The N-Terminal Domain of Transcription Factor IIB Is Required for Direct Interaction with the Vitamin D Receptor and Participates in Vitamin D-Mediated Transcription

Hisashi Masuyama, Stephen C. Jefcoat, Jr. and Paul N. MacDonald

St. Louis University Health Sciences Center Department of Pharmacological and Physiological Science St. Louis, Missouri 63104


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The interaction of the vitamin D receptor (VDR) with transcription factor IIB (TFIIB) represents a potential physical link between the VDR-DNA complex and the transcription preinitiation complex. However, the functional relevance of the VDR-TFIIB interaction in vitamin D-mediated transcription is not well understood. In the present study, we used site-directed mutagenesis to demonstrate that the structural integrity of the amino-terminal zinc finger of TFIIB is essential for VDR-TFIIB complex formation. Altering the putative zinc-coordinating residues (C15, C34, C37, or H18) to serines abolished TFIIB interaction with the VDR as assessed in a yeast two-hybrid system and by in vitro protein interaction assays. This N-terminal, VDR-interactive domain functioned as a selective, dominant-negative inhibitor of vitamin D-mediated transcription. Expressing amino acids 1–124 of human TFIIB (N-TFIIB) in COS-7 cells or in osteoblastic ROS17/2.8 cells effectively suppressed 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-induced transcription, but had no effect on basal or glucocorticoid-activated transcription. A mutant N-terminal domain [N-TFIIB(C34S:C37S)] that does not interact with VDR had no impact on 1,25-(OH)2D3-induced transcription. Interestingly, both in vitro and in vivo protein interaction assays showed that the VDR-TFIIB protein complex was disrupted by the 1,25-(OH)2D3 ligand. Mechanistically, these data establish a functional role for the N terminus of TFIIB in VDR-mediated transcription, and they allude to a role for unliganded VDR in targeting TFIIB to the promoter regions of vitamin D-responsive target genes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Vitamin D is essential for adequate intestinal absorption of dietary calcium and for the normal development and maintenance of the skeleton. In addition to mineral homeostasis, vitamin D also functions in fundamental cellular processes such as regulating the proliferation and differentiation of a variety of cell types (1, 2). The biologically active metabolite of vitamin D3 is 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), and its cellular actions are mediated through the vitamin D receptor (VDR), a member of the nuclear receptor superfamily for steroid hormones, thyroid hormone, and retinoids (3, 4). VDR is a ligand-activated transcription factor that forms heterodimers with retinoid X receptor, binds to vitamin D-responsive elements (VDRE) in the promoters of vitamin D-responsive genes, and alters the rate of transcription of selected genes (5, 6, 7).

The precise molecular details through which VDR affects the rate of RNA polymerase II (RNAP II)-directed transcription are largely unknown. As with other transcriptional regulatory proteins, one aspect of this mechanism likely involves selective interaction of VDR with components of the transcription preinitiation complex (PIC). These components include the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNAP II itself (8). The interaction of transactivators with these general transcription factors may be direct or it may occur indirectly through the action of bridging proteins such as the TATA-binding protein (TBP)-associated factors or various coactivator/corepressor proteins (8, 9, 10). One functional consequence of the direct interaction of transactivators with the general transcription factors is the recruitment of these basal components into the PIC and subsequent enhancement of the overall transcriptional process. For example, the transactivation domains of several proteins are known to facilitate TFIID, TFIID/TFIIA, and TFIIB assembly into the PIC (11, 12, 13, 14, 15). The prototypical example is Gal4-VP16, which interacts directly with TFIIB through its acidic-rich activation domain and recruits TFIIB into the PIC (14, 15). Importantly, mutants of TFIIB that do not interact with GAL4-VP16 are able to function in basal transcription, but they are not recruited to the PIC by GAL4-VP16, and they are not functional in GAL4-VP16-activated transcription (16). This strongly suggests that the interaction between an activator and TFIIB is one crucial aspect of the transcriptional mechanism mediated by some transactivating proteins.

TFIIB is also a central target for interaction with a variety of steroid hormone receptors, including the estrogen receptor, thyroid hormone receptor, progesterone receptor, and the VDR (17, 18, 19, 20, 21). A precise functional role for TFIIB interaction with the nuclear receptors in the mechanism of steroid-regulated transcription is not well understood. Recent data from two groups indicate that the thyroid hormone receptor (TR)-TFIIB interaction may be important for transcriptional silencing mediated by the unliganded TR (18, 19). In those studies, unliganded TR preferentially interacted with TFIIB and interfered with the formation of a functional PIC. A separate study suggested a positive role for TFIIB in vitamin D-mediated transcription in which TFIIB expression was shown to augment VDR-activated transcription of a reporter gene construct in P19 embryonal carcinoma cells (20). However, that study did not examine whether direct interactions between VDR and TFIIB were responsible for the observed synergistic transcriptional response.

Deletion analysis previously suggested that the amino-terminal domain of TFIIB is important for in vivo interaction with the VDR in the two-hybrid system (21). In contrast, the studies of Blanco et al. (20) indicated that this region was not required for VDR-TFIIB interaction in vitro. Therefore, in the present study, site-directed mutagenesis of TFIIB was used to probe the TFIIB-VDR interaction, and we establish that the structural integrity of the N-terminal domain of TFIIB is indeed required for direct interaction of TFIIB with the VDR. Moreover, expression of the TFIIB amino-terminal domain resulted in selective, dominant-negative inhibition of vitamin D-activated transcription in an osteoblast-like cell line, suggesting that VDR interaction with the N-terminal domain of TFIIB is one important aspect of the mechanism of vitamin D-mediated gene expression in bone cells. We also demonstrate that the 1,25-(OH)2D3 ligand specifically disrupts the formation of the VDR-TFIIB complex, thereby indicating a preferential interaction of TFIIB with unliganded VDR. Taken together, these studies establish the N terminus of TFIIB as a VDR-interactive domain that is important for VDR-mediated transcriptional responses and suggest an important role for VDR and the 1,25-(OH)2D3 ligand in the entry of TFIIB into the PIC on vitamin D/VDR target genes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of Amino Acid Residues in the N Terminus of TFIIB That Are Important for Interaction with VDR
As mentioned above, previous deletion analyses of TFIIB yielded conflicting evidence for a role of the N-terminal domain of TFIIB in mediating VDR-TFIIB complex formation (20, 21). Thus, to investigate the importance of the amino-terminal domain of TFIIB as a VDR contact site, several point mutations were introduced into this domain of TFIIB, and their effects on VDR-TFIIB interactions were examined using an in vivo two-hybrid assay and with in vitro protein interaction assays. The N terminus of TFIIB contains a putative zinc finger motif and a region of charged amino acids C-terminal to the zinc finger (Fig. 1Go, upper panel). Altering any of the four residues that are putatively involved in coordinating the zinc atom (C15, H18, C34, or C37) resulted in a marked impairment of TFIIB to interact with VDR in the two-hybrid system. The VDR-TFIIB interaction was reduced by approximately 70–80% when each of these four residues was changed to serine. In contrast, mutation of a number of other residues within and immediately adjacent to this domain had little effect on the interaction with VDR in this system (Fig. 1Go, lower panel).



View larger version (23K):
[in this window]
[in a new window]
 
Figure 1. Effects of Mutations in the Amino Terminus of TFIIB on Its Interaction with VDR in the Two-Hybrid System

A schematic illustration of the putative zinc finger motif in TFIIB is presented in the upper panel. Specific amino acid substitutions were generated in this domain, and the effect of each mutation on the interaction between VDR and TFIIB was examined in the Hf7c strain of yeast using a two-hybrid assay. Relative growth on histidine-deficient plates was assessed after 4 days at 30 C. VDR interaction with each TFIIB mutant was quantitated in a ß-galactosidase assay after overnight growth in a selection media (SC-leu-trp) in the absence of 1,25-(OH)2D3. Results are presented as the mean ± SD of triplicate independent cultures.

 
The effect of mutating the amino-terminal domain of TFIIB was also examined with purified proteins using an in vitro protein interaction assay. Wild type TFIIB and TFIIB(C34S:C37S) were expressed as glutathione S-transferase (GST) fusion proteins and purified with glutathione agarose and gel filtration chromatography. The purified wild type and mutant GST-TFIIB fusions were compared for their relative abilities to interact with increasing amounts of VDR (Fig. 2AGo). Whereas strong interactions were observed between VDR and wild type TFIIB (Fig. 2AGo, lanes 1–3), the interaction between VDR and the TFIIB(C34S:C37S) mutant was weak (Fig. 2AGo, lanes 4–6). When the relative stabilities of the VDR-TFIIB(WT) and the VDR-TFIIB(C34S:C37S) complexes were examined, we observed that the VDR-TFIIB(C34S:C37S) complex readily dissociated upon successive washes of the complex (Fig. 2BGo, lanes 4–6). This was in marked contrast to the VDR-TFIIB(WT) complex, which was quite stable under these wash conditions (Fig. 2BGo, lanes 1–3). These data indicate that the TFIIB(C34S:C37S) mutant exhibited a decreased relative affinity for the VDR compared with TFIIB(WT). These in vitro results with purified proteins agreed with the in vivo data generated in the two-hybrid interaction assay (Fig. 1Go). Thus, disrupting the putative zinc finger motif in the amino terminus of TFIIB reduced its ability to interact with VDR.



View larger version (30K):
[in this window]
[in a new window]
 
Figure 2. The Integrity of the Putative Zinc Finger Motif of TFIIB Is Essential for Interaction with VDR in an in Vitro Protein Interaction Assay

A, Various amounts of baculovirus-expressed full-length VDR (0.2, 1, 5 µg) were incubated with 2.5 µg purified wild type (WT) GST-TFIIB protein (lanes 1–3), or with identical concentrations of a purified mutant GST-TFIIB (C34S:C37S) protein (lanes 4–6), or with GST (lanes 7–9). Protein-protein complexes were precipitated with glutathione-agarose, washed extensively, and analyzed for VDR content by Western immunoblot analysis. The relative amount of VDR precipitated with GST-TFIIB represented 5–10% of the input VDR in this assay. B, GST-TFIIB(C34S:C37S) dissociates more readily from VDR compared with GST-TFIIB(WT). Baculovirus-expressed full-length VDR (1.3 µg) was incubated with 5.0 µg GST-TFIIB(WT) (lanes 1–3) or with 5.0 µg TFIIB(C34S:C37S) (lanes 4–6), and VDR-TFIIB complexes were analyzed by Western analysis after the glutathione-agarose beads were washed one, three, or five times.

 
TFIIB folds into a complex structure through the formation of extensive intramolecular contacts (i.e. the N terminus of TFIIB interacts with the C terminus) (22). It is possible that mutations in the zinc finger domain may disrupt the proper folding and drastically alter the overall tertiary structure of TFIIB, thereby affecting a distal site of TFIIB that is involved in VDR interaction. However, the following data argue strongly against this possibility. A C-terminal deletion mutant of TFIIB [TFIIB({Delta}124-298), also called N-TFIIB] interacted strongly with VDR. We observed approximately 3-fold higher ß-galactosidase activity with this C-terminal deletion mutant compared with wild type TFIIB (Fig. 3Go). Thus, removing the C terminus and eliminating intramolecular folding of TFIIB may actually enhance the accessibility of VDR to this amino-terminal domain. Importantly, introducing the C34S:C37S double mutant into the TFIIB({Delta}124-298) deletion construct virtually abolished the interaction of this amino-terminal domain with VDR (Fig. 3Go). Thus, in both the presence and absence of the C terminus of TFIIB, the C34S:C37S mutation disrupts TFIIB interaction with VDR, indicating a direct interaction of VDR with this N-terminal domain.



View larger version (13K):
[in this window]
[in a new window]
 
Figure 3. Disruption of VDR Interaction with the TFIIB(C34S:C37S) Mutation Is Independent of the C-Terminal Domain of TFIIB

TFIIB(WT), TFIIB({Delta}124-298), TFIIB(C34S:C37S), and TFIIB({Delta}124-298:C34S:C37S) were examined for interaction with AS1-VDR in the Hf7c strain of yeast in a two-hybrid interaction assay. The TFIIB constructs were inserted into the pGAD.GH vector and are illustrated schematically (left). Relative growth on histidine-deficient plates was assessed after 4 days at 30 C, and ß-galactosidase expression was quantitated after overnight growth in SC(-leu-trp) medium. Results are presented as the mean ± SD of triplicate independent cultures.

 
The N-Terminal Domain of TFIIB Suppresses the 1,25-(OH)2D3/VDR-Induced Transcription
To examine a functional role for VDR interaction with the amino terminus of TFIIB, we tested the effect of coexpressing N-TFIIB [TFIIB({Delta}124-298)] in a vitamin D-responsive transient gene expression system. The rationale was to overexpress that domain of TFIIB that interacted with VDR and determine whether VDR-mediated transcription was disrupted through the formation of nonproductive VDR/N-TFIIB complexes. To test the approach, we expressed and purified N-TFIIB and observed that increasing concentrations of the purified amino-terminal domain of TFIIB effectively reduced the ability of GST-TFIIB(WT) to interact with the VDR in the in vitro protein interaction assay (Fig. 4Go). Thus, N-TFIIB was indeed an effective competitor for wild type TFIIB binding to the VDR.



View larger version (42K):
[in this window]
[in a new window]
 
Figure 4. The N Terminus of TFIIB Competes with Full-Length TFIIB for Interaction with the VDR in Vitro

Wild type GST-TFIIB (1.5 µg) and baculovirus-expressed full-length VDR (1.3 µg) were incubated without (lane 1) or with 0.5, 2.5, and 12.5 µg of purified N-TFIIB [TFIIB({Delta}124-298)] (lanes 2–4) that was expressed and purified as described in Materials and Methods. VDR content was assessed as described in the legend to Fig. 2Go.

 
Therefore, a VDR expression plasmid (SG5-VDR) and vitamin D-responsive reporter gene constructs (VDRE4-TATA-GH and VDRE4-TK-GH) were introduced into COS-7 cells in the absence and presence of an expression vector that generates N-TFIIB (Fig. 5AGo). As predicted from the in vitro experiment in Fig. 4Go, expression of N-TFIIB in this transfection system effectively suppressed 1,25-(OH)2D3/VDR-dependent transactivation of both reporter gene constructs by more than 75% compared with similar transfections with the parent expression vector (pcDNA3). Importantly, expression of the amino-terminal mutation of TFIIB that does not interact with VDR [N-TFIIB(C34S:C37S)] had no effect on VDR-mediated reporter gene expression in this system. Moreover, the inhibitory effect of N-TFIIB was selective for vitamin D-responsive promoter activity since expression of the wild type amino terminus of TFIIB had no impact on glucocorticoid-induced transactivation of GRE2-TK-GH or on basal transcription of all of the reporter constructs (Fig. 5AGo). Under these conditions, N-TFIIB also did not alter basal expression of the TK-GH and TATA-GH reporter constructs that lack the hormone response elements (data not shown). The decrease in vitamin D-activated transcription by N-TFIIB expression was not due to differential expression of VDR in this transient expression assay since Western analysis revealed equivalent levels of VDR in COS-7 cells transfected with pcDNA3, pcDNA3-N-TFIIB, or pcDNA3-N-TFIIB(C34S:C37S) (Fig. 5BGo).



View larger version (21K):
[in this window]
[in a new window]
 
Figure 5. Suppression of 1,25-(OH)2D3/VDR-Induced Transactivation by the N-Terminal Domain of TFIIB

A, The effect of N-TFIIB expression in a vitamin D-responsive transient gene expression system in COS-7 cells. COS-7 cells were transfected with 2 µg of the VDRE4-TATA-GH or the VDRE4-TK-GH reporter gene construct together with 10 ng of the VDR expression plasmid. Another group of cells received the glucocorticoid-responsive reporter gene construct termed GRE2-TK-GH and 10 ng of the GR expression plasmid. These three groups also received 1 µg of pcDNA3 parent expression plasmid or pcDNA3 derivatives that express either N-TFIIB or N-TFIIB(C34S:C37S). Sixteen hours after addition of the calcium phosphate-DNA precipitate, the cells were washed, media were replaced, and the cells were treated with ethanol vehicle, with 10-8 M 1,25-(OH)2D3, or with 10-7 M dexamethasone. GH secreted into the media was quantitated 24 h following ligand addition using a RIA kit. The results represent the mean ± SD of triplicate determinations, and the number above each bar represents fold activation relative to the ethanol-treated control group. B, Relative level of expressed VDR in the presence and absence of N-TFIIB expression. Cells were transfected with the indicated DNAs, and expressed VDR was examined by Western blot analysis on extracts from cells transfected with pSG5-VDR and pcDNA3 derivatives. C, The effect of N-TFIIB expression in a vitamin D responsive osteoblast cell line, ROS17/2.8. ROS17/2.8 cells were cotransfected with 5 µg of VDRE4-TATA-GH, BGP(-1000)-GH, or BGP(-150)-GH reporter gene constructs and 2 µg of the N-TFIIB expression plasmids described in panel A. BGP(-1000)-GH contains approximately 1000 bp of 5'-flanking sequence of the native, vitamin D-responsive promoter from the osteocalcin gene. BGP(-150)-GH contains only 150 bp of this osteocalcin promoter and lacks the VDRE positioned around -450 bp. The cells were treated and GH secretion was quantitated as described in panel A. D, Concentration-dependent suppression of 1,25-(OH)2D3-induced transactivation with the increasing amounts of N-TFIIB expression plasmids. Various amounts of N-TFIIB expression vectors were transfected along with a constant amount of SG5-VDR (10 ng) and VDRE4-TATA-GH reporter (2 µg) in COS-7 cells. ROS 17/2.8 cells received 5 µg VDRE4-TATA-GH and the indicated concentrations of N-TFIIB expression plasmid. The cells were treated with 1,25-(OH)2D3, and GH secretion was determined. Suppression of 1,25-(OH)2D3-induced transactivation was calculated by measuring secreted GH with N-TFIIB relative to that of empty expression vector in the presence of 10-8 M 1,25-(OH)2D3.

 
This same inhibitory effect of N-TFIIB on vitamin D-mediated transcription was also observed in an authentic vitamin D-responsive osteoblast-like target cell line, ROS 17/2.8 (Fig. 5CGo). Vitamin D-mediated expression of both the artificial VDRE4-TATA-GH reporter and the reporter construct driven by the native osteocalcin promoter [BGP(-1000)-GH] was inhibited approximately 60% by N-TFIIB in ROS 17/2.8 cells. Again, this was not a generalized squelching phenomena since N-TFIIB did not influence expression of the native osteocalcin promoter deletion construct that lacks the VDRE [BGP(-150)-GH]. VDR-mediated reporter gene expression in ROS 17/2.8 cells also was not affected by expressing N-TFIIB(C34S:C37S), in agreement with the observation that this TFIIB mutant does not interact with VDR in vitro or in vivo (Fig. 5CGo). These inhibitory effects were concentration-dependent with increasing amounts of the N-TFIIB expression plasmid in both COS-7 cells and ROS 17/2.8 cells (Fig. 5DGo) and were apparent with two distinct vectors in which N-TFIIB expression was driven by either a cytomegalovirus promoter (pcDNA3) or an SV40 promoter (pSG5). Significant effects were apparent with 100 ng of either N-TFIIB expression plasmid. The excess of N-TFIIB expression vector compared with VDR expression vector that was required in these experiments may be related to the level of expression of each protein from these vectors, the stabilities of the expressed proteins, to the level of endogenous TFIIB that competes with N-TFIIB binding to VDR, or to the relative affinity of the VDR-TFIIB complex.

These data suggest that N-TFIIB interfered selectively with vitamin D-mediated transcription in a dominant negative fashion through the formation of a nonproductive VDR/N-TFIIB complex, and this inhibited the functional interaction of VDR with native TFIIB. Based on this model, the inhibitory effect of N-TFIIB should be reversible with the expression of additional VDR and/or additional TFIIB. The expression of additional full-length VDR (Fig. 6AGo) or additional, full-length WT TFIIB (Fig. 6BGo) reversed the negative impact of N-TFIIB on vitamin D-activated transcription in COS-7 cells. Thus, VDR and TFIIB were able to individually rescue the dominant-negative inhibition of vitamin D-activated transcription by N-TFIIB.



View larger version (20K):
[in this window]
[in a new window]
 
Figure 6. Rescue of N-TFIIB Effects on Vitamin D/VDR-Mediated Transcription

A, Rescue of the suppressed transactivation by VDR. COS-7 cells were cotransfected with 100 ng VDR together with 2 µg reporter construct (VDRE4-TATA-GH) and 100 ng N-TFIIB. The cells were treated and GH secretion was quantitated as described in Fig. 4Go. B, Rescue of the suppressed transactivation by full-length TFIIB. COS-7 cells were cotransfected with 100 ng full-length TFIIB together with 2 µg reporter construct (VDRE4-TATA-GH) and 100 ng N-TFIIB. The cells were treated and GH secretion was quantitated as described in Fig. 5Go.

 
The Effect of the 1,25-(OH)2D3 Ligand on the VDR-TFIIB Interactions
Having demonstrated the importance of the N terminus of TFIIB in VDR interaction in vitro and in vivo and the potential impact of that interaction on vitamin D-mediated transcription, the role of the 1,25-(OH)2D3 ligand in this process was examined. First, we investigated the effect of 1,25-(OH)2D3 on the interaction between VDR and TFIIB in the yeast two-hybrid protein interaction assay. Surprisingly, the interaction between VDR and TFIIB was decreased in a concentration-dependent fashion with increasing amounts of 1,25-(OH)2D3 (Fig. 7AGo). Significant effects were observed between 10-10-10-9 M 1,25-(OH)2D3, and the effect was maximal at 10-7 M 1,25-(OH)2D3. At this latter level of 1,25-(OH)2D3, VDR-TFIIB interaction was approximately 20% of control values in the absence of ligand as determined by a ß-galactosidase assay (Fig. 7AGo). This ligand-dependent decrease in VDR-TFIIB interaction was also apparent in the in vitro assay with purified components. Here, VDR interaction with GST-TFIIB was markedly diminished with increasing amounts of 1,25-(OH)2D3 (Fig. 7BGo, lanes 1–4). Nonspecific interaction of VDR with GST was not observed in the absence or presence of 1,25-(OH)2D3 in this assay (lanes 5–7).



View larger version (20K):
[in this window]
[in a new window]
 
Figure 7. Concentration-Dependent Inhibition of VDR Interaction with TFIIB by the 1,25-(OH)2D3 Ligand

A, Ligand disrupts in vivo interaction between VDR and TFIIB in the two-hybrid assay. Yeast expressing the AS1-VDR and GAD.GH-TFIIB two-hybrid plasmids were grown for 24 h at 30 C in the absence and presence of increasing concentrations of 1,25-(OH)2D3. VDR-TFIIB interaction was assessed in a ß-galactosidase assay. Results are presented as the mean ± SD of triplicate independent cultures. B, Ligand disrupts interactions between purified proteins in vitro. Baculovirus-expressed full-length VDR (1.3 µg) was incubated with 2.5 µg GST-TFIIB or GST in the absence or presence of 10-10, 10-8, or 10-6 M 1,25-(OH)2D3. Protein-protein complexes were precipitated with glutathione-agarose, washed five times, and analyzed for VDR by Western immunoblot analysis.

 
The inhibition by 1,25-(OH)2D3 on VDR interaction with TFIIB showed the appropriate ligand selectivity (Fig. 8Go). Only in the presence of 10-7 M 1,25-(OH)2D3 was a decrease in VDR-TFIIB interaction observed. Other vitamin D3 metabolites, such as 24,25-dihydroxyvitamin D3 (24, 25-(OH)2D3), 25-hydroxyvitamin D3 (25OHD3), and other ligands for nuclear receptors, such as 9-cis-retinoic acid (9-cis-RA) and dexamethasone, did not affect the interaction between VDR and TFIIB as assessed by the in vivo two-hybrid protein interaction assay and the in vitro GST fusion protein assay (Fig. 8Go, A and B). Thus, the two different systems that examined VDR-TFIIB interaction in vivo and in vitro indicated that the 1,25-(OH)2D3 ligand selectively disrupts VDR interaction with TFIIB.



View larger version (25K):
[in this window]
[in a new window]
 
Figure 8. Specific Effect of 1,25-(OH)2D3 on Disrupting VDR Interaction with TFIIB

A, The in vivo two-hybrid protein interaction assay. ß-Galactosidase expression was quantified in liquid culture after overnight culture in the absence or presence of 10-7 M 1,25-(OH)2D3, 24,25-(OH)2D3, 25OHD3, dexamethasone, or 9-cis-RA. Results are presented as the mean ± SD of triplicate independent cultures. B, The in vitro GST fusion protein interaction assay. Baculovirus-expressed full-length VDR (1.3 µg) was incubated with 2.5 µg GST-TFIIB in the absence or presence of 10-7 M 1,25-(OH)2D3, 24,25-(OH)2D3, 25OHD3, dexamethasone, or 9-cis-RA. The protein-protein complex were precipitated with glutathione-agarose, washed five times, and analyzed for VDR by Western immunoblot analysis.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We and others recently demonstrated that VDR interacts with TFIIB (20, 21), a key component of the general transcription machinery and a pivotal target for transactivator interaction with the PIC. This interaction represents a potential physical link between VDR bound at a distal enhancer sequence (i.e. the VDRE) and RNAP II activity at the core promoter element(s). However, a precise functional role for VDR interaction with TFIIB in vitamin D-mediated transcription is not well understood. In the current study, we developed a transcriptional interference assay to examine the potential importance of VDR-TFIIB interactions in vitamin D-activated transcription in mammalian cells. Toward this end, site-directed mutagenesis was used to firmly establish the requirement of the amino-terminal domain of TFIIB in the formation of the TFIIB-VDR complex in vitro and in vivo. Based on these data, a TFIIB derivative (N-TFIIB) was developed that contained the N-terminal, VDR-interactive domain, but lacked other regions of TFIIB that are required for general transcription. When N-TFIIB was expressed in mammalian cell lines to disrupt interactions between VDR and native TFIIB, this derivative interfered with VDR/vitamin D-activated transcription. These studies support a functional role for the interaction between VDR and the N terminus of TFIIB in the mechanism through which VDR alters target gene transcription in mammalian cells.

TFIIB is a single polypeptide that contains two functionally important regions, one in the N terminus and one in the C terminus (23, 24, 25). The crystal and solution structures for the C-terminal region of human TFIIB (residues 106–316) have been solved (26, 27). The C-terminal core region is required for interactions with the TFIID(TBP)-TATA element complex, making direct contacts with the C-terminal stirrup of TBP and with the phosphoribosyl backbone of the DNA both upstream and downstream of the TATA element (26, 27, 28, 29). Structural data on the amino-terminal region of TFIIB have not been reported. However, a putative zinc (Zn2+)-binding sequence (CysX2HisX17CysX2Cys) is located in this N-terminal region. Limited proteolysis and epitope mapping studies both indicate that the N terminus of the native molecule is exposed to solvent and readily accessible for macromolecular interactions (23, 30). This is supported by functional studies indicating that the N terminus is required for incorporation of TFIIF and RNAP II into the PIC, probably through interaction with the RAP30 subunit of TFIIF (23, 24, 25). However, this is not the only role that the amino-terminal domain plays. For example, Colgan et al. (31, 32) established the importance of the amino terminus of TFIIB in contacting the Ftz transactivator protein in vivo. Our current studies using the yeast two-hybrid system and in vitro interaction assays with purified proteins also establish the importance of this region in VDR-TFIIB interactions. Disrupting the structural integrity of the N terminus by mutating those residues putatively involved in coordinating the Zn2+ atom (C15, H18, C34, and C37 of human TFIIB) dramatically reduced TFIIB interaction with VDR in vivo and in vitro. The fact that these two diverse transactivators (Ftz and VDR) interact with the same region of TFIIB further support the hypothesis put forth by Colgan et al. (31, 32) that the N terminus of TFIIB, although important for RNAP II/TFIIF recruitment, may also have an important regulatory role in transcription mediated by activator proteins.

The transcriptional interference assay with N-TFIIB provides support for a role of the TFIIB amino terminus in regulated transcription by VDR. Under the conditions of the assay, N-TFIIB did not affect basal transcription of several different promoter constructs in both COS-7 cells and ROS 17/2.8 cells. It is likely that the relative level of N-TFIIB in these experiments was not sufficiently great enough to compromise RNAP II/TFIIF activity on basal promoters with comparatively low transcriptional throughput. Moreover, expression of N-TFIIB in this assay had no effect on glucocorticoid-activated transcription. This indicates that N-TFIIB did not limit the participation of RNAP II/TFIIF in higher order transcription elicited by all transactivating proteins, but of the two nuclear receptors examined, N-TFIIB was selective for VDR-activated transcription. The transcriptional interference was most likely due to N-TFIIB interaction with VDR because a mutant N-TFIIB that does not interact with VDR [N-TFIIB(C34S:C37S)] showed no ability to interfere with VDR-mediated transcription, and the effects of N-TFIIB were rescued by the expression of additional wild type VDR or TFIIB. Moreover, the in vitro competition assay clearly showed the ability of N-TFIIB to compete with wild type TFIIB for binding to the VDR. Taken together, these studies indicate that N-TFIIB interferes with vitamin D-activated transcription by disrupting the interaction between VDR and native TFIIB. As indicated by the contrasting studies of our group and Ozato’s group (20), it is possible that VDR interacts with multiple domains of TFIIB, one in the N terminus and another in the C terminus. However, our current data would clearly support a functional importance for VDR interaction with the N terminus of TFIIB in the mechanism of vitamin D-activated transcription.

What are the possible steps in the vitamin D transactivation cascade? Insight into potentially novel aspects of the mechanism of VDR-mediated transcription was realized with the remarkably potent, specific interference of the 1,25-(OH)2D3 ligand on VDR-TFIIB complex formation. Recently, Yen et al. (33) showed that unliganded VDR suppressed transcription of DR-3 and osteopontin VDRE-driven reporter gene constructs, suggesting that unliganded VDR may function as a transcriptional repressor, much like unliganded thyroid hormone receptor (TR). As suggested for thyroid hormone in disrupting TR-TFIIB interactions (18, 19), it is possible that part of the mechanism for VDR may involve unliganded VDR interaction with TFIIB to negatively influence the efficient formation of the PIC, resulting in a repressed transcriptional state. However, using osteocalcin VDRE-driven reporter constructs in both COS-7 cells and CV-1 cells, we do not observe repressed transcription by unliganded VDR, suggesting that the repressive effect may be response element or promoter specific. An alternative mechanism suggested by our data involves TFIIB recruitment to the immediate vicinity of vitamin D-responsive promoters through TFIIB interaction with unliganded VDR bound to the VDRE. This creates a "primed" state on that promoter in which limiting transcription factors such as TFIIB are more readily available for higher order transcription elicited by the hormone. The 1,25-(OH)2D3 ligand binds to VDR and releases TFIIB for assembly into the PIC forming on that particular vitamin D-responsive promoter. Here, the apoVDR-TFIIB interaction serves to sequester TFIIB (and possibly other limiting factors) around or near a particular vitamin D-responsive promoter area. Thus, it is not simply the formation of the VDR-TFIIB complex that leads to vitamin D-activated transcription, but VDR-directed targeting of TFIIB to vitamin D-responsive promoters and then a ligand-induced release of the sequestered TFIIB to the assembling PIC. In preliminary studies examining PIC assembly on a vitamin D-responsive promoter, we have observed a VDR-dependent and 1,25-(OH)2D3-dependent increase in the relative amount of TFIIB that assembles into the PIC (H. Masuyama and P. MacDonald, unpublished observation). This indicates that although 1,25-(OH)2D3 negatively impacts VDR-TFIIB interaction, the ligand has an overall positive influence on the relative level of TFIIB in the PIC. Such an observation is compatible with the mechanism discussed above, and it also supports previous observations by Ozato’s group showing synergistic activation of VDR-mediated transcription by exogenous TFIIB (20).

Although we have shown that VDR interaction with the N terminus of TFIIB is involved in the mechanism of vitamin D-mediated gene expression, it is clearly only a part of the transcriptional cascade. The nuclear receptors are known to interact with a variety of putative coactivator and corepressor proteins that also function in the mechanism of steroid-regulated gene transcription (34, 35, 36, 37, 38). For example, steroid receptor coactivator-1 (SRC-1), a putative coactivator for many of the steroid hormone receptors, is thought to mediate interactions between liganded receptors and the PIC (36). The nuclear receptor corepressor (N-CoR) and the silencing mediator for retinoid and thyroid hormone receptors (SMART) factors are nuclear corepressor proteins that interact with unliganded TRs and retinoid receptors to repress transcription of responsive genes in the absence of hormone (34, 35). Horlein et al. (35) proposed that certain nuclear receptor-regulated gene transcription may consist of a combination of hormone-dependent derepression mediated by corepressor proteins such as N-CoR and transactivation mediated through coactivators such as SRC-1. With specific regard to vitamin D-mediated transcription, the role of the known coactivator/corepressor proteins has not been established. Apparently, VDR does not interact with N-CoR (35), and its interaction with SRC-1 was not investigated (36). VDR has been reported to interact with thyroid hormone receptor interacting protein-1 (TRIP-1) (37), a putative cofactor that interacts in a ligand-dependent fashion with the AF-2 region of several nuclear receptors (37, 38). Indeed, our laboratory has isolated TRIP-1 as a ligand-dependent, VDR-interactive clone in a two-hybrid screen of an osteoblast cDNA library (H. Masuyama and P. MacDonald, unpublished observation). However, the precise role of TRIP-1 in steroid hormone-mediated transcription or its role as a nuclear receptor coactivator protein is still unresolved. Based on these observations, it is possible that VDR couples to the PIC through direct interactions with the N terminus of TFIIB as well as through interactions with novel coactivator/corepressor proteins that remain to be identified. Understanding the complex interplay of the receptor with TFIIB and with the various coactivator/corepressor proteins and the precise means through which these interactions result in efficient ligand-mediated transcription will be the focus of future research efforts.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Preparation of Two-Hybrid Expression Vectors
All two-hybrid plasmid constructs used the pAS1 and pGAD.GH yeast expression vectors (39, 40). The AS1-VDR construct, which contained the Gal4 DNA-binding domain (amino acids 1–147) and the carboxyl-terminal region of VDR (amino acids 93–427), was previously described (21). Point mutations were introduced into the human TFIIB cDNA with oligonucleotide-directed mutagenesis (41, 42). The N-terminal domain of TFIIB (N-TFIIB) was an NcoI/BglII deletion construct, termed TFIIB({Delta}124-296) (21). A mutated version of TFIIB({Delta}124-296) was also generated in which cysteine residues at amino acid positions 34 and 37 were altered to serine residues by site-directed mutagenesis. This construct was designated TFIIB({Delta}124-296;C34S:C37S) or more simply as N-TFIIB(C34S:C37S). All point mutants were confirmed by DNA-sequencing reactions, and each mutant was subcloned into the pGAD.GH vector to examine in the two-hybrid assay.

Yeast Transformation and ß-Galactosidase Assays
The pGAD-TFIIB wild type and mutant constructs were cotransformed with pAS1-VDR (93–427) into the yeast strain Hf7c, which was made competent with lithium acetate (43). Transformants were plated on media lacking leucine and tryptophan (SC-leu-trp) and were grown for 4 days at 30 C to select for yeast that had acquired both plasmids. Triplicate independent colonies from each plate were grown overnight in 2 ml of SC (-leu-trp). These samples were diluted to 0.02 OD600 units and were incubated overnight with or without the indicated concentrations of 1,25-(OH)2D3 or other hormones. Cells were harvested and assayed for ß-galactosidase activity as described (44).

Production and Purification of Glutathione S-Transferase (GST) Fusion Proteins and the in Vitro Protein Interaction Assay
The cDNAs for wild type TFIIB, TFIIB(C34S:C37S) and N-TFIIB [TFIIB({Delta}124-296)], were subcloned into pGEX-KT (45). GST-fusion proteins of wild type TFIIB, TFIIB(C34S:C37S), were expressed in the DH5{alpha} strain of bacteria and were purified by glutathione-agarose and Sephadex G-75 size exclusion chromatography. The N-TFIIB protein was purified by gluthatione-agarose chromatography and was separated from the GST moiety by thrombin cleavage (45). Proteins were analyzed by SDS-PAGE and stained with Coomassie blue to confirm their purity. Full-length VDR was expressed in a baculovirus expression system (46). The GST-TFIIB fusion proteins were incubated with 0.5 µg VDR in 100 µl of GBB buffer (20 mM Tris-Cl, pH 7.6, 200 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) for 1 h at 4 C. After the addition of 20 µl of a 50% slurry of glutathione agarose, the proteins were incubated for 30 min at 4 C. The glutathione-agarose and associated protein complexes were washed five times in 200 µl of GBB buffer, solubilized in SDS sample buffer, and analyzed for VDR content by Western blot analysis using monoclonal antibody 9A7{gamma} as described previously (21).

Transient Transfection Studies
The VDRE4-TATA-GH GH reporter plasmid contained four copies of the rat osteocalcin VDRE adjacent to the rat osteocalcin promoter fragment (-40 to +32) linked to a human GH reporter sequence in p0GH (47). VDRE4-TK-GH, BGP(-1000)-GH, and BGP(-150)-GH were described previously (46, 48). GRE2-TK-GH contained two copies of a consensus glucocorticoid-responsive element (GRE) upstream of the viral thymidine kinase promoter in the pTK-GH vector (47). N-TFIIB [TFIIB({Delta}124-296)] and the mutant N-TFIIB [TFIIB({Delta}124-296);C34S:C37S] were subcloned into the pcDNA3 expression vector (Invitrogen Co. San Diego CA). Full-length TFIIB was subcloned into the pSG5 expression vector (Stratagene, San Diego, CA). The pSG5-VDR and pSG5-GR expression plasmids were described previously (46, 49). COS-7 cells were cotransfected with reporter gene constructs (VDRE4-TATA-GH, VDRE4-TK-GH, or GRE2-TK-GH), receptor expression vectors (pSG5-VDR or pSG5-GR), and TFIIB expression vectors (N-TFIIB, mutant N-TFIIB, full-length TFIIB, or expression vector alone). ROS 17/2.8 cells were also cotransfected with reporter gene constructs [VDRE4-TATA-GH, BGP(-1000)-GH or BGP(-150)-GH), and TFIIB expression vectors (N-TFIIB, mutant N-TFIIB, or expression vector alone). In all transfections, the amount of total DNA was kept constant at 10 µg by adding pTZ18U (U.S. Biochemical, Cleveland, OH) as a carrier plasmid, and the cells were transfected by standard calcium phosphate coprecipitation procedures as described previously (46). Transfected cells were treated with 10-8 M 1,25-(OH)2D3, 10-7 M dexamethasone, or ethanol vehicle for 24 h, and the amount of secreted GH was determined with a RIA kit (Nichols Institute, San Juan Capistrano, CA).


    ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Peter Jurutka for the GRE2-TK-GH plasmid and Dr. Mark Haussler for his continued support and encouragement. We wish to thank Dr. Milan R. Uskokovic (Hoffmann-LaRoche, Nutley, NJ) for providing the 1,25-(OH)2D3 and Dr. Alex Brown for other vitamin D compounds. We also acknowledge Dr. Diane R. Dowd for experimental suggestions and for a critical reading of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Paul N. MacDonald, Ph.D., St. Louis University Health Sciences Center, Department of Pharmacological and Physiological Science, 1402 South Grand Boulevard, St. Louis, Missouri 63104.

This work was supported in part by NIH Grants R29DK-47293 and R01DK-50348 to P.N.M.

Received for publication June 3, 1996. Revision received October 18, 1996. Accepted for publication November 5, 1996.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Reichel H 1989 The role of the vitamin D endocrine system in health and disease. N Engl J Med 320:980–991[Medline]
  2. Abe E, Miyaura C, Sakagami H, Takeda M, Konno K, Yamazaki T, Yoshiki S, Suda T 1981 Differentiation of mouse myeloid leukemia cells induced by 1{alpha},25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 78:4990–4994[Abstract]
  3. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889–895[Medline]
  4. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  5. MacDonald PN, Dowd DR, Haussler MR 1994 New insight into the structure and functions of the vitamin D receptor. Semin Nephrol 14:101–118[Medline]
  6. Darwish H, DeLuca HF 1993 Vitamin D-regulated gene expression. Crit Rev Eukaryot Gene Expr 3:89–116[Medline]
  7. Whitfield GK, Hsieh JC, Jurutka PW, Selznick SH, Haussler CA, MacDonald PN, Haussler MR 1995 Genomic actions of 1,25-dihydroxyvitamin D3. J Nutr 125:1690S–1694S
  8. Zawel L, Reinberg D 1995 Common themes in assembly and function of eukaryotic transcription complexes. Annu Rev Biochem 64:533–561[CrossRef][Medline]
  9. Triezenberg SJ 1995 Structure and function of transcriptional activation domains. Curr Opin Genet Dev 5:190–196[CrossRef][Medline]
  10. Goodrich JA, Cutler G, Tjian R 1996 Contacts in context: promotor specificity and macromolecular interactions in transcription. Cell 84:825–830[Medline]
  11. Dynlacht BD, Hoey T, Tjian R 1991 Isolation of coactivators associated with TATA-binding protein that mediate transcriptional activation. Cell 66:563–575[Medline]
  12. Tanese N, Pugh BF, Tjian R 1991 Coactivators for a proline-rich activator purified from the multisubunit human TFIID complex. Genes Dev 5:2212–2224[Abstract]
  13. Lieberman PM, Berk AJ 1994 A mechanism for TAFs in transcriptional activation: activation domain enhancement of TFIID-TFIIA-promoter DNA complex formation. Genes Dev 8:995–1006[Abstract]
  14. Kim TK, Roeder RG 1994 Proline-rich activator CTF1 targets the TFIIB assembly step during transcriptional activation. Proc Natl Acad Sci USA 91:4170–4174[Abstract]
  15. Lin Y-S, Green MR 1991 Mechanism of action of an acidic transcriptional activator in vitro. Cell 64:971–981[Medline]
  16. Roberts SGE, Ha I, Maldonado E, Reinberg D, Green MR 1993 Interaction between an acidic activator and transcription factor IIb is required for transcriptional activation. Nature 363:741–744[CrossRef][Medline]
  17. Ing NH, Beekman JM, Tsai SY, Tsai M-J, O’Malley BW 1992 Members of the steroid hormone receptor superfamily interact with TFIIB (S300-II). J Biol Chem 267:17617–17623[Abstract/Free Full Text]
  18. Baniahmad A, Ha I, Reinberg D, Tsai S, Tsai M-J, O’Malley BW 1993 Interaction of human thyroid hormone receptor ß with transcription factor TFIIB may mediate target gene derepression and activation by thyroid hormone. Proc Natl Acad Sci USA 90:8832–8836[Abstract]
  19. Fondell JD, Roy AL, Roeder RG 1993 Unliganded thyroid hormone receptor inhibits formation of a functional preinitiation complex: implications for active repression. Genes Dev 7:1400–1410[Abstract]
  20. Blanco JCG, Wang I-M, Tsai SY, Tsai M-J, O’Malley BW, Jurutka PW, Haussler MR, Ozato K 1995 Transcription factor TFIIB and the vitamin D receptor cooperatively activate ligand-dependent transcription. Proc Natl Acad Sci USA 92:1535–1539[Abstract]
  21. MacDonald PN, Sherman DR, Dowd DR, Jefcoat SC, DeLisle RK 1995 The vitamin D receptor interacts with general transcription factor IIB. J Biol Chem 270:4748–4752[Abstract/Free Full Text]
  22. Roberts SGE, Green MR 1994 Activator-induced conformational change in general transcription factor TFIIB. Nature 371:717–720[CrossRef][Medline]
  23. Barberis A, Muller CW, Harrison SC, Ptashne M 1993 Deliniation of two discrete regions of transcription factor TFIIB. Proc Natl Acad Sci USA 90:5628–5632[Abstract]
  24. Buratowski S, Zhou H 1993 Functional domains of transcription factor IIB. Proc Natl Acad Sci USA 90:5633–5637[Abstract]
  25. Ha I, Roberts S, Maldonado E, Sun X, Kim L-U, Green M, Reinberg D 1993 Multiple functional domains of human transcription factor IIB: distinct interactions with two general transcription factors and RNA polymerase II. Genes Dev 7:1021–1032[Abstract]
  26. Nikolov DB, Chen H, Halay ED, Usheva AA, Hisatake K, Lee DK, Roeder RG, Burley SK 1995 Crystal structure of a TFIIB-TBP-TATA-element ternary complex. Nature 377:119–128[CrossRef][Medline]
  27. Bagby S, Kim S, Maldonado E, Tong KI, Reinberg D, Ikura M 1995 Solution structure of the C-terminal core domain of human TFIIB: similarity to cyclin A and interaction with TATA-binding protein. Cell 82:857–867[Medline]
  28. Lee S, Hahn S 1995 Model for binding of transcription factor TFIIB to the TBP-DNA complex. Nature 376:609–612[CrossRef][Medline]
  29. Hisatake K, Roeder RG, Horikoshi M 1993 Functional dissection of TFIIB domains required for TFIIB-TFIID-promoter complex formation and basal transcription activity. Nature 363:744–747[CrossRef][Medline]
  30. Thompson NE, Strasheim LA, Nolan KM, Burgess RR 1995 Accessibility of epitopes on human transcription factor IIB in the native protein and in a complex with DNA. J Biol Chem 270:4735–4740[Abstract/Free Full Text]
  31. Colgan J, Wampler S, Manley JL 1993 Interaction between a transcriptional activator and transcription factor IIB in vivo. Nature 362:549–553[CrossRef][Medline]
  32. Colgan J, Ashali H, Manley JL 1995 A direct interaction between a glutamine-rich activator and the N terminus of TFIIB can mediate transcriptional activation in vivo. Mol Cell Biol 15:2311–2320[Abstract]
  33. Yen PM, Liu Y, Sugawara A, Chin WW 1996 Vitamin D receptors repress basal transcription and exert dominant negative activity on triiodothyronine-mediated transcriptional activity. J Biol Chem 271:10910–10916[Abstract/Free Full Text]
  34. Chen JD, Evans RM 1995 A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377:454–457[CrossRef][Medline]
  35. Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa R, Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397–404[CrossRef][Medline]
  36. Onate SA, Tsai SY, Tsai M-J, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270: 1354–1357
  37. vom Baur E, Zechel C, Heery D, Heine MJS, Garnier JM, Vivat V, Le Douarin B, Gronemeyer H, Chambon P, Losson R 1996 Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1. EMBO J 15:110–124[Abstract]
  38. Lee JW, Ryan F, Swaffield JC, Johnston SA, Moore DD 1995 Interaction of thyroid-hormone receptor with a conserved transcriptional mediator. Nature 374:91–94[CrossRef][Medline]
  39. Durfee T, Becherer K, Chen P-L, Yeh S-H, Yang Y, Kilburn AE, Lee W-H, Elledge SJ 1993 The retinoblastoma protein associates with the protein phosphatase type I catalytic subunit. Genes Dev 7:555–569[Abstract]
  40. Hannon GJ, Demetrick D, Beach D 1993 Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins. Genes Dev 7:2378–2391[Abstract]
  41. Carter P 1987 Improved oligonucleotide-directed mutagenesis using M13 vectors. Methods Enzymol 154:382–403[Medline]
  42. Deng WP, Nickoloff JA 1992 Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem 200:81–88[Medline]
  43. Gietz D, Jean AS, Woods RA, Schiestl RH 1991 An improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425[Medline]
  44. Fagan R, Flint KJ, Jones N 1994 Phosphorylation of E2F-1 modulates its interaction with the retinoblastoma gene product and the adenoviral E4 19 kDa protein. Cell 78:799–811[Medline]
  45. Hakes DJ, Dixon JE 1992 New vectors for high level expression of recombinant proteins in bacteria. Anal Biochem 202:293–298[Medline]
  46. MacDonald PN, Dowd DR, Nakajima S, Galligan MA, Reeder MC, Haussler CA, Ozato K, Haussler MR 1993 Retinoid X receptors stimulate and 9-cis retinoic acid inhibits 1,25-dihydroxyvitamin D3-activated expression of the rat osteocalcin gene. Mol Cell Biol 13:5907–5917[Abstract]
  47. Selden RF, Howie KB, Rowe ME, Goodman HM, Moore DD 1986 Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol Cell Biol 6:3173–3179[Medline]
  48. Terpening CM, Haussler CA, Jurutka PW, Galligan MA, Komm BS, Haussler MR 1991 The vitamin D-responsive element in the rat bone gla protein is an imperfect direct repeat that cooperates with other cis-elements in 1,25-dihydroxyvitamin D3-mediated transcriptional activation. Mol Endocrinol 5:373–385[Abstract]
  49. Hsieh J-C, Jurutka PW, Galligan MA, Terpening CM, Haussler CA, Samuels DS, Shimizu Y, Shimizu N, Haussler MR 1991 Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function. Proc Natl Acad Sci USA 88:9315–9319[Abstract]