[Abstract unavailable

Two novel and related C 2 H 2 zinc finger proteins that are highly expressed in the brain, CTIP1 and CTIP2 (COUP TF-interacting proteins 1 and 2, respectively), were isolated and shown to interact with all members of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of orphan nuclear receptors. The interaction of CTIP1 with ARP1 was studied in detail, and CTIP1 was found to harbor two independent ARP1 interaction domains, ID1 and ID2, whereas the putative AF-2 of ARP1 was required for interaction with CTIP1. CTIP1, which exhibited a punctate staining pattern within the nucleus of transfected cells, recruited cotransfected ARP1 to these foci and potentiated ARP1-mediated transcriptional repression of a reporter construct. However, transcriptional repression mediated by ARP1 acting through CTIP1 did not appear to involve recruitment of a trichostatin A-sensitive histone deacetylase(s) to the template, suggesting that this repression pathway may be distinct from that utilized by several other nuclear receptors.COUP-TFI, 1 ARP1/COUP-TFII, and Ear2/COUP-TFIII have been grouped in the same subfamily of orphan nuclear receptors based on sequence similarity (1-3), evolutionary analysis (4), and a common capacity to repress ligand-dependent transcriptional activation of target genes mediated by other nuclear receptors, such as retinoic acid (5-10), thyroid hormone (8), estrogen (11)(12)(13)(14), and vitamin D 3 (9) receptors as well as peroxisome proliferator-activated receptor α (PPARα;Ref. 15).COUP-TFs play important roles in pattern formation in the developing nervous systems of Xenopus (16) and Drosophila (17). Deletion of the COUP-TFI gene in the mouse results in * This work was supported in part by American Heart Association Grant 9640219N; NIEHS, National Institutes of Health, Grants ES00210 and ES00040; the Oregon State University College of Pharmacy; and the Laboratory of Molecular Pharmacology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. ∥ An established investigator of the American Heart Association. To whom correspondence and reprint requests should be addressed: MATERIALS AND METHODS Yeast Two-hybrid Screening and cDNA CloningYeast two-hybrid screening was conducted as described previously (21) using the hinge region and putative ligand binding domain of ARP1 (amino acids 144-414) as a bait. Fragments corresponding to CTIP1 and CTIP2 (see Fig. 1A) were used to screen mouse cDNA libraries obtained from CLON-TECH and from Dr. René Hen (Columbia University), yielding several overlapping clones. These overlapping clones were then used to prepare a full-length CTIP1 construct that was inserted into the eukaryotic expression vector, pCDNA3+ (Invitrogen). Yeast β-Galactosidase Assays and GST Pull-down ExperimentsProtein-p...

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Chicken ovalbumin upstream promoter transcription factor-interacting proteins 1 and 2 (CTIP1 and CTIP2) are related transcriptional regulatory proteins. While overexpression of both of these proteins has been linked to the development of several lymphoid malignancies, lack of CTIP1 and CTIP2 expression results in defective lymphopoiesis and abnormal thymocyte development, respectively. Here, we describe the expression patterns of CTIP1 and CTIP2 during mouse embryogenesis and in the post-natal brain. Both CTIP1 and CTIP2 were expressed diffusely in the embryo at 10.5 days post-coitum (d.p.c.). However, the expression of both genes became increasingly restricted to the central nervous system (CNS) during the course of fetal development, culminating with high, but differential, expression levels throughout the hippocampal subregions, olfactory bulb and cortex, limbic system, basal ganglia and frontal cortex of the developing brain, and in dorsal cells of the spinal cord. The brain expression domains of CTIP1 and CTIP2 were maintained into adulthood. Outside the CNS, both genes exhibited differential expression within the facial mesenchyme at 12.5 d.p.c., and CTIP2 was selectively expressed from day 12.5 onwards in the olfactory epithelium and developing thymus, and to a lesser extent in oral and gut epithelia. Strong CTIP2 expression was maintained in the thymus at 18.5 d.p.c. These results support the selective contributions of both CTIP1 and CTIP2 in the development and function of both the central nervous and immune systems and the importance of future investigations to define the function(s) of both proteins. et al., 2000). Recruitment of either CTIP1 (Avram et al., 2000) or CTIP2 (Avram, unpublished studies) to the template by a COUP-TF family member was found to result in transcriptional repression of a reporter gene harboring a COUP-TF binding site. Additionally, CTIP1 and CTIP2 are sequence-specific DNA binding proteins that repress transcription via direct, COUP-TF-independent binding to a motif that is related to the canonical GC box (Avram et al., 2002). Thus, CTIPs may either be recruited to the template by a COUP-TF family member or bind directly to the template in a sequence-specific manner. In both cases, CTIP1 and CTIP2 appear to repress transcription in a manner that does not involve trichostatin A-sensitive histone deacetylation (Avram et al., 2000;Senawong et al., 2003). Although, the mechanistic basis of CTIP-mediated transcriptional repression has not been defined, overexpressed CTIP2 ( Expression of both CTIP1 and CTIP2 has recently been linked to the etiology of disease. Overexpression of CTIP1 following proviral integration led to the transformation of NIH3T3 cells and the development of myeloid leukemia in mice . This transformation event may be facilitated by a physical interaction of CTIP1 with BCL6, a known human B cell proto-oncogene product. Recently, an association between p53 and CTIP2 in mice has been implicated in the development of thymic lymphomas (Wakabayashi et al., ...

Retinoic acid receptor (RAR) and retinoid X receptor (RXR) form heterodimers and regulate retinoidmediated gene expression. We studied binding of RXR-and RAR-selective ligands to the RXR-RAR heterodimer and subsequent transcription. In limited proteolysis analyses, both RXR and RAR in the heterodimer bound their respective ligands and underwent a conformational change in the presence of a retinoic acidresponsive element. In reporter analyses, the RAR ligand (but not the RXR ligand), when added singly, activated transcription, but coaddition of the two ligands led to synergistic activation of transcription. This activation required the AF-2 domain of both RXR and RAR. Genomic footprinting analysis was performed with P19 embryonal carcinoma cells, in which transcription of the RAR␤ gene is induced upon retinoid addition. Paralleling the reporter activation data, only the RAR ligand induced in vivo occupancy of the RAR␤2 promoter when added singly. However, at suboptimal concentrations of RAR ligand, coaddition of the RXR ligand increased the stability of promoter occupancy. Thus, liganded RXR and RAR both participate in transcription. Finally, when these ligands were tested for teratogenic effects on zebra fish and Xenopus embryos, we found that coadministration of the RXR and RAR ligands caused more severe abnormalities in these embryos than either ligand alone, providing biological support for the synergistic action of the two ligands.Transcriptional responses to retinoids are mainly controlled by two classes of nuclear hormone receptors, retinoic acid (RA) receptors (RARs) and retinoid X receptors (RXRs), which form heterodimeric complexes, bind to RA-responsive elements (RAREs), and regulate gene expression in a retinoiddependent manner (reviewed in references 12, 25, 29, 30, 45, 46, 50, 57 and 60). The RAR-RXR heterodimer is activated by a number of naturally occurring retinoids that bind to RXR and/or RAR with different affinities (26,43,57). For example, RARs bind both all-trans-and 9-cis-RA stereoisomers, whereas RXRs bind only 9-cis-RA (3,4,26,43). The presence of multiple retinoids that are differentially distributed in various tissues and differentially bind to RARs and RXRs suggests that retinoid-dependent gene regulation and its biological effects are highly complex. There may be additional complexities depending on whether RAR and RXR in the heterodimer exhibit differential activities in response to retinoids. Given the complexity in retinoid action, it is important to study how each receptor in the heterodimer acts in response to various retinoids. Ligand binding and transcriptional activity of RXR may raise additional issues, as RXR forms heterodimers with several other nuclear receptors that respond to nonretinoid ligands (such as the thyroid hormone and vitamin D). In these cases, some retinoids, by binding to RXR, may elicit a modulatory effect on other ligands (12,38,45). However, making this possibility less attractive, it has been proposed that RXR does not play an active role in transcripti...