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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
31

Génomique intégrée des tumeurs bénignes corticosurrénaliennes / Integrated genomics of benign adrenocortical tumors

Faillot, Simon 28 November 2017 (has links)
Le cortex surrénalien produit des hormones stéroïdes, principalement le cortisol, l’aldostérone et des androgènes. Le cortex surrénalien peut être le siège de tumeurs –adénomes ou cancers-, hyperplasies et dysplasies. Ces lésions sont dans leur grande majorité bénignes. Elles peuvent être associés à une hypersécrétion d’hormone stéroïde, le plus souvent de cortisol (syndrome de Cushing) ou d’aldostérone. Il existe aussi des tumeurs non sécrétantes. Si des classifications moléculaires ont été établies pour les carcinomes, à ce jour il n’existe pas de classification pangénomique des tumeurs bénignes corticosurrénaliennes, qui pourrait renseigner sur les mécanismes de sécrétion autonome et de prolifération de ces lésions. Enfin le déterminisme génétique des dysplasies et hyperplasies n’est que partiellement connu. Au cours de ma thèse, j’ai pu analyser un jeu de données « omics » complet des lésions bénignes corticosurrénaliennes pour plus d’une centaine d’échantillons, incluant du séquençage haut-débit (exome/ciblé pour les mutations, RNA-seq pour l’analyse des microARNs), des puces transcriptome et méthylome, et des puces SNP pour la recherche d’altérations chromosomiques. J’ai pu identifier une classification moléculaire pangénomique relativement convergente entre les différentes « omics », qui concorde avec les types tumoraux et sécrétoires, mais identifie également des sous-groupes nouveaux au sein de ces lésions. Il ressort notamment que les mutations dans ces lésions sont des déterminants essentiels de la classification moléculaire. Ainsi sont regroupées les lésions selon la voie de signalisation ou le gène altéré, notamment la voie PKA/AMPc pour les lésions produisant du cortisol, la voie Wnt/beta-caténine pour les adénomes ne sécrétant pas ou peu de cortisol, et ARMC5 pour un sous-groupe d’hyperplasies macronodulaires. Ces groupes très distincts contiennent également des lésions sans mutation identifiée, avec vraisemblablement des mécanismes alternatifs d’altération de ces voies de signalisation. Dans le groupe des hyperplasies macronodulaires mutées ARMC5, la comparaison avec l’ensemble des autres lésions bénignes fait ressortir une signature d’expression ovarienne forte, marquée par l’expression de FOXL2 et de ses cibles CYP19A1 et PTHLH. Cette marque de différentiation spécifiquement gonadique au sein de la surrénale fait discuter une anomalie de développement. Cette analyse génomique intégrée identifie également des altérations épigénétiques de la stéroïdogenèse. Notamment les tumeurs sécrétant beaucoup de cortisol sont globalement hyperméthylées dans leurs îlots CpG. Par ailleurs l’hyperméthylation de CYP21A2 est vraisemblablement un mécanisme de déficits en 21-hydroxylase intratumoral. Des signatures de miRNA semblent également avoir un impact sur la stéroïdogenèse. Au cours de ma thèse j’ai également analysé l’exome des hyperplasies macronodulaires non mutées ARMC5. Je n’ai pas identifié de nouvelle mutation somatique récurrente. Au niveau de l’exome germinal, j’ai identifié plusieurs gènes candidats récurrents, qui ouvrent la voie à des analyses génétiques (extension de cohorte) et de biologie cellulaire complémentaires. Ce travail est la première caractérisation génomique d’envergure des lésions bénignes de la corticosurrénale. Même si tous les mécanismes ne sont pas élucidés dans le détail, ces données représentent une ressource importante pour orienter les recherches à venir dans la tumorigenèse surrénalienne bénigne et la stéroïdogenèse. / The adrenal cortex produces steroid hormones, mainly cortisol, aldosterone and androgens. The adrenal cortex can be the site of tumors - adenomas or cancers -, hyperplasias and dysplasias. These lesions are in their great majority benign. They may be associated with hypersecretion of steroid hormone, most commonly cortisol (Cushing's syndrome) or aldosterone. There are also non-secreting tumors. Although molecular classifications have been established for carcinomas, to date there is no genome-wide classification of benign adrenocortical tumors, which could provide information on the mechanisms of autonomic secretion and proliferation of these lesions. Finally, the genetic determinism of dysplasia and hyperplasia is only partially known. During my thesis, I analyzed a complete "omics" dataset of benign adrenocortical lesions for more than a hundred samples, including high-throughput sequencing (exome / targeted for mutations, RNA-seq for microRNA analysis), transcriptome and methylome microarrays, and SNP microarrays for chromosomal alterations. I was able to identify a relatively convergent genome-wide molecular classification between the different "omics", which is consistent with the tumor and secretory types, but also identifies new subgroups within these lesions. In particular, it appears that mutations in these lesions are essential determinants of molecular classification. Thus, the lesions are grouped according to the signaling pathway or the altered gene, in particular the PKA / cAMP pathway for lesions producing cortisol, the Wnt / beta-catenin pathway for adenomas that do not secrete little or no cortisol, and ARMC5 for a subgroup of macronodular hyperplasia. These very distinct groups also contain lesions with no identified mutation, presumably with alternative mechanisms of alteration of these signaling pathways. In the group of ARMC5 mutated macronodular hyperplasia, the comparison with all other benign lesions shows a strong ovarian expression signature, marked by the expression of FOXL2 and its targets CYP19A1 and PTHLH. This mark of specifically gonadal differentiation in the adrenal gland causes a development anomaly to be discussed. This integrated genomic analysis also identifies epigenetic alterations of steroidogenesis. In particular, tumors secreting a lot of cortisol are globally hypermethylated in their CpG islands. In addition, hypermethylation of CYP21A2 is probably a mechanism of intratumoral 21-hydroxylase deficiency. MiRNA signatures also appear to have an impact on steroidogenesis. During my thesis I also analyzed the exome of unmutated macronodular hyperplasia ARMC5. I did not identify a new recurrent somatic mutation. At the level of the germinal exome, I identified several recurrent candidate genes, which open the way for complementary genetic analyzes (cohort extension) and cell biology. This work is the first major genomic characterization of benign lesions of the adrenal cortex. Although not all mechanisms are fully elucidated, these data represent an important resource for guiding future research into benign adrenal tumorigenesis and steroidogenesis.
32

Reproductive toxicology of endocrine disruptors : effects of cadmium, phthalates and phytoestrogens on testicular steroidogenesis

Gunnarsson, David January 2008 (has links)
A number of investigations during the last two decades describe adverse trends in male reproductive health, which have been proposed to be caused by environmental factors with endocrine disrupting properties. In contrast to many other toxicants, endocrine disruptors often do not show linear dose-response relationships typical of those found in traditional toxicological studies. For many compounds, low-dose exposure causes effects opposite to the ones seen after high-dose exposure. In addition, the timing of exposure has been found to be critical. Hence, to correctly assess the impact of endocrine disruptors on reproductive health requires in-depth knowledge of their mechanisms of action. This thesis aimed at identifying the mechanisms underlying the effects of cadmium (Cd), phthalates and phytoestrogens on testicular steroidogenesis. For this purpose, in vitro as well as in vivo models were used. Cd was found to inhibit testosterone synthesis in vivo by down-regulating LH receptor gene expression and reducing the testicular levels of cAMP and StAR protein. In addition, Cd caused a pronounced increase in testicular prostaglandin F2ɑ (PGF2ɑ), suggesting that Cd exerts its suppressive effect on steroidogenesis also by inducing the inhibitory PKC pathway. Pre-treatment with zinc (Zn) protected completely against Cd-induced effects on testosterone and PGF2ɑ. Furthermore, we observed that Cd exposure increased glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in the testis. GAPDH is a potent coactivator of androgen receptor-mediated transcription and the up-regulation found in our study is probably a compensatory response to reduced testosterone concentrations. This finding is interesting since GAPDH has been proposed to have an important role in the regulation of apoptosis as well as sperm motility. We discovered that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the frequently used phthalate di-(2-ethylhexyl) phthalate (DEHP), stimulates Leydig cell steroidogenesis in vitro, by a cAMP- and StAR-independent mechanism. MEHP exposure caused a similar effect in granulosa cells. Gene expression analysis revealed that MEHP is likely to stimulate steroidogenesis by increasing the amount of cholesterol available for steroid synthesis. In the last investigation, we examined the effects of low-dose phytoestrogen exposure on testosterone synthesis during puberty in male goats. Isoflavones present in clover increased plasma concentrations of testosterone and free as well as total triiodothyronine (T3). T3 has previously been shown to induce testosterone synthesis and it is possible that an elevated T3 secretion underlies the increased plasma testosterone levels. Reduced fertility and reproductive tract malformations affect both the individual and the society. Hence, a sound knowledge of reproductive toxicants is of crucial importance. The findings presented in this thesis provide new insights into the reproductive toxicology of endocrine disruptors and may be valuable for risk assessment purposes.
33

Toxicity and mutagenicity of Upper Danube River sediments determined by chemical fractionation, the <i>Danio rerio</i> embryo assay, the Ames fluctuation test and the H295R assay.

Higley, Eric Bertram 18 November 2009
Declines in some fish populations in the Upper Danube River, Germany, have been reported during the past decades despite extensive stocking efforts. Many theories exist for why such declines have occurred including habitat change, dams, invasive species, disease and pollution. One of the factors of concern in the Upper Danube River is pollution because a number of studies have shown that sediments collected from this area were acutely and/or chronically toxic to fish. Although it can be difficult to link bioassay results to direct effects on the population level, bioassays can give us insight into the potential of exposure of wildlife including fish to sediment. In combination with other researchers a large battery of sediment testing on the Upper Danube River is being performed. Testing includes sediment testing of estrogen receptor mediated processes, dioxin-like responses and genotoxic effects. In this study, four sediment extracts from the Upper Danube River in Germany were used with a novel fractionation technique to characterize the sediment extracts and fractions for their ability to disrupt steroidogenesis, for their mutagenic activities and their teratogenic effects. Fractionation of each of the four sediment samples was performed by separating compounds according to their polarity, planarity, and the size of the aromatic ring system in an on-line fractionation procedure on coupled high performance liquid chromatography columns.<p> Mutagenic activity was measured in the raw sediment extracts and all 18 fractions using the Ames fluctuation assay and the Danio rerio embryo assay was used to assess lethal endpoints. Furthermore, disruptions of steroidogenesis were assessed by first establishing methods and a proof of concept of the H295R assay by exposing H295R cells to 7 model chemicals and measuring changes from a control in estradiol, testosterone and aromatase activity. Once methods were established all sediments and their fractions were analyzed using the Assay.<p> Specifically, in the <i>Danio rerio</i> assay, two raw sediment extracts killed 100% of <i>Danio rerio</i> embryos at a concentration of 33.3 mg sediment equivalents (SEQ)/ml, but none of the 18 fractions of these samples produced any measured toxicity at a concentration of 100 mg SEQ/ml. In the Ames fluctuation assay, significant mutagenic activity was measured in raw sediment extracts and in the fractions. Fraction 10 produced a significant mutagenic response in all sediment samples measured only in S9 bio-activated samples. Furthermore, fraction 15 produced a significant mutagenic response in all sediment samples measured only in non bio-activated samples.<p> All raw extracts tested in the H295R assay caused an increase in estradiol production up to 4-fold from controls. Testosterone production increased slightly from controls in only two of the raw extract samples. Of the 18 fractions, fractions 7, 10 and 15 increased estradiol in at least three of the samples studied (Sigmaringen2006, Opfingen2006, Lauchert2006 and Lauchert2004). Furthermore, fraction 7 significantly decreased testosterone production compared to controls in three of the four sediment samples.<p> Taken as a whole, these results show the value of using multiple bioassays and fractionation to characterize sediments that covers a variety of different biological endpoints. This study also demonstrates the usefulness of the H295R assay when combined with a new fraction technique to assess endocrine disrupting chemicals in sediment samples.
34

Toxicity and mutagenicity of Upper Danube River sediments determined by chemical fractionation, the <i>Danio rerio</i> embryo assay, the Ames fluctuation test and the H295R assay.

Higley, Eric Bertram 18 November 2009 (has links)
Declines in some fish populations in the Upper Danube River, Germany, have been reported during the past decades despite extensive stocking efforts. Many theories exist for why such declines have occurred including habitat change, dams, invasive species, disease and pollution. One of the factors of concern in the Upper Danube River is pollution because a number of studies have shown that sediments collected from this area were acutely and/or chronically toxic to fish. Although it can be difficult to link bioassay results to direct effects on the population level, bioassays can give us insight into the potential of exposure of wildlife including fish to sediment. In combination with other researchers a large battery of sediment testing on the Upper Danube River is being performed. Testing includes sediment testing of estrogen receptor mediated processes, dioxin-like responses and genotoxic effects. In this study, four sediment extracts from the Upper Danube River in Germany were used with a novel fractionation technique to characterize the sediment extracts and fractions for their ability to disrupt steroidogenesis, for their mutagenic activities and their teratogenic effects. Fractionation of each of the four sediment samples was performed by separating compounds according to their polarity, planarity, and the size of the aromatic ring system in an on-line fractionation procedure on coupled high performance liquid chromatography columns.<p> Mutagenic activity was measured in the raw sediment extracts and all 18 fractions using the Ames fluctuation assay and the Danio rerio embryo assay was used to assess lethal endpoints. Furthermore, disruptions of steroidogenesis were assessed by first establishing methods and a proof of concept of the H295R assay by exposing H295R cells to 7 model chemicals and measuring changes from a control in estradiol, testosterone and aromatase activity. Once methods were established all sediments and their fractions were analyzed using the Assay.<p> Specifically, in the <i>Danio rerio</i> assay, two raw sediment extracts killed 100% of <i>Danio rerio</i> embryos at a concentration of 33.3 mg sediment equivalents (SEQ)/ml, but none of the 18 fractions of these samples produced any measured toxicity at a concentration of 100 mg SEQ/ml. In the Ames fluctuation assay, significant mutagenic activity was measured in raw sediment extracts and in the fractions. Fraction 10 produced a significant mutagenic response in all sediment samples measured only in S9 bio-activated samples. Furthermore, fraction 15 produced a significant mutagenic response in all sediment samples measured only in non bio-activated samples.<p> All raw extracts tested in the H295R assay caused an increase in estradiol production up to 4-fold from controls. Testosterone production increased slightly from controls in only two of the raw extract samples. Of the 18 fractions, fractions 7, 10 and 15 increased estradiol in at least three of the samples studied (Sigmaringen2006, Opfingen2006, Lauchert2006 and Lauchert2004). Furthermore, fraction 7 significantly decreased testosterone production compared to controls in three of the four sediment samples.<p> Taken as a whole, these results show the value of using multiple bioassays and fractionation to characterize sediments that covers a variety of different biological endpoints. This study also demonstrates the usefulness of the H295R assay when combined with a new fraction technique to assess endocrine disrupting chemicals in sediment samples.
35

Alpha-class Glutathione Transferases from Pig: a Comparative Study

Fedulova, Natalia January 2011 (has links)
Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. This thesis contributes to knowledge about glutathione transferases from Sus scrofa (pig). The study is needed for better understanding of biochemical processes in this species and is desirable for drug development, for food industry research and in medicine. A primary role of GSTs is detoxication of electrophilic compounds. Our study presents porcine GST A1-1 as a detoxication enzyme expressed in many tissues, in particular adipose tissue, liver and pituitary gland. Based on comparison of activity and expression profiles, this enzyme can be expected to function in vivo similarly to human GST A2-2 (Paper II). In addition to its protective function, human GST A3-3 is an efficient steroid isomerase and contributes to the biosynthesis of steroid hormones in vivo. We characterized a porcine enzyme, pGST A2-2, displaying high steroid-isomerase activity and resembling hGST A3-3 in other properties as well. High levels of pGST A2-2 expression were found in ovary, testis and liver. The properties of porcine enzyme strengthen the notion that particular GSTs play an important role in steroidogenesis (Paper I). Combination of time-dependent and enzyme concentration-dependent losses of activity as well as the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurements of GSTs. Enzyme adsorption to surfaces was found to be the main explanation of high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Several approaches to improved functional comparison of highly active GSTs were proposed (Paper III). / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 733
36

Transcriptional Regulation of the Mouse Adrenal Cyclase Type 4 (Adcy4) in Y1 Adrenocortical Tumor Cells

Rui, Xianliang 20 May 2010 (has links)
Adenylyl cyclase (Adcy) is an important early effector of adrenocorticotrophin (ACTH) on the adrenal cortex; however, this enzyme consists of ten isozymes in mammalian cells and the factors governing the expression of different Adcy isozymes have not been well defined. The aim of this study is to investigate the regulation of mouse Adcy4, one of ten isozymes, in Y1 adrenocortical tumor cells and in mutant subclones derived from the Y1 cells. Adcy4 is expressed at a high level in brain but at lower levels in many other tissues including the Y1 cells. Moreover, this isozyme is specifically deficient in Y1 mutants with impaired steroidogenic factor 1 (SF1) activity. These observations support a hypothesis that Adcy4 expression is influenced by both ubiquitously expressed and tissue-specific transcription factors. My sequencing results indicate that mouse Adcy4 is highly homologous to the human and rat counterparts; its gene is located less than 1 kb downstream of Ripk3 and contains 26 exons. Primer extension and in silico analyses suggest that Adcy4 contains a TATA-less promoter and initiates transcription from multiple sites. Luciferase reporter gene assays indicate that Adcy4 promoter activity is mainly stimulated by the proximal GC-rich region but is inhibited by the first intron. This 124 bp GC-rich region is well conserved among several mammalian species and exhibits strong promoter activity in Y1 cells, which is functionally compromised in the Adcy4-deficient mutant. Within this region, three Sp1/Sp3- and one SF1-binding sites have been identified which bind the corresponding proteins Sp1 and Sp3 or SF1 in electrophoretic mobility shift assays (EMSAs). Site-directed mutagenesis reveals that the 5’-most Sp1/Sp3 site enhances Adcy4 promoter activity, whereas the middle Sp1/Sp3 and SF1 sites each repress this activity. In Y1 mutant cells, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with shRNA increases Adcy4 expression. All these data demonstrate that Adcy4 expression is under the control of the ubiquitous factors Sp1 and Sp3 and the tissue-specific factor SF1 and establish that SF1 is a repressor for Adcy4 promoter activity. This study is the first to demonstrate a repressor function for SF1 in certain promoter contexts.
37

THE REGULATION AND FUNCTION OF THE OVARIAN-DERIVED INSULIN-LIKE GROWTH FACTOR SYSTEM IN ZEBRAFISH (Danio rerio)

Irwin, David 13 December 2011 (has links)
Insulin-like growth factors (IGF) are known paracrine/autocrine regulators of ovarian development in teleosts. Initial studies investigated the hormonal and intracellular signal cascades involved in regulating the expression of ovarian-derived IGFs in zebrafish (Danio rerio). Quantitative real-time PCR was used to quantify the expression of igf3, igf2a, and igf2b in full grown immature (FG; 0.57-0.65 mm) and mid-vitellogenic (MV; 0.45-0.56 mm) follicles. Addition of the gonadotropin analogue human chorionic gonadotropin (hCG) and the adenylate cyclase activator forskolin increased igf3 expression in FG and MV follicles, but had no effect on igf2a or igf2b expression. The effects of hCG were blocked by the addition of the protein kinase A inhibitor H-89. Pituitary adenylate cyclase activating peptide stimulated a small increase in igf3 expression in FG follicles, while growth hormone and salmon gonadotropin releasing hormone had no effect on igf3, igf2a, or igf2b expression. Treatment with melittin, prostaglandin F2α, and prostaglandin E2 inhibited igf3 and igf2b expression in FG follicles whereas the protein kinase C activators, PMA and A23187, significantly inhibited igf3, igf2a, igf2b expression in FG and MV follicles. Secondary studies investigated the involvement of ovarian-derived IGFs in mediating the ovarian actions of gonadotropins on cell survival and steroidogenesis. Treatment of FG follicles with recombinant human IGF-I, hCG, or forskolin inhibited the induction of caspase-3/7 activity, which was used as a measure of apoptosis. The effects of hCG and forskolin on caspase-3/7 were attenuated by co-treatment with NVP-AEW54, an IGF-I receptor antagonist. hCG increased production of the maturation-inducing steroid 17α, 20β-dihydroxy-4-pregnen-3-one and co-treatment with NVP-AEW541 had no effect. These results suggest there is a high degree of hormonal specificity in regulating IGFs in the zebrafish ovary and the ovarian-derived IGFs, presumably IGF-III, are downstream mediators of gonadotropin-dependent cell survival, but are not involved in gonadotropin-induced steroidogenesis.
38

Gestational Form of Supplemental Selenium (Se) Affects Steroidogenic Gene Expression in the Newborn Calf Testis

Garbacik, Stefani R 01 January 2014 (has links)
Selenium (Se) is an important trace mineral in the diet of cattle. Our objective was to determine whether the form of supplemental Se fed to the dam would affect the expression of genes regulating steroidogenesis in the newborn testis. Twenty-four Angus-cross cows were assigned randomly (n=8) to individual ad libitum access of a mineral mix containing 35 ppm of Se supplied as sodium selenite (inorganic, ISe; Prince Se), Sel-Plex (organic, OSe; Sel-Plex, Alltech) or a 50/50 mix of ISe/OSe (MIX) 4 months prior to breeding and throughout gestation. All male calves were castrated within 2 days of birth and total testis RNA was subjected to microarray analysis using the Affymetrix Bovine 1.0 ST array. Ingenuity Pathway Analysis of differentially expressed genes, separated by one-way ANOVA (p<0.05) and a post-hoc LSD test, identified eight mRNAs associated with steroidogenesis. Specifically, mRNAs involved in the conversion of testosterone to estradiol were increased in the testis of OSe-supplemented when compared to ISe-supplemented animals, and mRNAs encoding genes regulating the conversion of androstenedione to testosterone or androstenedione to estradiol were decreased in the testis of MIX-supplemented when compared to ISe-supplemented cows.Expression in the neonate may equate to differences in fertility in the adult animal.
39

Steroidogenesis and steroidogenic gene expression in postnatal fetal rat Leydig cells

Weißer, Judith 08 July 2014 (has links) (PDF)
Die vorliegende Arbeit untersucht die Steroidogenese und die Expression Leydig-Zellspezifischer Gene in Kulturen postnataler fetaler Leydig-Zellen (PFLC). Die Stimulation von PFLC mit hCG und (Bu)2cAMP bewirkt eine Steigerung der Testosteronproduktion in vitro. Es wurde eine zeitabhängige Abschwächung der Testosteronproduktion durch (Bu)2cAMPstimulierte PFLC beobachtet. Diese war begleitet von einer Akkumulation von Progesteron im Kulturmedium und einer Suppression der Expression von P450c17 auf dem translatorischen Level. Während der Kultivierung verloren PFLC ihre Fähigkeit der Expression Leydig-Zell-spezifischer Gene (z.B. 3βHSD, P450c17, Insl3). Dieses Phänomen konnte durch Stimulation mit (Bu)2cAMP rückgängig gemacht werden. Außerdem zeigte sich, dass PDGFα allein und in Kombination mit (Bu)2cAMP signifikant die Proliferation der PFLC in vitro stimulierte. Die vorliegende Arbeit deutet darauf hin, dass cAMP-aktivierte Signalkaskaden eine wichtige Rolle in der Regulation von Differenzierung und Funktion von PFLC spielen.
40

Transcriptional Regulation of the Mouse Adrenal Cyclase Type 4 (Adcy4) in Y1 Adrenocortical Tumor Cells

Rui, Xianliang 20 May 2010 (has links)
Adenylyl cyclase (Adcy) is an important early effector of adrenocorticotrophin (ACTH) on the adrenal cortex; however, this enzyme consists of ten isozymes in mammalian cells and the factors governing the expression of different Adcy isozymes have not been well defined. The aim of this study is to investigate the regulation of mouse Adcy4, one of ten isozymes, in Y1 adrenocortical tumor cells and in mutant subclones derived from the Y1 cells. Adcy4 is expressed at a high level in brain but at lower levels in many other tissues including the Y1 cells. Moreover, this isozyme is specifically deficient in Y1 mutants with impaired steroidogenic factor 1 (SF1) activity. These observations support a hypothesis that Adcy4 expression is influenced by both ubiquitously expressed and tissue-specific transcription factors. My sequencing results indicate that mouse Adcy4 is highly homologous to the human and rat counterparts; its gene is located less than 1 kb downstream of Ripk3 and contains 26 exons. Primer extension and in silico analyses suggest that Adcy4 contains a TATA-less promoter and initiates transcription from multiple sites. Luciferase reporter gene assays indicate that Adcy4 promoter activity is mainly stimulated by the proximal GC-rich region but is inhibited by the first intron. This 124 bp GC-rich region is well conserved among several mammalian species and exhibits strong promoter activity in Y1 cells, which is functionally compromised in the Adcy4-deficient mutant. Within this region, three Sp1/Sp3- and one SF1-binding sites have been identified which bind the corresponding proteins Sp1 and Sp3 or SF1 in electrophoretic mobility shift assays (EMSAs). Site-directed mutagenesis reveals that the 5’-most Sp1/Sp3 site enhances Adcy4 promoter activity, whereas the middle Sp1/Sp3 and SF1 sites each repress this activity. In Y1 mutant cells, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with shRNA increases Adcy4 expression. All these data demonstrate that Adcy4 expression is under the control of the ubiquitous factors Sp1 and Sp3 and the tissue-specific factor SF1 and establish that SF1 is a repressor for Adcy4 promoter activity. This study is the first to demonstrate a repressor function for SF1 in certain promoter contexts.

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