Stephen A. Liebhaber - US grants

The ability to recognize that a genetic disease is caused by an abnormality in translational control must be based upon the definition of the mRNA structural features normally important in translation. Identification of such genetic defects may expand the understanding of pathogenesis in certain human genetic disorders such as the thalassemias and establish a scientific foundation for incorporating translational controls into the design of therapeutic approaches. The specific aim of the proposed program is to study the genetic control of translation by defining the structural features of the two highly homologous human Alpha-globin mRNAs which result in their different translational efficiencies. The translational efficiencies of each of the two Alpha-globin mRNA species will be established in vivo and in vitro by combining accurate measurements of Alpha 1- and Alpha 2-globin mRNA levels with genetic approaches which allow quantitation of Alpha 1- and Alpha 2-globin protein synthesis. The following potential differences between the two Alpha-globin mRNA will be investigated and related to their relative translational efficiencies: a) the subcellular distrubution (sequestration in mRNP particles, association with polysomes), b) translation initiation efficiencies, and c) post-transcriptional structural modifications (5 feet capping, 3 feet polyadenylation). The translation of the two Alpha-globin mRNAs will be compared in erythroid and non-erythroid systems in vitro to detect erythroid-specific translation factors. Such factors will be characterized by reciprocal mixing experiments between the two systems. These factors will be localized to the ribosomal and/or post-ribosomal fractions, and their final isolation will be approached by RNA affinity chromatography. The 3 feet nontranslated region of the Alpha-globin mRNA will be altered by introducing deletions of primary sequences and interruptions of secondary structure to directly test the effects of this region upon translation and to specifically characterize regulatory sequences.

The Oculocerebrorenal Syndrome of Lowe (OCRL; McKusick 309000) is an X- linked disorder characterized by mental retardation, congenital cataracts, and proximal renal tubule dysfunction. Since the last review of this application, the laboratory isolated a candidate gene (OCRL-1) for OCRL through positional cloning and found strong amino acid sequence homology to an enzyme HUMINP5P, one of at least three inositol polyphosphate-5-phosphatases (InP5P) known to exist in various human tissues. The homology suggests but does not prove that the OCRL-1 protein may be another inositol polyphosphate-5-phosphatase. This application is for continuation of genetic and molecular studies in Lowe Syndrome to address the following questions in the next grant period: 1. Does OCRL-1 encode another InP5P or some other enzymatic activity? 2. Do OCRL patients have a defect in InP5P activity? 3. What can we learn from the mutations in OCRL-1 found in OCRL patients? Research Design and Methods: 1. Characterize the OCRL-1 mRNA reading frame by cloning and sequencing additional cDNA. 2. Identify the enzymatic activity encoded by OCRL-1. Express the OCRL-1 protein to obtain highly purified OCRL-1 protein for enzymatic assay and as antigen for raising antisera. Identify the protein in normal cells by Western blotting and immunoprecipitation. 3. Investigate cells and tissues from normals and OCRL patients to determine whether probands have defects in the OCRL-1 gene and its product. 4. Determine the genomic structure of OCRL-1. 5. Identify mutations in OCRL patients. Examine OCRL patients' DNA by Southern blot for major alterations. Use polymerase chain reaction amplification of cDNA (RT-PCR) for single-stranded conformational polymorphism (SSCP) analysis in OCRL patients and normals and sequence the fragments showing SSCP alterations to search for mutations. Develop primers for all exons and intron/exon boundaries in genomic DNA. If OCRL is indeed a defect in inositol polyphosphate metabolism it represents the first human disease due to an inborn error of inositol metabolism, an important but very complex and incompletely understood area of human metabolism. Elucidation of the biochemical and molecular defects will provide important insights into three disease processes in man: mental retardation, cataract formation, and renal tubular dysfunction.

Sickle cell anemia can be a devastating disease. Amelioration its severity have been approached at many levels. The most fundamental approach involves altering the pattern of globin gene expression and hemoglobin synthesis. Such an effort necessitates a full understanding of the genetic determinants of erythroid differentiation and function. It is evident that this is a complex, multifaceted process involving transcriptional and post-transcriptional controls of a large number of genes. Our particular interest is role of mRNA stability in this process. Although globin gene expression is initiated by transcriptional activation, the accumulation of globin mRNAs to greater than 95% of total cellular mRNA is dependent on their unusual stabilities. Mutations in human alpha-globin mRNA that destroy its stability result in loss of gene expression and consequent disease (alpha-thalassemia). Studies which have been completed during the first three years of the current renewal of the Comprehensive Sickle Cell Center Grant have demonstrated that stability of human alpha-globin mRNA depends on a defined cis determinant in the 3 'UTR. The stability determinant functions via assembly of a multicomponent RNA-protein (RNP) complex. One of the proteins in the alpha-complex is a 39 kD cytoplasmic RNA binding protein with a polyC binding-specificity. This protein is necessary, but not sufficient, for alpha-complex formation. In this competitive renewal we propose to continue our studies of globin mRNA stability with three Specific Aims: (1) Identify the full complement of proteins that compose the human alpha-complex. Studies the include biochemical purification, genetic screens, and RNA/protein crosslinking studies. (2) Characterize the higher-order structure of the alpha-complex. This will include determination of its overall mass, the stoichiometry of its protein components, and the nature of the RNA-protein interactions. (3) Characterize the rate limiting steps in alpha-globin mRNA turnover. Analyses will be carried out on the degradation patterns of normal alpha- globin mRNA and mutant alpha-globin mRNAs which cannot form the stability complex. The goal of this project is to extend the understanding of globin gene expression and generate new tools for its therapeutic modulation.

Gene expression is a logical and increasingly feasible target for therapeutic efforts. The identification of novel molecular targets for new classes of drugs, specifically in the area of neucleic acid based therapeutics, will be based on advances in the detailed understanding of genetic mechanisms and controls. Contributions of posttranscriptional controls to gene expression are increasingly evident. MRNA stability is of particular importance for mRNAs with unusually short (oncogenes, cell growth factors) or long (structural proteins) half-lives. Mechanisms by which mRNA stability is controlled have in common a central role for interaction of sequences in the mRNA 3'-untranslated region (UTR) with cytosolic protein factors to form sequence-specific ribonucleoprotein (RNP) complexes. Alteration in the structure and/or assembly of these complexes may allow a predictable and potentially therapeutic increase or decrease of gene expression. Such approaches will utilize novel molecular targets and mechanisms of action for nucleic acid based therapies. Globin mRNA, a well documented protype of a long-lived mRNA and one of the few mRNAs for which the determinants of RNA stability have been studied in detail, will serve as the model system for this proposal. Alpha-globin mRNA stability is dependent on the interaction of defined sequences in the 3'-UTR with a set of cytosolic RNA binding proteins. We hypothesize that inhibition of this mRNA/protein interaction will result in selective mRNA destabilization. This hypothesis will be tested in an integrated set of in vitro, cell based, and transgenic studies to establish the efficacy, mechanisms, and feasability of this approach. A series of decoy nucleic acids will be tested in vitro and in intact cells in culture for their effects on 3'-UTR RNP complex formation and for corresponding effects on mRNA stability. Potential decoys will include polyC homopolymers, multimers of the alpha-globin stability motif (CCUCC), and segments of the alpha-globin mRNA 3'UTR. Variables of chemistry, structure, and delivery systems will be assessed for their effects on efficiency and selectivity of decoy action. Mechanisms and specificity of observed activities will be investigated. Transgenic mouse lines will be established expressing optimized decoy RNAs under the control of conditional promoters to test their efficacy and characterize their function(s) in vivo. This approach, once validated, optimized, and mechanistically defined in the model system, can be adapted to target a broad spectrum of gene systems including those which control cell transformation and oncogenesis.

DESCRIPTION: (Investigator's abstract) Normal levels and developmental control of globin synthesis in fetal and adult erythrocytes is critically dependent on the unusual stability of the encoding mRNAs. Our prior studies establish a link between stabilization of human (h)a-globin mRNA and formation of a sequence specific RNA-protein (RNP) complex ('a-Complex') at its 3' untranslated region. We hypothesize that this a-Complex, composed of a defined polypyrimidine tract bound by a sequence-specific RNA binding protein, aCP, stabilizes a-globin mRNA by controlling one or more rate-limiting steps in mRNA decay. These observations will be extended in this proposal by focusing on three Specific Aims. In Aim I we focus on characterization of structural determinants and mechanism(s) of ha-globin mRNA stabilization. Using a set of Tet-transactivator cell lines we will characterize and compare a-globin mRNA decay pathways in erythroid and nonerythroid environments, identify and characterize influences of 5'UTR and coding sequences on a-Complex function, and determine whether aCP is fully sufficient to mediate ha-globin mRNA stabilization. In Aim II we will characterize protein-protein interactions involved in a-Complex action. Interactions of aCP with candidate partner proteins will be assessed and their functional importance tested using an in vitro mRNA decay assay. In Aim m we will characterize the corresponding roles of nuclear and cytoplasmic aCP in assembly and function of the a-Complex. We will identify elements in aCP that dictate its subcellular localization, determine whether aCP associates with ha-globin mRNA in the nucleus, determine the site of cytoplasmic aCP action on ha-globin mRNA, and design dominant-negative mutations of aCP to probe its distinct nuclear and cytoplasmic functions. The characterization of the determinants and mechanisms involved in stabilization of ha-globin mRNA outlined in this proposal is central to a understanding of globin gene expression in health and disease and to the design of therapeutic approaches to a broad spectrum of hereditary anemias.

High-level stability of globin mRNAs is a major determinant of hemoglobin synthesis and erythrocyte function. The basis for selective stabilization of globin mRNAs during erythroid differentiation remains poorly understood. Our laboratory is using human ot-globin mRNA as a model for the study of this problem. Genetic, biochemical, and in vivo expression studies carried out over the present funding period point to a central role for a sequence-specific 3'UTR RNA-protein (RNP) complex ('c_ complex') in stabilizing ct- globin mRNA. Inactivation of the c_-complex by mutation of the C-rich binding motif or by blocking the binding of the ctCP protein results in an incremental loss of ot-globin mRNA stability. This loss of stability can be fully restored by artificially tethering ctCP to the 3'UTR. c_CPs are broadly distributed in tissues, suggesting that an erythroid- restricted role of the a-complex is dictated by specific modifications to c_CP or to interacting RNP components. The major ctCP isoforms are differentially localized in the nucleus and cytoplasm. Evidence suggests that the cytoplasmic role of etCPs in ct-globin mRNA stabilization is complemented by separate nuclear function(s) involved in enhancement of c_-globin mRNA processing. The pathways involved in selective stabilization of human c_-globin mRNA and the interrelationships between nuclear and cytoplasmic functions of aCPs in c_-globin gene expression will be explored in the proposed studies. Aim I. Identify interactions at the a complex that mediate ct-globin mRNA stabilization. Aim II. Define the mechanism(s) of _-globin mRNA stabilization and how a-globin mRNA evades decay in erythroid cells. Aim III. Determine how ctCPs enhance nuclear processing of ct-globin transcripts and how these nuclear events integrate with aCP-mediated cytoplasmic controls. These studies will extend our prior work on ct-globin gene expression, define novel pathways of mRNA decay, and establish a paradigm for coordinated nuclear and cytoplasmic post-transcriptional controls in erythroid gene expression.

DESCRIPTION (provided by applicant): The earliest known step in mammalian development is the formation of the trophoblast from the totipotential embryonic stem cell. The subsequent differentiation of the trophoblast stem cells (TS) to the placental syncytiotrophoblast (SynT) establishes the epithelial interface between fetal and maternal circulatory systems, mediating essential nutrient exchange and oxygen transport. The SynT also serves as the primary site of fetal hormone production that conditions maternal and fetal systems to sustain a successful gestation. The major hormones synthesized in the SynT are chorionic somatomammotropin (hCS) and growth hormone-variant (hGH-V). Both hormones are encoded in the multi-gene human growth hormone (hGH) gene cluster that also encompasses the pituitary growth hormone gene, hGH-N. We hypothesize that the selective and robust activation of the placental hCS and hGH-V genes during differentiation of TS to SynT reflects a defined progression of epigenetic modifications and alterations in chromatin structures that distinguish this pathway from that involved in activation of hGH-N in pituitary somatotropes. We further hypothesize that this developmental progression reflects global epigenetic controls that are fundamental to SynT differentiation and distinguish this lineage from the TS-derived lineage that leads to the formation of invasive trophoblast cells. The selective activation of the placentally-expressed genes from the hGH cluster thus presents an optimal model for understanding mechanisms of gene activation that underlie TS cell differentiation and that define function(s) of SynT cells in the placenta. Our proposal will address five Specific Aims: I. Establish mouse TS lines that model activation of the hGH locus during SynT differentiation. II. Define the structural alterations at the hGH locus that coincide with SynT-lineage commitment and the subsequent transcriptional induction of the hCS/hGH-V genes. III. Establish mechanistic linkages between SynT differentiation and hGH locus activation. IV. Identify structural features of the hGH locus required for activation in SynT. V. Define higher-order conformations at the hGH chromatin locus in SynT necessary for placenta-specific gene activation. These studies should expand our understanding of placental gene expression and development and expand our insights into the corresponding defects that impact on maternal and fetal health during gestation.

DESCRIPTION (provided by applicant): A distal locus control region (LCR) regulates the human growth hormone (hGH) gene cluster. Four DNaseI hypersensitive sites, HSI, II, III, and V located between 14.5 to 32 kb 5' to the pituitary hGH-N gene promoter, mark the determinants of this hGH LCR. These determinants act in concert to establish robust and appropriately regulated expression of hGH-N in pituitary somatotropes. HSI and HSII, at -14.5 to -15.5, are specific to pituitary chromatin and constitute the primary determinants of hGH-N transgene expression in somatotropes. The core elements of HSI, comprising an array of binding sites for the pituitary-enriched transfactor Pit-1, integrate multiple sets of epigenetic regulatory modifications in the process of hGH-N activation. These HSI-dependent activities include: 1) establishing of a 32kb domain of core histone hyperacetylation encompassing the hGH LCR and extending to the hGH-N promoter, 2) generation of a robust PolII 'domain of transcription' within the LCR and adjacent CD79b gene that is necessary for full levels of hGH- N expression, and 3) organization of higher-order chromatin structure that juxtaposes the LCR/CD79b domain of transcription and the hGH-N promoter during the process of hGH-N transcriptional enhancement. Remarkably, the powerful chromatin-based activities of the Pit-1 array at HSI can be functionally distinguished from the activity of the Pit-1 array at the hGH-N promoter. The HSI Pit-1 specificity is mediated, in part, by its unique binding site sequence. HSI activity appears to act in synergy with the adjacent HSII. HSII may be responsible for augmenting HSI activity via extension of epigenetic modifications or by enhancing PolII occupancy and non-coding transcription within the LCR. The role of the LCR in the activation and enhancement of hGH-N expression may be followed by an equally important role in maintaining high levels of hGH-N expression throughout adult life. In this proposal, individual components of LCR action will be characterized using a combination of in vitro, cell-based, and mouse transgenic methodologies. The temporal order of LCR-dependent alterations at the hGH locus will be determined in a novel set of tissue culture lines that are arrested at defined stages of somatotrope differentiation. A naturally occurring mutation of the human Pit-1 protein that selectively blocks hGH-N expression will be used to further extend our understanding of the basis for the specificity of Pit-1 action at the hGH LCR. The Specific Aims of this proposal focus on these major aspects of hGH LCR function with the ultimate goal of extending our understanding of hGH-N activation and expression and in generalizing our findings to pathways and mechanisms of long-range transcriptional activation in the eukaryotic genome. These findings will eventually be correlated with an array of pathologic defects in hGH expression, both inherited and acquired, and with physiologic alterations in hGH expression that occur in response to environmental stress and ageing.

DESCRIPTION (provided by applicant): The long-term goal of this grant is to establish a comprehensive understanding of post-transcriptional controls critical to mammalian erythroid differentiation. Erythroid differentiation is marked by a set of dramatic morphologic and functional changes, much of this process occurring in a transcriptionally-silent environment. For this reason erythroid differentiation is heavily dependent on post-transcriptional controls. RNA binding proteins (RBPs), the major regulators of post-transcriptional controls, impact on transcript processing in the nucleus as well as the stability and function of the mRNA in the cytoplasm. RBPs vary widely in their structures, binding specificities, and cellular compartmentalization and can modulate RNA functions by direct actions and/or via the recruitment of effector complexes. Identification of RBPs, their mRNA targets, and corresponding post-transcriptional controls have advanced our understanding of numerous developmental processes and have revealed unanticipated pathophysiologic pathways. By defining post-transcriptional controls in erythroid differentiation, we will expand our understanding of inherited and acquired disorders of erythropoiesis and establish a template for similar investigations in other systems. To achieve this goal, we will carry out a novel and state-of-the-art transcriptome-wide analyses to reveal the full range of mRNA-protein (mRNP) interactions that accompany the dynamic process of erythroid terminal differentiation. This approach combines biochemical methodologies with innovative informatic pipelines specifically designed for comprehensive descriptions of complex RNA-protein interactions. This unbiased analysis will be combined with a series of targeted in-depth mechanistic studies that focus on two sets of mRNA/protein interactions that we have identified to play central roles in erythroid differentiation. The combined output of these complementing approaches will establish the unique network of post-transcriptional controls in this developmentally robust and clinically relevant model system. This proposal encompasses three Specific Aims. Aim 1. Characterize the roles of the polyC-binding proteins, ?1 and ?2, as post- transcriptional integrators of erythroid differentiation. Aim 2. Identify the critical role(s) of the polyA binding protein, PABPC, in defining and sustaining the erythroid transcriptome. Aim 3. Map the global structure of mRNA/protein interactions in erythroid cells and define the dynamic nature of these interactions in the differentiation process. Combining transcriptome-wide analyses with targeted mechanistic studies will generate a powerful access to the complex array of RNA/protein interactions that constitute critical determinants of erythroid differentiation. Success in these studies will fundamentally alter our understanding of this intensively studied pathway; serve as a prototype for investigations of post- transcriptional controls in other systems, and present novel targets fo future diagnostic and therapeutic innovations.

Abstract The divergence of the somatotrope and lactotrope lineages in the anterior pituitary constitutes a highly informative, medically relevant, and well-characterized model of mammalian cellular differentiation. Remarkably, the differentiation of both lineages is dependent on the activity of the same pituitary-specific transcription factor, Pit-1 (POU1-F1). Landmark genes defining each of the two lineages, such as Growth Hormone (GH) and Prolactin (Prl), are under direct control of Pit-1 dependent cis-regulatory elements. Loss of Pit-1 expression results in absence of both cell types from the anterior pituitary in mice and humans with consequent combined hormone deficiency syndromes. Despite the central importance of Pit-1 to development and function of the anterior pituitary, the mechanisms and pathways that it activates to drive the differentiation and maintenance of the somatotrope and lactotrope lineages remain unclear. We hypothesize that Pit-1, acting in conjunction with differentially expressed transcription factors, controls the divergence of the somatotrope and lactotrope lineages by binding to lineage-defined cis-regulatory elements, mediating long- range transcriptional interactions, organizing nucleosomal architectures, and defining lineage-dependent three dimensional (3D) chromatin networks. In Aim 1, we test the hypothesis that Pit-1 occupancy at the major enhancer element within the human GH locus control region (HSI) activates a series of temporally-defined functions that are critical to the robust and selective activation of hGH-N transcription in the pituitary somatotrope and its reciprocal repression in the lactotrope lineage. In Aim 2 we test the hypothesis that the ability of Pit-1 to drive lineage divergence depends on its interactions with cooperating transcriptional factor(s). Candidate factors are identified by comparative transcriptome analyses of primary flow-sorted somatotropes and lactotropes and are validated by a set of compelling functional assays. In Aim 3 we test the hypothesis that distinct networks of 3D chromatin interactions are established throughout the somatotrope and lactotrope genomes to integrate and coordinate lineage-specific gene activation and repression programs and that these 3-D architectures are dependent on lineage-specific Pit-1 actions. All three Aims are based on analyses of primary cells isolated from the pituitaries of physiologically intact wild type or transgenic mouse lines. These studies will extend our understanding of pituitary function and will allow us to predict and define the basis for phenotypic variations in hormone expression, identify causative mutations in inherited and acquired endocrine disorders, and highlight targets for novel therapeutic interventions. Furthermore, this program will establish a paradigm of mammalian development in a landmark model and serve as a template for investigations of differentiation and genome regulation in a broad spectrum of experimental settings.

The Transgenic and Chimeric Mouse Core supports the overall mission of the Penn DRC to prevent, treat, and cure diabetes mellitus. The accelerating incidence of diabetes and metabolic disorders and the continuing high prevalence of endocrine disorders in the American population demands continued exploration of a broad array of corresponding mechanistic pathways, pathophysiologic sequelae, and potential therapeutic approaches. In many cases these investigations can be accomplished most efficiently using model systems established in intact animals. The use of mouse models in these pursuits is now well established for its power, feasibility, and enormous potential. The development of such models, by directed alterations of the mouse genome, while of high utility, remains a technically demanding and labor intensive component of an overall research effort in diabetes, obesity, and metabolic disorders. The Transgenic and Chimeric Mouse Core provides investigators of the Penn Diabetes Research Center (DRC) with the ability to carry out these studies in a cost effective and efficient manner. The TCMF applies state-of-the-art equipment and technology by a group of dedicated and highly skilled technical staff to this effort. The major services of the Core include generation of transgenic mice by pronuclear injection, creation of chimeric mice by blastocyst injections, assisted fertilization, cryopreservation, long-term cryostorage, and shipping of frozen embryos or sperm to other facilities. The Core uses multiple microinjection platforms, laser-assisted technologies, state-of-the-art cryopreservation of gametes and embryos, and in vitro fertilization based line re-derivation to facilitate these goals. The facility consists of a microinjection suite, an adjacent dedicated cage room and an off-site cryopreservation storage facility. All functions, from ordering services, to following work flow, to storing and sending out lines is now on-line and can be monitored in real-time. These efforts contribute substantially to the overall productivity of the members of the DRC and enhance the strength and relevance of their studies to intact mammalian systems. This maximizes the applicability of these studies to human disease and its therapeutics.

PROJECT SUMMARY ? Transgenic and Chimeric Mouse Facility The Transgenic and Chimeric Mouse Facility (TCMF) at the Abramson Cancer Center (ACC) has been continuously funded by the NCI CCSG since 1989 under the guidance of Dr. Stephen Liebhaber, Professor of Genetics and Medicine. Dr. Liebhaber is an experienced investigator with considerable expertise in technologies and experimental approaches that center on transgenic technologies, developmental biology, and mammalian gene targeting. An experienced technical team, led by Dr. Jean Richa, provides expertise in a full range of transgenic technologies, enabling the TCMF to regularly introduce new and improved services. Among the services provided by the Transgenic and Chimeric Mouse Facility are generation of genetically altered mice via direct genome editing (CRISPR/Cas9, and less frequently Zn finger and TALEN technologies), by DNA microinjections into fertilized oocyte to create transgenic lines, or by generation of chimeric mice via embryonic stem cell injection into blastocysts. The Transgenic and Chimeric Mouse Facility also carries out embryo re-derivation, embryo and sperm cryopreservation, in vitro fertilization (IVF), and centralized cryopreservation storage. The TCMF uses state-of-the-art laser conditioning of the zona to facilitate IVF and has intracytoplasmic sperm injection capability on-line to complement IVF services. Newly developed services during the current funding period include the major expansion of cryopreservation services with corresponding expansion of the cryopreservation facility, integration of CRISPR/Cas9 direct genome modifications with a newly established Perelman School of Medicine CRISPR core, and electroporation of DNA and RNA into embryos to increase throughput and decrease wait time for TCMF services. ACC members accounted for 29 of 71 investigators (41%) using the Shared Resource during the most recent reporting period (07/01/18- 06/30/19). Additional Institutional (non-CCSG) support is provided for equipment maintenance and facility infrastructure upgrades and maintenance. In one example of the supported high-impact research, the TCMF generated a series of mouse cell lines, which were used to demonstrate opposing effects of the LIN28B-IMP1 axis on post-transcriptional regulation of canonical WNT signaling, with implications for intestinal homeostasis, regeneration and tumorigenesis (Chatterji et al., Genes Dev, 2018). The TCMF provides cutting edge technologies to generate transgenic mice needed for rigorous cancer research studies conducted by ACC members.

Abstract The divergence of the somatotrope and lactotrope lineages in the anterior pituitary constitutes a highly informative, medically relevant, and well-characterized model of mammalian cellular differentiation. Remarkably, the differentiation of both lineages is dependent on the activity of the same pituitary-specific transcription factor, Pit-1 (POU1-F1). Landmark genes defining each of the two lineages, such as Growth Hormone (GH) and Prolactin (Prl), are under direct control of Pit-1 dependent cis-regulatory elements. Loss of Pit-1 expression results in absence of both cell types from the anterior pituitary in mice and humans with consequent combined hormone deficiency syndromes. Despite the central importance of Pit-1 to development and function of the anterior pituitary, the mechanisms and pathways that it activates to drive the differentiation and maintenance of the somatotrope and lactotrope lineages remain unclear. We hypothesize that Pit-1, acting in conjunction with differentially expressed transcription factors, controls the divergence of the somatotrope and lactotrope lineages by binding to lineage-defined cis-regulatory elements, mediating long- range transcriptional interactions, organizing nucleosomal architectures, and defining lineage-dependent three dimensional (3D) chromatin networks. In Aim 1, we test the hypothesis that Pit-1 occupancy at the major enhancer element within the human GH locus control region (HSI) activates a series of temporally-defined functions that are critical to the robust and selective activation of hGH-N transcription in the pituitary somatotrope and its reciprocal repression in the lactotrope lineage. In Aim 2 we test the hypothesis that the ability of Pit-1 to drive lineage divergence depends on its interactions with cooperating transcriptional factor(s). Candidate factors are identified by comparative transcriptome analyses of primary flow-sorted somatotropes and lactotropes and are validated by a set of compelling functional assays. In Aim 3 we test the hypothesis that distinct networks of 3D chromatin interactions are established throughout the somatotrope and lactotrope genomes to integrate and coordinate lineage-specific gene activation and repression programs and that these 3-D architectures are dependent on lineage-specific Pit-1 actions. All three Aims are based on analyses of primary cells isolated from the pituitaries of physiologically intact wild type or transgenic mouse lines. These studies will extend our understanding of pituitary function and will allow us to predict and define the basis for phenotypic variations in hormone expression, identify causative mutations in inherited and acquired endocrine disorders, and highlight targets for novel therapeutic interventions. Furthermore, this program will establish a paradigm of mammalian development in a landmark model and serve as a template for investigations of differentiation and genome regulation in a broad spectrum of experimental settings.

PROJECT SUMMARY (GENETICALLY MODIFIED MOUSE CORE) The significant incidence of digestive, liver and pancreatic diseases in the American population demands continued exploration of a broad array of corresponding mechanistic pathways, pathophysiologic sequelae, and potential therapeutic approaches. In many cases these investigations can only be accomplished, or can be accomplished most efficiently and relevantly, using model systems established in intact animals. The use of mouse models in these pursuits is now well established for their power, feasibility, flexibility, and enormous potential. Creation of such models, by targeted alterations of the mouse genome, is an essential component of an overall research effort in understanding normal functions and pathologic perturbations in the digestive tract, liver, and pancreas. The Genetically Modified Mouse Core (GMMC) provides investigators of the University of Pennsylvania Center for the Molecular Study of Digestive and Liver Diseases (CMSDLD) with the ability to carry out these technologically-demanding studies in a cost effective and efficient manner and enhance the rigor and relevance of these approaches. The GMMC has a dedicated and highly skilled staff that applies state-of-the-art equipment and techniques, and the facility consists of a microinjection suite, an adjacent dedicated cage room, and an off-site and highly secure cryopreservation storage facility. Major services available to CMSDLD investigators include the generation of transgenic mice by DNA pronuclear injection, creation of chimeric mice by ES cell injection into blastocysts, and direct genome mutation, editing, and modification via the use of targeted endonuclease (TALEN and Crispr-CAS technologies); these are complemented by the genotyping of founder mice, assisted (in vitro) fertilization, cryopreservation, long-term cryostorage, and shipping of frozen embryos or sperm to/from other facilities. To provide these services, the GMMC utilizes multiple microinjection platforms, laser-assisted technologies, state-of-the-art cryopreservation approaches, and highly efficient line re-derivation pipelines. All functions, from ordering services, to following workflow, to storing and sending out lines, are on-line and can be monitored in real-time. These efforts by the GMMC contribute substantially to the overall productivity of CMSDLD investigators and enhance the rigor and relevance of their studies to the mechanisms of digestive, liver, and pancreatic diseases in physiologically- intact mammalian systems. Moreover, the GMMC enhances interactions and collaborations for CMSDLD investigators and lowers the technical and financial barriers that would otherwise impede the application of these approaches for individual investigators. Thus the Genetically Modified Mouse Core contributes greatly to the research efforts of the CMSDLD.