YY1 Represses Vitamin D Receptor-Mediated 25-Hydroxyvitamin D3 24-Hydroxylase Transcription: Relief of Repression by CREB-Binding Protein

Mihali Raval-Pandya, Puneet Dhawan, Frank Barletta and Sylvia Christakos

Department of Biochemistry and Molecular Biology University of Medicine and Dentistry of New Jersey-New Jersey Medical School Newark, New Jersey 07103


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ying Yang transcription factor (YY1) can repress or activate transcription. 25-Hydroxyvitamin D3-24-hydroxylase [24(OH)ase], an enzyme involved in the catabolism of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], is up-regulated at the transcriptional level by 1,25-(OH)2D3 to self-induce its deactivation. Here we report that YY1 can repress 1,25-(OH)2D3-induced 24(OH)ase transcription in CV1 cells transfected with vitamin D receptor (VDR) expression vector or in LLCPK1 cells that contain VDR endogenously. With increasing amounts of YY1 DNA transfected (500 ng to 2 µg), ligand-dependent VDR activation of 24(OH)ase transcription was steadily repressed (maximum repression was 10-fold). Thus, YY1 may be a key modulator preventing activation at times that do not require the enzyme to be expressed. Relief of YY1 repression was observed in the presence of TFIIB or CBP (CREB binding protein) suggesting that YY1 may exert repression, in part, by sequestering TFIIB/CBP. Glutathione-S-transferase (GST) pull-down assays identified regions in the N and C termini of CBP that can bind YY1. In addition, the N-terminal region of CBP that interacts with YY1 can inhibit YY1 from binding to TFIIB. Thus, CBP may alleviate YY1-mediated repression, in part, by preventing YY1 from binding to TFIIB, which is required for VDR-mediated transcription. In summary, our results suggest that YY1 represses 24(OH)ase transcription, at least in part, by sequestering activator proteins involved in VDR-mediated transcription. In addition, our findings demonstrate a role for CBP in relief of repression of VDR-mediated transcription.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The vitamin D receptor (VDR) is a member of the family of steroid/nuclear receptors that, when bound to their respective ligands, are induced to act as transcription factors (1, 2). 1,25 Dihydroxyvitamin D3 [1,25-(OH)2D3], the hormonally active form of vitamin D, binds to the VDR resulting in the concentration of 1,25-(OH)2D3-VDR in the nucleus and the formation of a nuclear VDR/retinoid X receptor (RXR) heterodimer. The VDR/RXR heterodimer binds to hormone response elements [vitamin D response elements (VDREs)] and activates or represses the transcription of specific target genes in intestine, bone, and kidney, resulting in the maintenance of calcium homeostasis (3). In addition, VDR mediates other physiological functions of 1,25-(OH)2D3 including inhibition of cytokine production, effects on hormone secretion, and inhibition of proliferation of cancer cells (3). Recent studies have begun to address the mechanisms involved in mediating the effects of the VDR. Transcription factor IIB (TFIIB) has been reported to be important for VDR-mediated transcriptional responses (4, 5), and the 1,25-(OH)2D3 ligand has been suggested to play an important role in the recruitment of TFIIB into the basal components of the preinitiation complex on 1,25-(OH)2D3 target genes (5). In addition to TFIIB and other general transcription factors (6, 7), VDR is also known to interact with three members of a family of proteins known as p160 coactivators. They are steroid receptor coactivator 1 (SRC-1) (NCOA1), glucocorticoid receptor interacting protein 1 (GRIP-1) (TIF-2), and activator of thyroid and retinoic acid receptors (ACTR) (pCIP), which interact with VDR in a ligand- and AF-2-dependent manner (8, 9, 10, 11). It has been suggested that binding of SRC-1 or ACTR to other liganded hormone receptors recruits additional nuclear factors including CBP (CREB binding protein) that may result in synergistic coactivation or competitive interactions (9, 12). Since coactivators have histone acetylase activity, remodeling of chromatin by histone acetylation as well as recruitment of TFIIB and other transcription factors into the basal complex appear to be involved in transcriptional activation by steroid hormones including 1,25-(OH)2D3 (9, 12- 14).

YY1 is a ubiquitous transcriptional regulator that contains zinc fingers and has been shown to activate, repress, or initiate transcription (15, 16, 17). YY1 has been reported to interact with a number of proteins involved in RNA polymerase II transcription including TFIIB and p300 (18, 19). YY1 inhibition of VDR-mediated transcription of the osteocalcin gene in ROS 17/2.8 osteoblastic cells was previously shown by Guo et al. (20). To determine whether YY1 can affect the transcription of other vitamin D-dependent genes in cells other than osteoblastic cells, the role of YY1 in VDR-mediated transcriptional activation of 25-hydroxyvitamin D3-24-hydroxylase [24(OH)ase], the enzyme involved in the catabolism of 1,25-(OH)2D3 (21, 22), was examined in renal cells (LLCPK1 cells that contain VDR endogenously and in CV1 cells transfected with VDR). We show that YY1 represses VDR-mediated 24(OH)ase transcription, suggesting that YY1 may be a key modulator of 24(OH)ase transcription, preventing activation at times that do not require catabolism of 1,25-(OH)2D3. Overexpression of TFIIB can relieve this repression. In addition, we show that YY1 and CBP can directly interact with each other, that overexpression of CBP can relieve YY1 repression, and that a mutant of CBP that does not interact with YY1 fails to relieve YY1 repression. We also provide evidence of a weak YY1 binding site in the rat 24(OH)ase promoter, consistent with previous findings indicating that weak binding is important to the mechanism by which YY1 represses transactivation since this facilitates transactivating factors to interact with YY1 and relieve repression (23).

In summary, our results suggest that YY1 is a regulator not only of osteocalcin but also of 24(OH)ase transcription and that YY1 has a role in regulating vitamin D-dependent genes in tissues in addition to bone. In addition, our results demonstrate a role for CBP in relief of repression of VDR-mediated transcription.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
YY1 Represses 1,25-(OH)2D3-Induced 24(OH)ase Transcription
YY1 can function as an activator or repressor of transcription (15, 16, 17), and previous studies by Guo et al. (20) showed that YY1 can inhibit VDR-mediated transactivation of osteocalcin (a bone-specific calcium binding protein) in ROS 17/2.8 osteoblastic cells. To determine whether YY1 can affect the transcription of other vitamin D-responsive genes in cells other than osteoblastic cells, transcriptional activation of 24(OH)ase by 1,25-(OH)2D3 in the presence of YY1 was examined in CV1 cells transfected with VDR expression vector and in LLCPK1 cells, which contain VDR endogenously. Cotransfection in CV1 cells of pCMV-YY1 with the rat 24(OH)ase promoter (-1,367/+74) and the VDR expression vector was found to consistently repress 1,25-(OH)2D3-induced transcription of rat 24(OH)ase (Fig. 1AGo). Dose-dependent studies indicated a maximal repression in the presence of 2 µg pCMV-YY1 of 10 fold. Similarly, in LLCPK1 cells, which contain VDR endogenously, YY1 was also found to repress rat 24(OH)ase transcription in a dose-dependent manner (Fig. 1BGo). In addition to inhibition of rat 24(OH)ase transcription, YY1 was also found to repress VDR-mediated transcription of the human 24(OH)ase gene in LLCPK1 cells (Fig. 1CGo, left panel). The transcription of the pSV2CAT reporter construct, which contains the SV40 minimal promoter region, was not affected by YY1 (Fig. 1CGo, right panel). In addition, using the -777/+79 promoter construct and transfection in ROS 17/2.8 cells, vitamin D-induced transcription of the mouse osteopontin gene was unaffected by YY1 [data not shown; similar results were observed by Guo et al. (20)], suggesting that the YY1 inhibitory effect is gene specific. Overexpression of YY1 was also observed to inhibit the endogenous expression of 1,25-(OH)2D3-induced 24(OH)ase mRNA in LLCPK1 cells (Fig. 2AGo). The level of VDR was unaffected by overexpression of YY1 (Fig. 2BGo).



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Figure 1. Repression of 1,25-(OH)2D3 Induction of 24(OH)ase Promoter Activity by YY1

A, Dose-dependent repression of 1,25-(OH)2D3 induction of rat 24(OH)ase promoter activity by YY1 in CV1 cells. CV1 cells were cotransfected with CAT reporter plasmid (4 µg rat 24(OH)ase promoter CAT construct -1,367/+74, 4 µg VDR expression plasmid pAVhVDR, and increasing amounts of pCMV-YY1 (0.5–2 µg). Empty vector (pCMV) was used to keep the total DNA concentration the same. A representative autoradiogram is shown in the left panel. On the right, CAT activity is represented as fold induction (mean ± SE; n = 3–10 observations per group) from basal after treatment with 10-8 M 1,25-(OH)2D3. Similar results were also observed in COS cells (not shown). B, Dose-dependent repression of 1,25-(OH)2D3 induction of rat 24(OH)ase promoter activity by YY1 in LLCPK1 cells containing endogenous VDR. Conditions for transfection of LLCPK1 cells were the same as for CV1 cells (panel A) but in the absence of transfected pAVhVDR. A representative autoradiogram is shown in the left panel. On the right CAT activity is represented as fold induction (mean ± SE; n = 3–10 observations per group) after treatment with 10-8 M 1,25-(OH)2D3 and was quantitated by comparison to basal levels. C, Left panel, Repression of 1,25-(OH)2D3 induction of human 24(OH)ase promoter activity by YY1. LLCPK1 cells were transfected with 4 µg human 24(OH)ase promoter luciferase construct -5,000/-22 in the presence of empty vector (pCMV) or 2 µg pCMV-YY1. Luciferase activity is represented as fold induction (mean ± SE; n = 3–4 observations per group) after treatment with 10-8 M 1,25-(OH)2D3 and was quantitated by comparison to basal levels (in the absence of 1,25-(OH)2D3 and pCMV-YY1). Right panel, YY1 does not affect the transcription of the pSV2CAT reporter construct.

 


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Figure 2. YY1 Inhibits 1,25-(OH)2D3-Induced 24(OH)ase mRNA but Does Not Affect VDR Levels in LLCPK1 Cells

A, LLCPK1 cells transfected with vector alone or 2 µ g pCMV-YY1(+YY1) were treated with vehicle (-D) or 1,25-(OH)2D3 (10-7 M,+D) for 16 h. Northern blot was performed using 8 µg of poly(A+) RNA. The blot was probed for expression of 24(OH)ase mRNA and rehybridized with 32P-labeled 18S rRNA cDNA as a control for RNA loading. Results of three separate experiments indicated that in the presence of YY1 1,25-(OH)2D3-induced 24(OH)ase mRNA was 29.0 ± 6.0% of the 24(OH)ase mRNA levels induced by 1,25-(OH)2D3 in the absence of YY1. B, Western blot analysis was performed using 50 µg of nuclear proteins prepared from LLLCPK1 cells transfected with vector alone or 2 µg pCMV-YY1 (+YY1) and treated with vehicle (-D) or 1,25-(OH)2D3 (+D) for 16 h. Detection was by immunoblotting with a monoclonal anti-VDR antibody. Two additional experiments yielded similar results.

 
TFIIB Overexpression Relieves YY1-Mediated Repression of Rat 24(OH)ase Transcription
To gain insight into the mechanism of YY1-mediated repression of 24(OH)ase transcription, experiments were done in the presence of TFIIB, which can activate 1,25-(OH)2D3-dependent transcription (4, 5) and can bind to YY1 (18). When induction of 24(OH)ase transcription by 1,25-(OH)2D3 was suboptimal (2.5-fold activation), 1,25-(OH)2D3-induced transcription was significantly enhanced by TFIIB (1.8-fold, P < 0.05; Fig. 3AGo). The activity of the control promoter pSV2CAT was not enhanced by the addition of TFIIB (Fig. 3BGo). In the presence of TFIIB (1–2 µg pCGN-TFIIB) a 60–70% reversal of YY1 repression of rat 24(OH)ase transcription in LLCPK1 cells was observed. Shown in Fig. 3CGo is the reversal of YY1-mediated repression of rat 24(OH)ase transcription in LLCPK1 cells in the presence of 1.5 µg TFIIB expression vector. A similar relief of YY1 repression by TFIIB was observed when CV1 cells transfected with VDR were used (not shown). In cells transfected with an expression vector encoding a mutant YY1 that does not interact with TFIIB (pCMV-YY1{Delta} 334–414; 24) YY1-mediated repression was not observed (Fig. 3DGo). Since we found that TFIIB enhanced 1,25-(OH)2D3-induced 24(OH)ase transcription when reporter activation was suboptimal, YY1 repression of rat 24(OH)ase transcription may be due to sequestration of TFIIB by YY1 and thus reduced activation.



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Figure 3. TFIIB Enhances 1,25-(OH)2D3-Induced 24(OH)ase Transcription and Relieves YY1-Mediated Repression

A, LLCPK1 cells were transfected with 4 µg rat 24(OH)ase promoter CAT construct (-1,367/+74) in the presence or absence of 1.5 µg pCGN TFIIB. Cells were treated with 10-9 M 1,25-(OH)2D3, which resulted in suboptimal reporter activation. A representative autoradiogram is shown in the left panel. On the right CAT activity is represented as fold induction (mean ± SE, n = 3). B, LLCPK1 cells were transfected with 4 µg of a control reporter plasmid that contains the early promoter of the SV40 5'-flanking region (pSV2CAT) in the presence or absence of 1 or 1.5 µg pCGN TFIIB. Similar results were observed in a duplicate experiment. C, LLCPK1 cells were transfected with 4 µg rat 24(OH)ase promoter CAT construct -1,367/+74 in the presence or absence of 2 µg pCMV-YY1 or 2 µg pCMV-YY1 cotransfected with 1.5 µg pCGN TFIIB. Empty vectors were used to keep the total amount of DNA transfected equivalent. A representative autoradiogram is shown in the left panel. On the right CAT activity is represented as fold induction (mean ± SE; n = 3–6 observation per group) after treatment with 10-8 M 1,25-(OH)2D3 and was quantitated by comparison to basal levels. D, LLCPK1 cells were transfected with 4 µg of rat 24(OH)ase promoter CAT construct in the presence or absence of 1.5 µg pCMV-YY1 {Delta}334-414 (mutant pCMV-YY1 which does not bind to TFIIB) or 1.5 µg pCMV-YY1. Similar results were observed using 2.0 µg pCMV-YY1 {Delta} 334–414.

 
Relief of YY1-Mediated Repression of Rat 24(OH)ase Transcription by CBP
To further understand the mechanism of YY1-mediated repression, the effect of CBP, which has been reported to be essential for ligand-dependent transcription of a number of nuclear receptors (9, 12), was examined. As shown in Fig. 4Go, overexpression of wild- type CBP resulted in a 37–80% reversal of YY1-dependent repression in CV1 cells. In renal LLCPK1 cells, which contain VDR endogenously, a similar relief of YY1-mediated repression by CBP was observed (data not shown). Wild-type p300, a close homolog of CBP, was also found to relieve YY1-dependent repression of 1,25-(OH)2D3-induced 24(OH)ase transcription (not shown). Transfection of pCMV-CBP (1 µg) in CV1 cells in the absence of YY1 did not result in an enhancement of 1,25-(OH)2D3-induced transcription (Fig. 4Go, left panel, -YY1). Similar findings were also observed in CV1 cells in the absence of YY1 when reporter activation by 1,25-(OH)2D3 was suboptimal (+D, 5.2 ± 0.4 fold induction; +D + 650 ng CBP, 6.1± 1.0 fold induction; +D + 750 ng CBP, 4.6 ± 0.5 fold induction: P > 0.5, +D vs. +D +CBP). In further studies, glutathione-S-transferase (GST) pull-down assays identified two regions of CBP that can bind to YY1 (1–771 and 1,892–2,441; Fig. 5Go). The interaction of YY1 with the N terminus of CBP was stronger than the interaction of YY1 with the C terminus (Fig. 5Go). Mutant CBP (aa 1–1,109), which retains the stronger N-terminal YY1 binding site, was able to relieve YY1 mediated repression (Fig. 6AGo). CBP (aa 1–452) was unable to relieve YY1 repression (Fig. 6AGo), suggesting that the region within the N terminus between aa 452 and 771 is important for YY1 binding. Further GST pull-down assays demonstrated interaction between CBP aa 451–721 and YY1 but not between CBP aa 1–451 and YY1 (Fig. 6BGo). Figure 6CGo shows that the interaction between YY1 and TFIIB can be inhibited by the N-terminal region of CBP that retains the YY1 binding site. Thus, CBP may relieve repression in part by preventing YY1 from binding TFIIB, which is required for VDR-mediated transcription.



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Figure 4. CBP Relieves YY1 Repression of VDR-Mediated 24(OH)ase Transcription

Left panel, Representative autoradiogram: 4 µg of the rat 24(OH)ase promoter CAT construct were cotransfected in CV1 cells with 4 µg pAVhVDR in the presence or absence of 1 µg pRSV-CBP (-YY1) or in presence of 2 µg pCMV-YY1 and 750 ng pRSV-CBP (+YY1). The cells were treated with vehicle (Basal) or 10-8 M 1,25-(OH)2D3 (+D). Right panel, CV1 cells were transfected with the rat 24(OH)ase promoter CAT construct and pAVhVDR in the absence of YY1 (-YY1 + D) or in the presence of 2 µg pCMV-YY1 or 2 µg pCMV-YY1 cotransfected with increasing concentrations of pRSV-CBP (500 ng, 750 ng, and 850 ng). CAT activity is represented as fold induction (mean ± SE; n = 3–10 observations per group) after treatment with 1,25-(OH)2D3 and was quantitated by comparison to basal levels.

 


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Figure 5. YY1 Interacts with Both the N Terminus and the C Terminus of CBP

GST or GST fusion proteins CBP-(1–771), CBP-(706–1,069), and CBP-(1,892–2,441) were bound to glutathione-Sepharose beads, and equal amounts of fusion proteins were incubated with in vitro translated 35S-labeled YY1. After extensive washing, the bound proteins were eluted in SDS loading buffer and analyzed by SDS-PAGE and fluorography. Lower panel, Schematic representation of CBP showing the regions that bind YY1.

 


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Figure 6. Mutant CBP, Which Does Not Bind YY1, Is Unable to Relieve YY1-Mediated Repression

A, Upper region: Schematic representation of CBP and CBP mutants used for transfection in CV1 cells. Left panel, Representative autoradiograms: 4 µg of the rat 24(OH)ase promoter CAT construct were cotransfected in CV1 cells with 4 µg pAVhVDR and 2 µg pCMV-YY1 in the absence or presence of 750 ng or 850 ng pRSV-CBP mutant (aa 1–1,109), which retains the stronger N-terminal YY1 binding site (left autoradiogram) or in the presence of 750 ng or 1 µg pRSV CBP mutant (aa 1–452), which does not bind YY1 (right autoradiogram). The cells were treated with vehicle (Basal) or 10-8 M 1,25-(OH)2D3. Right panel, Graphic representation of results obtained from CAT assays in the presence of 0 (-YY1+D) or 2 µg YY1 or 2 µg YY1 cotransfected with increasing concentration of pRSV-CBP mutant aa 1–1,109 (750 ng and 850 ng) or pRSV-CBP mutant aa 1–452 (750 ng and 1 µg). CAT activity is represented as fold induction (mean ± SE, n = 3–10 observations per group) after treatment with 10-8 M 1,25-(OH)2D3 and was quantitated by comparison to basal levels. B, GST fusion proteins CBP (1–441), CBP (451–721), CBP (1–771), and CBP (706–1069) were bound to Sepharose beads, and equal amounts of fusion protein were incubated with in vitro translated 35S-labeled YY1. After washing bound proteins were eluted in SDS loading buffer and analyzed by SDS-PAGE and fluorography. C, CBP disrupts the interaction between YY1 and TFIIB. Ten percent in vitro translated TFIIB (lane 1) was incubated with glutathione agarose beads bound to GST (lane 2), or GST YY1 (lanes 3–5). N'-terminal CBP (1–771), which interacts with YY1, ablates YY1-TFIIB interaction (lane 4). CBP (706–1,069), which does not interact with YY1, does not affect YY1-TFIIB interaction (lane 5).

 
Electrophoretic Mobility Shift Assays
The rat and human 24(OH)ase promoters contain two functional VDREs (25, 26). For both the rat and the human gene, the proximal VDRE has been reported to contribute more to transcriptional activation by 1,25-(OH)2D3 than the distal VDRE (25, 26). Examination of sequences adjacent to the proximal rat 24(OH)ase VDRE suggests binding of YY1 by sequence homology. The nucleotide sequence that can bind YY1 is 5'-CCATNTT-3'. The rat 24(OH)ase promoter contains a CCAT sequence (TCCATCCTCTTCC) nine nucleotides downstream of the proximal VDRE (25). A nearly identical CCAT sequence (differing in only one nucleotide) is located exactly nine nucleotides downstream of the human 24(OH)ase proximal VDRE (TCCATCCTCCTTCC) (26), and YY1 repression sequences have been previously identified within the osteocalcin VDRE (20). Since we observed inhibition of 1,25-(OH)2D3-induced rat 24(OH)ase transcription by YY1, electrophoretic mobility shift assays (EMSAs) were used to test protein-DNA interactions using a 50-bp 32P labeled oligonucleotide including the rat proximal VDRE and the adjacent CCAT sequence (VDRE + YY1). The oligonucleotide 5'-GAATTCGCCCTCACTCACCTCGCTGACTCCATCCTCTTCCCACACCATGG-3' was used (VDRE is shown underlined by a solid line; the homologous YY1 site is shown in italics). Purified GST-YY1 (100 ng) was unable to bind the 32P-labeled oligonucleotide including the rat proximal VDRE and the adjacent sequence (data not shown), suggesting that YY1 may require other factors to stabilize its binding or may require a posttranslational modification that is absent in our preparation. Unlike the rat 24(OH)ase VDRE-YY1 site, purified YY1 was able to bind to the YY1 consensus site (Fig. 7AGo). Lower affinity binding of the rat 24(OH)ase site for YY1 binding compared with the YY1 consensus site was demonstrated by weak competition of the VDRE-YY1 competitor oligonucleotide compared with the consensus YY1 oligonucleotide for binding to purified YY1 (Fig. 7AGo) or to YY1 nuclear proteins extracted from K562 cells, a cell line expressing high levels of YY1 (Fig. 7BGo). An oligonucleotide containing a consensus CRE (TCACGTCA) was unable to affect the intensity of YY1 binding (Fig. 7BGo). EMSAs using 32P-labeled VDRE-YY1 oligonucleotide and nuclear extracts (Fig. 7CGo) suggest that YY1 binds weakly to this site. Although YY1 binding to this site is weak, since VDR/RXR can also bind (Fig. 7CGo, lane 6), it is possible that YY1, by binding activator proteins, may diminish the integrator complex needed for VDR-mediated transcription. The promoter specificity of the repressive effect of YY1 is indicated by transcription assays using a synthetic VDRE-tk CAT construct that lacks a YY1 binding site. Repressor effects of YY1 were not observed using this construct (Fig. 7DGo).



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Figure 7. EMSAs Indicate That YY1 Binds Weakly on the Rat 24(OH)ase Promoter: Repressive Effects of YY1 Are Not Observed Using a Synthetic VDRE tkCAT Construct

A, Purified YY1 (100 ng) was incubated with a 32P-labeled YY1 consensus oligonucleotide (YY1 cons) in the absence or in the presence of 50-fold molar excess unlabeled YY1 consensus oligonucleotide (YY1 + cold YY1) or in the presence of 50- fold molar excess unlabeled rat 24(OH)ase VDRE-YY1 oligonucleotide (YY1 + cold VDRE-YY1). B, Binding of YY1 nuclear proteins (5 µg), extracted from K562 cells, to 32P-labeled YY1 consensus oligonucleotide (lane 1, probe alone; lane 2, labeled probe was incubated with nuclear proteins alone or in the presence of YY1 antibody (lane 3, note supershifted complex), or competitor oligonucleotides (unlabeled mutant YY1, designed to abrogate the YY1 binding motif, 100-fold molar excess, lane 4; cold VDRE-YY1 oligonucleotide, 50-fold molar excess, lane 5; cold VDRE-YY1 100-fold molar excess, lane 6; cold CRE, 50-fold molar excess lane 7; cold CRE, 100-fold molar excess, lane 8; cold YY1 consensus oligonucleotide, 100-fold molar excess, lane 9. C, YY1 nuclear proteins and VDR/RXR bind to 32P-labeled VDRE-YY1. Lane 1, probe alone; lane 2, labeled probe incubated with nuclear proteins (5 µg) alone or in the presence of YY1 antibody (lane 3, note supershifted complex) or competitor oligonucleotide (cold YY1 consensus oligonucleotide, 100-fold molar excess, lane 4; cold mutant YY1, 100- fold molar excess, lane 5). In lane 6, labeled probe was incubated with 20 ng purified VDR in combination with 6.5 ng FLAG-hRXR{alpha}. D, Four micrograms of a synthetic VDRE-tk CAT construct [multimers of the VDRE, GGTTCA cga CGTTCA inserted upstream of the tk transcription start site] were cotransfected in COS cells with 4 µg pAVhVDR in the absence (-YY1) or presence of 2 µg pCMV-YY1, and cells were treated with vehicle (Basal) or 10-8 M 1,25-(OH)2D3 for 16 h. Similar results were observed in six separate assays. Similar results were also observed using LLCPK1 cells, which contain VDR endogenously.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
YY1-mediated repression of 1,25-(OH)2D3-induced transcription was shown for the first time for the bone-specific osteocalcin gene (20). In this report we have demonstrated repression by YY1 of the rat and human 24(OH)ase genes. The activity of YY1 as a transcriptional repressor has been reported for various genes including skeletal {alpha}-actin (27), serum amyloid (SAA; Ref. 28), ß-casein (23, 29), MoMuLV LTR (30), sterol regulatory element protein-responsive genes (31), and {epsilon}-globin (32). 24(OH)ase is the most transcriptionally responsive vitamin D-inducible gene identified to date (3, 25). It is transiently induced at higher concentrations of 1,25-(OH)2D3 (>10-8 M), preventing hypercalcemia by resulting in a reduction of 1,25-(OH)2D3. In our studies we observed maximal repression by YY1 when activation was maximal (at higher ligand concentrations; data not shown). Our binding studies suggest that the VDRE-YY1 binding site in the rat 24(OH)ase promoter binds YY1 weakly (Fig. 7Go, A–C). It is of interest that SAA, which is transiently induced several hundred fold in response to inflammation, is also repressed by YY1 and a weak YY1 site is present in the SAA promoter (28). Unlike SAA and 24(OH)ase, the {alpha}-actin gene, which is also repressed by YY1 (27), is not transiently induced. It is developmentally regulated, has more long-term effects, and has a stronger binding site for YY1 (27). Thus, as previously suggested (28), the weak binding of YY1 to the rat 24(OH)ase or the SAA promoter may allow for YY1 to be displaced more readily when these genes are transiently induced allowing for efficient activation.

Various mechanisms have been proposed for transcriptional repression by YY1 including competition with transcriptional activators for overlapping cis-acting elements. YY1 overlaps the nuclear factor{kappa}B (NF{kappa}B) site in the SAA1 promoter (28). In addition, the YY1 site in the fos promoter (33) and in the {alpha}-actin promoter (27) overlaps the binding site for the serum response factor (SRF). YY1 sites also overlap serum response elements in the M isozyme of the creatine kinase gene (34), and YY1 competes with erythroid-regulatory protein GATA-1 for overlapping sites in the promoter of the 44 {epsilon}-globin gene (32). YY1 and the VDR/RXR heterodimer compete for binding to the VDRE in the rat osteocalcin promoter (20). In our study the YY1 CCAT core sequence was not within the VDRE (unlike the osteocalcin gene) but adjacent to the VDR binding sites. Repression of transcription of the ß-casein gene by YY1 was also found to be mediated by a site, not overlapping, but rather adjacent to the mammary gland factor (MGF) binding site (23, 29). The cooperative interaction of proteins binding to the adjacent sites was suggested as a mechanism involved in the repression of transcription of the ß-casein gene by YY1 (23) and may also be a mechanism involved in inhibition of 24(OH)ase transcription by YY1. It is of interest that, similar to our observation in the human and rat 24(OH)ase promoter, the YY1 site with the same sequence was present in the promoter of both the rat and mouse ß-casein genes at the same distance from the MGF site (29, 35, 36).

YY1 may also repress transcription by binding activators, interfering with the binding to the promoter, or by interacting with the basic transcription machinery and preventing transcription initiation. Consistent with this proposal, TFIIB and CBP, which bind to YY1 and have been reported to be involved in the activation of 1,25-(OH)2D3-dependent transcription, are able to relieve YY1-mediated repression (Figs. 3Go and 4Go). Thus, YY1 can exert a repressive effect by both DNA binding and interaction with transactivators or the transcription machinery. More than one mechanism of repression by YY1 was reported for effects on SAA1 transcription (28). In addition, the ability of YY1 to affect the activation of the c-myc promoter has been reported to occur in the absence of YY1 sites (37). Thus, YY1-protein interactions are relevant for the function of YY1. The C-terminal region of YY1, which contains the zinc fingers, as well as the Gly/Ala-rich region, has been shown to be required for repression (24). It is of interest that these regions are also required for binding to transcription factors TFIIB and CBP, as well as the TATA box binding protein (TBP) and TAFII 55 (24). None of these interacting factors bind to the transactivation domain of YY1 composed of two acidic regions in the N terminus. These findings suggest, similar to our studies with TFIIB and CBP, that interaction of YY1 with these factors is relevant for repression rather than activation.

The repression by YY1 of VDR-mediated transcription and relief of repression by TFIIB and CBP, as well as the binding of these factors to YY1 (18, 24) and to VDR (4, 5, 38), suggest physical as well as functional associations among YY1, CBP, TFIIB, and VDR. The role of TFIIB in VDR-mediated transcription has been shown to be cell type specific (4). The authors suggested that cell type-specific accessory factors may function together with VDR and TFIIB to mediate cooperative activation. Recent studies by Masuyama et al. (5) indicated that VDR and TFIIB also interact directly and that the amino-terminal zinc finger of TFIIB (aa 1–124) is essential for the VDR-TFIIB interaction. The interaction of TFIIB is with unliganded VDR and the 1,25-(OH)2D3 ligand disrupts the VDR-TFIIB complex (5). These findings suggest that VDR prerecruits TFIIB and, in the presence of ligand, TFIIB is released for assembly into the preinitiation complex to facilitate activated transcription. The basic region of TFIIB (aa 124–297) is important for the TFIIB-YY1 interaction (20), and N terminus of TFIIB has been reported to be essential for the VDR-TFIIB interaction (5). Thus, it is also possible that repression occurs in the presence of YY1 since TFIIB may be held by VDR as a complex with YY1 preventing ligand-dependent release of TFIIB for assembly into the preinitiation complex. Cooperative interactions between factors that bind the VDRE for the repressive action of YY1 was suggested in this study by EMSAs [the VDRE-YY1 site can bind YY1 (weakly) as well as VDR and RXR] and by protein-protein interactions. YY1 alone was unable to bind to the VDRE+YY1 site. CBP, but not mutant CBP, which lacks the YY1 binding site, was able to relieve YY1-mediated repression. YY1 has been shown to recruit histone deacetylases, and another mechanism of repression has been suggested to be through histone modification (39, 40). YY1-mediated repression was not relieved with trichostatin A, a potent inhibitor of histone deacetylase (Raval-Pandya, M., unpublished data). In addition, mutant CBP (aa 1–1,109), which retained the stronger N-terminal YY1 binding site, but does not contain the region known to possess histone acetylase activity (41), was able relieve YY1-mediated repression (Fig. 6Go). These findings suggest that relief from YY1-mediated repression of 24(OH)ase transcription by CBP does not involve effects on acetylation of histones. We suggest that CBP can relieve YY1-mediated repression, in part, by its ability to bind YY1, which prevents YY1 from inhibiting the association of activator proteins. Phosphorylation may also affect repression by YY1 since YY1 was unable to repress 1,25-(OH)2D3-induced 24(OH)ase transcription in the presence of okadaic acid, an inhibitor of protein phosphatase (Raval-Pandya, M., unpublished data).

In summary, our results suggest that YY1 represses 24(OH)ase transcription, at least in part, by sequestering activator proteins involved in VDR-mediated transcription. Since the same cofactors that interact with VDR are also involved in transcriptional activation mediated by other steroid hormones, our findings suggest the possibility that YY1 may also modulate the activity of specific genes activated by other steroid receptors. Our findings also demonstrate a role of CBP in relief of repression of VDR-mediated transcription.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
[{gamma}-32P]ATP, [35S]methionine, and [14C] chloramphenicol were from Dupont/NEN Life Science Products (Boston, MA). Media for cell culture and DNA restriction and modifying enzymes were obtained from Life Technologies, Inc. (Gaithersburg, MD). Sera for cell cultures were purchased from Gemini (Calabasa, CA). Oligonucleotides were synthesized by the UMD Molecular Resource Facility (Newark, NJ). 1,25-(OH)2D3 was a generous gift from Dr. M. Uskokovic (Hoffmann-LaRoche Inc., Nutley, NJ). The YY1 Nushift Plus Kit containing a YY1 consensus oligonucleotide (5'-GGGGATCAGGGTCTCCATTTTGAAGCGGGATCTCCC-3'), a YY1 mutant oligonucleotide (5'-GGGGATCAGGGTCTTTGTTTTGAAGCGGGATCTCCC-3'), K562 cell nuclear extract, and YY1 affinity-purified polyclonal antiserum was obtained from Geneka Biotechnology Inc. (Montreal, Quebec, Canada). Glutathione-Sepharose 4B was purchased from Amersham Pharmacia Biotech AB (Upsala, Sweden). Antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Acetyl-coenzyme A as well as general reagents of molecular biology grade were purchased from Sigma (St. Louis, MO).

Plasmids
For transfection studies constructs of a chimeric gene in which the rat 24(OH)ase promoter (-1,367/+74) was linked to the chloramphenicol acetyltransferase (CAT) gene as previously described (25) were used. pCMV-YY1 and the pCMV vector lacking the YY1 insert used for transfection were provided by T. Shenk (Princeton University, Princeton, NJ). CMV-ß gal control plasmid was a gift of C.-G. Lee (UMDNJ-New Jersey Medical School, Newark, NJ). pRSV-CBP expression plasmid was described previously (42). pRSV control plasmid without insertions was purchased from Stratagene (La Jolla, CA). CBP mutant plasmids pRSV-CBP aa 1–1,109 and pRSV-CBP aa 1–452, a gift from R. P. Rehfuss (Bristol Meyers Squibb-Pharmaceutical Research Institute, Princeton, NJ), were described previously (43). The synthetic VDRE-tk CAT construct containing multiple copies of the VDRE, GGTTCA cga GGTTCA, was a gift of L. P. Freedman (Sloan Kettering Institute, New York, NY). The expression vector pCGNhTFIIB (44) was provided by W. P. Tansey and W. Herr (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The mouse osteopontin promoter CAT construct (-777/+79), described previously (45), was from D. Denhardt (UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ). pSV2CAT reporter construct was from M. Tocci (Merck, Rahway, NJ). J. Lian, J. Stein, and G. Stein provided pCMV-HA-YY1 (20) for in vitro transcription and translation. Plasmid pET11a containing phTFIIB (46), used to in vitro transcribe and translate TFIIB, was from D. Reinberg (UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ). hVDR (47) and FLAG-hRXR{alpha} were from L. P. Freedman (Sloan Kettering Institute, New York, NY). GST-CBP constructs (1–771; 706–1,069, and 1,842–2,441) were described previously (48). pCMVp300 (49) was obtained from R. Stein (Vanderbilt Medical Center, Nashville, TN). GST-CBP constructs (1–451, 451–721) as well as the expression plasmid for the YY1 mutant ({Delta} 334–414) were kindly provided by B. Luscher and were described previously (24).

Mammalian Cell Culture and Transient Transfection Assay
The African green monkey kidney cell lines CV1 and COS-7 as well as LLCPK1 cells (porcine renal epithelial cells) were obtained from ATCC (Manassas, VA). CV1 and COS-7 cells were maintained in DMEM supplemented with 10% heat-inactivated FBS. LLCPK1 cells were maintained in medium 199 containing 5% FBS. Cells were cultured at 37 C and 5% CO2/95% air. Twenty four hours before transfection, cells were plated at 70% confluence in 100-mm tissue culture dishes. Transfection was carried out by the calcium phosphate DNA precipitation method (50) or with lipofectamine (Life Technologies, Inc.), using reporter plasmid, expression plasmid, and the ß-galactosidase expression vector pCH110 (Pharmacia Biotech, Piscataway, NJ) as an internal control for transfection efficiency. Empty vectors were used to keep the total DNA concentration the same. Cells were transfected for 16 h, shocked for 1 min with 10% dimethyl sulfoxide-PBS, washed with PBS, and then cultured in medium supplemented with 2% charcoal-stripped serum. Cells were harvested 16 h after treatment with 10-8 M 1,25-(OH)2D3, trypsinized, pelleted, washed with PBS, resuspended in 50 µl 250 mM Tris (pH 7.2). Cells were lysed by pulse sonication two times at 10-sec intervals, and the supernatants were saved for CAT assays. The CAT assay was performed at constant ß-galactosidase as previously described (50, 51). CAT activity was quantitated by densitometric scanning of TLC autoradiograms (Shimadzu CS900 U densitometer, dual wavelength flying spot scanner, Shimadzu Scientific Instruments, Inc., Princeton, NJ). Several autoradiographic exposure times were needed in some cases for densitometric analysis to estimate changes in CAT activity. CAT activity was also quantitated by scanning TLC plates with the Packard instant imager system (Packard Instrument Co., Meriden, CT). Results are reported as the mean ± SE. Significance was determined by Students’ t test or Dunnets multiple comparison t statistic.

Northern Blot Analysis
Poly (A+) RNA was prepared and Northern blot analysis was performed as previously described (52). The 3.2-kb rat 24(OH)ase cDNA was obtained by EcoRI digestion and was a generous gift of K. Okuda (Hiroshima University School of Dentistry, Hiroshima, Japan). To detect any problems with transfer of RNA or differences in loading, blots were probed with 18S rRNA cDNA (obtained from R. Guntaka, University of Missouri, Columbia, MO).

VDR Western Blot Analysis
LLCPK1 cells were transfected with vector alone or 2 µg pCMV-YY1 and treated with vehicle or 1,25-(OH)2D3 (10-7 M) for 16 h and harvested by trypsinization; nuclear extracts were prepared by the method of Dignam et al. (53). After isolation of nuclear extracts, 50 µg of protein were separated by 10% SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Inc. Hercules, CA), the membrane was blocked with 0.5% Tween 20 in PBS (PBST) containing 5% nonfat milk for 16 h at 4 C and then incubated with rat monoclonal anti-VDR antibody (Affinity BioReagents, Inc., Neshanic Station, NJ) at 1:5,000 dilution for 2 h at 27 C. The membrane was then washed with PBST, incubated for an additional hour with goat antirat IgG conjugated to horseradish peroxidase (Sigma), and washed again with PBST; the enhanced chemiluminescent Western blotting detection system (NEN Life Science Products) was used to detect the antigen/antibody complex.

EMSAs
DNA fragments of the rat 24(OH)ase promoter (-151/-105) containing the proximal VDRE and the YY1 site (25) as well as a YY1 consensus oligonucleotide (5'-GGGGATCAGGGTCTCCATTTTGAAGCGGGATCTCCC- 3') were used as probes. Overlapping forward and reverse strands were heat denatured and annealed overnight, and 50 ng of duplex oligo were end-labeled with [{gamma}-32P] ATP using T4 polynucleotide kinase and purified by PAGE. The eluted probe was used for EMSA as previously described (54) using 5 µg of nuclear proteins. Gel retardation assays were also carried out with purified proteins. VDR and RXR were provided by Dr. L. P. Freedman (Sloan Kettering Institute, New York, NY) (47) and YY1, purified as previously described (17), was a gift from Y. Shi (Harvard Medical School, Boston, MA).

Protein-Protein Interaction Assay and Production of Recombinant Protein
GST fusion protein constructs were grown in E. coli DH5{alpha} cells for 3–4 h and induced with IPTG (0.5 mM-1 mM) for an additional 2 h. Cells were resuspended in 1/50 volume in PBS containing 0.1% Triton X-100, protease inhibitors (leupeptin, aprotinin, trypsin inhibitor, and phenylmethylsulfonyl fluoride), and extracts were generated by pulse sonication on ice (three times at 10-sec intervals). Cell debris was then pelleted at 10,000 rpm for 10 min at 4 C. Glutathione-Sepharose beads (500 µl) were washed with PBS containing 0.1% Triton X-100, and the supernatant (5 ml) obtained from the cell extracts was allowed to bind by gentle agitation for 2–3 h at 4 C in binding buffer (50 mM Tris, pH 7.2, 1 mM EDTA, 1 mM dithiothreitol, 150 mM NaCl, and 0.1% Triton X-100). The beads with the bound GST fusion protein were then pelleted and washed three times with 5 ml of the same buffer and analyzed by SDS-PAGE for amount of protein bound to the GST-beads. Coupled in vitro transcription/translation reactions (TNT Quick Coupled Transcription/Translation System, Promega Corp., Madison, WI) were used to produce 35S-labeled TFIIB and YY1 in rabbit reticulocyte lysate. 35S-methionine- labeled protein (4 µl of crude translated protein) was incubated with equal amounts of GST fusion protein bound to glutathione-Sepharose beads, washed, eluted, and fractionated by SDS-PAGE. The percent of starting material loaded in input lanes was 10 or 20%. For production of recombinant proteins, fusion proteins were eluted from glutathione-Sepharose by incubating the beads at 4 C for 10 min in 30 mM reduced glutathione. Proteins were dialyzed against 50 mM Tris, pH 7.2, analyzed by SDS-PAGE, and visualized by Coomassie blue staining.


    ACKNOWLEDGMENTS
 
We are grateful to the investigators who contributed reagents for this study (see Materials and Methods) and we acknowledge helpful discussions with J. Lian and G. Stein (University of Massachusetts Medical Center, Worcester, MA).


    FOOTNOTES
 
Address requests for reprints to: Dr. Sylvia Christakos, Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103-2714. E-mail: christak{at}umdnj.edu

This work was supported by NIH Grant DK-38961 to S.C.

Received for publication May 4, 2000. Revision received February 14, 2001. Accepted for publication March 8, 2001.


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