The adenoviral oncoprotein E1A induces progression through the cell cycle by binding to the products of the p300/CBP and retinoblastoma gene families. A new cellular p300/CBP-associated factor (P/CAF) having intrinsic histone acetylase activity has been identified that competes with E1A. Exogenous expression of P/CAF in HeLa cells inhibits cell-cycle progression and counteracts the mitogenic activity of E1A. E1A disturbs the normal cellular interaction between p300/CBP and its associated histone acetylase.

The anchor-away (AA) technique depletes the nucleus of Saccharomyces cerevisiae of a protein of interest (the target) by conditional tethering to an abundant cytoplasmic protein (the anchor) by appropriate gene tagging and rapamycin-dependent heterodimerization. Taking advantage of the massive flow of ribosomal proteins through the nucleus during maturation, a protein of the large subunit was chosen as the anchor. Addition of rapamycin, due to formation of the ternary complex, composed of the anchor, rapamycin, and the target, then results in the rapid depletion of the target from the nucleus. All 43 tested genes displayed on rapamycin plates the expected defective growth phenotype. In addition, when examined functionally, specific mutant phenotypes were obtained within minutes. These are genes involved in protein import, RNA export, transcription, sister chromatid cohesion, and gene silencing. The AA technique is a powerful tool for nuclear biology to dissect the function of individual or gene pairs in synthetic, lethal situations.

Nuclear receptors play important roles in the maintenance of the endocrine system, regulation of organ differentiation, and fetal development. Endocrine disruptors exert their adverse effects by disrupting the endocrine system via various mechanisms. To assess the effects of endocrine disruptors on nuclear receptors, we developed a high-throughput method for identifying activators of nuclear receptors. Using this system, we found that triphenyltin and tributyltin were activators of peroxisome proliferator-activated receptor (PPAR) ␥ and retinoid X receptor. Because PPAR␥ is a master regulator of adipocyte differentiation, we assessed the effect of organotin compounds on preadipocyte 3T3-L1 cells. We found that organotin compounds stimulated differentiation of 3T3-L1 cells as well as expression of adipocyte marker genes.

One of the urgent tasks in understanding endocrine disruptors (EDs) is to compile a list of suspected substances among the huge number of chemicals by using the screening test method. We developed a simple and rapid screening method using the yeast two-hybrid system based on the ligand-dependent interaction of nuclear hormone receptors with coactivators. To date, we have tested the estrogenic activity of more than 500 chemicals including natural substances, medicines, pesticides, and industrial chemicals. 64 compounds were evaluated as positive, and most of these demonstrated a common structure; phenol with a hydrophobic moiety at the para-position without bulky groups at the ortho-position. These results are expected to facilitate further risk assessment of chemicals.

Our previous work identified the inner basket of the NPC as a physical activation/protection station for force-tethered, epigenetically silenced genes. Here we show that a specific nucleopore-to-gene-promoter interaction (Nup-PI) is an early physiological event of gene activation. Nup-PI was discovered with chromatin endogenous cleavage (ChEC) experiments that mapped in vivo the genomic interaction sites of the nucleoporin Nup2p fused to microccocal nuclease (Nup2-MN). Strong Nup-PI, cleavage by Nup2-MN, is observed at the promoters of the GAL genes and at HXK1 upon activation of these genes with galactose. Nup-PI at the GAL locus requires Gal4p and the UASg and TATA box elements but not SAGA and active transcription. The physical, activation-dependent interaction of the GAL locus with the NPC basket was confirmed by imaging. Chromosome-wide ChEC studies indicated that Nup-PI occurs at numerous genes. The data identify the NPC basket as a new, integral participant in gene expression.

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The Drosophila 230-kDa TFIID subunit (dTAF230) interacts with the DNA binding domain of TATA boxbinding protein (TBP) which exists in the same complex. Here, we characterize the inhibitory domain in the yeast TAF145 (yTAF145), which is homologous to dTAF230. Mutation studies show that the N-terminal inhibitory region (residues 10 to 71) can be divided into two subdomains, I (residues 10 to 37) and II (residues 46 to 71). Mutations in either subdomain significantly impair function. Acidic residues in subdomain II are important for the interaction with TBP. In addition, yTAF145 interaction is impaired by mutating the basic residues on the convex surface of TBP, which are crucial for interaction with TFIIA. Consistently, TFIIA and yTAF145 bind competitively to TBP. A deletion of the inhibitory domain of yTAF145 leads to a temperaturesensitive growth phenotype. Importantly, this phenotype is suppressed by overexpression of the TFIIA subunits, indicating that the yTAF145 inhibitory domain is involved in TFIIA function.Transcription factor TFIID is a multisubunit protein complex found in various organisms including Drosophila melanogaster (17, 40), human (12,59,61,74), and more recently, the budding yeast Saccharomyces cerevisiae (23,(52)(53)(54). Holo-TFIID is composed of the highly conserved TATA box-binding polypeptide protein (TBP) and a number of associated polypeptides (TBP-associated factors [TAFs]). In vitro transcription studies revealed an important functional difference between holo-TFIID and TBP. Holo-TFIID mediates activator regulated transcription, whereas TBP itself mediates only basal levels of transcription. Thus, at least one or more TAFs included in holo-TFIID are essential for transmitting signals from various activators to the basal transcriptional machinery. Within the past 5 years, cDNAs encoding TAFs have been cloned from

Organotin compounds released from antifouling paints, such as tributyltin (TBT) and triphenyltin (TPT), are potent inducers of imposex (a superimposition of male genital tracts, such as penis and vas deferens, on females) in marine gastropods. Little is known about the induction mechanism of gastropod imposex. Here, we show that organotins bind the human retinoid X receptors (hRXRs) with high affinity and that injection of 9-cis retinoic acid (RA), the natural ligand of hRXRs, into females of the rock shell (Thais clavigera) induces the development of imposex. Cloning of the RXR homologue from T. clavigera revealed that the ligand-binding domain of rock shell RXR was very similar to vertebrate RXR and bound to both 9-cis RA and to organotins. These suggest that RXR plays an important role in inducing the development of imposex, namely, the differentiation and growth of male genital tracts in female gastropods.