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ABSTRACTRecently, we reported the unexpected finding that the monoubiquitination of histone H2B (H2Bub1) regulates inducible enhancers. Here, we propose a conceptual framework to reconcile the apparently discrepant roles of H2Bub1 in transcription initiation and elongation, and we discuss how H2Bub1 could regulate cellular processes linked to non-coding transcription.

Human papillomavirus type 16 (HPV16) and other oncogenic viruses have been reported to deregulate immunity by suppressing the function of the double-stranded DNA innate sensor TLR9. However, the mechanisms leading to these events remain to be elucidated. We show that infection of human epithelial cells with HPV16 promotes the formation of an inhibitory transcriptional complex containing NF-κBp50–p65 and ERα induced by the E7 oncoprotein. The E7-mediated transcriptional complex also recruited the histone demethylase JARID1B and histone deacetylase HDAC1. The entire complex bound to a specific region on the TLR9 promoter, which resulted in decreased methylation and acetylation of histones upstream of the TLR9 transcriptional start site. The involvement of NF-κB and ERα in the TLR9 down-regulation by HPV16 E7 was fully confirmed in cervical tissues from human patients. Importantly, we present evidence that the HPV16-induced TLR9 down-regulation affects the interferon response which negatively regulates viral infection. Our studies highlight a novel HPV16-mediated mechanism that combines epigenetic and transcriptional events to suppress a key innate immune sensor.

The higher incidence of thyroid carcinoma (TC) in women during reproductive years compared with men and the increased risk associated with the therapeutic use of estrogens have suggested a pathogenetic role exerted by these steroids in the development of TC. In the present study, we evaluated the potential of 17beta-estradiol (E2), genistein (G), and 4-hydroxyta-moxifen (OHT) to regulate the expression of diverse estrogen target genes and the proliferation of human WRO, FRO, and ARO thyroid carcinoma cells, which were used as a model system. We have ascertained that ARO cells are devoid of estrogen receptors (ERs), whereas both WRO and FRO cells express a single variant of ERalpha that was neither transactivated, modulated, nor translocated into the nucleus upon treatment with ligands. However, E2, G, and OHT were able either to induce the transcriptional activity of c-fos promoter constructs, including those lacking the estrogen-responsive elements, or to increase c-fos, cyclin A, and D1 expression. It is noteworthy that we have demonstrated that the G protein-coupled receptor 30 (GPR30) and the mitogen-activated protein kinase (MAPK) pathway mediate both the up-regulation of c-fos and the growth response to E2, G, and OHT in TC cells studied, because these stimulatory effects were prevented by silencing GPR30 and using the MEK inhibitor 2'-amino-3'-methoxyflavone (PD 98059). Our findings provide new insight into the molecular mechanisms through which estrogens may induce the progression of TC.

The role of oestrogens in the development of prostate cancer is poorly understood. However, a large body of evidence has suggested that oestrogenic hormones may be involved in prostatic malignancy. The localization of oestrogen receptor β (ERβ) in the secretory epithelium of the human prostate has raised the intriguing possibility that the action of oestrogen could be mediated, at least in part, by this receptor during the process of carcinogenesis. Hence, specific interference with oestrogen-activated and ERβ-mediated transcriptional activity could open new issues in the endocrine manipulation of prostate tumours. In the present study, we provide new insights into the role of ERβ in the context of an androgen-responsive prostate cancer cell line such as LNCaP, which was used as a model system together with steroid receptor negative HeLa cells. ERβ and the mutated androgen receptor (AR) T877A did not discriminate between oestrogen- or androgen-induced transactivation, whereas ERβ and AR transcriptional activity were inhibited only by the respective hormone antagonists ICI 182,780 and casodex. Furthermore, the nuclear localization of ERβ evaluated by immunocytochemistry confirmed the promiscuous response to hormones in addition to the specific inhibitory action of antagonists. Interestingly, ICI 182,780 and an ERβ antisense expression vector repressed the growth effects of both 17β-oestradiol and 5α-dihydrotestosterone, suggesting that ERβ has a key role in the proliferation induced by these steroids in LNCaP prostate cancer cells. Thus our findings implicate ERβ as a potential target for the treatment of prostate tumours.

High consumption of soybean products, such as phytoestrogens, has been hypothesized to contribute to a reduced incidence of prostate cancer in Southeast Asian people, although there have been inconsistent results among studies. Human LNCaP cells, extensively used as a model for androgen-dependent prostate tumor, express the androgen receptor (AR) mutant T877A promiscuously transactivated by estrogens and other ligands, which may further facilitate cancer progression. Here, for the first time to our knowledge, we demonstrate that genistein and quercetin, two phytoestrogens abundantly present in soybeans, activate either the AR mutant T877A in LNCaP or in transfected Chinese hamster ovary cells. This observation is supported by their capability to induce AR accumulation in the nuclear compartment of LNCaP together with mRNA down-regulation of the androgen target genes AR and PAP, and PSA up-regulation. Of interest, at concentrations eliciting transcriptional activity, both genistein and quercetin stimulate LNCaP cell growth, whereas at high levels, they become cytotoxic independently of AR expression, as ascertained in steroid receptor-negative Hela cells. The results of our study provide evidence that phytoestrogens may regulate several signaling processes in LNCaP cells; however, further studies are needed to assess their potential capability to restrain prostate tumor progression.

ABSTRACTBRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. Here, we identify a constitutive BRCA1-c-Abl complex and provide evidence for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. We show that the C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. We suggest a model in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity.

BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. Here, we identify a constitutive BRCA1-c-Abl complex and provide evidence for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. We show that the C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. We suggest a model in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity.

ABSTRACTThe highly abundant molecular chaperone Hsp90 functions with assistance from auxiliary factors, collectively referred to as Hsp90 cochaperones, and the Hsp70 system. Hsp104, a molecular chaperone required for stress tolerance and for maintenance of [psi+] prions in the budding yeastSaccharomyces cerevisiae, appears to collaborate only with the Hsp70 system. We now report that several cochaperones previously thought to be dedicated to Hsp90 are shared with Hsp104. We show that the Hsp90 cochaperones Sti1, Cpr7, and Cns1, which utilize tetratricopeptide repeat (TPR) domains to interact with a common surface on Hsp90, form complexes with Hsp104 in vivo and that Sti1 and Cpr7 interact with Hsp104 directly in vitro. The interaction is Hsp90 independent, as further emphasized by the fact that two distinct TPR domains of Sti1 are required for binding Hsp90 and Hsp104. In a striking parallel to the sequence requirements of Hsp90 for binding TPR proteins, binding of Sti1 to Hsp104 requires a related acidic sequence at the C-terminal tail of Hsp104. While Hsp90 efficiently sequesters the cochaperones during fermentative growth, respiratory conditions induce the interaction of a fraction of Hsp90 cochaperones with Hsp104. This suggests that cochaperone sharing may favor adaptation to altered metabolic conditions.

The highly abundant molecular chaperone Hsp90 functions with assistance from auxiliary factors, collectively referred to as Hsp90 cochaperones, and the Hsp70 system. Hsp104, a molecular chaperone required for stress tolerance and for maintenance of [psi+] prions in the budding yeastSaccharomyces cerevisiae, appears to collaborate only with the Hsp70 system. We now report that several cochaperones previously thought to be dedicated to Hsp90 are shared with Hsp104. We show that the Hsp90 cochaperones Sti1, Cpr7, and Cns1, which utilize tetratricopeptide repeat (TPR) domains to interact with a common surface on Hsp90, form complexes with Hsp104 in vivo and that Sti1 and Cpr7 interact with Hsp104 directly in vitro. The interaction is Hsp90 independent, as further emphasized by the fact that two distinct TPR domains of Sti1 are required for binding Hsp90 and Hsp104. In a striking parallel to the sequence requirements of Hsp90 for binding TPR proteins, binding of Sti1 to Hsp104 requires a related acidic sequence at the C-terminal tail of Hsp104. While Hsp90 efficiently sequesters the cochaperones during fermentative growth, respiratory conditions induce the interaction of a fraction of Hsp90 cochaperones with Hsp104. This suggests that cochaperone sharing may favor adaptation to altered metabolic conditions.

Phytoestrogens are a chemically diverse group of compounds made by plants that can have estrogenic effects in animals. Both tumorigenic and antitumorigenic effects have been reported. Although estrogens stimulate the growth of many breast tumors, there is a negative correlation between the incidence of breast cancer and the phytoestrogen-rich diet of certain Asian populations. To begin to resolve this paradox, we have analyzed the estrogenic properties of genistein and quercetin, two flavonoid phytoestrogens particularly abundant in soybeans. Trans-activation experiments with a transfected reporter gene for nuclear estrogen receptors (ER) show strong activation of the endogenous ERαby both phytoestrogens in two MCF7 human breast cancer cell lines. This is supported by the observation that the two phytoestrogens induce the down-regulation of ERαmRNA and protein levels. Using chimeric proteins consisting of the hormone binding domains of ERαand ERβfused to the Gal4 DNA binding domain, we have established that genistein and quercetin are full estrogenic agonists of both ER isoforms. Ligand binding experiments with purified ERαand ERβconfirm that the two phytoestrogens are ER ligands. At concentrations that are sufficient to obtain substantial transcriptional activity, they stimulate the proliferation of two ERα-dependent breast cancer cell lines. At high concentrations, such as those reached with a soy-rich diet, genistein and quercetin are strong cytotoxic agents that even kill ER-independent HeLa cells. Thus, the mode of action of phytoestrogens and the balance between being risk or chemopreventive factors for breast cancer may depend on the dietary load.

The molecular chaperone Cdc37 is thought to act in part as a targeting subunit of the heat‐shock protein 90 (Hsp90) chaperone complex. We demonstrate here that Cdc37 is required for activity of the kinase Ste11 in budding yeast. A cdc37mutant strain is defective in Ste11‐mediated pheromone signaling and in accumulation and functional maturation of the constitutively active Ste11 version Ste11ΔN. Moreover, Cdc37, Ste11ΔN and Hsp90 coprecipitate pairwise. Thus, Hsp90 and Cdc37 may transiently associate with Ste11 to promote proper folding and/or association with additional regulatory factors. Our results establish Ste11 as the first endogenous Cdc37 client protein in yeast.

Atomically precise gold nanoclusters (AuNCs) are an emerging class of quantum-sized nanomaterials with well-defined molecular structures and unique biophysical properties, rendering them highly attractive for biological applications. We set out to study the impact of different ligand shells of atomically similar nanoclusters on cellular recognition and response. To understand the effects of atomically precise nanoclusters with identical composition on cells, we selected two different water-soluble gold nanoclusters protected with captopril (Capt) and glutathione (GSH): Au25(Capt)18(CNC) and Au25(GSH)18(GNC), respectively. We demonstrated that a change of the ligand of the cluster completely changes its biological functions. Whereas both nanoclusters are capable of internalization, only CNC exhibits remarkable cytotoxicity, more specifically on cancer cells. CNC shows enhanced cytotoxicity by inhibiting the OXPHOS of mitochondria, possibly by inhibiting the ATP synthase complex of the electron transport chain (ETC), and by initiating the leakage of electrons into the mitochondrial lumen. The resulting increase in both mitochondrial and total cellular ROS triggers cell death indicated by the appearance of cellular markers of apoptosis. Remarkably, this effect of nanoclusters is independent of any external light source excitation. Our findings point to the prevailing importance of the ligand shell for applications of atomically precise nanoclusters in biology and medicine.

The response to endocrine therapy of breast cancer is not entirely predictable from hormone receptor status alone since some point mutated or splicing variants of the estrogen receptor (ER) show altered biological activities. In order to characterize the activities of all forms of ER in a heterogeneous breast tumor, a functional assay in Saccharomyces cerevisiae was developed. Total RNA isolated from breast cancer cells and one breast cancer specimen was reverse trans‐cribed and the ER cDNA was amplified by PCR. The products were then cloned into an expression vector by in vivo homologous recombination in yeast. The yeast strain carries a reporter gene (ADE2) coupled to an estrogen response element. Activation of the reporter by ER yielded white colonies whereas lack of ER activity produced red colonies. This permitted the testing for functionality of individual ER molecules and subsequent analysis by rescuing of the ER expression plasmids and complete DNA sequencing. This simple visual test allows discrimination between wild‐type ER, constitutively active ER and inactive ER.

The aminosteroid U73122 is generally used as a specific inhibitor of phosphoinositide specific phospholipase C (PLC) and typically, the structurally related compound U73343 is used as control, since it lacks PLC inhibitory activity. We have found that both compounds possess strong estrogenic activity and that this activity is mediated by the estrogen receptors (ER) α and β. Although no direct evidence for binding of U73122 and U73343 to the ER could be provided, the estrogenic activity of the aminosteroids requires an intact ER hormone binding pocket. Given the chemical structure of the two aminosteroids, they may be converted to an estrogenic derivative by chemical degradation or an enzymatic metabolization reaction. Our data indicate that additional care should be taken in the interpretation of the effects of U73122 in cells expressing ER.

ABSTRACTThe protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZreporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

AbstractThe budding yeast Saccharomyces cerevisiaehas been used extensively as a biological ‘test tube’ to study the regulation of the human estrogen receptor (ER) α. However, anti-estrogens, which are of great importance as therapeutic agents and research tools, fail to antagonize the activation by estrogen in yeast. Here, we have surveyed the antagonistic potential of five different anti-estrogens of diverse chemical nature. While they all act as agonists for wild-type ERα we have established a novel yeast assay system for anti-estrogens, in which at least the commonly used anti-estrogen hydroxytamoxifen is a potent antagonist.

The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZreporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

The heat-shock protein 90 (Hsp90) is a cytosolic molecular chaperone that is highly abundant even at normal temperature. Specific functions for Hsp90 have been proposed based on the characterization of its interactions with certain transcription factors and kinases including Raf in vertebrates and flies. We therefore decided to address the role of Hsp90 for MAP kinase pathways in the budding yeast, an organism amenable to both genetic and biochemical analyses. We found that both basal and induced activities of the pheromone-signaling pathway depend on Hsp90. Signaling is defective in strains expressing low levels or point mutants of yeast Hsp90 (Hsp82), or human Hsp90β instead of the wild-type protein. Ste11, a yeast equivalent of Raf, forms complexes with wild-type Hsp90 and depends on Hsp90 function for accumulation. For budding yeast, Ste11 represents the first identified endogenous “substrate” of Hsp90. Moreover, Hsp90 functions in steroid receptor and pheromone signaling can be genetically separated as the Hsp82 point mutant T525I and the human Hsp90β are specifically defective for the former and the latter, respectively. These findings further corroborate the view that molecular chaperones must also be considered as transient or stable components of signal transduction pathways.

Most inducible expression vectors for the budding yeast Saccharomyces cerevisiaeare based on galactose‐inducible promoters. Yeast has been increasingly used to study vertebrate steroid receptors because of its powerful genetics. In principle, both regulatory systems are compatible and can be combined in the same strain. However, we found that commercial galactose can be contaminated by bioactive estrogen and progesterone at concentrations that are sufficient to fully activate their cognate receptors. Since steroids can elicit biological responses in pathogenic fungi and possibly other microorganisms, such contaminants in a commonly used fermentable carbon source may need to be screened for.

Overexpression of c‐Abl tyrosine kinase can be growth inhibitory in certain fibroblast cell lines. Using a series of conditional chimeras between Abl and Src, we have now further dissected the Abl protein to determine which domains are required for this function. We found that growth inhibition, unlike transformation by oncogenic forms of Abl, is dependent on the presence of the cognate SH2 and tyrosine kinase domains. Since growth inhibition correlates with low tyrosine kinase activity, it may involve highly specific interactions of target proteins with both domains without the processivity of phosphorylation associated with oncogenic Abl.

The reliance of many cancers on aerobic glycolysis has stimulated efforts to develop lactate dehydrogenase (LDH) inhibitors. However, despite significant efforts, LDH inhibitors (LDHi) with sufficient specificity and in vivoactivity to determine whether LDH is a feasible drug target are lacking. We describe an LDHi with potent, on-target, in vivoactivity. Using hyperpolarized magnetic resonance spectroscopic imaging (HP-MRSI), we demonstrate in vivoLDH inhibition in two glycolytic cancer models, MIA PaCa-2 and HT29, and we correlate depth and duration of LDH inhibition with direct anti-tumor activity. HP-MRSI also reveals a metabolic rewiring that occurs in vivowithin 30 min of LDH inhibition, wherein pyruvate in a tumor is redirected toward mitochondrial metabolism. Using HP-MRSI, we show that inhibition of mitochondrial complex 1 rapidly redirects tumor pyruvate toward lactate. Inhibition of both mitochondrial complex 1 and LDH suppresses metabolic plasticity, causing metabolic quiescence in vitroand tumor growth inhibition in vivo.

During differentiation of lymphocytes into antibody-producing cells, an immunoglobulin κ variable-region gene is transcriptionally activated by rearrangement linking it to a κ constant (Cκ) region gene which is already transcribed prior to somatic rearrangement1,2. The presence of a transcriptional enhancer element within the large intron of the κ light-chain gene has been postulated to explain this mode of activation, supported by evidence of a chromatin region which is preferentially accessible to DNase I3–5and restriction enzymes5. This DNA region contains a segment of about 130 base pairs (bp) which is strongly conserved between mouse, rabbit and man6. Moreover, no transcripts are detectable from a κ gene, which is truncated within the large intron7. Recently, a lymphocyte-specific enhancer has been identified downstream of the joining region in immunoglobulin heavy-chain genes8–10. We now show direct evidence for a functionally similar enhancer within the large κ gene intron of the mouse. It is, however, less active than the heavy-chain gene enhancer. In contrast, no enhancer was found to be associated with a cloned λI light-chain gene.

Le livre « Réduire les pertes d’azote dans l’élevage» s’attache à identifier les moyens de lutter efficacement contre les risques de pollution de l’environnement par les composés azotés réactifs dus à cette production. Les formes d’azote réactif sont décrites, leurs sources identifiées, leurs flux mesurés. Les moyens de réduire ces flux et les politiques à conduire pour y parvenir sont analysés.

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A transcriptional enhancer is thought to play a major role in determining tissue-specific expression of the immaunoglobulin heavy chain (IgH) gene (1–3). However in three B-lymaphoid cell lines the Ig enhancer has been lost due to a spontaneous deletion, yet heavy chain synthesis persists at a high level (4–6). In the case of the enhancerless delta chain gene (5) we wanted to test whether the IgH enhancer is no longer necessary for maintenance of transcription or whether perhaps a new enhancer was created de novo by the deletion process. To this end we have cloned the relevant portion of the variant IgH gene and transfected it into myeloma and hybridoma cells. We find that the reintroduced gene segment is not expressed unless it is linked again to an enhancer. The results suggest that the IgH enhancer is necessary for the onset of transcription, presumably by organizing the gene into stable transcription complexes, but is dispensable at later stages once the transcription unit is activated.

Most inducible expression vectors for the budding yeast Saccharomyces cerevisiae are based on galactose-inducible promoters. Yeast has been increasingly used to study vertebrate steroid receptors because of its powerful genetics. In principle, both regulatory systems are compatible and can be combined in the same strain. However, we found that commercial galactose can be contaminated by bioactive estrogen and progesterone at concentrations that are sufficient to fully activate their cognate receptors. Since steroids can elicit biological responses in pathogenic fungi and possibly other microorganisms, such contaminants in a commonly used fermentable carbon source may need to be screened for.