Leonard P. Freedman

Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex.

Nuclear receptors modulate the transcription of genes in direct response to small lipophilic ligands. Binding to ligands induces conformational changes in the nuclear receptors that enable the receptors to interact with several types of cofactor that are critical for transcription activation (transactivation). We previously described a distinct set of ligand-dependent proteins called DRIPs, which interact with the vitamin D receptor (VDR); together, these proteins constitute a new cofactor complex. DRIPs bind to several nuclear receptors and mediate ligand-dependent enhancement of transcription by VDR and the thyroid-hormone receptor in cell-free transcription assays,. Here we report the identities of thirteen DRIPs that constitute this complex, and show that the complex has a central function in hormone-dependent transactivation by VDR on chromatin templates. The DRIPs are almost indistinguishable from components of another new cofactor complex called ARC, which is recruited by other types of transcription activators to mediate transactivation on chromatin-assembled templates,. Several DRIP/ARC subunits are also components of other potentially related cofactors, such as CRSP, NAT, SMCC and the mouse Mediator, indicating that unique classes of activators may share common sets or subsets of cofactors. The role of nuclear-receptor ligands may, in part, be to recruit such a cofactor complex to the receptor and, in doing so, to enhance transcription of target genes.

Increasing the Complexity of Coactivation in Nuclear Receptor Signaling

Although the sheer number of coactivators and the novel mechanisms by which they are recruited to receptors might appear mind-numbing, a recently characterized multisubunit complex that binds to vitamin D receptor (VDR) (Rachez et al. 1998xRachez, C., Suldan, Z., Ward, J., Chang, C.P., Burakov, D., Erdjument-Bromage, H., Tempst, P., and Freedman, L.P. Genes Dev. 1998; 12: 1787–1800Crossref | PubMedSee all ReferencesRachez et al. 1998), thyroid hormone receptor (TR) (Fondell et al. 1996xFondell, J.D., Ge, H., and Roeder, R.G. Proc. Natl. Acad. Sci. USA. 1996; 93: 8329–8333Crossref | PubMed | Scopus (402)See all ReferencesFondell et al. 1996), and, most likely, many other members of the nuclear receptor family, points to the ultimate generality of transactivation. This complex, called DRIP or TRAP, is recruited to the LBD AF2 core in response to ligand-binding through a single subunit (DRIP205/TRAP220) via one LXXLL motif in much the same manner as the p160 coactivators. DRIP205 anchors the other 14–16 proteins comprising the DRIP/TRAP complex to the receptor, thereby conferring hormone-dependent recruitment of what appears to be a preformed complex. DRIP/TRAP is required for ligand-dependent transcription activation in vitro, which notably has not yet been demonstrated for the p160 coactivators. Several DRIP/TRAP subunits are homologous to proteins described as components of Mediator (7xGu, W., Malik, S., Ito, M., Yuan, C.-X., Fondell, J.D., Zhang, X., Martinez, E., Qin, J., and Roeder, R.G. Mol. Cell. 1999; 3: 97–108Abstract | Full Text | Full Text PDF | PubMed | Scopus (224)See all References, 13xRachez, C., Suldan, Z., Ward, J., Chang, C.P., Burakov, D., Erdjument-Bromage, H., Tempst, P., and Freedman, L.P. Genes Dev. 1998; 12: 1787–1800Crossref | PubMedSee all References), a complex that together with SRB proteins associates with RNA polymerase II (Pol II) through its large subunit’s carboxy-terminal repeat domain (CTD). This suggests that DRIP/TRAP, perhaps through Mediator/SRB subunits, might function in part by targeting Pol II holoenzyme to promoters (Figure 2BFigure 2B). The generality of this complex stems from the unexpected observation that other activators unrelated to nuclear receptors, including Sp1, recruit the DRIP/TRAP complex or subcomplexes (called CRSP in Ryu et al. 1999xRyu, S., Zhou, S., Ladurner, A.G., and Tjian, R. Nature. 1999; 397: 446–450Crossref | PubMed | Scopus (282)See all ReferencesCRSP in Ryu et al. 1999), and that several of the DRIP/TRAP subunits are present in two similar, if not identical, SRB-interacting complexes, NAT and SMCC (18xSun, X., Zhang, Y., Cho, H., Rickert, P., Lees, E., Lane, W., and Reinberg, D. Mol. Cell. 1998; 2: 213–222Abstract | Full Text | Full Text PDF | PubMedSee all References, 7xGu, W., Malik, S., Ito, M., Yuan, C.-X., Fondell, J.D., Zhang, X., Martinez, E., Qin, J., and Roeder, R.G. Mol. Cell. 1999; 3: 97–108Abstract | Full Text | Full Text PDF | PubMed | Scopus (224)See all References). As studies on the DRIP/TRAP complex progress, it is expected that their role in transcriptional regulation will be further generalized; many classes of activators are likely to recruit coactivator complexes and subcomplexes containing the shared subunits of DRIP/TRAP and SRB/Mediator. This additionally suggests that nuclear receptors utilize a general mechanism for transactivation, but with inducibility imposed by ligand binding, which is required for the recruitment of the DRIP/TRAP complex.How the DRIP/TRAP complex interfaces with the p160/CBP/pCAF system, if at all, is perhaps one of the key questions regarding nuclear receptor coactivation presently. They may act independently or they may act in sequence, where the HAT coactivators remodel chromatin to then allow the DRIP/TRAP complex to recruit RNA polymerase II holoenzyme (Figure 2Figure 2). In this model, what influences the dissociation of the first complex and the binding of the second is unknown. What is certain is that as the number of coactivators and the complexes they form grow, the challenge of understanding their underlying molecular functions will continue to generate surprises and inspire insights into how gene transcription is so exquisitely regulated.

Transcriptional repression of the interleukin-2 gene by vitamin D3: direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor.

T-lymphocyte proliferation is suppressed by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, and is associated with a decrease in interleukin 2 (IL-2), gamma interferon, and granulocyte-macrophage colony-stimulating factor mRNA levels. We report here that 1,25(OH)2D3-mediated repression in Jurkat cells is cycloheximide resistant, suggesting that it is a direct transcriptional repressive effect on IL-2 expression by the vitamin D3 receptor (VDR). We therefore examined vitamin D3-mediated repression of activated IL-2 expression by cotransfecting Jurkat cells with IL-2 promoter/reporter constructs and a VDR overexpression vector and by DNA binding. We delineated an element conferring both DNA binding by the receptor in vitro and 1,25(OH)2D3-mediated repression in vivo to a short 40-bp region encompassing an important positive regulatory element, NF-AT-1, which is bound by a T-cell-specific transcription factor, NFATp, as well as by AP-1. VDR DNA-binding mutants were unable to either bind to this element in vitro or repress in vivo; the VDR DNA-binding domain alone, however, bound the element but also could not repress IL-2 expression. These results indicate that DNA binding by VDR is necessary but not sufficient to mediate IL-2 repression. By combining partially purified proteins in vitro, we observed the loss of the bound NFATp/AP-1-DNA complex upon inclusion of VDR or VDR-retinoid X receptor. Order of addition and off-rate experiments indicate that the VDR-retinoid X receptor heterodimer blocks NFATp/AP-1 complex formation and then stably associates with the NF-AT-1 element. This direct inhibition by a nuclear hormone receptor of transcriptional activators of the IL-2 gene may provide a mechanistic explanation of how vitamin derivatives can act as potent immunosuppressive agents.

Mechanisms of gene regulation by vitamin D3 receptor: a network of coactivator interactions

The vitamin D(3) receptor regulates transcription in direct response to its cognate hormonal ligand, 1,25(OH)(2)D(3). Ligand binding leads to the recruitment of coactivators. Many of these factors, acting in large complexes, have emerged as chromatin remodelers partly through intrinsic histone modifying activities. In addition, other ligand-recruited complexes appear to act more directly on the transcriptional apparatus, suggesting that transcriptional regulation by VDR and other nuclear receptors may involve a process of both chromatin alterations and direct recruitment of key initiation components at regulated promoters.

Transcriptional Activation of the Human p21WAF1/CIP1 Gene by Retinoic Acid Receptor CORRELATION WITH RETINOID INDUCTION OF U937 CELL DIFFERENTIATION

We reported previously that the induced differentiation of the myelomonocytic cell line U937 by vitamin D3 is facilitated by the transcriptional induction of the p21WAF1/CIP1 gene by the vitamin D3 receptor (Liu, M., Lee, M.-H., Cohen, M., and Freedman, L. P. (1996) Genes Dev. 10, 143-153). Retinoic acid (RA), a physiological metabolite of vitamin A, is also a potent inducer of differentiation of several cell types, including myeloid leukemic cells. Like vitamin D3, RA acts through a subfamily of nuclear hormone receptors, RARs and RXRs (retinoid X receptors), which regulate the expression of target genes by binding to specific DNA elements and modulating transcription initiation. In this report we demonstrate that the gene encoding p21 is also a RA-responsive target gene, and we describe a functional RA response element in this genes promoter which is required to confer RA induction through RARRXR heterodimers. These results correlate the RA induction of monocytic differentiation of U937 cells with the transcriptional activation of the p21 gene and suggest a role for this cyclin/cyclin-dependent kinase complex inhibitor in facilitating this differentiation pathway.

Mediator complexes and transcription.

Over the past decade, various components of the transcription machinery have been identified as potential targets for activators. Recently, metazoan versions of yeast Mediator have been isolated and found to act as key coactivators to many transcription factors. Recent work has defined the composition, function and biology of metazoan mediator complexes, which has led us to propose a new nomenclature for the variously named versions of the mediator complex.

DIFFERENTIAL REGULATION OF GLUCOCORTICOID RECEPTOR TRANSCRIPTIONAL ACTIVATION VIA AF-1-ASSOCIATED PROTEINS

The hormone‐activated glucocorticoid receptor (GR), through its N‐ and C‐terminal transcriptional activation functions AF‐1 and AF‐2, controls the transcription of target genes presumably through interaction(s) with transcriptional regulatory factors. Utilizing a modified yeast two‐hybrid approach, we have identified the tumor susceptibility gene 101 (TSG101) and the vitamin D receptor‐interacting protein 150 (DRIP150) as proteins that interact specifically with a functional GR AF‐1 surface. In yeast and mammalian cells, TSG101 represses whereas DRIP150 enhances GR AF‐1‐mediated transactivation. Thus, GR AF‐1 is capable of recruiting both positive and negative regulatory factors that differentially regulate GR transcriptional enhancement. In addition, we show that another member of the DRIP complex, DRIP205, interacts with the GR ligand binding domain in a hormone‐dependent manner and facilitates GR transactivation in concert with DRIP150. These results suggest that DRIP150 and DRIP205 functionally link GR AF‐1 and AF‐2, and represent important mediators of GR transcriptional enhancement.

Repression of transcription of the p27 Kip1 cyclin-dependent kinase inhibitor gene by c-Myc

Upon engagement of the B Cell Receptor (BCR) of WEHI 231 immature B cells, a drop in c-Myc expression is followed by activation of the cyclin-dependent kinase inhibitor (CKI) p27Kip1, which induces growth arrest and apoptosis. Here, we report inverse patterns of p27 and c-Myc protein expression follow BCR engagement. We present evidence demonstrating, for the first time, that the p27Kip1 gene is a target of transcriptional repression by c-Myc. Specifically, the changes in p27 protein levels correlated with changes in p27 mRNA levels, and gene transcription. Induction of p27 promoter activity followed BCR engagement of WEHI 231 cells, and this induction could be repressed upon co-transfection of a c-Myc expression vector. Inhibition of the TATA-less p27 promoter by c-Myc was also observed in Jurkat T cells, vascular smooth muscle, and Hs578T breast cancer cells, extending the observation beyond immune cells. Consistent with a putative Inr element CCAGACC (where +1 is underlined) at the start site of transcription in the p27 promoter, deletion of Myc homology box II reduced the extent of repression. Furthermore, enhanced repression was observed upon transfection of the c-Myc ‘super-repressor’, with mutation of Phe115 to Leu. The sequences mediating transcriptional activity and c-Myc repression were mapped to bp −20 to +20 of the p27 gene. Finally, binding of Max was shown to facilitate c-Myc binding and repression of p27 promoter activity. Overall, these studies identify the p27 CKI gene as a new target whereby c-Myc can control cell proliferation, survival and neoplastic transformation.

1,25-Dihydroxyvitamin D3 Inhibits IFN-γ and IL-4 Levels During In Vitro Polarization of Primary Murine CD4+ T Cells

Following their activation, naive CD4+ T cells can differentiate into one of two effector cell subsets, Th1 and Th2. These two subsets have different cytokine secretion patterns and thus mediate separate arms of the immune response. It has been established that the fat-soluble vitamin D3 metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its nuclear receptor, the vitamin D receptor, play an important role in the immune system primarily through the transcriptional inhibition of cytokine genes that either are required for Th1 differentiation or are products of differentiated Th1 cells. Therefore, we wanted to test directly the ability of 1,25(OH)2D3 to alter the Th differentiation process. Our results indicate that 1,25(OH)2D3 inhibits not only the Th1 cytokine IFN-γ but also the Th2 cytokine IL-4 in naive CD62 ligand+CD4+ T cells during their in vitro polarization. This effect is most dramatic when the ligand is present from the onset of the differentiation process. If the ligand is added after the polarization has ensued, the inhibition is significantly diminished. In activated (CD62 ligand−CD4+) T cells, 1,25(OH)2D3 is still able to inhibit IFN-γ but has no effect on IL-4 production. Our results also indicate that inhibition of these two cytokines in naive cells by vitamin D receptor and its ligand is neither a result of a cell cycle block nor an inhibition of Th1 or Th2 transcription factor expression but, rather, at least in the case of Th2 differentiation, an attenuation of IL-4 transcription by the receptor.

Estrogen protects bone by inducing Fas ligand in osteoblasts to regulate osteoclast survival

Estrogen deficiency in menopause is a major cause of osteoporosis in women. Estrogen acts to maintain the appropriate ratio between bone‐forming osteoblasts and bone‐resorbing osteoclasts in part through the induction of osteoclast apoptosis. Recent studies have suggested a role for Fas ligand (FasL) in estrogen‐induced osteoclast apoptosis by an autocrine mechanism involving osteoclasts alone. In contrast, we describe a paracrine mechanism in which estrogen affects osteoclast survival through the upregulation of FasL in osteoblasts (and not osteoclasts) leading to the apoptosis of pre‐osteoclasts. We have characterized a cell‐type‐specific hormone‐inducible enhancer located 86 kb downstream of the FasL gene as the target of estrogen receptor‐alpha induction of FasL expression in osteoblasts. In addition, tamoxifen and raloxifene, two selective estrogen receptor modulators that have protective effects in bone, induce apoptosis in pre‐osteoclasts by the same osteoblast‐dependent mechanism. These results demonstrate that estrogen protects bone by inducing a paracrine signal originating in osteoblasts leading to the death of pre‐osteoclasts and offer an important new target for the prevention and treatment of osteoporosis.