Michael G. Rosenfeld

A CBP Integrator Complex Mediates Transcriptional Activation and AP-1 Inhibition by Nuclear Receptors

Nuclear receptors regulate gene expression by direct activation of target genes and inhibition of AP-1. Here we report that, unexpectedly, activation by nuclear receptors requires the actions of CREB-binding protein (CBP) and that inhibition of AP-1 activity is the apparent result of competition for limiting amounts of CBP/p300 in cells. Utilizing distinct domains, CBP directly interacts with the ligand-binding domain of multiple nuclear receptors and with the p160 nuclear receptor coactivators, which upon cloning have proven to be variants of the SRC-1 protein. Because CBP represents a common factor, required in addition to distinct coactivators for function of nuclear receptors, CREB, and AP-1, we suggest that CBP/p300 serves as an integrator of multiple signal transduction pathways within the nucleus.

Molecular determinants of resistance to antiandrogen therapy.

Using microarray-based profiling of isogenic prostate cancer xenograft models, we found that a modest increase in androgen receptor mRNA was the only change consistently associated with the development of resistance to antiandrogen therapy. This increase in androgen receptor mRNA and protein was both necessary and sufficient to convert prostate cancer growth from a hormone-sensitive to a hormone-refractory stage, and was dependent on a functional ligand-binding domain. Androgen receptor antagonists showed agonistic activity in cells with increased androgen receptor levels; this antagonist-agonist conversion was associated with alterations in the recruitment of coactivators and corepressors to the promoters of androgen receptor target genes. Increased levels of androgen receptor confer resistance to antiandrogens by amplifying signal output from low levels of residual ligand, and by altering the normal response to antagonists. These findings provide insight toward the design of new antiandrogens.

Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-gamma.

The peroxisome proliferator-activated receptor-γ (PPAR-γ) is a ligand-dependent transcription factor that is important in adipocyte differentiation and glucose homeostasis and which depends on interactions with co-activators, including steroid receptor co-activating factor-1 (SRC-1). Here we present the X-ray crystal structure of the human apo-PPAR-γ ligand-binding domain (LBD), at 2.2 Å resolution; this structure reveals a large binding pocket, which may explain the diversity of ligands for PPAR-γ. We also describe the ternary complex containing the PPAR-γ LBD, the antidiabetic ligand rosiglitazone (BRL49653), and 88 amino acids of human SRC-1 at 2.3 Å resolution. Glutamate and lysine residues that are highly conserved in LBDs of nuclear receptors form a ‘charge clamp’ that contacts backbone atoms of the LXXLL helices of SRC-1. These results, together with the observation that two consecutive LXXLL motifs of SRC-1 make identical contacts with both subunits of a PPAR-γ homodimer, suggest a general mechanism for the assembly of nuclear receptors with co-activators.

The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function

The functionally conserved proteins CBP and p300 act in conjunction with other factors to activate transcription of DNA. A new factor, p/CIP, has been discovered that is present in the cell as a complex with CBP and is required for transcriptional activity of nuclear receptors and other CBP/p300-dependent transcription factors. The highly related nuclear-receptor co-activator protein NCoA-1 is also specifically required for ligand-dependent activation of genes by nuclear receptors. p/CIP, NCoA-1 and CBP all contain related leucine-rich charged helical interaction motifs that are required for receptor-specific mechanisms of gene activation, and allow the selective inhibition of distinct signal-transduction pathways.

A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) has essential roles in adipogenesis and glucose homeostasis, and is a molecular target of insulin-sensitizing drugs. Although the ability of PPAR-γ agonists to antagonize inflammatory responses by transrepression of nuclear factor kappa B (NF-κB) target genes is linked to antidiabetic and antiatherogenic actions, the mechanisms remain poorly understood. Here we report the identification of a molecular pathway by which PPAR-γ represses the transcriptional activation of inflammatory response genes in mouse macrophages. The initial step of this pathway involves ligand-dependent SUMOylation of the PPAR-γ ligand-binding domain, which targets PPAR-γ to nuclear receptor corepressor (NCoR)–histone deacetylase-3 (HDAC3) complexes on inflammatory gene promoters. This in turn prevents recruitment of the ubiquitylation/19S proteosome machinery that normally mediates the signal-dependent removal of corepressor complexes required for gene activation. As a result, NCoR complexes are not cleared from the promoter and target genes are maintained in a repressed state. This mechanism provides an explanation for how an agonist-bound nuclear receptor can be converted from an activator of transcription to a promoter-specific repressor of NF-κB target genes that regulate immunity and homeostasis.

RXRβ: A coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements

The retinoic acid receptor (RAR) requires coregulators to bind effectively to response elements in target genes. A strategy of sequential screening of expression libraries with a retinoic acid response element and RAR identified a cDNA encoding a coregulator highly related to RXR alpha. This protein, termed RXR beta, forms heterodimers with RAR, preferentially increasing its DNA binding and transcriptional activity on promoters containing retinoic acid, but not thyroid hormone or vitamin D, response elements. Remarkably, RXR beta also heterodimerizes with the thyroid hormone and vitamin D receptors, increasing both DNA binding and transcriptional function on their respective response elements. RXR alpha also forms heterodimers with these receptors. These observations suggest that retinoid X receptors meet the criteria for biochemically characterized cellular coregulators and serve to selectively target the high affinity binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate DNA response elements.

Coactivator and corepressor complexes in nuclear receptor function.

The nuclear hormone receptors constitute a large family of transcription factors. The binding of the hormonal ligands induces nuclear receptors to assume a configuration that leads to transcriptional activation. Recent studies of retinoic acid and thyroid hormone receptors revealed that, upon ligand binding, a histone deacetylase (HDAC)-containing complex is displaced from the nuclear receptor in exchange for a histone acetyltransferase (HAT)-containing complex. These observations suggest that ligand-dependent recruitment of chromatin-remodeling activity serves as a general mechanism underlying the switch of nuclear receptors from being transcriptionally repressive to being transcriptionally active.

A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype

Multiple related cis-active elements required for cell-specific activation of the rat prolactin gene appear to bind a pituitary-specific positive transcription factor(s), referred to as Pit-1. DNA complementary to Pit-1 mRNA, cloned on the basis of specific binding to AT-rich cell-specific elements in the rat prolactin and growth hormone genes, encodes a 33 kd protein with significant similarity at its carboxyl terminus to the homeodomains encoded by Drosophila developmental genes. Pit-1 mRNA is expressed exclusively in the anterior pituitary gland in both somatotroph and lactotroph cell types, which produce growth hormone and prolactin, respectively. Pit-1 expression in heterologous cells (HeLa) selectively activates prolactin and growth hormone fusion gene expression, suggesting that Pit-1 is sufficient to confer a characteristic pituitary phenotype. The structure of Pit-1 and its recognition elements suggests that metazoan tissue phenotype is controlled by a family of transcription factors that bind to related cis-active elements and contain several highly conserved domains.

Nuclear receptor coactivators

Retinoic acid, steroid and thyroid hormones regulate complex programs of gene expression by binding to intracellular receptors that are members of the nuclear receptor superfamily of ligand-dependent transcription factors. Recent studies have led to the identification and cloning of genes encoding coactivator molecules that appear to play important roles in mediating ligand-dependent transcription by members of this family. The identification of these coactivator molecules suggests a point of entry into the general transcriptional machinery that is common to several other classes of regulated transcription factors.

Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription

With the recent recognition of non-coding RNAs (ncRNAs) flanking many genes, a central issue is to obtain a full understanding of their potential roles in regulated gene transcription programmes, possibly through different mechanisms. Here we show that an RNA-binding protein, TLS (for translocated in liposarcoma), serves as a key transcriptional regulatory sensor of DNA damage signals that, on the basis of its allosteric modulation by RNA, specifically binds to and inhibits CREB-binding protein (CBP) and p300 histone acetyltransferase activities on a repressed gene target, cyclin D1 (CCND1) in human cell lines. Recruitment of TLS to the CCND1 promoter to cause gene-specific repression is directed by single-stranded, low-copy-number ncRNA transcripts tethered to the 5′ regulatory regions of CCND1 that are induced in response to DNA damage signals. Our data suggest that signal-induced ncRNAs localized to regulatory regions of transcription units can act cooperatively as selective ligands, recruiting and modulating the activities of distinct classes of RNA-binding co-regulators in response to specific signals, providing an unexpected ncRNA/RNA-binding protein-based strategy to integrate transcriptional programmes.