Identification of a cis-regulatory element that directs prothoracicotropic hormone gene expression in the silkworm Bombyx mori

In the silkworm Bombyx mori and other insects, prothoracicotropic hormone (PITH) plays a central role in controlling molting and metamorphosis by stimulating the prothoracic glands to synthesize and release the molting hormone ecdysone. Using an AcNPV (Autographa californica nucleopolyhedrovirus)mediated transient gene transfer system, we identified a cis-regulatory element that participates in the decision to switch expression of PTTH on or off in PTTH-producing neurosecretory cells (PTPCs). The nucleotide sequence of this cis-regulatory element is similar to a cis-regulatory element that participates in direction of expression of diapause hormone-pheromone biosynthesis activating neuropeptide gene (DH-PBAN) (Shiomi et al., 2007). Furthermore, we found that B. mori Pitx (BmPitx), a bicoid-like homeobox transcription factor, binds the element and activates PITH expression. Therefore, we propose that the cell-specific expression of two neuropeptide hormone genes, PITH and DH-PBAN, is activated by the Pitx transcription factor, which may act as a pan-activator in the insect neuroendocrine system and in vertebrate pituitary cells. (C) 2011 Elsevier Ltd. All rights reserved.ArticleINSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY. 41(6):356-361 (2011)journal articl

Sequence-Specific DNA Recognition by Steroidogenic Factor 1: A Helix at the Carboxy-Terminus of the DNA Binding Domain is Necessary for Complex Stability

Abbreviated title: Structure-Function of SF1-DNA Interactions Key words: SF1; nuclear hormone receptor; DNA recognition; NMR; solution structure Disclosure of Potential Conflicts of Interest: K.E.M. has consulted for World Book Science Inc., has equity interests in Ligand Pharmaceuticals Inc., and received lecture fees from Serono Inc., but has no conflicts with entities directly related to the material being published. All other authors have nothing to disclose. This is an un-copyedited author manuscript copyrighted by The Endocrine Society. This may not be duplicated or reproduced, other than for personal use or within the rule of "Fair Use of Copyrighted Materials" (section 107, Title 17, U.S. Code) without permission of the copyright owner, The Endocrine Society. From the time of acceptance following peer review, the full text of this manuscript is made freely available by The Endocrine Society at http://www.endojournals.org/. The final copy edited article can be found at http://www.endojournals.org/. The Endocrine Society disclaims any responsibility or liability for errors or omissions in this version of the manuscript or in any version derived from it by the National Institutes of Health or other parties. base-pair DNA sequences as a monomer. Here we describe the solution structure of the SF1 DBD in complex with an atypical sequence in the proximal promoter region of the inhibingene that encodes a subunit of a reproductive hormone. SF1 forms a specific complex with the DNA through a bipartite motif binding to the major and minor grooves through the core DBD and the N-terminal segment of the FTZ-F1 box, respectively, in a manner previously described for two other monomeric receptors, NGFI-B and ERR2. However, unlike these receptors, SF1 harbors a helix in the C-terminal segment of the FTZ-F1 box that interacts with both the core DBD and DNA and serves as an important determinant of stability of the complex. We propose that the FTZ-F1 helix along with the core DBD serves as a platform for interactions with coactivators and other DNA-bound factors in the vicinity.

The Roles of PIN1 in the Pituitary Ganadotropes

Ph.DDOCTOR OF PHILOSOPH

Role of the orphan nuclear receptor NR5A2 in ovarian function

Le récepteur nucléaire orphelin NR5A2, également connu sous le nom de liver receptor homolog 1 (LRH-1), est exprimé dans l’ovaire, spécifiquement au niveau des cellules de granulosa. L’activation et le développement des follicules ovariens sont caractérisés par la multiplication des cellules de granulosa, des follicules primordiaux et leur unique rang de cellules de prégranulosa, aux follicules préovulatoires avec de nombreuses couches de cellules de granulosa cuboïdales. NR5A2 est un acteur bien connu de la régulation de la prolifération cellulaire dans des organes comme l’intestin et des pathologies comme le cancer du sein. L’hypothèse explorée est que NR5A2 régule le développement folliculaire en agissant sur la prolifération des cellules de granulosa et la régulation de l’activation des follicules primordiaux de la réserve ovarienne. Pour étudier ce facteur de transcription, un modèle de souris transgéniques caractérisées par la déplétion conditionnelle de NR5A2 dans les cellules de granulosa de tous les follicules (génotype Nr5a2f/f; Amhr2Cre+) a été développé. Les souris femelles mutantes, désignées comme cKO (pour conditional knock-out) sont infertiles suite à l’absence d’ovulation. La première partie de l’étude a mis en évidence, et ce aussi bien in vivo qu’in vitro, une déplétion significative du nombre de cellules en phase S, phase du cycle cellulaire au cours de laquelle a lieu la réplication de l’ADN. Cette diminution du nombre de cellules en division s’accompagne d’une déplétion significative de l’expression de gènes essentiels pour la prolifération des cellules de granulosa, parmi lesquels les cyclines D et E. De plus, démonstration a été faite que la régulation de la multiplication cellulaire passait en partie par l’interaction de NR5A2 avec b-caténine et que l’impact négatif de la déplétion de NR5A2 sur la prolifération des cellules de granulosa ne pouvait être secouru par l’adjonction d’estrogènes, un puissant mitogène des cellules ovariennes. Pour ce qui est du mécanisme, il a été déterminé que la régulation négative de CDKN1A, un inhibiteur du cycle cellulaire, est interropue en cas de déplétion de NR5A2. Ce gène a de ce fait été identifié comme un des principaux médiateurs de l’abrogation de la prolifération des cellules de granulosa gonadotropine-dépendante observée dans les souris cKO. La seconde partie de l’étude s’intéresse à l’impact de la déplétion de NR5A2 sur la population de follicules primordiaux. Dans ce même modèle murin, la déplétion de NR5A2 engendre la multiplication des follicules primordiaux et coincide avec la surexpression de marqueurs liés à leur formation et à la survie cellulaire. Ces changements s’accompagnent également de la diminution de la quantité de transcrits codant pour des gènes favorisant l’activation folliculaire. Dans des souris de type sauvage, l’analyse de la localisation de NR5A2 par immunocytochimie a confirmé l’existence de deux sous-populations de follicules primordiaux, l’une exprimant NR5A2 dans les cellules de granulosa est considérée comme prête à s’activer, l’autre caractérisée par l’absence de ce même facteur de transcription est constituée de follicules primordiaux quiescents. Chez les souris cKO une augmentation significative dans l’abondance de la quantité de transcrits de gènes favorisant la quiescence a aussi été établie, ce qui plaide en faveur du concept des deux populations de follicules primordiaux. En conclusion, NR5A2 joue un rôle dans la formation des follicules primordiaux, leur survie et leur activation. Pris dans leur ensemble, ces résultats démontrent que le récepteur nucléaire orphelin NR5A2 est un des régulateurs clé des évènements pré et peri-ovulatoires en agissant en particulier sur l’activation des follicules primordiaux et la prolifération des cellules de granulosa.The orphan nuclear receptor NR5A2, also known as liver receptor homolog 1 (LRH1), is expressed in the ovary, specifically in granulosa cells. Activation and development of ovarian follicles are characterized by the multiplication of granulosa cells, from primordial with only one layer of pregranulosa cells, to pre-ovulatory stage with multiples layers of cuboidal granulosa cells. NR5A2 is a well known actor in regulation of cell proliferation in organs such as the intestine and in conditions such as breast cancer. The hypothesis that NR5A2 regulates follicular development by acting on granulosa cells proliferation and the regulation of the primordial follicle pool activation was tested. To study this transcription factor, a knock-out mouse model characterized by the conditional depletion of NR5A2 in granulosa cells of all follicles (genotype Nr5a2f/f, Amrh2Cre/+) was developed. These female mutant mice, designated as cKO (conditional knock-out), were infertile due to the absence of ovulation. The first part of the study highlighted, in vivo and in vitro, a significant decrease of the number of cells reaching the S phase, where the DNA is replicated. This decrease in the dividing cells was accompanied by significant depletion of genes essential for granulosa cell proliferation, the cyclins D and E among others. Moreover, an effect of the depletion of NR5A2 on granulosa cell proliferation, via its interaction with b-catenin, was demonstrated and proliferation could not be rescued by the ovarian mitogen, estrogen. In terms of the mechanism, it was shown that the expected negative regulation of CDKN1A, a cell cycle inhibitor, did not occur following the depletion of NR5A2. This gene was therefore identified as the main mediator of the abrogation of gonadotropin dependent granulosa cell proliferation observed in the cKO mice. The impact of NR5A2 depletion on primordial follicles population was next studied. In this mouse genotype, there was a several fold increase in the number of primordial follicles, with concomitant upregulation of markers linked to their formation and cell survival. These changes were accompanied by a decrease in the transcripts coding for genes linked to follicle activation. In wild type mice, quantitative analysis by immunocytochemistry confirmed that there are two subpopulations of primordial follicles, one where NR5A2 is expressed in granulosa cells and one where it is absent, the former was judged to be poised for activation, while the latter was quiescent. The cKO mice are characterized by a significant increase in transcript abundance of genes promoting quiescence, support for this concept. We conclude that Nr5a2 plays a role in primordial follicle formation, survival and activation. Taken together these results demonstrate that the orphan nuclear receptor NR5A2 plays a key role in pre and peri-ovulatory events, acting particularly on primordial follicles activation and granulosa cells proliferatio

LRH-1 as a Key Regulator of Estrogen Responses in Breast Cancer Cells

Liver receptor homolog-1 (LRH-1; NR5A2) is an orphan member of the Ftz-F1 family of nuclear receptors, which comprises four members (NR5A1-NR5A4). LRH- 1 has been linked to a number of key developmental, metabolic and proliferative processes and is known to play an important role in the regulation of cholesterol biosynthesis, lipid homeostasis and the control of steroid aromatisation. In this respect, LRH-1 is recognised to play an important role in breast cancer, where it acts to regulate aromatase activity, leading to the paracrine production of estrogen. Recent findings have also suggested a direct role for LRH-1 in cancer, where LRH-1 is found to be involved in the induction of intestinal tumours and LRH-1 has been found by immunohistochemistry in tumour cells of human mammary ductal carcinomas. Recently, a gene expression microarray analysis of estrogen responses in an engineered breast cancer cell line, where Estrogen Receptor-α (ERα) activity can be conditionally repressed, provisionally identified LRH-1 as an estrogen responsive gene that may be important in the estrogen-regulated growth of breast cancer cells. Based on this initial observation, I have gone on to study the role of LRH-1 in the estrogen response in breast cancer cells. Using ERα-positive breast cancer cell lines, I have confirmed that LRH-1 levels increase in response to estrogen and are inhibited by anti-estrogens (tamoxifen and ICI 182,780). Using 5`RNA Ligase Mediated Rapid Amplification of cDNA Ends (5`RLM-RACE) to characterise the 5’ end of the LRH-1 mRNA, I have found that the estrogen regulation of LRH-1 is mediated through a previously undescribed gene promoter, which results in the production of a variant LRH-1 mRNA species that initiates transcription just upstream of exon 2 of the LRH-1 gene. Further, reverse transcriptase polymerase chain reaction (RT-PCR) showed that the newly identified variant LRH-1 transcript is expressed in tissues in which LRH-1 expression has previously been described. Moreover, this variant was seen to be the major form of LRH-1 expressed in breast cancer cell lines. Having established the estrogen regulation of LRH-1, studies were carried out to investigate the role of LRH-1 in growth and gene expression in breast cancer cells. siRNA-mediated inhibition of LRH-1 expression inhibited proliferation of MCF-7, ZR-75-1 and T47-D breast cancer cell lines but did not inhibit BT474 and MDAMB- 231 breast cancer cells, in which LRH-1 is not expressed. Further, a group of recently described synthetic agonists for LRH-1 stimulated the growth of breast cancer cell lines expressing LRH-1 in a dose dependent manner, but did not stimulate growth in those breast cancer cell lines which do not express LRH-1. Finally, RNA interference experiments directed against LRH-1 identified ERα as an important LRH-1 regulated gene. These results show that LRH-1 potentially plays a key role in regulating estrogen responses in ERα–positive breast cancer cells, primarily through the direct regulation of ERα gene expression. These new findings, taken together with the previously described role for LRH-1 in regulating aromatase gene expression identify LRH-1 as a potentially important target for the development of new therapies for the treatment of breast cancer