Multicellular organisms express many genes in a cell-typespecific fashion. The mechanisms governing cell-specific gene expression are becoming increasingly understood at two levels. First, there are numerous examples in which the binding of nuclear transcription factors to cis elements within gene regulatory regions has been shown to govern cell-specific activation of gene transcription. A second level of regulation has been proposed to be mediated by the remodeling of chromatin organization both globally and at individual gene loci. Chromatin remodeling itself encompasses many types of changes, including nucleosome phasing, alterations of chromosomal protein content (histone and nonhistone), and posttranslational modification of chromosomal proteins by acetylation, phosphorylation, poly(ADP-ribosyl)ation, methylation, and ubiquitination (6). Although it is widely acknowledged that both levels must govern the cell-specific expression of individual genes, there has been little information as to mechanisms that might integrate these two levels of regulation. Recently, however, histone acetyltransferases have been shown to bind to specific trans-acting factors (recently reviewed in references 22, 29, and 55). Such protein-protein binding suggests a mechanism by which chromatin histones can be modified locally (as opposed to globally) through binding of histone acetyltransferases to transcription factors that are in turn bound to DNA cis elements within the regulatory regions of specific genes. We report here a new mechanism by which these two levels of gene regulation can be integrated; through direct binding of the chromatin-modifying protein, poly(ADP-ribose) polymerase (PARP), to DNA sequences within MCAT1 regulatory elements.
Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) represses the transcriptional activity of a number of nuclear receptors, including that of retinoid receptors (RAR and RXR) and thyroid hormone receptors (TR). Since COUP-TF is capable of binding to DNA in vitro either as a homodimer or as a heterodimer with RXR or TR, it has not been possible to distinguish between competitive DNA binding and heterodimer formation as a mechanism to account for the repression. Using a two-hybrid system we have investigated the dimerisation properties of COUP-TF II in intact cells. In conditions where COUP-TF II homodimers and RXR alpha-RAR alpha heterodimers were formed we were unable to detect the formation of heterodimers between COUP-TF II and RXR alpha. Moreover, we were unable to detect an interaction between COUP-TF II and RXR alpha on DNA. Similarly COUP-TF II homodimers and RXR alpha-TR beta heterodimers are favoured over COUP-TF II-TR beta heterodimers. We conclude that the formation of functionally inactive heterodimers is unlikely to represent a general mechanism by which COUP-TF represses the transcriptional activity of nuclear receptors and favour a model in which repression is mediated by COUP-TF homodimers competing for binding to DNA.
A subset of nuclear receptors, including those for thyroid hormone (TR), retinoic acid, vitamin D 3 , and eicosanoids, can form heterodimers with the retinoid X receptor (RXR) on DNA regulatory elements in the absence of their cognate ligands. In a mammalian twohybrid assay, we have found that recruitment of a VP16-RXR chimera by a Gal4-TR ligand-binding domain fusion is enhanced up to 50-fold by thyroid hormone (T 3 ). This was also observed with a mutant fusion, Gal4-TR(L454A), lacking ligand-inducible activation function (AF-2) and unable to interact with putative coactivators, suggesting that the AF-2 activity of TR or intermediary cofactors is not involved in this effect. The wild-type and mutant Gal4-TR fusions also exhibited hormonedependent recruitment of RXR in yeast. Hormone-dependent recruitment of RXR was also evident with another Gal4-TR mutant, AHTm, which does not interact with the nuclear receptor corepressor N-CoR, suggesting that ligand-enhanced dimerization is not a result of T 3 -induced corepressor release. Finally, we have shown that the interaction between RXR and TR is augmented by T 3 in vitro, arguing against altered expression of either partner in vivo mediating this effect. We propose that ligand-dependent heterodimerization of TR and RXR in solution may provide a further level of control in nuclear receptor signaling.The thyroid hormone receptor (TR) 1 is a member of the nuclear receptor superfamily of ligand-inducible transcription factors. A subset of receptors, including those for estrogen and glucocorticoid, bind to regulatory sequences in target gene promoters as homodimers, with cooperative DNA binding and dimerization mediated by regions within their DNA-and ligand-binding domains (1-7). The thyroid hormone receptor is also capable of binding to thyroid hormone response elements (TREs) as a monomer or homodimer, but the latter complex is known to dissociate in the presence of thyroid hormone (T 3 ) (8 -11). More recently, it has been shown that TR as well as receptors for vitamin D 3 , retinoic acid (RAR), and eicosanoids form heterodimeric complexes with an auxiliary factor, the retinoid X receptor (RXR) (12-22). The TR⅐RXR heterodimer binds TREs with higher affinity than TR alone and remains stable in the presence of T 3 (8 -11), suggesting that this complex mediates ligand-dependent transcriptional activation.The isolated DNA-binding domains (DBDs) of TR and RXR are capable of both cooperative binding to and discrimination of direct repeat TREs (23). The dimerization interface between DBDs was mapped initially by mutational analyses (24) and then elucidated from the crystal structure of TR⅐RXR DBDs bound to a DR4 TRE (25). A separate dimerization interface in the carboxyl-terminal domains of nuclear receptors was first delineated as a series of nine conserved hydrophobic heptad repeats (26) predicted to form ␣-helices with the potential to mediate a coiled-coil protein interaction. A 40-amino acid region encompassing the ninth heptad has been shown to be critical for heter...
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