6), whereas no interaction was apparent with the AS1-SNF1 and GAD-TFIIB combination (area 5).

The Carboxyl-terminal Domain of VDR Interacts with the Amino-terminal Region of TFIIB

To identify the region of VDR that contacts TFIIB, several amino- and carboxyl-terminal deletion mutants of AS1-VDR were examined (Fig. 2A). Comparison of the AS1-VDR full-length construct to the AS1-VDR(93-427) and the AS1-VDR(116-427) mutants demonstrated that the zinc finger DNA binding domain of VDR is not critical for VDR-TFIIB interactions. However, eliminating the sequence between Leu and Ser of VDR abolished the interaction of VDR with TFIIB. Deleting the last 40 amino acids from the carboxyl-terminal tail in AS1-VDR(93-387) also eliminated VDR-TFIIB interactions. Thus, the extreme amino-terminal(116-165) and carboxyl-terminal(387-427) regions of the ligand binding domain comprised at least two of the domains in VDR essential for TFIIB-VDR association.


Figure 2: The ligand binding domain of VDR interacts with the amino-terminal domain of TFIIB. A, amino- and carboxyl-terminal deletion mutants of AS1-VDR were tested against GAD-TFIIB in the two-hybrid system. Relative growth on His-deficient plates was assessed after 4 days, and beta-galactosidase expression was quantitated in liquid cultures. Results are presented as the mean (± standard deviation) of triplicate cultures. The DNA box in the figure represents the DNA binding domain of VDR. B, amino-terminal and internal deletion mutants of TFIIB were constructed in the GAD.GH vector. Each TFIIB construct was examined for interaction with AS1-VDR by monitoring expression of the HIS3 and lacZ reporter genes in the two-hybrid system. Openboxes in the illustration represent the imperfect direct repeats in the amino acid sequence of TFIIB.



A similar analysis of TFIIB is presented in Fig. 2B. Elimination of most of the carboxyl terminus of TFIIB (Delta125-297 and Delta66-294) did not interfere with VDR-TFIIB interactions. In fact, these mutants appeared to interact with VDR better than full-length TFIIB, possibly due to the removal of interfering domains in TFIIB (23) . However, deleting the amino-terminal regions (Delta1-42 and Delta1-123) eliminated beta-galactosidase expression and growth on His-deficient media. Importantly, addition of the most extreme amino-terminal 43 residues to the Delta1-123 mutant partially restored activity, suggesting that this amino-terminal domain is sufficient to mediate TFIIB-VDR interactions.

Specificity of TFIIB Interactions

Next, we tested the specificity of this interaction with similar carboxyl-terminal domains in the related receptors, RARalpha and RXRalpha. As illustrated in Fig. 3, interactions between AS1-VDR and the GAD-RXR or GAD-TFIIB constructs were readily apparent (leftplate). However, similar regions of RXR or RAR did not interact with TFIIB as indicated by the inability of the AS1-RXR or AS1-RAR fusion proteins to couple with the GAD-TFIIB fusion and activate HIS3 expression (middle and rightplates, respectively). However, these same protein domains of RXR and RAR mediated strong interactions of RXR with VDR (Fig. 3, middleplate) and of RAR with RXR (Fig. 3, rightplate), both of which are authentic protein:protein associations previously demonstrated(5) . These data indicated that TFIIB coupled specifically with the carboxyl-terminal domain of VDR and not with the carboxyl-terminal regions of RARs of RXRs.


Figure 3: Specificity of the interaction between VDR and TFIIB. HF7c yeast were transformed with AS1-VDR (leftplate), AS1-RXRalpha (middleplate), or AS1-RARalpha (rightplate) together with the indicated GAD.GH constructs. Individual colonies were streaked on His-deficient plates to identify plasmid combinations that result in HIS3 gene expression. Specific interactions were noted with AS1-VDR/GAD-RXR, AS1-VDR/GAD-TFIIB, AS1-RXR/GAD-VDR, and AS1-RAR/GAD-RXR.



VDR and TFIIB Proteins Interact in Vitro

Finally, the interaction of VDR and TFIIB was examined in vitro (Fig. 4). The carboxyl-terminal region of human RXRalpha and full-length TFIIB were expressed as fusion proteins with GST. The purified fusion proteins were incubated with VDR in the presence and absence of the 1,25(OH)(2)D(3) ligand. Protein complexes were precipitated with glutathione-agarose and washed extensively; the beads were analyzed for VDR content by Western analysis. Fig. 4A shows that both GST-RXRalpha (lanes8 and 9) and GST-TFIIB (lanes6 and 7) efficiently precipitated VDR while a 4-fold molar excess of GST did not (lanes4 and 5). Interestingly, GST-RXR precipitated over 8-fold more VDR when the latter was complexed with 1,25(OH)(2)D(3) (lane9) compared with unliganded VDR (lane8). This response was similar to 1,25(OH)(2)D(3)-enhanced solution heterodimers of VDR and RAF previously reported(4) . In contrast, VDR-TFIIB interactions were relatively independent of ligand (compare lanes6 and 7).


Figure 4: Interaction of VDR with TFIIB and with RXR in vitro. A, ligand-dependent interactions. Baculovirus-expressed full-length VDR (1.3 µg) was incubated with 15 µg of GST (lanes4 and 5), 7.5 µg of GST-TFIIB (lanes6 and 7), or 7.5 µg of GST-RXRalpha (lanes8 and 9) in the absence or presence of 0.8 times 10M 1,25(OH)(2)D(3). Protein:protein complexes were precipitated with glutathione-agarose, washed extensively, and analyzed for human VDR by Western immunoblot analysis. Lanes1 and 2 represent 2.5% of the total baculovirus-expressed human VDR present in the interaction assay. B, role of the TFIIB amino terminus in VDR-TFIIB interactions. Wild-type GST-TFIIB (lane 1), GST-TFIIB(Delta45-124) (lane 2), and GST-TFIIB(Delta1-124) (lane 3) were examined for their ability to interact with VDR as described above.



Fig. 4B illustrates the effect of several TFIIB deletion mutants in the in vitro VDR interaction assay. In agreement with the results of the two-hybrid system, the TFIIB(Delta1-124) mutation did not interact appreciably with VDR, while addition of amino acids 1-43 to this construct (lane2) partially restored interaction with VDR compared with wild-type TFIIB (lane1). Thus, these data indicate that the amino terminus of TFIIB mediates interactions with the VDR both in vivo and in vitro.


DISCUSSION

A central question in eukaryotic transcription concerns the mechanism by which site-specific activators stimulate the transcriptional process. One current hypothesis suggests that activators function by contacting components of the RNA polymerase II preinitiation complex. The basal transcription factor TFIIB is important in this regard. TFIIB assembly into the preinitiation complex is a rate-limiting step that is increased by direct interaction with a transactivator such as VP16(13, 14) . Furthermore, a TFIIB mutant that does not interact with VP16 still functions in basal transcription but not in VP16-activated transcription(24) . Thus, interaction between an acidic activator and TFIIB is necessary for transcriptional activation in some systems. In the present study, we demonstrate that TFIIB forms a highly specific protein:protein contact with the VDR. The interaction was apparent both in vivo in the yeast two-hybrid interaction system and in vitro in protein interaction assays with purified TFIIB and VDR. Thus, TFIIB may be a pivotal link in the communication between VDR and the transcription preinitiation complex during vitamin Dmediated transcription. However, functional analysis of VDR and/or TFIIB mutants will be required to test this hypothesis.

The carboxyl-terminal region of TFIIB contains two imperfect repeats of approximately 75 amino acids with a cluster of basic residues in the first repeat that may fold into an amphipathic alpha-helix(25, 26) . A conserved cysteine-rich sequence is present in the amino terminus that may form a zinc binding motif(27) . Limited proteolysis and deletion analysis reveal that these two domains are functionally distinct(28, 29) . The amino terminus may be required for recruitment of RNA polymerase II and/or TFIIF, while the carboxyl-terminal region interacts with the TATA binding protein-DNA complex. The bipartite nature of TFIIB is also evident in its interactions with VDR in that the carboxyl-terminal domain of TFIIB is dispensable for VDR coupling while the amino terminus is absolutely required. In contrast, VP16 couples to the putative amphipathic alpha-helix of TFIIB(24) , and this region is not involved in VDR-TFIIB interactions. Thus, the activation domains of different classes of transcription factors may ultimately influence gene expression by interacting with distinct domains on TFIIB.

Several other steroid-thyroid receptors interact with TFIIB, and different regions of each receptor mediate this interaction(16, 17) . TFIIB interacts with the carboxyl-terminal ligand binding domain of the estrogen receptor(17) , with two distinct domains of the thyroid hormone receptor (16) and with the DNA binding domain of the progesterone receptor(17) . In comparison, the amino-terminal DNA binding domain of VDR was not crucial for VDR-TFIIB interactions (Fig. 2A). Multiple requisite domains in the carboxyl terminus of VDR were essential. Removal of either Leu-Ser or Leu-Ser completely abolished VDR-TFIIB interactions, and neither region on its own was sufficient to promote strong interactions with TFIIB. Both regions are rich in hydrophobic and charged amino acid residues, and, specifically, the region between Leu and Tyr has the propensity to form an amphipathic alpha-helix that may be potentially important in VDR-TFIIB interactions.

Recent mutagenesis of VDR suggests that several widely spaced regions of the ligand binding domain also mediate VDR-RXR and VDR-RAF interactions(8) . The extreme carboxyl-terminal residues of the VDR are also critical for VDR-RXR interactions, indicating that TFIIB and RXR may interact with a common domain in VDR. However, one aspect of the present study suggests that distinct domains of VDR interact with TFIIB and RXR since VDR-RXR interaction was enhanced by 1,25(OH)(2)D(3), whereas the VDR-TFIIB interaction was ligand independent (Fig. 4). It is conceivable that a ligand-induced conformational change exposes a domain of the VDR involved in heterodimerization with RXR, and this domain is not directly involved in VDR interactions with TFIIB. More refined mutagenesis and binding studies are required to test this hypothesis.

With regard to specificity, we noted a clear preference of TFIIB for the carboxyl terminus of VDR. TFIIB did not interact to the same extent with the carboxyl terminus of RXR (Fig. 3) nor did GST-TFIIB interact appreciably with murine RXRbeta in our in vitro assay (data not shown). Our studies do not rule out the possibility that TFIIB interacts with other isoforms or with more amino-terminal domains of RARs or RXRs. However, a nearly full-length RXRalpha construct (AS1-RXRalpha (Glu-Thr)) showed no interaction with the TFIIB fusion in the two-hybrid system (data not shown), supporting the concept that RXR does not interact appreciably with TFIIB under these conditions. It is an intriguing possibility that the RXR portion of the heterodimer may contact the preinitiation complex through a distinct protein, such as another general transcription factor or a bridging protein. Regardless, these data indicate that fundamental differences exist in the mechanisms leading to transcriptional regulation by the nuclear receptors for retinoids and vitamin D.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant R29DK47293 (to P. N. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Saint Louis University School of Medicine, Dept. of Pharmacological and Physiological Science, 1402 S. Grand Blvd., St. Louis, MO 63104. Tel.: 314-268-5318; Fax: 314-577-8233.

(^1)
The abbreviations used are: 1,25(OH)(2)D(3), 1,25-dihydroxyvitamin D(3); VDR, vitamin D receptor; RXR, retinoid X receptor; TFIIB, transcription factor IIB; RAF, receptor auxiliary factor; RAR, retinoic acid receptor; GST, glutathione S-transferase.


ACKNOWLEDGEMENTS

We gratefully acknowledge Gregory J. Hannon and David Beach for the GAD.GH vector, the GAD.GH-HeLa cDNA library, and the HF7c strain of yeast. We also recognize Stephen J. Elledge for providing the pAS1, pSE1111, and pSE1112 plasmids and Ronald Evans for the retinoid receptor cDNAs. We thank Mark R. Haussler and Carol A. Haussler for the VDR cDNA, for several cell lines, and for cell culture advice. We acknowledge Mark Johnston and Dan Goldberg for helpful discussions and support.


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