Biomedical subjects

B D Lemon

Publications and source records attributed to B D Lemon.

6 recordsLinked to original sources

PubMed2001

Transcriptional coactivator complexes.

The last two decades have witnessed a tremendous expansion in our knowledge of the mechanisms employed by eukaryotic cells to control gene activity. A critical insight to transcriptional control mechanisms was provided by the discovery of coactivators, a diverse array of cellular factors that connect sequence-specific DNA binding activators to the general transcriptional machinery, or that help activators and the transcriptional apparatus to navigate through the constraints of chromatin. A number of coactivators have been isolated as large multifunctional complexes, and biochemical, genetic, molecular, and cellular strategies have all contributed to uncovering many of their components, activities, and modes of action. Coactivator functions can be broadly divide into two classes: (a) adaptors that direct activator recruitment of the transcriptional apparatus, (b) chromatin-remodeling or -modifying enzymes. Strikingly, several distinct coactivator complexes nonetheless share many subunits and appear to be assembled in a modular fashion. Such structural and functional modularity could provide the cell with building blocks from which to construct a versatile array of coactivator complexes according to its needs. The extent of functional interplay between these different activities in gene-specific transcriptional regulation is only now becoming apparent, and will remain an active area of research for years to come.

Acetyltransferases↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

Nuclear receptor cofactors as chromatin remodelers.

Nuclear receptors regulate transcription in direct response to their cognate hormonal ligands. Ligand binding leads to the dissociation of corepressors and the recruitment of coactivators. Many of these factors, acting in large complexes, have emerged as chromatin remodelers through intrinsic histone modifying activities or through other novel functions. In addition, other ligand-recruited complexes appear to act more directly on the transcriptional apparatus, suggesting that transcriptional regulation by nuclear receptors may involve a process of both chromatin alterations and direct recruitment of key initiation components at regulated promoters.

Adenosine Triphosphate↗

PubMed1997

Retinoid X receptor:vitamin D3 receptor heterodimers promote stable preinitiation complex formation and direct 1,25-dihydroxyvitamin D3-dependent cell-free transcription.

The numerous members of the steroid/nuclear hormone receptor superfamily act as direct transducers of circulating signals, such as steroids, thyroid hormone, and vitamin or lipid metabolites, and modulate the transcription of specific target genes, primarily as dimeric complexes. The receptors for 9-cis retinoic acid and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], RXR and VDR, respectively, as members of this superfamily, form a heterodimeric complex and bind cooperatively to vitamin D responsive elements (VDREs) to activate or repress the transcription of a multitude of genes which regulate a variety of physiological functions. To directly investigate RXR- and VDR-mediated transactivation, we developed a cell-free transcription system for 1,25(OH)2D3 signaling by utilizing crude nuclear extracts and a G-free cassette-based assay. Transcriptional enhancement in vitro was dependent on purified, exogenous RXR and VDR and was responsive to physiological concentrations of 1,25(OH)2D3. We found that RXR and VDR transactivated selectively from VDRE-linked templates exclusively as a heterodimeric complex, since neither receptor alone enhanced transcription in vitro. By the addition of low concentrations of the anionic detergent Sarkosyl to limit cell-free transcription to a single round and the use of agarose gel mobility shift experiments to assay factor complex assembly, we observed that 1,25(OH)2D3 enhanced RXR:VDR-mediated stabilization or assembly of preinitiation complexes to effect transcriptional enhancement from VDRE-linked promoter-containing DNA.

Base Sequence↗

PubMed1996

Selective effects of ligands on vitamin D3 receptor- and retinoid X receptor-mediated gene activation in vivo.

Steroid/nuclear hormone receptors are ligand-regulated transcription f factors that play key roles in cell regulation, differentiation, and oncogenesis. Many nuclear receptors, including the human 1,25-dihydroxyvitamin D3 receptor (VDR), bind cooperatively to DNA either as homodimers or as heterodimers with the 9-cis retinoic acid (RA) receptor (retinoid X-receptor [RXR]). We have previously reported that the ligands for VDR and RXR can differentially modulate the affinity of the receptors' interaction with DNA in vitro, primarily by modulating the dimerization status of these receptors. These experiments suggested a complex interaction between VDR and RXR and their respective ligands on inducible target genes in vivo. To examine these effects in cells, we used a transient-transfection strategy whereby we simultaneously introduced two different reporter plasmids that are selectively inducible by each ligand. Although VDR can bind as a homodimer to the osteopontin gene vitamin D response element, we find that a RXR-VDR heterodimer must be the transactivating species from the element in vivo, since RXR enhances and 9-cis RA and other RXR-specific ligands attenuate this induction. Conversely, when VDR is overexpressed, vitamin D3 attenuates 9-cis RA induction from an RXR-responsive element. These effects, however, appear to be very sensitive to both the relative ratios of the two receptors and their respective target elements. Functional RXR-VDR complexes are strictly dependent on the DNA-binding polarity. Chimeric versions of VDR and RXR were also constructed to examine the putative activities of homodimeric receptors; a VDR chimera can transactivate in the absence of RXR, demonstrating that VDR has intrinsic transactivation properties. Taken together, these results establish a complex, sensitive cross talk in vivo between two ligands and their receptors that signal through two distinct endocrine pathways.

Animals↗

PubMed1995

Vitamin D3-retinoid X receptor dimerization, DNA binding, and transactivation are differentially affected by analogs of 1,25-dihydroxyvitamin D3.

A number of analogs of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have been synthesized that act as more potent inducers of cellular differentiation and inhibitors of cell growth than the natural ligand; at the same time, many of the analogs have reduced hypercalcemic properties. This combination makes these compounds attractive candidates for clinical use. The mechanism by which the analogs act, however, is unclear. Potentially, the analogs could be taken up more readily, be more slowly catabolized, or have higher binding affinities for the vitamin D receptor (VDR). Analogs of 1,25-(OH)2D3 could also differentially modulate one or more of the activities of VDR, namely dimerization, DNA binding, and/or transcriptional regulation. To directly examine this latter possibility, we used a sensitive assay for the kinetics of dimerization and DNA binding, surface plasmon resonance, and report here that three 1,25-(OH)2D3 analogs, 1,25-(OH)2-16-ene-23-yne-D3, 1,25-(OH)2-16-ene-23-yne-26,27-di home-D3, and 1,25-(OH)2-26,27-hexafluoro-16-ene-23-yne-D3, all confer distinct rate and equilibrium constants for VDR-retinoid X receptor heterodimerization and DNA binding to a specific vitamin D response element relative to the natural ligand. In response to the hexafluoro analog, the apparent Kd for DNA binding by VDR was significantly lower than that for 1,25-(OH)2D3, and correspondingly, in vivo transactivation from a responsive reporter was greater. Interestingly, solution heterodimerization was not affected by this analog. These results suggest that vitamin D analogs do indeed confer biological effects by acting directly and differentially at the level of VDR, and that specific vitamin D analogs can act on distinct receptor functions.

Base Sequence↗