Ligand-dependent Transcriptional Regulators Research Articles

Photocaged Agonist for an Analogue‐Specific form of the Vitamin D Receptor

Nuclear hormone receptors (NHRs) represent a diverse class of ligand-dependent transcriptional regulators. NHRs that have been rendered functionally inactive due to mutations that abrogate proper ligand binding can often be rescued by appropriately designed hormone analogues. The analogue-specific receptor-ligand pairs provide an ideal platform from which to develop new chemogenomic tools for the spatial and temporal control of gene expression. Here, we describe the synthesis and in vitro assessment of a photocaged VDR agonist specific to a mutant NHR that is associated with vitamin D-resistant rickets. The results provide insight into the utility of the agonist as a potential tool for photoinduced gene patterning.

FoxG1, a member of the forkhead family, is a corepressor of the androgen receptor

The androgen receptor (AR) is a ligand-dependent transcriptional regulator which belongs to the nuclear receptor superfamily. The basal transcriptional activity of the androgen receptor is regulated by interaction with coactivator or corepressor proteins. The exact mechanism whereby comodulators influence target gene transcription is only partially understood, especially for corepressors. Whereas several coactivators are described for the AR, only a few corepressors are known. Here, we describe the discovery of a new androgen receptor corepressor, FoxG1, which belongs to the forkhead family. By using a fragment of the AR (aa 325–919) as bait in a yeast two hybrid screen, the C-terminal region (aa 175–489) of FoxG1 (also known as BF1), was identified as AR-interacting protein. Binding of AR to the FoxG1 fragment was verified by one- and two-hybrid assays, and pull-down experiments. In addition, we show that the full-length form of FoxG1 functions as a strong corepressor in the AR-mediated transactivation. The FoxG1 expression profile in adult individuals is restricted to brain and testis in human and decreases during aging in the rodent brain. Both AR and FoxG1 expression are developmentally regulated. Besides its reported role in neurogenesis, the strong expression of FoxG1 in AR-abundant areas of the adult brain suggests possible involvement in neuroendocrine regulation. Taken together, the data presented suggest that, in addition to repression of transcription by direct binding to DNA, FoxG1 may interact with AR in vivo, thereby targeting its repressor function specifically to sex hormone signaling.

Circumventing Anti-Androgen Resistance by Molecular Design

Nuclear/steroid hormone receptors (NHRs) function as ligand-dependent transcriptional regulators of diverse sets of genes involved in development and homeostasis. Mutations to the androgen receptor (AR), a member of NHRs, have been linked to the failure of hormonal therapy in the treatment of prostate cancer (PCa). It has been proposed that AR mutations such as Thr877→Ala, Trp741→Leu, and Trp741→Cys that cause anti-androgens such as flutamide and bicalutamide to function as agonists are likely associated with anti-androgen withdrawal syndrome in PCa therapy. The recently solved crystal structure of bicalutamide was used to design analogues that would complement the Trp741→Leu mutation, effectively restoring antagonist action of the ligand. Three out of the six designed analogues showed potent antagonistic activity in all three mutations as well as wild-type, suggesting that these analogues may be considered “pan-antagonists” of AR.

Glucocorticoid modulation of cytokine signaling

Cytokine signaling is essential for intercellular communication and affects cell proliferation, differentiation, and survival. In the immune system, cytokines coordinate the activities of many cell types ultimately leading to both innate and adaptive immune responses. Dysregulation of these processes can result in a wide spectrum of diseases ranging from defective host responses to invading pathogens to autoimmunity. Most cytokines signal through the Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathway initiated by the cytokine binding to its cell surface receptor, which leads to the activation of STAT proteins, their binding to response elements near target promoters ultimately changing the transcription of STAT-responsive genes. STAT proteins do not exist in isolation but act in concert with other transcription factors and cofactors which can either stimulate or inhibit their activity. One such factor is a ligand-dependent transcriptional regulator termed the glucocorticoid (GC) receptor (GR), which transduces the information conveyed by GC hormones and their synthetic analogs. GR is known for its anti-inflammatory and immunosuppressive properties; GC-like molecules have been used as drugs for inflammatory, autoimmune and lymphoproliferative diseases since the 1950s. In contrast, cytokines frequently promote activation of the immune system. In last several years, functional interactions have been described between virtually every member of the STAT family and GR or its cofactors. This review focuses on the recent literature on the modes and levels of interactions between these seemingly unrelated regulators and potential biological implications of STAT : GR cross-talk.

BAF60a mediates critical interactions between nuclear receptors and the BRG1 chromatin-remodeling complex for transactivation.

Nuclear hormone receptors are ligand-dependent transcriptional regulators that modulate chromatin structure. However, the precise molecular mechanisms by which receptors recruit chromatin-remodeling activity are not fully elucidated. We show that in the absence of its ligand-binding domain, the glucocorticoid receptor (GR) is able to interact with both nuclear receptor coactivators and the BRG1 chromatin-remodeling complex in vivo. Individually, the GR makes direct interactions with BRG1-associated factor 60a (BAF60a) and BAF57, but not with BRG1, BAF155, or BAF170. Further, BAF60a possesses at least two interaction surfaces, one for GR and BRG1 and a second for BAF155 and BAF170. A GR mutant, GR(R488Q), that fails to interact with BAF60a in vitro has reduced chromatin-remodeling activity and reduced transcriptional activity from the promoter assembled as chromatin in vivo. Stable expression of a BAF60a truncation mutant, BAF60a4-140, caused chromatin-specific loss of GR functions in vivo. In the presence of the BAF60a mutant, the GR fails to interact with the BRG1 complex and consequently is also deficient in its ability to activate transcription from chromatin. Thus, in addition to previously identified BAF250, BAF60a may provide another critical and direct link between nuclear receptors and the BRG1 complex that is required for promoter recruitment and subsequent chromatin remodeling.

Functions of RARs and RXRs in vivo: Genetic dissection of the retinoid signaling pathway

Retinoids, the active metabolites of vitamin A, regulate complex gene networks involved in vertebrate morphogenesis, growth, cellular differentiation, and homeostasis. They are used for the treatment of skin disorders and as chemopreventive agents for certain cancers. Molecular biology and genetic studies performed during the last 15 years in vitro, using either acellular systems or transfected cells, have shown that retinoid actions are mediated through heterodimers between the 8 major RARα, β, and γ; isoforms and the 6 major RXRα, β and γ isoforms that belong to the nuclear receptor (NR) superfamily, and act as ligand-dependent transcriptional regulators. Furthermore, RXRs not only heterodimerize with RARs, but also with numerous other members of the NR superfamily. As in vitro studies are carried out under nonphysiological conditions, they only indicate what is possible, but not necessarily what is actually occurring in vivo. Therefore, mutations have been introduced by homologous recombination (HR) in F9 embryonal carcinoma (EC) cells, a cell-autonomous system that differentiates in the presence of RA, in order to disrupt RAR and RXR genes and establish their cellular and molecular functions in RA-induced differentiation. However, genetic approaches in the animal should be used to determine the function of retinoid receptors under truly physiological conditions. HR in embryonic stem (ES) cells, has therefore been used to generate null mutations of the various RARs and RXRs in the mouse germline. As reviewed here, the generation of such RAR and RXR germline mutations, combined with pharmacological approaches to block the RA signaling pathway, has provided many valuable insights on the developmental functions of RA receptors. However, due to (i) the complexity in "hormonal" signaling through transduction by the multiple RARs and RXRs, (ii) the functional redundancies (possibly artefactually generated by the mutations) within receptor isotypes belonging to a given gene family, and (iii) in utero or postnatal lethality of certain germline null mutations, these genetic studies through germline mutagenesis have failed to reveal many of the physiological functions of RARs and RXRs, notably in adults. We conclude that spatio-temporally controlled somatic mutations generated in animal models in given cell-types/tissues and at chosen times during pre- and postnatal life, are required to reveal the physiological and pathophysiological functions of the receptor genes involved in the retinoid signaling pathway throughout the life of the mouse.

Structure-based design of selective agonists for a rickets-associated mutant of the vitamin d receptor.

The nuclear and steroid hormone receptors function as ligand-dependent transcriptional regulators of diverse sets of genes associated with development and homeostasis. Mutations to the vitamin D receptor (VDR), a member of the nuclear and steroid hormone receptor family, have been linked to human vitamin D-resistant rickets (hVDRR) and result in high serum 1,25(OH)(2)D(3) concentrations and severe bone underdevelopment. Several hVDRR-associated mutants have been localized to the ligand binding domain of VDR and cause a reduction in or loss of ligand binding and ligand-dependent transactivation function. The missense mutation Arg274 --> Leu causes a >1000-fold reduction in 1,25(OH)(2)D(3) responsiveness and is, therefore, no longer regulated by physiological concentrations of the hormone. In this study, computer-aided molecular design was used to generate a focused library of nonsteroidal analogues of the VDR agonist LG190155 that were uniquely designed to complement the Arg274 --> Leu associated with hVDRR. Half of the designed analogues exhibit substantial activity in the hVDRR-associated mutant, whereas none of the structurally similar control compounds exhibited significant activity. The seven most active designed analogues were more than 16 to 526 times more potent than 1,25(OH)(2)D(3) in the mutant receptor (EC(50) = 3.3-121 nM). Significantly, the analogues are selective for the nuclear VDR and did not stimulate cellular calcium influx, which is associated with activation of the membrane-associated vitamin D receptor.

Androgen-induced NH2- and COOH-terminal Interaction Inhibits p160 coactivator recruitment by activation function 2.

The androgen receptor undergoes an androgen-specific NH(2)- and COOH-terminal interaction between NH(2)-terminal motif FXXLF and activation function 2 in the ligand binding domain. We demonstrated previously that activation function 2 forms overlapping binding sites for the androgen receptor FXXLF motif and the LXXLL motifs of p160 coactivators. Here we investigate the influence of the NH(2)- and COOH-terminal interaction on androgen receptor function. Specificity and relative potency of the motif interactions were evaluated by ligand dissociation rate and the stability of chimeras of transcriptional intermediary factor 2 with full-length and truncated androgen or glucocorticoid receptor. The results indicate that the androgen receptor activation function 2 interacts specifically and with greater avidity with the single FXXLF motif than with the LXXLL motif region of p160 coactivators, whereas this region of the glucocorticoid receptor interacts preferentially with the LXXLL motifs. Expression of the LXXLL motifs as a fusion protein with the glucocorticoid receptor resulted in loss of agonist-induced receptor destabilization and increased half-time of ligand dissociation. The NH(2)- and COOH-terminal interaction inhibited binding and activation by transcriptional intermediary factor 2. We conclude that the androgen receptor NH(2)- and COOH-terminal interaction reduces the dissociation rate of bound androgen, stabilizes the receptor, and inhibits p160 coactivator recruitment by activation function 2.

The Human Homologue of the Yeast DNA Repair and TFIIH Regulator MMS19 Is an AF-1-specific Coactivator of Estrogen Receptor

Steroid/nuclear hormone receptors are ligand-dependent transcriptional regulators that control gene expression in a wide array of biological processes. The transcriptional activity of the receptors is mediated by an N-terminal ligand-independent transcriptional activation function AF-1 and a C-terminal ligand-dependent transcriptional activation function AF-2. The nuclear receptor coactivator RAC3 (also known as AIB1/ACTR/pCIP/TRAM-1/SRC-3) is amplified in breast cancer cells, where it forms a complex with estrogen receptor (ER) and enhances AF-2 activity of the receptor. Here, we identify a putative human homologue of the yeast DNA repair and transcriptional regulator MMS19 as a RAC3-interacting protein. The human MMS19 interacts with the N-terminal PAS-A/B domain of RAC3 in vivo and in vitro through a conserved C-terminal domain. Interestingly, the human MMS19 also interacts with estrogen receptors in a ligand-independent manner but not with retinoic acid receptor or thyroid hormone receptor. Overexpression of the interacting domain of hMMS19 strongly inhibits ER-mediated transcriptional activation, indicating a dominant negative activity. In contrast, over expression of the full-length hMMS19 enhances ER-mediated transcriptional activation. We find that hMMS19 stimulates the AF-1 activity of ERalpha, but not the AF-2 activity, suggesting that hMMS19 may be an AF-1-specific transcriptional coactivator of estrogen receptor.

Selective regulation of gene expression by an orthogonal estrogen receptor–ligand pair created by polar-group exchange

Background: The nuclear and steroid hormone receptors function as ligand-dependent transcriptional regulators in eukaryotes. Hormone receptors have been engineered to selectively respond to synthetic ligands and used as remote regulators of gene expression for the study of gene function and as potential regulators of gene therapies. Results: In this work, a new ligand–receptor engineering strategy called ‘polar-group exchange’ is used to create a mutant form of the estrogen receptor, ER(Glu353→Ala), which lacks a carboxyl group critical for high-affinity binding of estradiol, but is able to transactivate in response to nanomolar concentrations of a carboxylate-functionalized estrogen analog, ES8. ES8 activates ER(Glu353→Ala) at concentrations that do not appreciably activate the ‘wild-type’ receptor ER(wt). Two similar carboxylate-functionalized ligands, ES6 and ES7, do not induce transactivation function. Similar selectivities are observed in ligand-binding assays in vitro, which follow the trends predicted by molecular modeling. Conclusions: Polar-group exchange is an effective strategy for rationally engineering ligand–receptor pairs. The ER(E353A)/ES8 ligand–receptor pair should constitute a unique and functionally orthogonal ligand-dependent transcriptional regulator.

Ligand-Independent Activation with Ets-1

Nuclear receptors of the steroid hormone receptor family are typically considered ligand-dependent transcriptional regulators. Tolón et al. found that if the Ets-1 transcription factor was co-expressed with the estrogen receptor (ER), the vitamin D receptor (VDR), or the peroxisome-proliferator-activated receptor (PPAR), then these receptors acted with the prolactin-specific transcription factor GHF-1 to cause ligand-independent activation of a reporter construct containing the prolactin promoter. Without Ets-1, a ligand (for example vitamin D for the VDR) was required in order to activate transcription. Ligand-independent activation by Ets-1 did not occur with the retinoid X receptor suggesting specificity to the Ets-1 interaction. This response was not limited to the prolactin promoter, but also occurred with the estrogen response element, the vitamin D response element, and the peroxisome-proliferator response element when Ets-1 and the cognate nuclear receptor were coexpressed with the reporter gene. Ets-1 directly interacted with the ER, VDR, and PPAR in coimmunoprecipitation and glutathione- S -transferase pull down experiments. The importance of this interaction in vivo was confirmed by the ability of the VDR and Ets-1 to coprecipitate from pituitary cells in both the presence and absence of vitamin D. Ets-1 belongs to a family of transcription factors regulated by mitogen-activated protein kinase (MAPK) cascade. Thus, this Ets-1-induced transcriptional activity represents one mechanism by which the MAPK signals can influence nuclear receptor activity even in the absence of the hormone. Tolón, R.M., Castillo, A.I., Jiménez-Lara, A.M., and Aranda, A. (2000) Association with Ets-1 causes ligand- and AF2-independent activation of nuclear receptors. Mol. Cell. Biol. 20 : 8793-8802. [Abstract] [Full Text]

Chemically Regulated Zinc Finger Transcription Factors

Ligand-dependent transcriptional regulators were generated by fusion of designed Cys(2)-His(2) zinc finger proteins and steroid hormone receptor ligand binding domains. To produce novel DNA binding domains, three-finger proteins binding specific 9-base pair sequences were constructed from modular building blocks. Fusion of these zinc finger proteins to a transcriptional activation domain and to modified ligand binding domains derived from either the estrogen or progesterone receptors yielded potent ligand-dependent transcriptional regulators. Together with optimized minimal promoters, these regulators provide 4-hydroxytamoxifen- or RU486-inducible expression systems with induction ratios of up to 3 orders of magnitude. These inducible expression systems are functionally independent, and each can be selectively switched on within the same cell. The potential use of zinc finger-steroid receptor fusion proteins for the regulation of natural promoters was also explored. A gene-specific six-finger protein binding an 18-base pair target sequence was converted into a ligand-dependent regulator by fusion with either two estrogen receptor ligand binding domains or one ecdysone receptor and one retinoid X receptor ligand binding domain. These single-chain receptor proteins undergo an intramolecular rearrangement, rather than intermolecular dimerization and are functional as monomers. Thus, the ability to engineer DNA binding specificities of zinc finger proteins enables the construction of ligand-dependent transcriptional regulators with potential for the regulation of virtually any desired artificial or natural promoter. It is anticipated that the novel chemically regulated gene switches described herein will find many applications in applied and basic research, where the specific modulation of gene expression can be exploited.

Structural role of a detergent molecule in retinoic acid nuclear receptor crystals

The human nuclear receptor of retinoic acid hRARgamma is a ligand-dependent transcription regulator. The presence of a completely ordered dodecyl-alpha-D-maltoside molecule in the crystal structure of the hRARgamma ligand-binding domain (LBD) refined at 1. 3 A resolution is reported. The non-ionic detergent is required for stabilization and crystallization of the hRARgamma LBD and mediates a crystal contact in the region where coactivator proteins bind. Its dodecyl moiety is buried in a hydrophobic channel, whereas the maltoside head group is hydrogen bonded to water molecules and polar residue side chains.

The Crystal Structure of the Nuclear Receptor for Vitamin D Bound to Its Natural Ligand

The action of 1α,25-dihydroxyvitamin D 3 is mediated by its nuclear receptor (VDR), a ligand-dependent transcription regulator. We report the 1.8 Å resolution crystal structure of the complex between a VDR ligand-binding domain (LBD) construct lacking the highly variable VDR-specific insertion domain and vitamin D. The construct exhibits the same binding affinity for vitamin D and transactivation ability as the wild-type protein, showing that the N-terminal part of the LBD is essential for its structural and functional integrity while the large insertion peptide is dispensable. The structure reveals the active conformation of the bound ligand and allows understanding of the different binding properties of some synthetic analogs.

Carotenoids and retinoids as suppressors on adipocyte differentiation via nuclear receptors

The adipocyte differentiation program is regulated by the sequential expression of transcriptional activators, mainly peroxisome proliferator activated receptor (PPAR) families. In the present study, we have decided to systematically examine the effects of vitamin A and its precursors, carotenoids and retinoids, on terminal differentiation from preadipocytes to adipocytes on the cellular and molecular aspects. The effects of active form of vitamin A, retinoic acid (RA), are believed to be mediated by specific nuclear receptor proteins [retinoic acid receptor (RAR)] which are members of the steroid and thyroid/retinoid receptor superfamily of ligand dependent transcriptional regulators, RARalpha, RARgamma, RXRalpha, and RXRbeta mRNA were abundant in adipose tissue and 3T3-L1 adipose cells. The autoregulated amplification of RARgamma mRNA was observed by these own ligands in 3T3-L1 cells. And, RA inhibited PPARgamma2 expression more effectively and caused concomitantly a greater inhibition of adipocyte differentiation. These results suggest that the inhibitory action of adipocyte differentiation by carotenoids and retinoids are exhibited through the RAR up-regulation and the suppression of PPARgamma2. The nature of the cross talk of vitamin A actions between the RARs, RXRs and PPARs via co-activator in adipose tissue will likely prove to be important for understanding the process of adipogenesis.

Physical and functional interactions between cellular retinoic acid binding protein II and the retinoic acid-dependent nuclear complex.

Two sorts of proteins bind to, and mediate the developmental and homeostatic effects of, retinoic acid (RA): the RAR and RXR nuclear receptors, which act as ligand-dependent transcriptional regulators, and the cellular RA binding proteins (CRABPI and CRABPII). CRABPs are generally known to be implicated in the synthesis, degradation, and control of steady-state levels of RA, yet previous and recent data have indicated that they could play a role in the control of gene expression. Here we show for the first time that, both in vitro and in vivo, CRABPII is associated with RARalpha and RXRalpha in a ligand-independent manner in mammalian cells (HL-60, NB-4, and MCF-7). In the nucleus, this protein complex binds the RXR-RAR-specific response element of an RA target gene (RARE-DR5). Moreover, in the presence of retinoids that bind both the nuclear receptors and CRABPII, enhancement of transactivation by RXRalpha-RARalpha heterodimers is observed in the presence of CRABPII. Thus, CRABPII appears to be a novel transcriptional regulator involved in RA signaling.

Retinoid receptors in health and disease: co-regulators and the chromatin connection

Retinoid receptors behave as ligand-dependent transcriptional regulators, repressing transcription in the absence of ligand and activating transcription in its presence. The different effects on transcription are carried out through recruitment of co-regulators: unliganded receptors bind corepressors (NCoR and SMRT) that are found within a complex containing histone deacetylase (HDAC) activity, whereas liganded receptors recruit coactivators with histone acetylase activity (HATs). Chromatin remodeling activities have also shown to be required, suggesting a hierarchy of promoter structure modifications in RA target genes carried out by multiple coregulatory complexes. In this review, we examine the experimental evidence for the model just sketched. We focus on recent findings highlighting new molecular details in receptor*b1coregulator interactions, including the discovery and initial characterisation of novel complexes with multiple chromatin modifying activities. Finally, we look at the role of aberrant recruitment of the NCoRHDAC complex by altered retinoid receptors in the pathogenesis of acute promyelocytic leukemia. These results point to a crucial role for control of transcription factor coregulator interactions in the regulation of cellular processes, and suggest new molecular targets for cancer therapy.

Peroxisome Proliferator-activated Receptor γ Ligands Are Potent Inhibitors of Angiogenesis in Vitro and in Vivo

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that functions as a transcription factor to mediate ligand-dependent transcriptional regulation. Activation of PPARgamma by the naturally occurring ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), or members of a new class of oral antidiabetic agents, e.g. BRL49653 and ciglitizone, has been linked to adipocyte differentiation, regulation of glucose homeostasis, inhibition of macrophage and monocyte activation, and inhibition of tumor cell proliferation. Here we report that human umbilical vein endothelial cells (HUVEC) express PPARgamma mRNA and protein. Activation of PPARgamma by the specific ligands 15d-PGJ2, BRL49653, or ciglitizone, dose dependently suppresses HUVEC differentiation into tube-like structures in three-dimensional collagen gels. In contrast, specific PPARalpha and -beta ligands do not affect tube formation although mRNA for these receptors are expressed in HUVEC. PPARgamma ligands also inhibit the proliferative response of HUVEC to exogenous growth factors. Treatment of HUVEC with 15d-PGJ2 also reduced mRNA levels of vascular endothelial cell growth factor receptors 1 (Flt-1) and 2 (Flk/KDR) and urokinase plasminogen activator and increased plasminogen activator inhibitor-1 (PAI-1) mRNA. Finally, administration of 15d-PGJ2 inhibited vascular endothelial cell growth factor-induced angiogenesis in the rat cornea. These observations demonstrate that PPARgamma ligands are potent inhibitors of angiogenesis in vitro and in vivo, and suggest that PPARgamma may be an important molecular target for the development of small-molecule inhibitors of angiogenesis.

RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF2.

Steroids, thyroid hormones, vitamin D3, and retinoids are lipophilic small molecules that regulate diverse biological effects such as cell differentiation, development, and homeostasis. The actions of these hormones are mediated by steroid/nuclear receptors which function as ligand-dependent transcriptional regulators. Transcriptional activation by ligand-bound receptors is a complex process requiring dissociation and recruitment of several additional cofactors. We report here the cloning and characterization of receptor-associated coactivator 3 (RAC3), a human transcriptional coactivator for steroid/nuclear receptors. RAC3 interacts with several liganded receptors through a mechanism which requires their respective ligand-dependent activation domains. RAC3 can activate transcription when tethered to a heterologous DNA-binding domain. Overexpression of RAC3 enhances the ligand-dependent transcriptional activation by the receptors in mammalian cells. Sequence analysis reveals that RAC3 is related to steroid receptor coactivator 1 (SRC-1) and transcriptional intermediate factor 2 (TIF2), two of the most potent coactivators for steroid/nuclear receptors. Thus, RAC3 is a member of a growing coactivator network that should be useful as a tool for understanding hormone action and as a target for developing new therapeutic agents that can block hormone-dependent neoplasia.

Isolation of a Genomic DNA Fragment Having Negative Vitamin D Response Element

The vitamin D receptor (VDR) binds to the vitamin D response element (VDRE) in the promoter region of target genes and acts as a ligand-dependent transcriptional regulator. In order to identify novel VDREs and new genes that are regulated by the active form of vitamin D [1,25-(OH)2D3], rat genomic DNA fragments bound by VDR were isolated. One of these fragments, designated as VBF5, mediated 1,25-(OH)2D3-dependent negative regulation. Moreover, a genomic DNA region around VBF5 was transcribed and the transcript was down-regulated by 1,25-(OH)2D3. These data strongly indicate that VBF5 may contain a VDRE regulating negatively an unidentified gene expression.