Valerie J. Peterson

P300 FUNCTIONS AS A COACTIVATOR FOR THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA

The integrator protein, p300, was demonstrated to interact with mouse peroxisome proliferator-activated receptor α in a ligand-enhanced manner. The PPARα-interacting domain of p300 was mapped to amino acids 39–117 which interacted strongly with PPARα but did not interact with retinoic acid receptor-γ or retinoid X receptor-α. Amino acids within the carboxyl terminus of PPARα as well as residues within the hinge region were required for ligand-dependent interaction with p300. p300 enhanced the transcriptional activation properties of PPARα and, therefore, can be considered a bona fide coactivator for this nuclear receptor. These observations extend the group of p300-interacting proteins to include mPPARα and further characterize the molecular mechanisms of PPARα-mediated transcriptional regulation.

Identification of nuclear receptor corepressor as a peroxisome proliferator-activated receptor alpha interacting protein.

Nuclear receptor corepressor (NCoR) was demonstrated to interact strongly with peroxisome proliferator-activated receptor α (PPARα), and PPARα ligands suppressed this interaction. In contrast to the interaction of PPARα with the coactivator protein, p300, association of the receptor with NCoR did not require any part of the PPARα ligand binding domain. NCoR was found to suppress PPARα-dependent transcriptional activation in the context of a PPARα·retinoid X receptor α (RXRα) heterodimeric complex bound to a peroxisome proliferator-responsive element in human embryonic kidney 293 cells. This repression was reversed agonists of either receptor demonstrating a functional interaction between NCoR and PPARα·RXRα heterodimeric complexes in mammalian cells. NCoR appears to influence PPARα signaling pathways and, therefore, may modulate tissue responsiveness to peroxisome proliferators.

Involvement of the Histone Deacetylase SIRT1 in Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF)-interacting Protein 2-mediated Transcriptional Repression

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 (CTIP1 and CTIP2) enhance transcriptional repression mediated by COUP-TF II and have been implicated in hematopoietic cell development and malignancies. CTIP1 and CTIP2 are also sequence-specific DNA-binding proteins that repress transcription through direct, COUP-TF-in-dependent binding to a GC-rich response element. CTIP1- and CTIP2-mediated transcriptional repression is insensitive to trichostatin A, an inhibitor of known class I and II histone deacetylases. However, chromatin immunoprecipitation assays revealed that expression of CTIP2 in mammalian cells resulted in deacetylation of histones H3 and/or H4 that were associated with the promoter region of a reporter gene. CTIP2-mediated transcriptional repression, as well as deacetylation of promoter-associated histones H3/H4 in CTIP2-transfected cells, was reversed by nicotinamide, an inhibitor of class III histone deacetylases such as the mammalian homologs of yeast Silent Information Regulator 2 (Sir2). The human homolog of yeast Sir2, SIRT1, was found to interact directly with CTIP2 and was recruited to the promoter template in a CTIP2-dependent manner. Moreover, SIRT1 enhanced the deacetylation of template-associated histones H3/H4 in CTIP2-transfected cells, and stimulated CTIP2-dependent transcriptional repression. Finally, endogenous SIRT1 and CTIP2 co-purified from Jurkat cell nuclear extracts in the context of a large (1–2 mDa) complex. These findings implicate SIRT1 as a histone H3/H4 deacetylase in mammalian cells and in transcriptional repression mediated by CTIP2.

Ligand-induced Peroxisome Proliferator-activated Receptor α Conformational Change

Structurally diverse peroxisome proliferators and related compounds that have been demonstrated to induce the ligand-dependent transcriptional activation function of mouse peroxisome proliferator-activated receptor α (mPPARα) in transfection experiments were tested for the ability to induce conformational changes within mPPARα in vitro. WY-14,643, 5,8,11,14-eicosatetraynoic acid, LY-171883, and clofibric acid all directly induced mPPARα conformational changes as evidenced by a differential protease sensitivity assay. Carboxyl-terminal truncation mutagenesis of mPPARα differentially affected the ability of these ligands to induce conformational changes suggesting that PPAR ligands may make distinct contacts with the receptor. Direct interaction of peroxisome proliferators and related compounds with, and the resulting conformational alteration(s) in, mPPARα may facilitate interaction of the receptor with transcriptional intermediary factors and/or the general transcription machinery and, thus, may underlie the molecular basis of ligand-dependent transcriptional activation mediated by mPPARα.

CTIP2 Associates with the NuRD Complex on the Promoter of p57KIP2, a Newly Identified CTIP2 Target Gene

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2), also known as Bcl11b, is a transcriptional repressor that functions by direct, sequence-specific DNA binding activity or by recruitment to the promoter template by interaction with COUP-TF family members. CTIP2 is essential for both T cell development and axonal projections of corticospinal motor neurons in the central nervous system. However, little is known regarding the molecular mechanism(s) by which CTIP2 contributes to either process. CTIP2 complexes that were isolated from SK-N-MC neuroblastoma cells were found to harbor substantial histone deacetylase activity, which was likely conferred by the nucleosome remodeling and deacetylation (NuRD) complex. CTIP2 was found to associate with the NuRD complex through direct interaction with both RbAp46 and RbAp48, and components of the NuRD complex were found to be recruited to an artificial promoter template in a CTIP2-dependent manner in transfected cells. Finally, the NuRD complex and CTIP2 were found to co-occupy the promoter template of p57KIP2, a gene encoding a cyclin-dependent kinase inhibitor, and identified herein as a novel transcriptional target of CTIP2 in SK-N-MC cells. Therefore, it seems likely that the NuRD complex may be involved in transcriptional repression of CTIP2 target genes and contribute to the function(s) of CTIP2 within a neuronal context.

Heterodimeric Interactions between Chicken Ovalbumin Upstream Promoter-Transcription Factor Family Members ARP1 and Ear2

Members of the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) subfamily of orphan nuclear receptors, which minimally includes COUP-TFI and ARP1, are highly expressed in brain and are generally considered to be constitutive repressors of transcription. We have used a yeast two-hybrid system to isolate proteins expressed in brain that interact with ARP1. One of the proteins isolated in this screen was Ear2, another orphan receptor that has been suggested to be a member of the COUP-TF subfamily. Here we demonstrate that ARP1 and Ear2 form heterodimers in solution and on directly repeated response elements with high efficiency and a specificity differing from that of homodimeric complexes composed of either receptor. ARP1 and Ear2 were observed to interact in mammalian cells, and the tissue distribution of Ear2 transcripts was found to overlap precisely with the expression pattern of ARP1 in several mouse tissues and embryonal carcinoma cell lines. Heterodimeric interactions between ARP1 and Ear2 may define a distinct pathway of orphan receptor signaling.

Identification of a unique binding protein specific for a novel retinoid inducing cellular apoptosis.

The retinoid 6‐[3‐(1‐adamantyl)‐4‐hydroxyphenyl]‐2‐naphthalenecarboxylic acid (AHPN, CD437) induces apoptosis in a variety of cell types, many of which are cancer cells that resist the antiproliferative and/or differentiating effects of retinoids. While the retinoids exert their effects by binding to the retinoic acid nuclear receptors (RARs) or retinoid X receptors (RXRs), AHPN (CD437) binds to another protein with different ligand specificity. In nuclear extracts from HL‐60R cells the binding of AHPN (CD437) was only minimally competed by either retinoic acid (tRA)or 9‐cis‐retinoic acid (9‐cis‐RA), the natural ligands for the RARs and RXRs, respectively. Moreover, AHPN (CD437) was unable to compete with either tRA or 9‐cis‐RA for binding to endogenous retinoid receptors in nuclear extracts from the MDA‐MB‐468 breast carcinoma cell line. Size exclusion chromatography revealed AHPN binding to a 95 kDa protein(s) which is neither an RAR or RXR. Our results suggest that apoptosis induction by AHPN (CD437) may occur through interaction with another protein and is independent of the RAR/RXR‐signaling pathways. Int. J. Cancer 86:474–479, 2000.

Catecholaminergic CATH.a cells express predominantly δ-opioid receptors

Abstract CATH.a cells are a catecholaminergic cell line of neuronal origin. The opioid receptor complement expressed by CATH.a cells was defined pharmacologically and by reverse transcription-polymerase chain reaction (RT-PCR). CATH.a cells were found to express mRNA encoding all three of the major subtypes of opioid receptors. The relative abundance of CATH.a cell opioid receptor transcripts was δ > κ > μ . Pharmacological and functional data were in agreement with the results of RT-PCR inasmuch as δ -opioid receptor was identified as the most abundant opioid receptor subtype expressed by CATH.a cells. In addition, at least one of the opioid signalling pathways, inhibition of adenylyl cyclase activity, was found to be operant in this cell line. CATH.a cells should be of general utility for the study of opioid receptor signalling mechanisms in the context of catecholaminergic neurons.

Mass-spectrometric analysis of agonist-induced retinoic acid receptor γ conformational change

Apo and holo forms of retinoic acid receptors, and other nuclear receptors, display differential sensitivity to proteolytic digestion that likely reflects the distinct conformational states of the free and liganded forms of the receptor. We have developed a method for rapid peptide mapping of holo-retinoic acid receptor gamma that utilizes matrix-assisted laser-desorption-ionization time-of-flight MS to identify peptide fragments that are derived from the partially proteolysed holo-receptor. The peptide maps of retinoic acid receptor gamma bound by four different agonists were identical, suggesting that all four ligands induced a similar conformational change within the ligand-binding domain of the receptor. In all cases, this agonist-induced conformational change promoted the direct association of retinoic acid receptor gamma with the transcriptional co-activator p300 and inhibited interaction of the receptor with the nuclear receptor co-repressor. SR11253, a compound previously reported to exert mixed retinoic acid receptor gamma agonist/antagonist activities in cultured cells, was found to bind directly to, but only weakly altered the protease-sensitivity of, the receptor and failed to promote interaction of the receptor with p300 or induce dissociation of receptor-nuclear receptor co-repressor complexes. This technique should be generally applicable to other members of the nuclear receptor superfamily that undergo an induced structural alteration upon agonist or antagonist binding, DNA binding and/or protein-protein interaction.