Many transcription coactivators interact with nuclear receptors in a ligandThe nuclear receptor superfamily is a group of proteins that regulate, in a ligand-dependent manner, transcriptional initiation of target genes by binding to specific DNA sequences named hormone response elements (reviewed in reference 23). Functional analysis of nuclear receptors has shown that there are two major activation domains. The N-terminal domain (AF1) contains a ligand-independent activation function, whereas the ligand-binding domain (LBD) exhibits ligand-dependent transactivation function (AF2). The AF2 core region, located at the extreme C terminus of the receptor LBDs, is conserved among nuclear receptors and undergoes a major conformational change upon ligand binding (23). This region has been shown to play a critical role in mediating transactivation by serving as a ligand-dependent interaction interface with many different coactivators (reviewed in reference 9). These coactivators, including the p160 family members (i.e., SRC-1, SRC-2/GRIP1/TIF2, and SRC-3/ACTR/pCIP/AIB1/ RAC3/TRAM1), CBP/p300, p/CAF, TRAP/DRIP, activating signal cointegrator 2 (ASC-2), and many others, bridge nuclear receptors and the basal transcription apparatus and/or remodel the chromatin structures (9).Chromatin, the physiological template of all eukaryotic genetic information, undergoes a diverse array of posttranslational modifications that largely impinge on histone amino termini, thereby regulating access to the underlying DNA (reviewed in reference 12). SRC-1 and the p160 family member ACTR, along with CBP and p300, were recently shown to contain histone acetyltransferase (HAT) activities and associate with yet another HAT protein, p/CAF (9). In contrast, SMRT and N-CoR, nuclear receptor corepressors, form complexes with Sin3 and histone deacetylase proteins (9). These results are consistent with the notion that the acetylation of histones destabilizes nucleosomes and relieves transcriptional repression by allowing transcription factors to access recognition elements, whereas deacetylation of the histones stabilizes the repressed state. More recently, the histone arginine methyltransferases CARM1 and PRMT1 were newly defined as transcriptional coactivators of nuclear receptors (4, 40). NSD1 and

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ASC-2, a recently isolated transcriptional coactivator molecule, stimulates transactivation by multiple transcription factors, including nuclear receptors. We generated a potent dominant negative fragment of ASC-2, encompassing the N-terminal LXXLL motif that binds a broad range of nuclear receptors. This fragment, termed DN1, specifically inhibited endogenous ASC-2 from binding these receptors in vivo, whereas DN1/m, in which the LXXLL motif was mutated to LXXAA to abolish the receptor interactions, was inert. Interestingly, DN1 transgenic mice but not DN1/m transgenic mice exhibited severe microphthalmia and posterior lenticonus with cataract as well as a variety of pathophysiological phenotypes in many other organs. Our results provide a novel insight into the molecular and histopathological mechanism of posterior lenticonus with cataract and attest to the importance of ASC-2 as a pivotal transcriptional coactivator of nuclear receptors in vivo.The nuclear receptor superfamily is a group of proteins that regulate, in a ligand-dependent manner, transcriptional initiation of target genes by binding to specific DNA sequences named hormone response elements (for a review, see reference 15). Genetic studies indicated that transcription coactivators without specific DNA-binding activity are essential for transcriptional activation, which led to the identification of many proteins interacting with the C-terminal ligand-dependent transactivation domain of nuclear receptors (for reviews, see references 1 and 19). These coactivators, including the p160 family, CBP/p300, p/CAF, TRAP/DRIP, and many others, bridge transcription factors and the basal transcription apparatus and/or remodel the chromatin structures.ASC-2, also named AIB3, TRBP, RAP250, NRC, and PRIP, is a recently isolated transcriptional coactivator molecule which is gene amplified and overexpressed in human cancers and stimulates transactivation by nuclear receptors, AP-1, NF-B, SRF, and numerous other transcription factors (1, 10-12, 14, 24). In particular, the single-cell microinjection results with ASC-2 antibody demonstrated that endogenous ASC-2 is required for transactivation by nuclear receptors and AP-1 (11, 12). More recently, ASC-2 was found to exist in a steady-state complex of approximately 2 MDa that contains histone H3-lysine 4-specific methyltransferase enzymes, and chromatin immunoprecipitation experiments demonstrated that this complex specifically binds to the retinoid-responsive ␤-retinoic acid response element and p21 WAF1 promoter regions in a liganddependent manner (Goo et al., unpublished data).Interestingly, ASC-2 contains two nuclear receptor interaction domains (12), both of which are dependent on the integrity of their core LXXLL sequences (16,19). The C-terminal LXXLL motif specifically interacts with the liver X receptors (LXR␣ and LXR␤), whereas the N-terminal motif binds a broad range of nuclear receptors (12).In this report, we show that inhibition of ASC-2 recruitment to nuclear receptors by using a dominant negative fragm...

Gonadotropins (GTHs), FSH and LH, play central roles in vertebrate reproduction. Here, we report the production of biologically-active recombinant FSH (r-mtFSH) and LH (r-mtLH) of an endangered salmon species, Manchurian trout (Brachymystax lenok), by baculovirus in silkworm (Bombyx mori) larvae. The biological activities of the recombinant hormones were analyzed using COS-7 cell line transiently expressing either amago salmon FSH or LH receptor. The steroidogenic potency of the r-mtFSH and r-mtLH was examined by a culture system using rainbow trout follicles in vitro. In vivo, bioactivity was assessed by measuring ovarian weight, oocyte diameter, and plasma steroid hormone levels in female rainbow trout. Moreover, inducing potency of milt production were examined in vivo using goldfish. Our results demonstrated that the r-mtFSH and r-mtLH were successfully produced in the baculovirus-silkworm system and recognized by their cognate receptors specifically in vitro. The production of estradiol-17b (E2) and testosterone (T) was stimulated by the r-mtFSH and r-mtLH respectively, from the full-grown follicles of rainbow trout, whereas both E2 and T were increased by relatively higher doses of the recombinant hormones from the follicles of the maturing stage. In in vivo assay, injection of the r-mtFSH but not r-mtLH increased ovarian weight, oocyte diameter, and plasma E2 levels in immature rainbow trout. Injection of both r-mtFSH and r-mtLH induced milt production in male goldfish. In conclusion, the present study strongly suggests that the r-mtFSH and r-mtLH have distinct biological properties, such as a specific responsiveness for the cognate receptor, steroidogenic, and vitellogenic activities for ovarian follicles in salmonids. These recombinant FSH and LH may be applied for future studies on the gonadal development and maturation in fishes as well as the endangered salmon species.

For the orphan nuclear receptor subfamily that includes Nur77 (NGFI-B), Nurr1, and NOR-1, no transcriptional coregulators have been identified thus far. In this report, we found that Ca 2؉ /calmodulin-dependent protein kinase IV enhances Nur77 transactivation in cotransfections either alone or in synergy with AF2-dependent coactivator ASC-2, whereas corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) is repressive. Interestingly, Nur77 interacted with SMRT but did not directly bind ASC-2, and accordingly, the putative AF2 core domain of Nur77 did not affect the Nur77 transactivation. SMRT harbors transferable repression domains that associate with various histone deacetylases. Surprisingly, histone deacetylase inhibitor trichostatin A was unable to block the repressive effect of SMRT while dramatically stimulating the Nur77 transactivation. These results suggest that SMRT and ASC-2 are specific coregulators of Nur77 and that SMRT may dynamically compete with a putative adaptor molecule, which links ASC-2 to Nur77, for the identical binding sites within Nur77 in vivo.The nuclear receptor superfamily is a group of ligand-dependent transcriptional regulatory proteins that function by binding to specific DNA sequences named hormone response elements in the promoters of target genes (for a review, see Ref.1). The superfamily includes receptors for a variety of small hydrophobic ligands, such as steroids, T3, and retinoids, as well as a large number of related proteins that do not have known ligands referred to as orphan nuclear receptors. Functional analysis of nuclear receptors has shown that the ligand-binding domain (LBD) 1 exhibits ligand-dependent transcriptional activation function referred to as activation function-2 (AF2). Notably, this region has been shown to play a critical role in mediating transactivation by serving as a ligand-dependent interaction interface with many different coactivators (for reviews, see Refs. 2 and 3). Transcriptional coactivators either bridge transcription factors and the components of the basal transcriptional apparatus and/or remodel the chromatin structures. In particular, cAMP-response element-binding protein (CREB)-binding protein (CBP) and its functional homologue p300, steroid receptor coactivator-1 (SRC-1) and its family members, and activating signal cointegrator-2 (ASC-2) were shown to be essential for the activation of transcription by a large number of regulated transcription factors, including many members of the nuclear receptor superfamily (2, 3). Interestingly, SRC-1 and its family member activator of thyroid and retinoic acid receptors (ACTR) along with CBP and p300 were recently shown to contain histone acetyltransferase activities and associate with another histone acetyltransferase protein p300/CBP-associated factor (P/CAF) (2, 3). In contrast, nuclear receptor corepressor (N-CoR) and its homologue silencing mediator of retinoid and thyroid hormone receptors (SMRT) harbor transferable repression domains that can associate with va...

Summary.This paper reports on the distribution, relationship and seasonal variations of neuropeptide Y (NPY)-like and gonadotropin-releasing hormone (GnRH)-like immunoreactants in the brain and hypophysis of the bony fish, the ayu Plecoglossus altivelis. NPY-like immunoreactivity was widely distributed in the brain: labeled cells were found in the nervus terminalis, the nucleus entopeduncularis, the habenula, the nucleus preopticus periventricularis, the nucleus tuberis lateralis, the mediodorsal hypothalamus, the dorsal tegmentum, and other sites. NPY fibers were considerably dense in the telencephalon and hypothalamus, and innervated the hypophysis. GnRH-positive cells occurred in the nervus terminalis and were sparsely distributed in the preoptic and tuberal areas of the hypothalamus. GnRH fibers were found in various regions of the brain. They were relatively dense in the hypothalamus, showing a local concentration in the middle region of the neurohypophysis.The GnRH-positive cells and fibers in the hypothalamo-hypophyseal complex increased in density around the spawning season. In tandem with gonadal maturation, NPY labeling in the cells of the nucleus tuberis lateralis became intense concomitantly with an increase in the labeled varicosities in the middle region of the neurohypophysis. Double immunostaining showed that NPY fibers were closely apposed to GnRH cells in the preoptic area. These results suggest a correlative involvement of NPY and GnRH in the control of the hypophyseal gonadotropic function of the ayu.

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