Glucocorticoid receptor (GR) exerts anti-inflammatory action in part by antagonizing proinflammatory transcription factors such as the nuclear factor kappa-b (NFKB). Here, we assess the crosstalk of activated GR and RELA (p65, major NFKB component) by global identification of their binding sites and target genes. We show that coactivation of GR and p65 alters the repertoire of regulated genes and results in their association with novel sites in a mutually dependent manner. These novel sites predominantly cluster with p65 target genes that are antagonized by activated GR and vice versa. Our data show that coactivation of GR and NFKB alters signaling pathways that are regulated by each factor separately and provide insight into the networks underlying the GR and NFKB crosstalk.

In higher eukaryotes, the large subunit of the general transcription factor TFIIA is encoded by the single TFIIA␣␤ gene and posttranslationally cleaved into ␣ and ␤ subunits. The molecular mechanisms and biological significance of this proteolytic process have remained obscure. Here, we show that TFIIA is a substrate of taspase 1 as reported for the trithorax group mixed-lineage leukemia protein. We demonstrate that recombinant taspase 1 cleaves TFIIA in vitro. Transfected taspase 1 enhances cleavage of TFIIA, and RNA interference knockdown of endogenous taspase 1 diminishes cleavage of TFIIA in vivo. In taspase 1 ؊/؊ MEF cells, only uncleaved TFIIA is detected. In Xenopus laevis embryos, knockdown of TFIIA results in phenotype and expression defects. Both defects can be rescued by expression of an uncleavable TFIIA mutant. Our study shows that uncleaved TFIIA is transcriptionally active and that cleavage of TFIIA does not serve to render TFIIA competent for transcription. We propose that cleavage fine tunes the transcription regulation of a subset of genes during differentiation and development.In eukaryotes, initiation of RNA polymerase II transcription requires the assembly of a preinitiation complex. Specific binding of TBP to promoters is a key step in the formation of PIC, which is followed by recruitment of general transcription factors and polymerase II. The basal transcription factor TFIIA interacts with TBP and stabilizes its binding to DNA (26,28). TFIIA has also been shown to interact with several activators (11,12,18,27) and is required for transcriptional activation of certain genes (10,13,14,20,21).In higher eukaryotes, purified TFIIA is composed of three subunits, ␣, ␤, and ␥. TFIIA␣␤ is encoded by a single gene and cleaved posttranslationally into ␣ and ␤ subunits. The ␥ subunit is conserved among different species, whereas sequence similarity in TFIIA␣␤ is limited mostly to the N-terminal region of the ␣ subunit and the C terminus covering most of the ␤ subunit (19). Recently, the cleavage recognition site (CRS) that is essential for TFIIA cleavage has been identified as QVDG (amino acids [aa] 272 to 275), and the N terminus of the ␤ subunit was determined to be at D278, located 3 amino acids downstream of the CRS (Fig. 1B) (6). The CRS is remarkably similar in different evolutionarily distinct species and is embedded in an otherwise nonconserved and probably unstructured region (1, 4, 24). The germ cell-specific TFIIA-like factor ALF, a TFIIA variant that contains the CRS, was also shown to be cleaved (5, 6). TFIIA cleavage was first reported more than a decade ago (26), and it has been generally assumed that uncleaved TFIIA is the precursor and cleavage occurs to activate TFIIA for transcription. Both uncleaved ␣␤ and the cleaved ␣ and ␤ subunits can be found in association with the TFIIA␥ subunit in vivo (15, 16), and both forms interact with TBP on DNA and support transcription to similar extents in vitro and in reporter assays (6,22). TFIIA is mainly found in the cleaved form (␣ plus ␤ plu...

Engineered protein-based sensors of ligand binding have emerged as attractive tools for the discovery of therapeutic compounds through simple screening systems. We have previously shown that engineered chimeric enzymes, which combine the ligand-binding domains of nuclear hormone receptors with a highly sensitive thymidylate synthase reporter, yield simple sensors that report the presence of hormone-like compounds through changes in bacterial growth. This work describes an optimized estrogen sensor in Escherichia coli with extraordinary reliability in identifying diverse estrogenic compounds and in differentiating between their agonistic/antagonistic pharmacological effects. The ability of this system to assist the discovery of new estrogen-mimicking compounds was validated by screening a small compound library, which led to the identification of two structurally novel estrogen receptor modulators and the accurate prediction of their agonistic/antagonistic biocharacter in human cells. Strong evidence is presented here that the ability of our sensor to detect ligand binding and recognize pharmacologically critical properties arises from allosteric communication between the artificially combined protein domains, where different ligand-induced conformational changes in the receptor are transmitted to the catalytic domain and translated to distinct levels of enzymic efficiency. To the best of our knowledge, this is one of the first examples of an engineered enzyme with the ability to sense multiple receptor conformations and to be either activated or inactivated depending on the nature of the bound effector molecule. Because the proposed mechanism of ligand dependence is not specific to nuclear hormone receptors, we anticipate that our protein engineering strategy will be applicable to the construction of simple sensors for different classes of (therapeutic) binding proteins.

Bisphenol A (BPA) is an estrogen-mimicking endocrine disruptor. Early-life exposures to low doses of BPA exert long-lasting effects on animals' reproductive and brain physiology. However, little is known about the effects of BPA on the stress-response system. Given the interaction of sex and stress hormones, we examined the effect of a low perinatal BPA exposure on the function of the hypothalamic-pituitary-adrenal (HPA) axis at rest and upon application of acute stress. Throughout pregnancy and lactation rats received daily 40 mg BPA/kg body weight orally via cornflakes. We studied the effect of this low but chronic exposure to BPA in the male and female offspring at puberty. BPA exposure led to abnormal adrenal histology including reduced zona reticularis especially in male offspring, hyperplasia of zona fasciculata in both sexes, and increased adrenal weight in female offspring. BPA-treated females had increased basal corticosterone and reduced hypothalamic glucocorticoid receptors (GR) levels. Stressed BPA-exposed females exhibited anxiety-like behavioral coping, a less rigorous corticosterone response, and did not downregulate GR in the hypothalamus, compared with control females. BPA-exposed males exhibited a heightened corticosterone stress response compared with females; they also displayed increased pro-opiomelanocortin mRNA levels and retained the prestress levels of pituitary corticotropin-releasing hormone-receptor 1, compared with control males. We found that perinatal chronic exposure to a low dose of BPA perturbs the basal and stress-induced activity of the HPA axis in a sexually dimorphic manner at adolescence. Exposure to BPA might contribute to increased susceptibility to stress-related disorders in later life.

The transcription factor TFIIA is encoded by two genes, TFIIAab and TFIIAc. In higher eukaryotes, the TFIIAab is translated as a precursor and undergoes proteolytic cleavage; the regulation and biological implications of the cleavage have remained elusive. We determined by Edman degradation that the TFIIAb subunit starts at Asp 278. We found that a cleavage recognition site (CRS), a string of amino acids QVDG at positions À6 to À3 from Asp 278, is essential for cleavage. Mutations in the CRS that prevent cleavage significantly prolong the half-life of TFIIA. Consistently, the cleaved TFIIA is a substrate for the ubiquitin pathway and proteasome-mediated degradation. We show that mutations in the putative phosphorylation sites of TFIIAb greatly affect degradation of the b-subunit. We propose that cleavage and subsequent degradation fine-tune the amount of TFIIA in the cell and consequently the level of transcription.

Transcription of TATA box-containing genes by RNA polymerase II is mediated by TBP-containing and TBP-free multisubunit complexes consisting of common and unique components. We have identified a highly stable TBP-TFIIA-containing complex, TAC, which is detectable in embryonal carcinoma (EC) cells but not in differentiated cells. TAC contains the TFIIAgamma subunit and the unprocessed form of TFIIAalphabeta, although the processed TFIIAalpha and TFIIAbeta subunits are present in EC cells. TAC mediates transcriptional activation by RNA polymerase II in vivo, even though it does not contain classical TAFs. Formaldehyde cross-linking revealed that in EC but not in differentiated cells, association of TBP with chromatin is strongly enhanced when complexed with TFIIA in vivo. Remarkably, the TFIIAalphabeta precursor is preferentially, if not exclusively, associated with chromatin as compared to the processed subunits present in "free" TFIIA in EC cells.

No abstract

Although deoxybenzoins are intermediates in the synthesis of isoflavones, their estrogenic activity has not been investigated. Eleven deoxybenzoins were synthesized and their estrogenicity was evaluated. While their affinities for estrogen receptors (ER) ERalpha and ERbeta were found grossly comparable to those of daidzein, some exhibited considerable selectivity and transcriptional bias toward ERbeta, which appeared to allow for enhancement of ER-mediated transcription via deoxybenzoin binding of ERbeta. Their activity to stimulate the proliferation of ER-positive breast cancer cells and regulate the expression of endogenous and stably transfected reporter genes differed considerably, with some inhibiting cell proliferation while effectively inducing gene expression at the same time. Molecular modeling confirmed that deoxybenzoins fit well in the ligand binding pocket of ERbeta, albeit with different orientations. Our data support the view that deoxybenzoins constitute a promising new class of ERbeta-biased phytoestrogens.