James L. Jameson - US grants

Follicle-stimulating hormone (FSH) is important for gametogenesis and gonadal steroid biosynthesis in both males and females. The hormonal regulation of FSH biosynthesis is complex and at present, is poorly understood. In addition to gonadotropin- releasing hormone (GnRH) and gonadal steroids, several recently characterized hypothalamic (GnRH-associated peptide) and gonadal peptides (Inhibins A and B, FSH-releasing peptide) have been shown to be potent regulatory factors for FSH secretion. This proposal focuses on the regulation of human FSH alpha and beta subunit biosynthesis at the level of gene expression. To complement studies of the previously isolated alpha-gene, the human FSHbeta gene and a heterogenous group of FSHbeta cDNAs will be isolated and characterized. Initial studies will use deletion mutagenesis and analyses of expression in transfected cell lines to define the structural features of the alpha and FSHbeta genes that confer cell-specific expression and hormone responsivity. In parallel, interactions between putative gene regulatory elements and intracellular trans-acting factors will be assessed by (1) cotransfection competition assays for binding of intracellular activators or repressors of gene expression by putative regulatory sequences, and (2) analysis of in vitro binding of nuclear proteins to regulatory sequences using gel retardation assays and DNase 1 footprinting studies. These studies will allow detailed analyses of the regulatory regions of the alpha and FSHbeta genes and initial characterization of the intracellular factors that interact with these regulatory elements.

Chorionic gonadotropin (CG) is a placental hormone that stimulates gonadal steroidogenesis and is important for maintenance of the corpus luteum of pregnancy. After implantation of the fertilized ovum, CG levels increase through the first trimester, after which they gradually decline. CG is also produced ectopically by a variety of neoplasms. The long-term goal of this proposal is to understand the cellular mechanisms that regulate CG biosynthesis at the level of gene expression. CG is a heterodimer consisting of nonidentical alpha and beta subunits that are encoded by separate genes. The alpha-subunit is encoded by a single copy gene, whereas the beta-subunit genes comprise a multigenic family that includes six CG beta genes and a single copy of the structurally-related LH beta gene. This project will address: 1) Determination of the DNA regulatory sequences of the alpha, CG beta, and LH beta genes using deletion mutagenesis and analyses of gene expression in transfected cells. These studies will focus on the cis-acting DNA sequences that confer cell-specific expression and hormone-responsiveness as well as the regulatory elements that result in coordinant expression of alpha and beta subunit genes. In addition, the regulatory DNA sequences of the recently diverged LH beta/CG beta gene pair will be exchanged onto the heterologous beta-subunit promoter to examine the mechanisms that regulate placental-specific expression of the CG beta gene and pituitary-specific expression of the LH beta gene. Similarly, the gene regulatory elements involved in ectopic gene expression will be sought by defining the DNA sequences that confer the capacity to express the CG alpha and beta genes in ectopic hormone-producing cell lines. 2) The trans-acting cellular factors that interact specifically with the alpha and beta gene regulatory elements will be examined using gel-retardation assays and DNA footprinting studies. These assays will also provide a means to monitor the purification of these DNA binding proteins during a series of isolation steps. Competition assays in transfected cells will be used to examine the cellular factors that interact with DNA regulatory elements. These studies will include experiments to assess interactions of alpha and beta regulatory sequences with shared regulatory factors that may mediate coordinant gene expression. 3) In parallel with the studies of transfected fusion genes, expression of the endogenous gonadotropin alpha and beta subunit genes will be examined in pituitary and placental cell lines as well as in pituitary tumors. These studies will focus on coordinant expression of the alpha and beta subunits, and characterization of defects in biosynthesis and secretion that occur in pituitary tumors.

Steroid hormones are involved in a wide array of physiological processes such as cellular differentiation during development and maintenance of homeostasis in the adult. Steroid hormones act by binding to intracellular receptors that subsequently interact with DNA regulatory elements to activate or repress the transcription of specific genes. cDNAs encoding a large family of thyroid and steroid hormone receptors have been cloned, providing new approaches for understanding steroid receptor action. The glycoprotein hormones (TSH, LH, FSH,hCG) are regulated by thyroid and steroid hormones in classic feedback fashion, thereby providing an ideal model system for examining mechanisms by which these hormones modulate gene expression. This proposal will specifically address interaction of thyroid and glucocorticoid receptors with hormone response elements in the human glycoprotein hormone alpha-gene. Preliminary studies indicate that the alpha-gene contains a negative thyroid response element that resides adjacent to the TATA box. The alpha-gene also contains positive and negative glucocorticoid response elements that overlap previously characterize enhancer elements. These findings indicate that the thyroid and glucocorticoid receptors converge with other cellular transcription factors at common or overlapping DNA target sites, providing a potentially powerful mechanism for modulating alpha-gene transcription. The functional properties of glucocorticoid and thyroid receptors (including thyroid receptor variants) will be explored using gene transfer studies in receptor deficient cell lines. Extensive mutagenesis of the alpha-promoter will be used to define the DNA sequence determinants for the thyroid and glucocorticoid response elements. The convergence of steroid receptor and other cellular signalling pathways at common promoter regions will be dissected using mutational analyses and heterologous constructions. Receptor interaction with cognate DNA response elements will be examined using in vitro binding studies to determine the specificity and affinity of receptor interactions. The DNA sequence determinants for receptor binding will be correlated with mutations that alter hormone responsiveness. In vitro transcription assays will be used to further examine interactions of receptors with partially purified or cloned transcription factors. As a result of these studies, the alpha-gene will provide a valuable model system for understanding multihormonal regulation of gene expression. The mechanims derived from these studies should be applicable to other genes that are regulated by thyroid and steroid hormones.

Hypogonadotropic hypogonadism (HH) is characterized by deficiency in the production of the gonadotropins. LH and FSH. and can be a reversible form of infertility. Causes of HH include deficient production or regulation of hypothalamic GnRH as well as abnormal responsiveness of the pituitary gonadotrope cell. Remarkably, studies of pathways that regulate expression of the gonadotropin genes are now pointing the way towards disorders that underlie reproductive dysfunction. Transcription factors such as SF-1 and DAX-1 appear to play a key role in the development of the normal gonadotrope phenotype. Mutations in DAX-1 have been shown to cause X-linked adrenal hypoplasia congenita (AHC) and associated HH. A gene knockout of SF-1 results in a phenotypically similar disorder in a mouse model. In both cases, there is evidence for abnormalities in GnRH production and gonadotrope responsiveness. Preliminary studies suggest that these transcription factors regulate the expression of an array of gonadotrope-specific genes including the GnRH receptor and the alpha and beta-subunit genes for LH and FSH. The goal of this project is to perform an integrated series of studies that define the effects of DAX-1 and SF-1 mutations at a clinical level. in animal models, and in terms of how these proteins function at a cellular level. The specific aims are to: 1) Identify DAX-1 and SF-1 gene mutations as causes of hypogonadotropic hypogonadism in humans and to characterize the clinical phenotypes using detailed studies of the hypothalamic-pituitary-gonadal axis. 2) Examine the developmental and functional relationships of SF-1 and DAX-1 in the pituitary and hypothalamus. The spaciotemporal patterns of SF-1 and DAX-1 expression will be determined and a DAX-1 knockout mouse model will be created to evaluate the functional and developmental role of this transcription factor in an animal model. 3) Identify and characterize the DNA-binding and functional properties of SF-1 and DAX-1 with respect to target genes expressed in gonadotrope cells. These studies will provide new insights into the genetic and cellular basis of gonadotropin deficiency syndromes and will provide a platform for rational therapeutic interventions.

Thyroid hormone (T3) regulates growth and differentiation during development and modulates the activity of a wide variety of metabolic pathways in adults. Disorders that result in low (hypothyroid) or high (hyperthyroid) levels of T3 are common, and result in a multitude of clinical effects owing to the broad distribution of T3 receptors. Thyroid hormone receptors (TRs) function as transcription factors to increase or decrease levels of gene expression. Although there has been great progress in our understanding of how T3 stimulates gene transcription, the mechanism by which it turns genes off is not well- understood. The physiologic role of negative regulation by T3 is most clearly illustrated by a classical negative feedback loop in which T3 inhibits the production of pituitary thyroid-stimulating hormone (TSH). The inhibition TSH by T3 represents a critical ~set-point~ that establishes the metabolic status of an individual. In this proposal, we will continue our studies of the TSH alpha and beta genes as a model for how T3 turns off gene transcription. Recent studies have identified a family of transcriptional co-repressors and co-activators that interact with the TR. We propose that these transcriptional co- factors play a critical role in T3 mediated repression. In the case of negatively regulated genes, we hypothesize that in the absence of T3, the unliganded TR recruits co-repressors away from the promoter resulting in transcriptional activation . Upon T3 binding to the receptor, a cascade of events is proposed to culminate in T3-dependent repression. These events include the displacement of co-repressors from TR to other proteins on the target promoter, and the ability of the activated TR to bind key co-investigate the role of Nuclear Co- repressors (NcoRs) in the control of genes that are repressed in response to T3; 2) To examine the interactions of transcriptional co- factors with the CREB/CBP transcription factors as a mechanism for T3-induced repression. These studies will address a fundamental question of how T3 acts as a switch to turn off a selected array of genes. In addition to the implications for thyroid hormone action, analogous mechanisms are likely to occur for other nuclear receptors.

DESCRIPTION: (Adapted from applicant's description) This research is based on the hypothesis that, in gonadotropes, a cellular protein, FKBP12 may be a regulatory protein for IP3 receptors and be controlled via GnRH signaling. The broad goal of the proposal is to evaluate the role of FKBP12 in the regulation of IP3 Receptor function and GnRH-induced LH secretion. The study proposes to quantify the contribution of FKBP12 to GnRH-induced LH secretion using in vitro and in vivo models and to determine whether expression, assembly, or phosphorylation of the IP3-Receptor complex is regulated by hormones that alter sensitivity to GnRH. The projects should provide information about the GnRH stimulated PKC pathway leading to calcium release from the endoplasmic reticulum. It will use a drug, FK506, to test the role of FKBP12. FK506 is an immunosuppressive drug that is widely used in organ transplants. Thus, information about its potential role in the reproductive system would also be obtained.

FSH, acting primarily through the protein kinase A pathway, regulates follicular development and function. Recent studies reveal convergence of the FSH/cAMP pathway with a group of transcription factors (SF-1, DAX-1) that control target genes in the ovary. SF-1 and DAX-1 are orphan nuclear receptors that play a key role in the development and function of the adrenal, gonads, and pituitary gonadotropes. A gene knock-out of SF-1 prevents the development of these organs, and SF-1 response elements have been identified in promoters of many different ovarian genes. Mutation of the X-linked DAX-1 gene causes the syndrome of adrenal hypoplasia congenital. It is characterized by the absence of the adult zone of the adrenal cortex with hypogonadotropic hypogonadism with apparently normal testicular Leydig cell function. Duplication of DAX-1 causes sex reversal in genetic males, and DAX-1 has been postulated to foster ovarian development. We have shown that DAX-1 modulates SF-1 action and play a critical role in ovarian development and function. We have shown that DAX- 1 modulates SF-1 action and hypothesize that the balance of DAX-1 and SF-1 expression in the ovary regulates its development, and subsequently, the function of various target genes like inhibin-alpha and steroidogenic enzyme genes. We propose an integrated group of in vitro and in vivo studies to further define the functions of SF-1 and DAX-1 in the ovary. There are three specific aims: 1) To examine mechanisms by which the cAMP and SF-1 pathways interact to regulate ovarian genes. We hypothesize that transcriptional co-factors, such as CBP, provide a basis for convergence of the FSH signaling pathway with the nuclear receptor, SF-1; 2) To determine the basis of divergent effects of DAX-1 on SF-1-mediated transcription of different ovarian target genes. We hypothesize that DAX-1 promoters will be studied as representative examples of SF-1 responsive genes that are regulated differentially by DAX-1; 3) To determine the effect of targeted deletion and over-expression of DAX-1 on ovarian gene expression. These studies will test in vivo whether DAX-1 plays a role in ovarian development and in the regulation of ovarian gene expression.

DESCRIPTION (provided by applicant): Sex determination in mammals is governed by a series of genetic switches that influence cell fate and differentiation during critical periods of development. Remarkably, the primordial fetal gonad is bipotential and the precursor gonadal cells can develop along either a male (testis) or female (ovary) pathway, depending on which genes are expressed. The main goal of this project is to identify key genes that regulate gonadal development and phenotypic sex. To this end, the investigators propose to screen mice for sex-reversal, manifest as XY phenotypic females or as XX phenotypic males, taking advantage of the existing Northwestern University Genome Wide ENU Mutagenesis Center. The specific aims of this project are: Aim 1: To screen progeny of ENU-mutagenized mice for sex reversal. Mice will be characterized genetically for the presence or absence of the Y-chromosomal Sry gone. At age 21 days, they will be classified as phenotypically male or female. This screen will include at least 10,000 mice per year. Aim 2: To characterize the gonadal phenotype of mice with sex-reversal. Morphologic, hormonal, histologic, and developmental analyses will be used to characterize the gonadal (testis and ovary) defects associated with sexreversal. Functional analyses will assess spermatogenesis in males or ovulation in females. These initial studies will be followed by more detailed characterizations of altered gene and protein expression using in situ hybridization, quantitative RT-PCR, immunohistology, and western blot studies. Microarray analyses will be used to identify altered genetic pathways, particularly during key stages of gonadal development. Aim 3: To perpetuate germline transmission of mutations associated with sex-reversal and gonadal dysgenesis and to map the genetic locus of the defect. Aim 4: To act as a national resource for mouse mutants by providing access to phenotypic screening analyses "online". In addition to listing identified phenotypes online, putative mutants that are heritable will be cryopreserved and made available to the scientific community directly from the Center and through arrangements with an established national distribution center. Gonadal development provides an excellent opportunity to identify genes involved in differential organogenesis. In addition to providing new information about basic mechanisms that regulate gonad development, these studies are also likely to enhance our understanding of gonadal dysgenesis and infertility in humans.

DESCRIPTION (provided by applicant): Over the last 8 years, we identified and characterized patients with naturally occurring mutations in the orphan nuclear receptors, DAX1 and SF1, and demonstrated that DAX1 acts in part by inhibiting SF1- mediated transcription. Using targeted mutagenesis, we developed a murine Dax1 knockout (KO) model and identified roles for Dax1 in gonadal determination, testis development, and adult testis function. In this grant, we propose to extend these studies, which have provided unexpected insight into mechanisms of gonadal development. We propose 3 inter-related aims that focus on Dax1 structure and function as a transcriptional repressor, identify genetic pathways regulated by Dax1, and develop animal models to unravel the interplay of Dax1 with other genes involved in testis development and function. Specifically, in Aim 1 we will identify molecular partners that mediate DAX1 transcriptional repression. Naturally occurring DAX1 mutations will be identified and characterized to elucidate key structural domains required for DAX1 function in vivo. Candidate proteins identified in a yeast two-hybrid screen of an embryonic gonadal library will be further characterized. The goal of Aim 2 is to identify the genetic pathways regulated by Dax1 using microarray analyses of genes expressed in wild type versus Dax1-deficient embryonic gonads at 12 dpc, a timepoint when Dax1 expression diverges in males and females. Aim 3 is designed to explore the interaction of Dax1 with other genetic pathways in vivo. Using mice that lack Dax1, we will explore the gonadal phenotypes of mice with selective rescue of Dax1 in various cell types. In addition, Dax1-deficient mice will be crossed to other strains with alterations in genetic pathways proposed to intersect with Dax1 (e.g., Sf1, Sry, Ptc). Success in these aims should provide a critical link between DAX1 and transcriptional repression pathways that link a variety of other transcription factors involved in gonadal development.

Estrogen plays a critical role in female reproduction. Within the ovary, estrogen acts together with FSH and a variety of paracrine factors, such as IGF-1 and activin, to induce ovarian follicle maturation. Estrogen action is mediated through nuclear estrogen receptors (ER) alpha and beta, which act by altering gene expression and by modifying signaling pathways. ERalpha is expressed primarily in ovarian theca and stroma cells whereas ERbeta is predominant in granulosa cells. The estrogen receptor modulates gene transcription by two fundamentally different mechanisms: (1) via a classical estrogen response element (ERE)-mediated pathway and (2) via a non-classical or tethered pathway that involves ER interactions with other transcription factors present on target genes. We have created a novel knock-in mouse model in which two amino acids in the DNA-binding domain of ERalpha were mutated to Ala (ER[AA]), thereby selectively eliminating classical pathway signaling. Heterozygous females containing one mutant ER allele and one wild-type ER allele (ER AA/WT) are infertile-- they are anovulatory, exhibit increased follicular atresia, and have increased numbers of lipid-laden stromal cells. We hypothesize that the phenotypic features of this mouse model reflect the selective loss of gene expression via the classical ERE-mediated pathway. This unique genetic model will be crossed to ER knockout mice (ER -/-) to examine the distinct roles of the ERalpha classical and nonclassical pathways in ovarian function. Aim 1 will examine gene dosage effects of the ER AA mutation on follicular development and ovulation in the following six ERalpha genotypes: ER WT/WT, ER AA/WT, ER AA/AA, ER WT/-, ER AA/-, and ER -/-. Aim 2 will use microarrays in combination with analyses of genes known to regulate ovarian function to characterize genes regulated by the classical and nonclassical pathways. Aim 3 will examine the mechanism by which the ER AA mutant alters cellular function by using a combination of in vitro transfection studies and analyses of gene regulation in cells derived from the various ER genotypes. Aim 4 will examine how the ER AA mutation alters ER-mediated neuroendocrine signaling and gonadotropin stimulation of the ovary. Taken together, these proposed studies will use the ER AA genetic model to gain insight into how estrogen acts by classical and nonclassical pathways to regulate ovarian function.

DESCRIPTION (provided by applicant): Over the last 8 years, we identified and characterized patients with naturally occurring mutations in the orphan nuclear receptors, DAX1 and SF1, and demonstrated that DAX1 acts in part by inhibiting SF1- mediated transcription. Using targeted mutagenesis, we developed a murine Dax1 knockout (KO) model and identified roles for Dax1 in gonadal determination, testis development, and adult testis function. In this grant, we propose to extend these studies, which have provided unexpected insight into mechanisms of gonadal development. We propose 3 inter-related aims that focus on Dax1 structure and function as a transcriptional repressor, identify genetic pathways regulated by Dax1, and develop animal models to unravel the interplay of Dax1 with other genes involved in testis development and function. Specifically, in Aim 1 we will identify molecular partners that mediate DAX1 transcriptional repression. Naturally occurring DAX1 mutations will be identified and characterized to elucidate key structural domains required for DAX1 function in vivo. Candidate proteins identified in a yeast two-hybrid screen of an embryonic gonadal library will be further characterized. The goal of Aim 2 is to identify the genetic pathways regulated by Dax1 using microarray analyses of genes expressed in wild type versus Dax1-deficient embryonic gonads at 12 dpc, a timepoint when Dax1 expression diverges in males and females. Aim 3 is designed to explore the interaction of Dax1 with other genetic pathways in vivo. Using mice that lack Dax1, we will explore the gonadal phenotypes of mice with selective rescue of Dax1 in various cell types. In addition, Dax1-deficient mice will be crossed to other strains with alterations in genetic pathways proposed to intersect with Dax1 (e.g., Sf1, Sry, Ptc). Success in these aims should provide a critical link between DAX1 and transcriptional repression pathways that link a variety of other transcription factors involved in gonadal development.

EXCEED THE SPACE PROVIDED. This is a competing renewal grant proposal for the General ClinicalResearch Center (GCRC) at Northwestern University and Northwestern Memorial Hospital. The GCRC has been continuouslyfunded since 1961 and is a six bed discrete unit. Lewis L. Landsberg, MD, Dean of Northwestern University Medical School, is the new Principal Investigator. William L. Lowe, Jr., MD is the newly appointed Program Director. Gerhard P. Baumann, MD continues as Associate Program Director responsible for the Core Laboratory, while Ram Yogev, MD and Boyd Metzger, MD continue as Associate Program Directors, responsible for Pediatrics and Training respectively. Over the past few years, research activities at Northwestern University Medical School have increased substantiallyboth in the clinical and the basic sciences. Successful recruitments of research-oriented Chairs of Pediatrics, Neurology, Neurological Surgery, Surgery, Molecular Pharmacology, and Physical Therapy contribute to an environment that fosters the current and future increased utilization of the GCRC. This renewal application contains 52 protocols from 37 different principal investigators representing 7 Departments (Medicine, Pediatrics, Surgery, Neurological Surgery, Obstetrics/Gynecology, Neurology, and Preventive Medicine), and 9 divisions within the Department of Medicine. The renewal also includes a continuation of the satellite inpatient and outpatient CRC facilities at Children's Memorial Hospital. The breadth of science is demonstrated by the protocols to be presented at the Site Visit. These include studies of the genetics and phenotype of gestational diabetes mellitus, use of hematopoietic stem cell transplantation for the treatment of autoimmune diseases, association of blood factor levels and genetic variations of genes encoding blood factors with peripheral arterial disease outcome, new innovations in P-cell transplantation for the treatment of type 1 diabetes mellitus, racial differences in the metabolism of sodium and potassium, role of neuromuscular and mechanical factors in knee osteoarthritis progression, the mechanismsof underlying age-related changes in sleep and circadian rhythmsand the effectiveness of behavioral interventions to improve sleep anddaytime neuropsychological

[unreadable] DESCRIPTION (provided by applicant): This application requests support to establish a BIRCWH Program at Northwestern University titled, Career Development in Women's Health (CDWH). This program will be used to develop a group of independent, tenure track scientists with backgrounds in clinical medicine or basic science disciplines whose research will address high priority areas relevant to women's health. We have identified five focus areas that have been historically strong within Northwestern and that are fundamental to the understanding and treatment of women's health and disease - differences in cardiovascular disease risk; ovarian biology; obstetrical and gynecological disorders; sex differences in sleep and rheumatology and osteoporosis. In order to develop expertise outside the Ob/Gyn specialty, faculty members who have interdisciplinary training in basic reproductive science and gender-specific disease research must be cultivated. Northwestern has a long- standing and rich tradition of interdisciplinary excellence in the reproductive sciences and in disorders that affect women, providing a strong foundation upon which the CDWH Program will be developed. In achieving our goal, the Career Development in Women's Health Program will create a focused curriculum, provide strong mentorship and engaged faculty development activities. The junior faculty trained through this program will be in the vanguard of women's health research scholarship and it is anticipated will expand the training to an ever widening group of clinicians and investigators. The program at Northwestern University is robust, timely and rigorous. Three key ingredients drive this application and will underlie the success of the candidates: high quality research programs, high expectations, and a robust training environment. The CDWH program at Northwestern University is dedicated to these criteria and to the next generation of clinical investigators and translational scientists who will improve health and allay disease in women and men. [unreadable] [unreadable] [unreadable] [unreadable]