Here we report the use of fluorescence recovery after photobleaching (FRAP) to examine the intranuclear dynamics of fluorescent oestrogen receptor-alpha (ER). After bleaching, unliganded ER exhibits high mobility (recovery t1/2 < 1 s). Agonist (oestradiol; E2) or partial antagonist (4-hydroxytamoxifen) slows ER recovery (t1/2 approximately 5-6 s), whereas the pure antagonist (ICI 182,780) and, surprisingly, proteasome inhibitors each immobilize ER to the nuclear matrix. Dual FRAP experiments show that fluorescent ER and SRC-1 exhibit similar dynamics only in the presence of E2. In contrast to reports that several nuclear proteins show uniform dynamics, ER exhibits differential mobility depending upon several factors that are linked to its transcription function.

Hotspot mutations in the spliceosome gene SF3B1 are reported in ∼20% of uveal melanomas. SF3B1 is involved in 3′-splice site (3′ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear. Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1R625/K666 mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3′ss. Modelling the differential junctions in SF3B1WT and SF3B1R625/K666 cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3'ss-sequence context. SF3B1WT knockdown or overexpression do not reproduce the SF3B1R625/K666 splice pattern, qualifying SF3B1R625/K666 as change-of-function mutants. Mutagenesis of predicted branchpoints reveals that the SF3B1R625/K666-promoted splice pattern is a direct result of alternative branchpoint usage. Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease.

The RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins that are involved in gene-expression regulation; however, their in vivo targets and activities in biological processes such as cell differentiation, which requires reprogramming of gene-expression programs at multiple levels, are not well characterized. Here, we uncovered a mechanism by which DDX5 and DDX17 cooperate with heterogeneous nuclear ribonucleoprotein (hnRNP) H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We then observed that downregulation of DDX5 and DDX17 protein expression during myogenesis and epithelial-to-mesenchymal transdifferentiation contributes to the switching of splicing programs during these processes. Remarkably, this downregulation is mediated by the production of miRNAs induced upon differentiation in a DDX5/DDX17-dependent manner. Since DDX5 and DDX17 also function as coregulators of master transcriptional regulators of differentiation, we propose to name these proteins "master orchestrators" of differentiation that dynamically orchestrate several layers of gene expression.

Pre-mRNA splicing is functionally coupled to transcription, and genotoxic stresses can enhance alternative exon inclusion by affecting elongating RNA polymerase II. We report here that various genotoxic stress inducers, including camptothecin (CPT), inhibit the interaction between Ewing's sarcoma proto-oncoprotein (EWS), an RNA polymerase II-associated factor, and YB-1, a spliceosome-associated factor. This results in the cotranscriptional skipping of several exons of the MDM2 gene, which encodes the main p53 ubiquitin ligase. This reversible exon skipping participates in the regulation of MDM2 expression that may contribute to the accumulation of p53 during stress exposure and its rapid shut-off when stress is removed. Finally, a splicing-sensitive microarray identified numerous exons that are skipped in response to CPT and EWS-YB-1 depletion. These data demonstrate genotoxic stress-induced alteration of the communication between the transcriptional and splicing machineries, which results in widespread exon skipping and plays a central role in the genotoxic stress response.

Background: Thermococcus gammatolerans was isolated from samples collected from hydrothermal chimneys. It is one of the most radioresistant organisms known amongst the Archaea. We report the determination and annotation of its complete genome sequence, its comparison with other Thermococcales genomes, and a proteomic analysis.

Anti-Müllerian hormone (AMH) is secreted by immature testicular Sertoli cells. Clinical studies have demonstrated a negative correlation between serum AMH and testosterone in puberty but not in the neonatal period. We investigated AMH regulation using mouse models mimicking physiopathological situations observed in humans. In normal mice, intratesticular, not serum, testosterone repressed AMH synthesis, explaining why AMH is downregulated in early puberty when serum testosterone is still low. In neonatal mice, AMH was not inhibited by intratesticular testosterone, due to the lack of expression of the androgen receptor in Sertoli cells. We had shown previously that androgeninsensitive patients exhibit elevated AMH in coincidence with gonadotropin activation. In immature normal and in androgen-insensitive Tfm mice, follicle stimulating hormone (FSH) administration resulted in elevation of AMH levels, indicating that AMH secretion is stimulated by FSH in the absence of the negative effect of androgens. The role of meiosis on AMH expression was investigated in Tfm and in pubertal XXSxr b mice, in which germ cells degenerate before meiosis. We show that meiotic entry acts in synergy with androgens to inhibit AMH. We conclude that AMH represents a useful marker of androgen and FSH action within the testis, as well as of the onset of meiosis. ( J. Clin.

Estrogen receptor ␣ (ER) is a member of the nuclear receptor (NR) superfamily and regulates transcription of specific target genes in response to ligand binding and phosphorylation (2,19,36). Functional domains involved in ER transcription function have been mapped and include a centrally located DNA binding domain (DBD); in addition, two activation function domains (AFs) have also been identified, including an N-terminal domain, AF-1, and a C-terminal domain, AF-2, containing the ligand binding domain (LBD) (14,15,35).ER functionally interacts with a large group of proteins referred to as steroid receptor coregulators, including both coactivators and corepressors (reviewed in reference 20). Coactivators such as steroid receptor coactivator 1 (SRC-1) and CREB-binding protein (CBP/p300) interact with ER in an agonist-dependent manner to increase transcription function (5, 12, 13, 24), in part due to intrinsic histone acetyltransferase activity (23, 30). SRC-1 and CBP act synergistically to enhance steroid receptor-based transcription (28), suggesting that they are in the same molecular complex with steroid receptors. In the presence of agonist only, SRC-1 redistributes to ER foci bound to insoluble nuclear structures (33). The molecular mechanisms underlying interactions between ER and coactivators involve structural rearrangements in the LBD that occur upon hormone binding (4, 27). These structural changes involve the repositioning of helix 12 (amino acids [aa] 538 to 546) of the ER LBD, allowing for coactivator interactions in the presence of agonist. In vitro experiments have shown that helix 12 and key amino acids found in the coactivator binding pocket are essential for transcriptional activity and coactivator interactions (7,10,18).Many assays used to analyze interactions between steroid receptors and coregulators are performed in vitro or with yeast two-hybrid systems; both of which fail to recapitulate the elegant organization of the mammalian nucleus (22). Furthermore, experiments to characterize transactivator function often involve the use of transient transfection assays and exogenous templates that only partially reflect the complex organization of steroid receptors in the context of nuclear architecture (29). Recently, a functional green fluorescent protein-ER fusion protein (GFP-ER) was shown to undergo ligand-dependent intranuclear reorganization (11,33). Furthermore, this reorganization correlates with nuclear matrix (NM) association (33), suggesting that the NM plays a role in the subnuclear organization of ER and agonist-dependent SRC-1 binding. In contrast to a report showing high mobility of several intranuclear proteins (25), our recent photobleaching and biochemical data demonstrate that ligand and, surprisingly, proteasome activity regulate the intranuclear mobility-NM association between ER and SRC-1 (34). This work supports the notion that a highly organized and dynamic subnuclear environment provides a framework for receptor function (1,6,31,32).Integrated DNA segments containing multi...

Both epigenetic and splicing regulation contribute to tumor progression, but the potential links between these two levels of gene-expression regulation in pathogenesis are not well understood. Here, we report that the mouse and human RNA helicases Ddx17 and Ddx5 contribute to tumor-cell invasiveness by regulating alternative splicing of several DNA- and chromatin-binding factors, including the macroH2A1 histone. We show that macroH2A1 splicing isoforms differentially regulate the transcription of a set of genes involved in redox metabolism. In particular, the SOD3 gene that encodes the extracellular superoxide dismutase and plays a part in cell migration is regulated in an opposite manner by macroH2A1 splicing isoforms. These findings reveal a new regulatory pathway in which splicing factors control the expression of histone variant isoforms that in turn drive a transcription program to switch tumor cells to an invasive phenotype.