Corepressors are involved in gene silencing by various transcriptional repressor proteins such as MAD/MAX and MxiI (2), YY1 (74), KRAB domain proteins (27), NGF1-A, KROX 20 (59, 63) and some members of the nuclear hormone receptor (NHR) superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR) (8,19,38,50). Both TR and RAR repress gene activity in the absence of hormone in vivo (3, 4, 21, 73) and in vitro (28, 67, 68). This repression is mediated by a silencing domain in the carboxy terminus, encompassing about 250 amino acids (aa) (3,33,46,58). In addition to the silencing function, TR and RAR harbor several other functions C-terminal to their DNA binding domain (DBD) including dimerization, hormone binding and hormone-dependent transactivation. These activities can be transferred to heterologous proteins and therefore represent functional domains (for reviews, see references 6, 50, and 65).Gene silencing by NHRs is relieved by addition of the cognate ligand, which induces a conformational change and transforms the receptor into a transcriptional activator. In this way, both hormone binding and the small conserved receptor activation domain, AF2/AF2-AD/4/c (8, 11, 12, 22, 49, 50), representing helix 12 (14, 38, 55, 57, 71), are required to dissociate corepressors from the receptors (8,9,19,38). It is yet unknown why so many different coactivators are involved in transcriptional activation by NHRs. Gene silencing by TR, RAR, Rev-erbA␣, and COUP-TF is mediated, at least in part, by corepressors in vivo (8, 10, 19, 25, 38, 61) and in vitro (68), which bind to the unliganded (apo) receptors. Only one class of nuclear receptor corepressors has been identified, which exhibit hormone-sensitive interaction. This class contains two related members, 38). These corepressors were isolated by the yeast twohybrid system and bind to the silencing domains of TR and RAR only in the absence of ligand. Hormone binding by the receptor leads to dissociation of these corepressors. Furthermore, SMRT and N-CoR are localized in the cell nucleus and harbor an autonomous silencing function when bound to DNA (19,38). The mechanism of repression by the SMRT/N-CoR class involves interaction with SIN3 and a histone deacetylase function (1,35,52).Here, we describe a novel corepressor, Alien, which is unrelated to SMRT and N-CoR and is highly conserved from humans to Drosophila. Conserved sequences are even found in Ricinus communis and Caenorhabditis elegans. Alien interacts with TR only in the absence of hormone and does not interact with RAR, retinoid X receptor (RXR), or glucocorticoid
DAX-1 is an unusual member of the nuclear hormone receptor (NHR) superfamily. Lack of DAX-1-mediated silencing leads to adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Gene silencing through NHRs such as the thyroid hormone receptor (TR) is mediated by corepressors. We have previously characterized a novel corepressor, termed Alien, which interacts with TR and the ecdysone receptor but not with the retinoic acid receptors RAR or RXR. Here, we show that DAX-1 interacts with the corepressor Alien but not with the corepressor SMRT. This interaction is mediated by the DAX-1-silencing domain. Naturally occurring mutants of the DAX-1 gene fail to interact with Alien and have lost silencing function. Because the silencing domain of DAX-1 is unusual for NHRs, we mapped the interaction of Alien with DAX-1 and with TR. We show that Alien exhibits different binding characteristics to DAX-1 and TR. Furthermore, Northern experiments demonstrate that Alien is expressed in the adrenal gland and testis in tissues where DAX-1 is specifically expressed. Interestingly, a novel adrenal gland-specific mRNA of Alien was discovered. Thus, the impairment of Alien binding seems to play an important role in the pathogenesis mediated by DAX-1 mutants.The gene superfamily of nuclear hormone receptors (NHRs) 1 represents a large class of transcription factors including receptors for steroids, nonsteroids, and members for which no ligand has yet been identified, the so-called orphan receptors (for reviews, see Refs. 1 and 2). Gene silencing is mediated by a few members such as receptors for thyroid hormone (TR (NR1A)) (3) and retinoic acid (RAR (NR1B)) (4 -10). Thereby, the receptor is associated with corepressors, and the complex of receptor and corepressors mediates target gene repression (2, 9 -12). Among corepressors, N-CoR and SMRT represent one class, and Alien represents another class, which interacts in a hormone-sensitive manner with NHR (10 -13). The silencing domain of NHRs is localized in the C terminus and overlaps with the hormone-binding domain to a great extent. In the case of TR and RAR, the silencing domain encompasses 230 amino acids (5,14). Hormone binding by NHRs leads to a conformational change, dissociation of corepressors, subsequent binding of coactivators, and gene activation (for review, see Ref. 15). The transcriptional properties of nuclear receptors can be transferred to heterologous proteins and therefore represent functional domains (for review, see Refs. 2,16,and 17).DAX-1 (NR0B1) is an unusual member of the NHR superfamily (18,19). The DNA-binding domain is unrelated to other nuclear receptors and is composed of four repeat sequences, which are able to recognize and bind to hairpin structures (20). The C terminus, however, is homologous to members of the NHR superfamily (18). Mutations in the DAX-1 gene cause the X-linked disorder of adrenal hypoplasia congenita and the associated hypogonadotropic hypogonadism (18,19,21,22). DAX-1 is predominantly expressed in adrenal gland and testis (18), w...
ING1 was identified as an inhibitor of growth and has been described as a tumor suppressor. Furthermore, the expression of ING1 is induced in senescent cells and antisense ING1 extends the proliferative life span of primary human fibroblasts. Cooperation of p33ING1 with p53 has been suggested to be an important function of ING1 in cell cycle control. Intriguingly, it has been shown that p33ING1 is associated with histone acetylation as well as with histone deacetylation function. Here we show that p33ING1 is a potent transcriptional silencer in various cell types. However, the silencing function is independent of the presence of p53. By use of deletion mutants two potent autonomous and transferable silencing domains were identified, but no evidence of an activation domain was found. The amino (N)-terminal silencing domain is sensitive to the histone deacetylase inhibitor trichostatin A (TSA) whereas the carboxy-terminal silencing function is resistant to TSA, suggesting that p33ING1 confers gene silencing through both HDAC-dependent and -independent mechanisms. Interestingly, the presence of oncogenic Ras, which is able to induce premature senescence, increases the p33ING1-mediated silencing function. Moreover, ING1-mediated silencing was reduced by coexpressing dominant-negative Ras or by treatment with the mitogen-activated protein kinase inhibitor PD98059 but not by treatment with SB203580, an inhibitor of the p38 pathway. In addition, we show that both silencing domains of ING1 are involved in cell cycle control, as measured by inhibition of colony formation of immortalized cells and by thymidine incorporation of primary human diploid fibroblasts (HDF). Interestingly, p33ING1 expression induces features of cellular senescence in HDFs.
Metastasis comprises several subsequent steps including local invasion and intravasation at the primary site, then their adhesion/arrest within the vessels of host organs followed by their extravasation and infiltration into the target organ stroma. In contrast to previous studies which have used aspartate-glycine-arginine (RGD) peptides and antibodies against integrins, we used rare collagen- and laminin-antagonizing integrin inhibitors from snake venoms to analyze the colonization of the liver by tumor cells both by intravital microscopy and in vitro. Adhesion of liver-targeting tumor cells to the sinusoid wall components, laminin-1 and fibronectin, is essential for liver metastasis. This step is inhibited by lebein-1, but not by lebein-2 or rhodocetin. Both lebeins from the Vipera lebetina venom block integrin interactions with laminins in an RGD-independent manner. Rhodocetin is an antagonist of alpha2beta1 integrin, a collagen receptor on many tumor cells. Subsequent to tumor cell arrest, extravasation into the liver stroma and micrometastasis are efficiently delayed by rhodocetin. This underlines the importance of alpha2beta1 integrin interaction with the reticular collagen I-rich fibers in liver stroma. Antagonists of laminin- and collagen-binding integrins could be valuable tools to individually block the direct interactions of tumor cells with distinct matrix components of the Disse space, thereby reducing liver metastasis.
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