Debra J. Wolgemuth - US grants

This research proposal will extend our recent studies elucidating the role of a new, testis-specific, homeo box-containing gene in the process of spermatogenesis and ongoing investigations examining DNA modifications. A cDNA containing sequences homologous to the homeo box region of genes important in the development of organisms as diverse as Drosophila and humans has been identified in a cDNA library prepared from mature mouse testis. This new homeo box-containing gene will be characterized at the molecular level by (a) obtaining a full-length cDNA for the homeo box-containing transcript and characterizing the clone by DNA sequence analysis; (b) isolating the gene; and (c) mapping to the appropriate chromosome. Examination of its tissue-, cellular-, and developmental stage-specific expression will be undertaken by Northern blot hybridization, nuclear run-off transcription, and in situ hybridization. Determination of function will ask if homeo-box containing transcripts are translated, and if so, when during the process of differentiation of the germ cells. Polysomes will be prepared from germ cells and tissues and assayed for the presence of homeo box-containing transcripts. Characterization of the size and cellular distribution of the protein products by cell-free translation of the homeo box-containing transcripts and by production of an antibody to amino acid sequences of the homeo box-containing polypeptide will be carried out. A study of the effect on germ cell development of interfering with the expression of the homeo box-containing gene, specifically within the germ line, will be accomplished by producing transgenic mice containing exogenous DNA composed of a construct of a testis cell specific promoter(s) and a portion of the homeo box-containing cDNA clone in an anti-sense orientation. Current studies determining the role of the pattern of methylation and DNase I hypersensitivity of single or low-copy genes during spermatogenesis and the regulation of their expression will be continued by examining specific sites of methylation, especially those at the 5' end of the genes, in premeiotic, meiotic, and post-meiotic cells.

Several mouse genes containing the homeobox domain have been shown to exhibit precisely regulated patterns of expression in the embryo and in certain tissues of the adult, suggesting that these genes may be important in development and differentiation in mammals. We have recently demonstrated that abnormal development of the gut and enteric nervous system, resulting in a condition resembling congenital megacolon, is associated with overexpression of the homeobox-containing gene Hox-1.4 in transgenic mice. Preliminary morphological analysis indicates that the mice are hypoganglionic in the distal region of the colon and that the affected region exhibits other structural alterations involving abnormal innervation. We want to determine the cellular and molecular mechanisms responsible for this mutant phenotype in order to elucidate the potential functions of homeobox-containing genes in development of the mammalian gut. We will begin by comparing the morphology of neural elements, smooth muscle, and extracellular matrix of the normal and abnormal segments of the colon of three lines of the transgenic mice which exhibit varying degrees of severity of the megacolon phenotype. Both adults and fetuses will be examined using immunocytochemistry and electron microscopy. The results will be compared to the observations made on mice that are homozygous for the lethal spotted (ls) mutation and those heterozygous for Dominant megacolon (Dom). To begin to identify the cells responsible for the abnormal phenotype, we will determine if normal neural crest-derived cells can colonize the presumptive abnormal gut of the transgenic mice and can differentiate into neurons appropriate for the enteric nervous system in vitro, or conversely, to determine if transgenic crest-derived cells can colonize a segment of bowel from control animals. Finally, we will address the functional redundancy of murine Hox genes, particularly with respect to gut development, by producing transgenic mice carrying multiple copies of the Hox-2 paralogue of Hox-1.4, namely Hox-2.6, under the Hox-1.4 promoter. These studies will provide new insight into the regulation of expression and function of homeobox-containing genes during mammalian development, their potential role in regulating the complex pathways responsible for proper differentiation and innervation of the mammalian gut, and their potential role in the etiology of congenital megacolon.

We have used the homeobox domain, first identified in Drosophila development-regulating genes, to identify genes in the mammalian genome that may be involved in development and differentiation. This proposal will investigate the regulation of expression and function of several members of the Hox-A cluster of homeobox genes and test the hypothesis that these genes function in the development of the male reproductive system and in gametogenesis, using mouse as a model system. We have focused most of our previous efforts on the gene Hoxa-4 because of its abundant and developmentally restricted expression in the male germ line in the adult animal. In the present proposal, we will continue certain novel aspects of the regulation of the expression and function of this gene in the testis and assess the effects of a complete null mutation on gametogenesis. We will also extend our studies to include experiments analyzing the expression and function of members of the Abdominal B class of Hox=A cluster genes, the mutation of one of which (Hoxa-11) has resulted in both male and female sterility. Specifically, we will: [1] Determine the role of Hoxa-4 in testicular function by i) elucidating the molecular basis of a novel Hoxa-4 transcript that is found in mice in which the endogenous Hoxa-4 gene has been mutated by the insertion of a Neo cassette; and ii) generating mice that will completely lack the Hoxa-4 gene, by deletion of the endogenous gene via targeted recombination in ES cells, and examining the effect on gametogenesis and reproductive function; [2] Delineate the regulatory elements specifically responsible for the expression of Hoxa-4 in the germ line, as compared to its embryonic expression, using reporter constructs in transgenic mice; [3] Determine the role of another member of the Hox-A cluster, Hoxa-A cluster, Hoxa-11, in male reproductive system development and function by examining several aspects of the male sterile phenotype that results from targeted mutagenesis of the Hoxa-11 gene; (4) Examine the expression of two other Abd-B class Hox genes which are adjacent to Hoxa-11 in the cluster, namely Hoxa-9 and Hoxa-10, with particular focus on the reproductive system in the embryo and adult. These experiments will include a detailed molecular analysis of their complex transcripts; and [5] Determine the role of Hoxa-9 and Hoxa-10 during embryonic and adult development by generating mutations in these genes by homologous recombination in ES cells. These studies will provide new insight into the function of homeobox-containing genes during mammalian development, and in particular, their potential role in regulating the complex differentiation pathways during development and in the function of the reproductive system.

Our initial efforts to elucidate the genetic program controlling mitosis and meiosis in germ cells have focused on four classes of genes known to be involved with cell cycle control: the cyclins, the cyclin-dependent kinases (Cdks), and the mammalian homologues of the fission yeast phosphatase Cdc25 and tyrosine kinase Wee1. We have demonstrated remarkable cell-cycle specificity of expression of several of these genes and have established the existence of sex-specific differences in the lineage specificity of expression of several genes. We have furthermore recently identified a germ-line specific gene, Cyclin Al, a member of a class that has yet to be identified in simpler eukaryotes. These observations have underscored the fact that there exist unique regulatory features of meiosis that are not found in less complex eukaryotes nor in mitotic lineages, and which differ between the male and female germ cells. In this proposal, we will investigate the role of the cyclin A~s in particular, in meiosis. We will test the hypothesis that CycA1 functions during the first meiotic division of spermatocytes by examining the effects on the progression of meiosis in male mice carrying a null mutation in the Cyclin A1 gene produced by targeted mutagenesis and homologous recombination in ES cells and the generation of chimeric mice from the targeted cells. To examine the role of cyclin A2 during meiotic maturation of oocytes, we will interfere with the function of Cyclin A2 by injection of antisense oligonucleotides and possibly antibodies into germinal vesicle intact oocytes and monitoring the progression of meiosis. The potential catalytic partners of Cyclin A1 and Cyclin A2 in testicular and ovarian cells will be identified by two strategies. In the first, co-immunoprecipitation with antibodies to Cyclin A1 and cyclin A2 and to cyclin dependent kinases (Cdk~s) known to be expressed in these cells followed by immunoblot analysis will identify whether such potential interacting Cdk~s actually associate with CycA1 or A2 in vivo. Second, potential new catalytic partners will be identified by using the yeast 2-hybrid screen with CycA1 and CycA2 cDNAs as the baits to screen testicular and ovarian cDNA libraries, respectively. Finally, we will begin to explore the role of potential activators of the CycA/Cdk complexes, namely, the Cdc25 family of dual specificity phosphatases, during gametogenesis. These studies will provide insight as to the in vivo functions of the cyclin A~s and their interacting proteins in mammalian cells in general and in germ cells in particular.

Homeobox-containing genes of the Hox class have been implicated in patterning and specification during embryonic development and in the development and function of specific tissues and structures. We have performed preliminary in situ hybridization experiments that demonstrate the expression of the endogenous Hoxa4 gene in the skin of fetal and newborn mice, notably in hair follicles. We wish to pursue these various preliminary observations with the following series of pilot project aims: 1) we will examine the temporal, spatial, and cellular distribution of expression of Hoxa4 in the murine skin, with particular emphasis on hair follicles, during embryonic and post-natal development and in adult animals; 2) will initiate studies to identify regions of the Hoxa4 gene that regulate its expression in the skin using transgenic approaches; and 3) we will investigate the possible modulation of Hoxa4 expression by retinoic acid. This will be accomplished by treatment of in vitro cultures of dissected hair follicles with retinoic acid and monitoring of gene expression of the endogenous gene expression by in situ hybridization. The goal of these pilot studies will be to provide evidence for the hypothesis that Hoxa4 is involved in hair follicle development and function and to begin to elucidate how this gene is regulated during this process. As Hox genes have been implicated in both proliferative and differentiative events, understanding their function in normal skin will be important for elucidating their potential role in skin disorders and diseases.

The importance of dietary retinol (vitamin A) for normal spermatogenesis and other aspects of male fertility has been recognized for many years. Animals deprived of vitamin A in their diet exhibit a variety of abnormalities, including male sterility. The requirement for dietary retinol raise interesting question concerning the function of retinoids in the testis, with regard to both their targets which are required for spermatogenesis to occur as well as to their metabolism with various testicular compartments. The ability to mutate endogenous genes involved in the pathways by which vitamin A elicits its effects in the mouse model system has enhanced our understanding of the diverse functions of the retinoids and has opened the opportunity for understanding the molecular basis of their functions. Studies generating mutations in specific receptors for vitamin A metabolites have clearly shown a role for the retinoid receptor RAR-alpha and RXR-beta genes in spermatogenesis. To further our understanding of the role of vitamin A in controlling spermatogenesis, this project will 1) Characterize the phenotypic abnormalities resulting in male sterility in the recently generated strains of mice mutated in RAR-alpha and RXR-beta, with particular emphasis on the developmental etiology of the abnormalities in the testis; 2) Test the hypothesis that the effects of the RAR-alpha and RXR-beta mutations on spermatogenesis are essentially phenocopied by vitamin A- deficiency in mice by comparing the spermatogenic abnormalities in the mutant strain mice with mice which have been vitamin A-deficient from birth, and 3) Explore the role of the RAR-alpha gene in the progression of spermatogenesis, specifically in germ cells. The hypothesis to be tested is that RAR-alpha has distinct functions in the germ line versus somatic cells. Furthermore, it is hypothesized that within the germ line lineage, RAR-alpha may have different functions at different stages of the developmental pathway. To distinguish these functions, it will be necessary to interfere with the function of the RAR-alpha gene selectively in one or the other cell types. To mutate its function in the germ line, conditional knockout mutations will be generated in RAR-alpha uniquely in germ cells at specific stages of development. This will be accomplished by mating mice carrying RAR-alpha flanked by loxP sites with mice carrying a transgene with Cre recombinase driven by regulatory elements driving expression specifically in spermatogonia, early pachytene spermatocytes, and early spermatids, respectively.

The central focus of this Program Project is to understand the mechanisms by which vitamin A acts to maintain optimal growth and health of the organism. For vitamin A to act in maintaining growth and health, it must be obtained from the diet; undergo transport, storage and mobilization processes; and be metabolized to active forms. Finally, its active forms must interact with transcription factors and the transcription machinery to regulate gene expression. Diets deficient in vitamin A result in characteristic abnormal development and disease in virtually all animals, including humans. However, the molecular mechanisms of action of vitamin A following its provision in the diet are not well understood. To elucidate the role of vitamin A in maintaining health, it is necessary to integrate an understanding of dietary vitamin A intake, vitamin A metabolism and the molecular events important to regulating responsive genes. The five individuals research projects which compose the Program Project will explore in the mouse model different aspects of vitamin A transport, storage and metabolism, the factors and processes which influence vitamin A uptake by cells and tissues or the molecular processes through which vitamin A acts to regulate gene expression and how intake of different levels of vitamin A from the diet influences each of these. Together, the five projects constitute a research program which has a major objective delineating a comprehensive and unified view of vitamin A physiology and vitamin A actions. One of the themes for this Program Project is exploring tissue-specific differences both in vitamin A delivery and processing and in the mechanisms through which vitamin A acts to regulate cellular homeostasis. A second them is understanding the role of vitamin A and its metabolites in the regulation of gene expression, again, in a tissue-specific manner. A third common theme for the Program is to ask how the modulation of dietary intake of vitamin A through purified diets will affect various aspects of vitamin A physiology and actions in the mouse model. Each project employs induced mutant, both transgenic and knockout, mice in its investigations: thus, the use of genetic approaches in mouse models is a fourth common and central theme for this Program. This Program is unique in the breadth of scope in which the function of dietary retinol will be addressed--from the uptake and metabolism of a micronutrient to its function at the level of specific target genes.

Our initial efforts to elucidate the genetic program controlling mitosis and meiosis in germ cells have focused on four classes of genes known to be involved with cell cycle control: the cyclins, the cyclin-dependent kinases (Cdks), and the mammalian homologues of the fission yeast phosphatase Cdc25 and tyrosine kinase Wee1. We have demonstrated remarkable cell-cycle specificity of expression of several of these genes and have established the existence of sex-specific differences in the lineage specificity of expression of several genes. We have furthermore recently identified a germ-line specific gene, Cyclin Al, a member of a class that has yet to be identified in simpler eukaryotes. These observations have underscored the fact that there exist unique regulatory features of meiosis that are not found in less complex eukaryotes nor in mitotic lineages, and which differ between the male and female germ cells. In this proposal, we will investigate the role of the cyclin A~s in particular, in meiosis. We will test the hypothesis that CycA1 functions during the first meiotic division of spermatocytes by examining the effects on the progression of meiosis in male mice carrying a null mutation in the Cyclin A1 gene produced by targeted mutagenesis and homologous recombination in ES cells and the generation of chimeric mice from the targeted cells. To examine the role of cyclin A2 during meiotic maturation of oocytes, we will interfere with the function of Cyclin A2 by injection of antisense oligonucleotides and possibly antibodies into germinal vesicle intact oocytes and monitoring the progression of meiosis. The potential catalytic partners of Cyclin A1 and Cyclin A2 in testicular and ovarian cells will be identified by two strategies. In the first, co-immunoprecipitation with antibodies to Cyclin A1 and cyclin A2 and to cyclin dependent kinases (Cdk~s) known to be expressed in these cells followed by immunoblot analysis will identify whether such potential interacting Cdk~s actually associate with CycA1 or A2 in vivo. Second, potential new catalytic partners will be identified by using the yeast 2-hybrid screen with CycA1 and CycA2 cDNAs as the baits to screen testicular and ovarian cDNA libraries, respectively. Finally, we will begin to explore the role of potential activators of the CycA/Cdk complexes, namely, the Cdc25 family of dual specificity phosphatases, during gametogenesis. These studies will provide insight as to the in vivo functions of the cyclin A~s and their interacting proteins in mammalian cells in general and in germ cells in particular.

9910500
Wolgemuth

This award supports a 2-year collaborative research project between Dr. Debra J. Wolgemuth, Columbia University and Dr. Kunsoo Rhee, Seol National University, Korea. The proposed collaboration aims to explore the function of a human NIMA-like kinase, Nek2 during development of germ cells and other cell types. The long-term goal of the research effort is to elucidate the genetic program controlling the mitotic and meiotic divisions of the germ line cells, with particular consideration of the evolutionary conservation of the regulation of these processes.

This collaborative proposal outlines genetic and biochemical experiments to begin to determine the role of Nek2, a putative mammalian homologue of aspergillus nimA. The results can provide insight into the function of the NIMA pathway in mammalian meiosis, providing new understanding of the control of meiosis in higher organisms as well as information on the evolutionary conservation of cell division processes.

This project will be performed in conjunction with Dr. Kunsoo Rhee at Korea under the U.S.-Korea Cooperative Science Program. The collaborative interaction will provide for sharing of expertise and resources. This project is relevant to the objectives of the U.S.-Korea Cooperative Science Program, which seeks to increase the level of cooperation between U.S. and Korean scientists and engineers through the exchange of scientific information, ideas, skills, and techniques and through collaboration on problems of mutual benefit. Korean participation is supported by the Korea Science and Engineering Foundation (KOSEF).

The WWMeB meeting will bring together biologists and software developers who are working to develop a World Wide Meiosis Database. Meiosis is the process by which egg and sperm cells are generated, the gametes. Two previous meetings have been held. At this meeting, the organizerrs will incorporate two new gender-specific databases that have been developed for describing events in the development of eggs and sperm, respectively. The meeting will produce a plan for coordinating international efforts in an area of biology where the general processes have been highly conserved.

DESCRIPTION (provided by applicant): We had previously identified a new mammalian A-type cyclin, cyclin A1, in addition to the already known cyclin A2. Both A-type cyclins are expressed in the mouse testis. Expression of the cyclin A1 gene, Ccna1, is germ cell-specific and restricted to late meiotic prophase spermatocytes while Ccna2 is expressed more broadly, in both somatic cells and in spermatogonia and pre-leptotene spermatocytes. Targeted mutagenesis of Ccna1 resulted in viable progeny but male sterility, while females were fully fertile. Spermatocytes in cyclin A1-deficient mice cannot enter into the first meiotic division, the activation of MPF is deficient and the cells undergo apoptosis. In contrast, cyclin A2-deficient mice are embryonic lethal. We now wish to understand the unique regulation and function of cyclin A1 in spermatocytes, to explore the function of cyclin A2 in the male germ line, and to dissect the regulatory pathways that are driven by cyclin A1 and its catalytic Cdk partners during male meiosis. We will also investigate the extraordinarily robust induction of apoptosis that occurs in the cyclin A1- and Cdk2-deficient models. We will identify regulatory elements and corresponding transcription factors required for the proper in vivo expression of Ccna1. This will involve DNase 1 hypersensitivity mapping, electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP), expression of reporter constructs in transgenic mice, sequence analysis, and a novel transcription factor screen to identify the regulatory network responsible for both activating and repressing its expression. We will also examine the changes in the events in the cell cycle, chromosome dynamics, and phosphorylation status of putative substrates that result in the arrest at diplotene and apoptosis in cyclin A1 and Cdk2-defident spermatocytes. This will also involve a novel phosphorylation screen to identify substrates of cyclin A1-complexes. Finally, we will determine the affect of targeted conditional mutagenesis of cyclin A2 in spermatogonia. These studies will provide new and novel insight into the function of the mammalian A-type cyclins, information that cannot be obtained from simpler model systems such as yeast, which lack this class of cyclins, or Drosophila, which contain only a single A-type cyclin. They will further enhance our understanding of the control of meiosis as it relates to male infertility and possible new targets for contraception.

DESCRIPTION (provided by applicant): We have identified a novel mammalian A-type cyclin, cyclin A1, which we have shown to be expressed at highest levels if not exclusively in the testis in mice and humans. Targeted mutagensis in mice revealed that during normal development, cyclin A1 functions uniquely in the progression of male germ cells into the first meiotic division. Human cyclin A1 is also highly expressed in leukemic cells from patients with acute myeloid leukemia. To test the hypothesis that the aberrant high levels of cyclin A1 were causal in the leukemic phenotype, i.e., acting as an oncogene, we generated transgenic mice in which cyclin A1 was expressed under the direction of the human cathepsin G (hCG) promoter in myeloid precursor cells. The transgenic animals exhibited abnormal myelopoiesis and developed acute myeloid leukemia. We have also recently observed high levels of cyclin Al expression in testicular tumors of the highly invasive embryonal carcinoma class but not in the more common and less invasive seminoma. We propose that cyclin A1 represents a novel target for drug intervention because of: (i) its causal role in myeloid leukemia, which we have demonstrated; (ii) its elevated expression in a restricted subset of invasive testicular tumors; and (iii) importantly, its remarkable tissue specificity of function (testis) during normal development. That is, if interference with cyclin A1 function in leukemic cells can revert their tumorigenic phenotype, the lack of its function would be predicted to not affect any other systemic organ other than the testis in men and to have no systemic effects in women. The aims of this present application are therefore: (1) To ask if inhibiting the expression of cyclin A1 in leukemic cells in vitro and in vivo, initially using antisense approaches, can revert the leukemic phenotype; and (2) to develop efficient in vitro kinase assays for screening pharmacological targets for inhibition specifically for cyclin A1/Cdk function. These experiments will initially involve identifying the preferred in viva Cdk partner for cyclin A1 in leukemic cells and then characterization of the cyclin A1/Cdk complex on selected substrates to optimize an in vitra kinase assay suitable for large-scale screening for inhibitors of the kinase assay.

DESCRIPTION (provided by applicant): We have identified a novel mammalian A-type cyclin, cyclin AI, that our targeted mutagenesis in mice revealed to be essential for the progression of male germ cells into meiosis. Human cyclin AI is also highly expressed in myeloid leukemia cell lines and in leukemic cells from patients with acute myeloid leukemias, in the promyelocytic form (APL) in particular. We have tested the hypothesis that the aberrant high levels of cyclin AI were causal in the leukemic phenotype, i.e., acting as an oncogene. Transgenic mice in which cyclin AI was expressed under the control of the human cathepsin G promoter in myeloid precursor cells were generated. They exhibited abnormal myelopoiesis and developed acute myeloid leukemia with low penetrance and long latency. Interestingly, in the transgenic mouse model and in human NB4 cells, the localization of cyclin A1 is predominantly cytoplasmic, distinct from its nuclear localization in germ cells. We wish to understand the cellular mechanisms in myelopoiesis that are altered in the presence of elevated levels of cyclin A1 that is now mostly cytoplasmic. The distinct cytoplasmic localization of cyclin A1 will be studied, testing the hypothesis that this property contributes to the tumorigenesis. We will also address the role of cyclin A1 during normal hematopoiesis by studying hematopoietic parameters in mice that are null for the cyclin A1 gene. The hypothesis that cyclin A1 will have distinct Cdk partners, other interacting partners, and substrates in normal versus leukemic cells will be tested using immunoprecipitation and a yeast 2-hybrid screen. As high levels of cyclin A1 protein have been shown to be characteristic of APL, we will ask whether manipulating the expression of cyclin A1 will affect the development of the leukemia. We will test this idea by performing genetic studies in which we will manipulate the expression of cyclin A1 in the fusion oncogene X-RARalpha transgenic animal models of APL. The question is whether these mice will be more resistant to the development of leukemia in the absence of cyclin AI. These studies will provide important insight into the etiology of myeloid leukemia, the role of cell cycle control in the oncogenic process, and the development of new and potentially highly tissue-specific target molecules for pharmacologic intervention.

[unreadable] DESCRIPTION (provided by applicant): The bromodomain is an evolutionarily conserved motif that binds acetylated lysines in histones and other proteins. The BET sub-class of mammalian bromodomain-containing proteins is unique in that its members contain two bromodomains (BD1 and BD2) and an extra terminal (ET) domain. There are four BET family members in mouse (and human) - Brd2, Brd3, Brd4, and Brdt - and they are expressed in a striking and dynamic pattern in the male germ line. We have generated a mutation in the mouse Brdt gene, designated Brdt BD1, that yields a truncated protein lacking the first of the two bromodomains. Homozygous Brdt BD1 progeny are viable but the males are sterile, producing fewer sperm that are morphologically abnormal. Aim 1 will test the hypotheses that i) Brdt functions as part of a transcription complex that regulates a set of genes whose expression is essential for spermatogenesis, and ii) that the BD1 of Brdt is required for this regulation. We will identify genes whose expression is changed in the absence of BD1 by microarray analysis and concomitantly, examine the chromatin modification status of the H1t promoter as a model for Brdt-complex binding regions. Aim 2 will test the alternative, but not mutually exclusive, hypothesis that Brdt-containing complexes function to mark regions of the spermatid genome for subsequent recognition by complexes that are involved in the unique changes in chromatin structure during spermiogenesis. ChIP with anti-Brdt antibodies followed by genome-wide sequencing using the Solexa/Illumina 1 G technology will be used. Aim 3 will test the hypotheses that first, the two bromodomains of Brdt have distinct functions in modulating transcription and/or chromatin re-modeling during spermiogenesis; second, that Brdt may function in several stages and processes of spermatogenesis, in addition to spermiogenesis; and third, that the Brdt BD1-mutant allele is a hypomorphic allele. We will generate a mutant allele producing Brdt protein containing BD1 but lacking BD2 (Aim 3a) and a mutant Brdt allele completely lacking functional protein (Aim 3b). We predict that the resulting phenotypes will overlap in part but will be distinct from the BD1-deficient mutant. Understanding the function of Brdt during spermatogenesis will provide a powerful developmental model system for elucidating the role of the BET genes during normal differentiation. Importantly, spermatogenesis is also a physiologically relevant system in which histone acetylation is clearly linked to chromatin remodeling. PUBLIC HEALTH RELEVANCE: Brdt is a member of a sub-family of bromodomain-containing proteins which have recently been shown to have essential functions in diverse basic cellular functions from DNA replication to transcription to chromatin remodeling. Our targeted mutational analysis has shown that deletion of the first of the two bromodomains in Brdt in the mouse model leads to male sterility, but the animals are otherwise viable and the females are fertile. Our studies will provide important insight into the potential mis- function of human BRDT in cases of unexplained (or idiopathic) infertility in men and may provide a new and novel target for male contraception. [unreadable] [unreadable] [unreadable]

The long-term goal of the Cancer Genetics and Epigenetics (CGE) Program is to pursue basic research on fundamental cellular processes relevant to cancer biology and to seek opportunities for translating the resulting information into clinical use. To this end, the following Specific Goals will be pursued: 1. Identify the molecular processes by which genomic instability is generated and contributes to oncogenesis; 2. Explore how epigenetic modifications of DMA and chromatin influence tumor initiation and progression; and 3. Elucidate the mechanisms underlying control of cell division and ascertain how these mechanisms are abrogated in cancer. The CGE Program is one of the two Basic Science Programs of the HICCC. In replacing the former Developmental Biology & Genetics Program it has been restructured to increase cancer relevance, and the heightened cancer focus of the new CGE Program is reflected by a 400% increase in NCI funding. The Program pursues its scientific goals by promoting interactions among CGE investigators and with other HICCC members, encouraging collaborative research projects and joint grant proposals, and providing a forum in which CGE investigators share their latest discoveries and consider the clinical value of their basic research findings. Potential clinical applications include identification and analysis of environmental toxins, modified therapeutic regimens to accommodate radiation bystander effects, development of biodosimetry, use of nanofluidic cassettes (biochips) in diagnostic/predictive laboratory assays (including monitoring therapeutic responses), high-throughput screening to identify small molecules that modulate malignant processes, and pre-clinical testing of these molecules for therapeutic effects. The CGE Program consists of 32 members (all full members of the HICCC) from eleven departments at Columbia University. The Program is supported by several collaborative efforts, including a recently renewed, five-year $5.2M (direct costs) program project grant from the NCI entitled Radiation Bystander Effects: Mechanisms (P.I., Tom Hei). For the last budget year of the grant (July 1, 2006 - June.30, 2007), the CGE Program received a total of $17.12M (direct costs) in cancer-relevant grant support, including $3.69M (direct costs) in NCI funding, $12.95M (direct costs) in other cancer-related peer-reviewed funding, and $0.48M (direct costs) in cancer-related non-peer-reviewed funding. The total number of cancer-related publications by the current Program members since the previous submission (i.e., 2003-present) was 330, with 17.0% inter-programmatic and 12.4% intra-programmatic publications.

DESCRIPTION (provided by applicant): Juvenile Myoclonic Epilepsy (JME) is among the most common types of syndromes of Idiopathic Generalized Epilepsy (IGE) and has a complex genetic inheritance. Although several putative JME susceptibility genes have been identified, the functions of these genes in the brain and their roles in the pathogenesis of seizures are unknown. Linkage and association analysis led us to the bromodomain-containing gene BRD2. BRD2 is a member of BET subfamily of double bromodomain-containing genes implicated in regulating gene expression. We have shown that BRD2 is expressed in developing and adult brain, and recently, that Brd2-null embryos die at mid-gestation with central nervous system malformations. Further, our preliminary data show that heterozygous Brd2 mice exhibit a reduction in the number of GABAergic interneurons in the frontal cortex and, strikingly, heterozygous Brd2 mice exhibit an enhanced sensitivity to chemically-induced seizures. Our working hypothesis is that mis-expression of BRD2 contributes to seizure susceptibility. We have also discovered that intron 2 of BRD2 contains a highly conserved, alternatively spliced exon, which introduces a premature termination codon and we have identified polymorphisms in the lengths of CA-repeats in intron 2 that are associated with JME. Variation in the length of CA-repeats has been shown to affect alternative splicing events in other experimental models. We now wish to test the hypothesis that the polymorphisms in intron 2, in particular variants in CA repeat-length that we have identified in patient DNA that associate with JME, affect splicing of the alternative exon and the levels of the corresponding transcripts, and hence, function of BRD2 products. In Aim 1, we will initially examine the effect of variable CA-repeat lengths on the relative levels of the two alternatively spliced BRD2 transcripts by transient expression of 'mini-gene'constructs containing [exon 2]-[intron 2]-[exon3] of human BRD2 with varying CA-repeat lengths and measuring the ratio of the resulting two transcripts. In Aim 2, we will begin to elucidate the functional significance of the alternatively spliced transcripts by determining whether the alternative transcripts are translated and if so, whether the proteins made affect homodimerization or sub-cellular localization. PUBLIC HEALTH RELEVANCE: Juvenile Myoclonic Epilepsy (JME) is among the most common types of syndromes of Idiopathic Generalized Epilepsy (IGE) and is characterized by adolescent onset and life-long affliction, requiring continual medication to suppress seizures. The proposed experiments will demonstrate whether the JME- associated variants in intron 2 of BRD2 play a direct role in the frequency of alternative splicing, and further, will provide insight into the possible physiological significance of the alternative transcripts, outcomes that are highly relevant to the stated goals of the RFA and the R03 funding mechanism.

DESCRIPTION (provided by applicant): The importance of dietary retinol (vitamin A) and retinoid signaling for normal development and differentiation in the testis has been recognized for many years. Signaling is effected in part through the retinoic acid receptors (RARs), of which there are three isoforms, ?, ß, and ?. We and others have shown the importance of signaling via the RAR? receptor in particular during spermatogenesis by gene targeting: mice deficient in RAR? (Rara-/-) are viable but the males are sterile, exhibiting defects in the seminiferous epithelium resembling those seen VAD diet. We have also shown that mice treated with an orally bioavailable pan-RAR antagonist become sterile, with similar testicular abnormalities. We have further shown that this induced sterility is reversible even with daily treatments for as long as 4 months upon cessation of drug treatment. Importantly, no detectable side effects were observed at these low doses and normal progeny were sired by fathers after restoration of fertility. This suggested that RAR-antagonists have potential as novel, non-steroidal compounds for male contraception. We propose to pursue this potential as outlined in the following specific aims. Specific Aim 1. Given the successful use of the pan-RAR antagonist compound 9 to induce male sterility and the recognition that it is RAR? that is critical for regulating spermatogenesis, we propose to test RAR?-selective antagonists that are immediately available and are being developed by our medicinal chemist collaborators in inhibiting spermatogenesis. Specific Aim 2. RAR? functions as a transcription factor, activating or repressing downstream target genes and antagonists function to block this transcriptional regulation. We therefore propose that such downstream target genes may themselves represent targets for interfering with spermatogenesis and inducing sterility, particularly if they are either testis-specific or function in a testisspecific manner. As spermiation appears to be a cellular process that is exquisitely sensitive to modulation of retinoid signaling, we will therefore focus on elucidating the mechanisms responsible for the improper anchorage of spermatids relative to the basal compartment, failure to translocate from basal compartment to the tubular lumen, and failure to disengage for spermiation, etc. at the cellular and molecular levels. These experiments will involve examining the expression of candidate genes known to be important in these processes and the identification of new genes by state-of-the-art RNA-Seq analysis. Specific Aim 3. To pursue RAR-antagonists ultimately as contraceptives in men, we propose to undertake a small scale trial in a non-human primate model (the common marmoset). The goal is to set the stage for future pre-clinical trials by examining the efficacy of antagonists with regard to induction of sterility and restoration of fertility. These studies will be done in collaboration with scientistsat the Southwest National Primate Research Center in San Antonio. Our prediction is that the pan-antagonist compound 9 will inhibit spermatogenesis in a reversible manner at doses that will not induce undesirable side effects.

? DESCRIPTION (provided by applicant): We are requesting continued funding of our T32 training grant DK007647 Graduate Training in Nutrition to support for our predoctoral training program in Nutritional and Metabolic Biology. The overall goal of our program is to train individuals to become leading investigators in the field of nutritional sciences who will contribut substantially to modern biomedical research. The program consists of a highly structured didactic component and a mentored research component. Support from this grant was the key ingredient that allowed this training program in nutrition to grow from 6 PhD students and 11 faculty in 1989 to its present steady-state size of ~30 PhD students and 31 faculty. For the previous grant period, we received support for 6 Ph.D. students per year (plus supplemental support for two underrepresented minority students). Because of the growth and achievements of our training program, we are requesting support for 7 PhD students per year in this renewal application. This training program is broadly focused on the nutritional and metabolic sciences. The required didactic training consists of graduate level basic science and nutrition courses and all PhD students participate in the Doctoral Seminar and Reviews in Nutrition course throughout their residence in the training program. All the training faculty have laboratory-based basic science research programs focused on nutrients or nutrition- related diseases such as diabetes, obesity, or cardiovascular disease. Although training focused on basic nutritional research, the program also provides a broad education in clinical and public health nutrition. The initial stages of the program provide comprehensive, structured training in modern biomedical research with a focus on nutrition and nutrition-related questions. Next, trainees are provided with rigorous mentored research training. This research training takes place in the research groups of one of the productive and well- funded independent scientists who comprise the training faculty. The data and narrative provided within this application demonstrate that we are very successfully training individuals committed to careers in research, teaching, and related professions with the fundamental knowledge, skills, and experience that are needed for developing successful, independent nutritional sciences research careers in the 21st Century.

DESCRIPTION (provided by applicant): This proposal requests support for a new, interdisciplinary post-doctoral training program at Columbia University Medical Center (CUMC). The program will be led by the Institute of Human Nutrition (IHN) and the Department of Epidemiology (EPI), and will be co-directed by Dr. Debra Wolgemuth (IHN) and Dr. Ezra Susser (EPI). The proposed mentors are well-funded and well-published investigators with considerable experience in training Masters and predoctoral students and postdoctoral fellows. A major theme of this training program is to train investigators with translational skills applicable to defining the effects of nutrients and nutrition early in the lifecycle on health outcomes later in life. The proposed program will capitalize on the unique strengths of these two partners within CUMC relevant to this theme: i) basic research in nutritional and metabolic biology in the IHN, and ii) population health science in EPI. The goal of the program is to provide postdoctoral training for research scientists with doctorate or equivalent degrees in a basic science (usually nutritional science), medicine, or in a population health science (usually epidemiology) through didactic and research experience in the complementary discipline. During the minimum two-year training period, the candidates will pursue course work and concomitant research projects in the complementary discipline under the direction of a mentor and co-mentor from each field. The trainees will have the opportunity to obtain an MS degree in the complementary discipline as part of their training. The trainees will retain their original disciplinary focus in basic science or population health science, but their training in the complementary discipline will facilitate engagement in interdisciplinary research which requires partners from both fields, and will make this interdisciplinary research much more effective. Thus trainees of the program will be uniquely prepared to enter translational research environments where bridging disciplines is an essential requirement for cutting edge research to enhance human health.

DESCRIPTION (provided by applicant): We had previously identified a new mammalian A-type cyclin, cyclin A1, in addition to the already known cyclin A2. We showed that cyclin A1 is testis-specific and by targeted mutagenesis, that it is essential for spermatogenesis--spermatocytes arrest at the diplotene to metaphase 1 transition. As knockouts of the more ubiquitously expressed cyclin A2 resulted in early embryonic lethality, we generated a conditional knockout model (A2CKO) and discovered unexpected and important functions of cyclin A2 in hematopoietic and embryonic stem cells. We have now also generated A2CKO models lacking cyclin A2 expression in undifferentiated type A spermatogonia, which results in a severe disruption of spermatogenesis and sterility. In Aim 1 of this renewal, we will determine the effects of loss of cyclin A2 function in different stages of spermatogonial development and in primordial germ cells (PGCs). We will determine which cell types require cyclin A2 for proliferation, where in the cell cycle the arrest occurs, and the fate of spermatogonia lacking cyclin A2 (Aim 1a). We will then extend these observation by ablating cyclin A2 in PGCs and in embryonic and post- natal gonocytes (Aim 1b) and in later stages of post-natal spermatogonial differentiation (Aim 1c), using our floxed Ccna2 mice and selected Cre deleter lines. We will begin to address the fundamental question of why higher organisms have evolved two A-type cyclins by asking if cyclin A1 and cyclin A2 are functionally redundant, using both genetic and biochemical approaches. We previously showed that loss of cyclin A1 results in a very specific and completely penetrant arrest of meiotic prophase spermatocytes just before the first meiotic division and have begun to identify key regulatory regions of the Ccna1 gene. We therefore propose to knock-in cyclin A2 coding sequences into the Ccna1 locus and examine the effects on meiotic progression, that is, can cyclin A2 function in place of cyclin A1 if expressed at the correct time? Finally, to further address the function of cyclin A1 in spermatocytes and to extend assessment of functional redundancy, in Aim 3 we will identify candidate testicular substrates of cyclin A1 and cyclin A2 complexed with their catalytic partners CDK1 and CDK2. This will be accomplished by fractionating lysates from spermatocytes and immature testes and subjecting them to phosphorylation by active cyclin A1- and A2-CDK complexes in which the ATP-binding pocket of the CDK is mutated so that it can readily accept modified ATP analogues, which endogenous kinases cannot (the Shokat strategy). These studies will provide novel insights into the function of the mammalian A-type cyclins, information that cannot be obtained from simpler model systems such as yeast, which lack this class of cyclins, or Drosophila, which contain only a single A-type cyclin. Our findings will enhance our understanding of the control of meiosis as it relates to male infertility, possibly suggest new targets for contraception, and provide critical insight into the unique properties of cell cycle regulation in stem cells, which i highly relevant in both normal development and in oncogenesis.

PROJECT SUMMARY Animals deprived of vitamin A in their diet (VAD) exhibit male sterility. All trans retinoic acid (ATRA), an active metabolite of vitamin A, functions by binding to the nuclear retinoic acid receptors (RARs), of which there are three isoforms ?, ?, and ?. We and others have shown the importance of ATRA signaling via the RAR? receptor in particular during spermatogenesis by gene targeting. Rara-/- mice are viable and healthy but the males are sterile, with defects in spermatogenesis similar to that seen in mice maintained on a VAD diet. Following up on observations of ?testicular toxicity? in rats treated with a pan-RAR antagonist, we have shown that indeed, spermatogenesis is inhibited in male mice after treatment with this orally bioavailable compound, and importantly, that the induced sterility is reversible. Our toxicology studies did not reveal any detectable side effects and normal progeny were sired after restoration of fertility. This suggests that antagonizing RAR signaling and RAR? in particular, has potential as a novel and non-steroidal hormone-based approach to male contraception. The objective in this application is to identify retinoids with that selectively antagonize RAR ? and to evaluate their therapeutic efficacy and reversibility. In Specific Aim 1, we propose to design, synthesize, and evaluate the in vitro activity, binding, and ADMET properties of RAR?-selective antagonists. Binding studies will utilize isothermal titration calorimetry, differential scanning fluorimetry, surface plasmon resonance, microscale thermophoresis, and X-ray crystallography; activity studies will use a luciferase reporter assay; and pharmacological profiles will be assessed. Specific Aim 2 will then assess the in vivo effects of the most promising RAR?-selective antagonists on spermatogenesis. After the initial morphological analysis, we will conduct fertility studies and develop dosing regimens for the most promising candidate molecules to determine the lowest doses and longest dosing periods at which efficient induction of sterility with complete reversibility could be achieved. Specific Aim 3 will then move to testing the lead candidate drug in a non-human primate, the common marmoset, model in a small scale trial to set the stage for future pre-clinical trials. Finally, we have observed in both our genetic and pharmacological models that one of the physiological processes in spermatogenesis that is extremely sensitive to altered retinoid signaling is spermiation. In Specific Aim 4, we therefore propose to determine both the quantitative global proteome and the quantitative phospho-proteome in particular during spermiation using dissected segments of testicular tubules and analysis by mass spectrometry.