Gerald Crabtree - US grants

Recent evidence indicates that cells become malignant when the normal control mechanisms over cell division and proliferation malfunction. The purpose of the studies described in this proposal will be to obtain a better understanding of the mechanisms used by IL-2 and its receptor to promote T-cell proliferation and to define possible sites at which this process may malfunction in malignancy. Normal T-cell growth and clonal proliferation probably occur by an autocrine pathway in which the cell both makes and responds to its own growth factor. Normally induction of IL-2 and its receptor is controlled by the presence of antigen; thus T-cell growth is limited to a subpopulation of antigen-specific cells. However, the autocrine nature of this process suggests that it requires delicate regulation, and we hypothesize that perturbations of the control mechanisms over IL-2 or its receptor may produce a transformed cell. To test this hypothesis we plan to introduce the gene for IL-2 into the IL-2 dependent cell lines CT-6 and CTLL, which constitutively make the IL-2 receptor but not IL-2. If these cells become IL-2 independent, as our preliminary studies indicate, we will test for a transformed phenotype by injecting the cells into mice of a genetic background which is identical to that of the cell line. If tumors cannot be established or if the cells cannot be made to become IL-2 independent, we will change the regulatory regions of the gene to bring it under constitutive control, and determine if the modified gene is capable of conferring the malignant phenotype. In addition we plan to investigate the generality of the growth-promoting properties of the IL-2 receptor by introducing the IL-2 receptor gene into a variety of non-T lymphocytes and other cells and testing for the ability of IL-2 to promote proliferation of the cells receiving the gene. Finally, we will examine the role of the IL-2 receptor in the proliferation of human T-cell lymphomas which express the IL-2 receptor constitutively. We will test the effect of a plasmid producing an anti-sense copy of the IL-2 receptor mRNA on the growth of a HTLV infected cell line which constitutively makes the IL-2 receptor. We will also examine the function of this isolated receptor gene. We anticipate that these studies will help determine if IL-2 or the IL-2 receptor have a role in the pathogenesis of human T-cell malignancies.

The studies described in this proposal are designed to provide a better understanding of the factors that control fibrinogen production by influencing the activity of the three linked genes for fibrinogen. Fibrinogen production in the liver is controlled by an indirect feedback mechanism in which the degradation products of fibrinogen interact with monocytes and Kupffer cells to induce a peptide, possibly IL-1, which acts directly on hepatocytes to stimulate fibrinogen production. Since previous work from our laboratory indicates tht fibrinogen production is controlled at the level of transcription, our studies will concentrate on the factors influencing and coordinating transcription of these three linked genes and will employ two model systems: the activation of fibrinogen gene transcription in the livers of intact animals during the acute phase reaction and the constitutive and induced production of fibrinogen in hepatocyte lines. We will complete the sequencing of the three fibrinogen genes in rats and humans, and search for sites of DNase sensitivity within the fibrinogen gene complex. In addition we will locate transcriptional enhancing elements within the fibrinogen gene complex and determine if one, two, or three transcriptional enhancing elements are necessary to activate three closely-linked genes. If an enhancing element is found neighboring each fibrinogen gene, as preliminary studies indicate, we will attempt to determine if the enhancing elements are using the same regulatory molecule by examining competition among the enhancing elements for cellular factors, after transfection into differentiated hepatoma cell lines. Since regional chromatin structure may be important in controlling the fibrinogen genes, we will test the hypothesis that transcriptional activation of the three linked fibrinogen genes occurs by a regional change in chromatin structure conducted along double-stranded DNA by superhelical torsional stress. In these studies we will analyze the function of the genes after gamma radiation of intact cells at doses calculated to make from 1 to 10 single-stranded nicks within the 70 kb gene complex and thereby relax torsional stress. We anticipate that our studies will provide specific information on the control of fibrinogen production by the liver as well as general information on the control of linked families of genes.

Recent evidence indicates that cells become malignant when the normal control mechanisms over cell division and proliferation malfunction. The purpose of the studies described in this proposal will be to obtain a better understanding of the mechanisms used by IL-2 and its receptor to promote T-cell proliferation and to define possible sites at which this process may malfunction in malignancy. Normal T-cell growth and clonal proliferation probably occur by an autocrine pathway in which the cell both makes and responds to its own growth factor. Normally induction of IL-2 and its receptor is controlled by the presence of antigen; thus T-cell growth is limited to a subpopulation of antigen-specific cells. However, the autocrine nature of this process suggests that it requires delicate regulation, and we hypothesize that perturbations of the control mechanisms over IL-2 or its receptor may produce a transformed cell. To test this hypothesis we plan to introduce the gene for IL-2 into the IL-2 dependent cell lines CT-6 and CTLL, which constitutively make the IL-2 receptor but not IL-2. If these cells become IL-2 independent, as our preliminary studies indicate, we will test for a transformed phenotype by injecting the cells into mice of a genetic background which is identical to that of the cell line. If tumors cannot be established or if the cells cannot be made to become IL-2 independent, we will change the regulatory regions of the gene to bring it under constitutive control, and determine if the modified gene is capable of conferring the malignant phenotype. In addition we plan to investigate the generality of the growth-promoting properties of the IL-2 receptor by introducing the IL-2 receptor gene into a variety of non-T lymphocytes and other cells and testing for the ability of IL-2 to promote proliferation of the cells receiving the gene. Finally, we will examine the role of the IL-2 receptor in the proliferation of human T-cell lymphomas which express the IL-2 receptor constitutively. We will test the effect of a plasmid producing an anti-sense copy of the IL-2 receptor mRNA on the growth of a HTLV infected cell line which constitutively makes the IL-2 receptor. We will also examine the function of this isolated receptor gene. We anticipate that these studies will help determine if IL-2 or the IL-2 receptor have a role in the pathogenesis of human T-cell malignancies.

During the immune response, foreign antigens present on invading organisms or transplanted tissues interact with the antigen receptor of T lymphocytes to induce a complex series of changes which result in T cell proliferation and the acquisition of immunologic competence. Our objectives over the next five years are to gain insight into the genetic regulatory events which underly T cell activation. Specifically, we hope to understand how events initiated at the antigen receptor are transmitted to the nucleus and result in the activation of a group of genes including those for IL-2, the IL-2 receptor, and gamma interferon. Each of the specific aims of this proposal describes an approach to characterizing the molecules which transmit these signals. We will begin our investigation by characterizing two nuclear proteins, NFIL2A and E, that we have recently discovered which bind to essential sequences within the IL-2 enhancer as well as the HIV LTR. NFIL2E is present only in activated T lymphocytes. It appears within 20 minutes of the binding of antigen to the antigen receptor and precedes IL-2 gene activation by 10-15 minutes. The second protein, NFIL2A, is constitutive but its recognition sequence is able information gained in an initial characterization of these molecules to obtain antibodies and cDNA clones for these proteins. These materials will be used to answer several specific questions including: what is the nature of the antigen-receptor dependent activation of these molecules? how is this process inhibited by cyclosporin? and finally are these proteins transcriptional factors or do they function by another mechanism? Our goal will be to learn how these molecules are involved in T cell activation and T cell proliferation. The last specific aim describes a novel approach to selecting mutations at various levels in the normal pathways of T cell activation. We will prepare transgenic mice using the T-cell specific and activation-specific transcription enhancer of the IL- 2 gene directing transcription stimulus each time the antigen receptor is triggered, but should not escape antigenic control unless a mutation develops in the normal pathways of T cell activation. These mutations in T cell activation will be identified and the site and biologic behavior of the mutations characterized using the reagents derived in Specific Aims 1 and 2.

Recent studies conducted in several countries have come to the unanimous conclusion that fibrinogen levels are a major risk factor for cardiovascular disease. The risk associated with elevated levels equals or exceeds the risk for serum cholesterol, high blood pressure, smoking, diabetes and obesity. Furthermore, the epidemiologic data suggest that elevated fibrinogen levels play a role in the pathogenesis of cardiovascular disease and correlate with a restriction polymorphism in the fibrinogen locus. Production of fibrinogen is in large part controlled by the transcription factor, hepatocyte nuclear factor 1 or HNF-1, which interacts with the control regions for the fibrinogen genes as well as other liver-specific genes. We have isolated a cDNA for HNF-1 and found that it contains a homeodomain similar to that found in genes responsible for pattern formation in invertebrates, and also is a distant member of the POU family of transcriptional activators. Our goal is to understand how this protein functions and how it is developmentally regulated. Specifically, we will begin by completing the sequencing of the cDNA and gene. This information will be used to dissect the functional regions of HNF-1 by analysis of alanine-substitution mutations and by using portions of HNF-1 to complement the function of well- characterized mutations is known transcriptional activators. The role of HNF-1 in hepatocyte differentiation and development will be explored by determining if expression of the protein in dedifferentiated cells leads to expression of endogenous fibrinogen genes. In a second approach we will determine if the inductive influences that are responsible for the formation of the liver and other organs operate by directly controlling HNF-1 n the developing embryo. Finally, we will make use of the observation that retinoic acid induces the production of HNF-1 in the F9 cell line to define the events that are required for the developmental regulation of HNF-1 and the molecules which cooperate with HNF-1 to activate liver-specific genes. At the end of this five year period, we hope to have characterize the cellular and molecular events that regulate HNF-1 during developing and to have a precise understanding of its role in organogenesis and the activation of the fibrinogen and other liver- specific genes.

Recent studies conducted in several countries have come to the unanimous conclusion that fibrinogen levels are a major risk factor for cardiovascular disease. The risk associated with elevated levels equals or exceeds the risk for serum cholesterol, high blood pressure, smoking, diabetes and obesity. Furthermore, the epidemiologic data suggest that elevated fibrinogen levels play a role in the pathogenesis of cardiovascular disease and correlate with a restriction polymorphism in the fibrinogen locus. Production of fibrinogen is in large part controlled by the transcription factor, hepatocyte nuclear factor 1 or HNF-1, which interacts with the control regions for the fibrinogen genes as well as other liver-specific genes. We have isolated a cDNA for HNF-1 and found that it contains a homeodomain similar to that found in genes responsible for pattern formation in invertebrates, and also is a distant member of the POU family of transcriptional activators. Our goal is to understand how this protein functions and how it is developmentally regulated. Specifically, we will begin by completing the sequencing of the cDNA and gene. This information will be used to dissect the functional regions of HNF-1 by analysis of alanine-substitution mutations and by using portions of HNF-1 to complement the function of well- characterized mutations is known transcriptional activators. The role of HNF-1 in hepatocyte differentiation and development will be explored by determining if expression of the protein in dedifferentiated cells leads to expression of endogenous fibrinogen genes. In a second approach we will determine if the inductive influences that are responsible for the formation of the liver and other organs operate by directly controlling HNF-1 n the developing embryo. Finally, we will make use of the observation that retinoic acid induces the production of HNF-1 in the F9 cell line to define the events that are required for the developmental regulation of HNF-1 and the molecules which cooperate with HNF-1 to activate liver-specific genes. At the end of this five year period, we hope to have characterize the cellular and molecular events that regulate HNF-1 during developing and to have a precise understanding of its role in organogenesis and the activation of the fibrinogen and other liver- specific genes.

T lymphocytes capable of responding to virtually any antigen on invading pathogens or transplanted tissue are produced in the thymus as a result of selection and intrathymic differentiation. These cells migrate to the peripheral lymphoid organs and upon encountering foreign antigen undergo an orderly sequence of events that results in proliferation and acquisition of immunologic function. During this process they coordinate the activity of other cells involved in the immune response by controlling cell fate decisions and proliferation of B cells, macrophages and granulocytes through the production of cytokines and cell-cell interactions. We propose to explore the pathways used by the T cell antigen receptor to communicate with genes necessary for immunologic activation. Most of our attention will be directed toward the least understood part of the pathway which connects Ca2+-dependent events and tyrosine kinase substrates to the activation of certain genes, such as IL-2 that are critical for proliferation. A comprehensive and unbiased genetic approach has been devised to produce mutations in the proteins carrying signals from the antigen receptor to the IL-2 gene. Cells having these mutations will be placed into complementation groups by analysis of heterokaryons formed between the different clones. Each complementation group will be characterized to localize the site of action of the defective protein. Genes able to complement the mutant phenotype will be cloned by transfecting cDNA expression libraries into the mutants and rescuing plasmids able to restore signal transmission. This genetic approach will be enhanced by an analysis of the nuclear events essential for IL-2 gene activation using an in vitro transcription system developed in our laboratory that faithfully reflects the complex requirements for the activation of the IL-2 gene. We will use this system to identify proteins (kinases, proteases, etc) necessary for the activation of two transcription factors, OAP (Oct-1 Associated Protein) and NF-AT (Nuclear Factor of Activated T cells), which are required for IL-2 gene activation and specifically activated by triggering the antigen receptor. By continuing this process of working backward from proteins essential IL-2 gene activation we expect, by the end of the 5 year granting period, to define the processes used by the antigen receptor to communicate with genes in the nucleus.

The introduction of cyclosporin as an immunosuppressive agent revolutionized the treatment of transplant rejection and also aided in the treatment of certain autoimmune diseases such as juvenile onset diabetes. Remarkably these therapeutic advances are achieved with inhibition of only 50% of the activity of calcineurin, the target of both cyclosporin and FK506, indicating that calcineurin is a particularly critical molecules in the development of the normal immune response. The use of cyclosporin is limited by renal, CNS, and pancreatic toxicity all of which are now thought to be due to a block in the actions of calcineurin. In the present proposal we will construct transgenic animals containing a mutant cyclophilin (Cph/t-) expressed only in lymphocytes that interacts with a modified harmless cyclosporin (CsA/t-). In this mice we can completely block the function of calcineurin in selective cell types. We will then determine if the development of autoimmune diabetes in NOD mice can be completely prevented by suppression of the Ca2+/calcineurin pathway in T cells. Since cyclosporin treatment also appears to result in the development of long-term tolerance to transplanted tissues we will determine if complete suppression of the Ca/2+/calcineurin/NF-AT pathway enhances the development of tolerance. If successful, these experiments will direct efforts to treat auto immunity to the development of specific inhibitors of the calcineurin/NF-AT pathway and lay the groundwork for treatment of graft-versus-host disease by inserting the modified cyclophilin gene into stem cells at the time of transplantation and treating with the modified cyclosporin A.

DESCRIPTION (provided by applicant): Developing biological vascular grafts can potentially revolutionize the treatment of patients who need new blood vessels. The most obvious way to assemble vessels for therapeutic use is to recapitulate the developmental processes that lead to vessel assembly. My first goal will be to obtain an understanding of vascular development so that the goal of therapeutic assembly can be realized. The signaling roles of blood vessel components and the biological molecules involved in vascular assembly and patterning will contribute to the engineering of biological vascular grafts. Conceivably, these molecules can be applied in bioengineering to promote assembly of blood vessels and to define the boundary of vascular growth once the vascular graft is implanted into a site in vivo. Ca2*/Calcineurin/NF-ATc pathway is crucial for proper vascular development. Disruption of this pathway leads to disassembly of endothelial and smooth muscle cell layers of the blood vessel wall, and aberrant vascular invasion into tissues that normally form the anatomic vascular boundary. To address the spatiotemporal regulation of calcineurin/NF-ATc signaling during angiogenesis, mouse genetic models in which the calcineurin signaling is disrupted only in endothelial cells or vascular smooth muscle cell progenitors will be generated to study the contribution of this signaling pathway in either cell type to the recruitment of smooth muscle cells to the vascular wall. Signaling roles of calcineurin B in vascular patterning and development of arterial and venous systems will also be assessed using these mouse models. In addition, DNA microarray technology will be employed to identify novel genes regulated by the Ca2+/Calcineurin/NFATc pathway during angiogenesis. Emphasis will be placed on genes that promote the vascular assembly and genes that define the anatomic vascular paths. The expression profile of candidate genes will be studied using in situ hybridization, and their functions will be studied with various in vitro angiogenesis assays.

DESCRIPTION (provided by applicant): Following specification of neuronal fates, transcriptional mechanisms are thought to contribute to the production of a wide range of different neuronal morphologies. In mammals relatively little is known about how these morphogenic programs are regulated. Recent genetic evidence indicates that signaling by Ca2+, calcineurin and NFATc proteins is essential to generate a variety of specific axonal morphologies in mice. The studies described in this proposal are directed at understanding the mechanisms used by these molecules to generate specific patterns of axonal outgrowth that are essential for normal neural development. We will begin by defining the cell types in which the pathway functions. We will then define the developmental time period that the pathway is needed for specific patterns of axonal outgrowth. Since the phenotypes of mice with defects in this pathway suggest that it might respond to novel axonal guidance cues, we will define new ligands and receptors that activate NFAT in neurons. Axonal outgrowth is often accompanied by the production of complex architectures or specialized sensory structures suggesting some means of communicating local information from the growth cone to the nucleus to activate the genes essential for these specialized structures. NFATc proteins rapidly move from the axon and growth cone to the cell body after Ca2+ stimuli, implying that they could convey positional cues to the nucleus. We will define the mechanism of this transport and determine if it could allow rapid transcriptional monitoring of positional cues needed for specific axonal morphologies. Finally, we will develop a general method to rapidly and reversibly control the activity of any protein of interest in a mouse. We will then use this method to understand the functions of calcineurin and NFAT at critical times in the formation of specific axon morphologies. After completing our studies we expect to have defined a pathway beginning with ligand-receptor interactions through Ca2+, calcineurin and NFAT to the activation of target genes critical for specialized neuronal morphologies. We believe that this pathway, its target genes and its many modulators will be a rich source of new molecules for therapeutic intervention in neurologic diseases.

[unreadable] DESCRIPTION (provided by applicant): Perhaps few questions in development have been so intractable as the means by which different cell fates are produced in response to a gradient of signals. These analogue-to-digital switches underlie the determination of cell fates in many organisms. T Lymphocyte development may be one of the best systems to understand this general problem because the critical signals are given at a stage where biochemical, genetic and cell biologic methods can all be brought to play. In lymphocytes, weak or transient signals are thought to produce positive selection (differentiation and proliferation) of T cells capable of reacting to self-MHC on thymic stromal cells. On the other hand, strong signals produced by self-antigen lead to death of cells responding to self-antigen. The pro-apoptotic protein Bim is required for negative selection but is not necessary for positive selection. Conversely, we have recently found that calcineurin is essential in T cells for positive selection, but dispensable for negative selection. Surprisingly calcineurin specifically controls the activation of ERK but not other MAP kinases or IkB, suggesting a revision of the accepted signaling pathways of thymocyte selection. These observations set the stage for a biochemical march from Bim and calcineurin to the molecule(s) that divert signals from positive to negative selection with increasing signal intensity. Current studies support several possible mechanisms by which signals of different intensity could control selection. To avoid the difficulties encountered with forward analysis of biochemical pathways we will work backward from Bim and calcineurin to define the biochemical pathways that control their activity in CD4+, CD8+ thymocytes. Our goal in these studies will be to define the lowest common mediator necessary for activation of both Bim and calcineurin and hence positive and negative selection. We will then determine the mechanism by which this molecule is induced to channel high intensity signals to Bim and low intensity signals to calcineurin. We will also define the processes downstream of calcineurin that mediate positive selection including the mechanism of NFATc nuclear import and export, the set of genes that are dependent on calcineurin activity in positive selection and how these genes give rise to a population of immunologically competent peripheral lymphocytes. Defining these mechanisms should lead to a more complete understanding of immune defense and provide useful information for development of new therapies. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Studies in both vertebrates and invertebrates have defined signaling pathways, such as Notch, Wnt and BMP as well as transcription factors such as the HLH and Sox families that are essential for neurogenesis and neural differentiation. These and other regulators appear to operate within a developmental context, which is in part defined by a neural chromatin landscape regulating genomic accessibility to ubiquitous signaling and developmental pathways. Recent studies have shown that neurons contain a highly specific polymorphic family of ATP-dependent chromatin remodeling complexes, which resemble yeast SWI/SNF complexes. ATP- dependent chromatin remodeling complexes are thought to use the energy of ATP hydrolysis to control transitions between stable epigenetic states by exchanging or mobilizing nucleosomes thereby altering histone codes. Neural progenitors have a distinct complex (npBAF) distinguished by its subunit composition. The npBAF complex is essential for self renewal of neural progenitor/stem cells. Near the last mitotic division of neurons, two of the subunits (BAF45a and BAF53a) are removed from npBAF complexes and replaced by homologous subunits (BAF45b and BAF53b) to give rise to a new family of neural-specific complexes (neural BAF or nBAF), which are required for dendrite morphogenesis and other post mitotic functions. This exchange of ATP-dependent chromatin remodeling complexes also occurs in the course of directed differentiation of ES cells to neurons. Our studies are designed to understand the mechanism and biologic meaning of the switch between these two essential epigenetic states. Because the expression of the neural progenitor subunits of the complexes (BAF45a and BAF53a) is mutually exclusive with the expression of the subunits of the neural complexes (BAF45b and BAF53b), negative feedback mechanisms are a possible contributor to the switch. We will define the intrinsic regulators that bring about this essential switch in subunit composition. In the second specific aim we will use this information to define the extrinsic regulators using an unbiased genetic screen. We will then test the role of these polymorphic complexes and their regulators by preparing mice carrying a conditional deletion of the neural-specific subunits and also mice in which the subunit composition is altered. One family of subunits (BAF45) contains two PHD domains and a Kruppel domain and hence might contribute to the specific retargeting of these complexes to different genomic sites at different developmental stages. The role of these subunits will be tested using the conditional mutant mice described above as well as transgenic animals in which the deleted subunits are replaced with chimeric genes. To help understanding targeting and the mechanism of action of these neural specific ATP-dependent chromatin remodeling complexes we will develop a generally useful technique for precise temporal targeting of proteins that interact with DNA or chromatin and use it to understand the actions of the neural progenitor and neural chromatin remodeling complexes. At the conclusion of our studies we will have defined the mechanisms used by an essential epigenetic switch to regulate specific aspects of neural development. These studies should contribute to a fundamental understanding of neural development and also help with the goal of production of specific cell types from pluripotent cells for uses in regenerative medicine. PUBLIC HEALTH RELEVANCE: One of the major challenges of modern biology and medicine is to repair damaged or diseased tissues. Avenues to accomplish this have grown from an understanding of the fundamental processes controlling the development of the embryo. One of these processes is the control of accessibility of our genetic material to regulatory mechanisms that allow an orderly use of genes to make tissues and organs such as the heart, lungs, immune system and brain. We hope to understand how the accessibility of genetic material is controlled during the formation of the brain and how it differs from other tissues. Our studies might provide new avenues for the production of tissues and cells for regeneration of diseased or damaged tissues.

[unreadable] DESCRIPTION (provided by applicant): The most common human aneuploidy is complete or partial trisomy of human chromosome 21 (HSA21), which results in Down Syndrome (DS). Trisomy 21 occurs at a frequency of 1 in 43 spontaneous abortions and 1 in 750 live births. Despite its high prevalence and intensive investigation, the molecular mechanisms leading to the development of phenotypic changes are poorly understood. The characteristics of DS include certain constant phenotypic characteristics such as mental retardation, craniofacial features, and immunodeficiency as well as inconstant characteristics such as congenital heart disease, placental vascular insufficiency, muscular hypotonia, and gastrointestinal malformations. The observation that a 1.5-fold increase in gene dosage can produce significant developmental effects suggest that the genes at fault might function synergistically. Recently, synergy between two genes DYRK1a and DSCR1 within the critical region of chromosome 21 was shown to reduce the nuclear occupancy of the NFATc proteins leading to the misregulation of genes critical to neural, skeletal, and immune development and function. NFATc proteins are both transcription factors and signaling proteins and are essential to many aspects of vertebrate development and morphogenesis. This signaling and transcriptional pathway is required for signaling by neurotrophins, netrins, FGF, VEGF, RANKL, the T lymphocyte receptor and Ca++ channels. Remarkably, the phenotypes of NFATc1, c2, c3 and c4 mutant mice reproduce severe forms of many or most of the characteristic features of Down syndrome. Feedback loops within the Calcineurin/NFAT pathway produce extraordinary sensitivity to a 1.5-fold increase of DYRK1a and DSCR1, which have been found to be over- expressed in tissues of patients with DS in virtually all published studies. Mathematical modeling predicts that increased DSCR1 and DYRK1a reduce nuclear occupancy of the NFATc proteins and lead to a failure to activate critical target genes and thereby to features of DS. Preliminary observations of mice with segmental trisomy and fetuses with trisomy have been consistent with this Trisomy Synergy Model. We propose to test this model of the pathogenesis of DS and if confirmed use this model to develop therapeutic leads to treat pathologic features of Down syndrome. Initially we will determine if samples from DS human fetuses, cord blood hematopoietic stem cells and T lymphocytes have the expected biochemical and genetic features of reduced NFAT activity and reduced target gene activation. We will formally test the role of DYRK1a and DSCR1 in murine models of DS by determining if normalizing the dosage of DYRK1a and DSCR1 rescues the DS-like characteristics in these mice. We will develop quantitative mathematical models of the effects of trisomy on the NFAT genetic circuit and expand these models to include interactions with other trisomic genes. This Trisomic Synergy Model will be useful for predicting potential sites of therapeutic intervention as well as predicting differences between gene dosage effects in mice and humans. Finally, we will develop small molecule screens for activators of NFAT-dependent transcription and test the ability of any molecules found to rescue the defects in DS T lymphocytes. At the conclusion of our studies we expect to have clearly defined the role of NFAT dysfunction in producing the phenotypic features of Down syndrome and to have discovered therapeutic leads for the treatment of non-developmental aspects of Down syndrome. Project Narrative: Down Syndrome is a common disorder caused by an additional copy of chromosome 21. Recent studies indicate that the extra chromosome imbalances a pathway controlling the development and function of the brain, skeleton, cardiovascular and immune systems. We will test this hypothesis and if correct find molecule that correct the balance and thereby develop new treatments for some of the disabling characteristics of Down Syndrome. [unreadable] [unreadable] [unreadable]

Program Director/Principallnvestigator (Last, First, First, Middle): Crabtree, Gerald 2 R01 Program Director/Principal Investigator (Last, Middle) : Crabtree , Gerald 2 ROl E-, A1060037-06 AI060037 -06 shape repertoire lymphocytes has been one the most The mechanisms that shape the repertoire of T lymphocytes has been one of the most enduring mysteries in immunology. The process of repertoire selection is critical to the enduring mysteries in immunology. The process of repertoire selection is critical to the development of effective immunity to foreign antigens, transplant rejection, antigens, transplant rejection, immunosuppression and also to the pathogenesis of autoimmune disease. Substantial Substantial immunosuppression and also the autoimmune evidence mechanism in which signals the antigen experimental evidence supports a mechanism in which intense signals at the antigen receptor lead to the elimination of clones of T cells bearing receptors binding self to elimination cells bearing receptors binding antigen, while weaker signals mediated by TCR's that match MHC molecules lead to the lead to antigen, mediated cells able to mount immune responses to foreign antigen survival and differentiation of T cells able to mount immune responses to foreign antigen. . distinguished and These observations indicate that signals of different intensity must be distinguished and sorted to produce distinct cell fates. The problem of discriminating signals of different problem sorted to produce distinct cell fates. signals different unsolved question in biology and evidence indicates that signal intensity is a general and unsolved question in biology and evidence indicates that signal intensity at several different morphogen receptors also determines cell fate. Lymphocyte receptors also determines cell fate. Lymphocyte development is well suited to solve this general problem as biochemical, cell biologic well suited this general problem biochemical, cell and genetic approaches can all be effectively combined. Recent evidence indicates that can combined. indicates negative selection in response to intense signals at the antigen receptor is mediated by signals mediated by the transcriptional activation of BIM by a Signaling pathway requiring PKCa. In contrast, BIM In contrast, transcriptional activation signaling pathway requiring positive selection appears to arise when early signaling by calcineurin leads to a brief selection when signaling by calcineurin leads to period of ERK hypersensitivity allowing differential responses to weak signals at the allowing differential responses to signals the alpha/betaTCR in CD4/CD8 T cells or Erk alpha/betaTCRin CD4+/CD8+ T cells.. Mutations in components of the calcineurin or Erk pathways produce selective defects in positive selection with no apparent defect in selective in selection with in negative selection, while mutation of Bim leads to selective defects in negative selection of Bim leads to selective defects in negative selection with no defect in positive selection . These observations indicate that the mechanisms with no in positive selection. These observations indicate that the mechanisms sort signals to distinct fates in T cells must operate upstream of Bim, that operate to sort signals to distinct fates in T cells must operate upstream of Bim, Calcineurin and Erk, yet downstream of CD3, LAT, VAV and other molecules required of CD3, LAT, VAV and other molecules required Calcineurin for both positive and negative selection . We have revised our plans to focus on the and negative selection. We have revised focus on the comments of the reviewers or our application . Hence we will focus on the strong points comments the reviewers application. Hence we will focus on the strong points including using a genetic strategy to identify the targets of calcineurin signaling in CD4 including using genetic strategy to identify the targets of calcineurin signaling in CD4 ICD8- thymocytes that modulates Erk activity and are essential for positive selection.. /CD8 thymocytes that modulates Erk activity and are essential for positive selection be first with deletion The genes identified will be tested 'first with simple RNAi methods and later with deletion to define their role in T cell development. As the reviewers suggested we will document cell development. reviewers suggested will the role of signaling pathway between PKCa and BIM using additional models of between on negative selection based on other antigens and also as the reviewers suggested focus and also reviewers suggested focus on using a "on-time" deletion model of negative selection. Proteomic methods will be "on-time" deletion model negative selection. Proteomic methods will be in bim regulators dependent upon negative selection used to define modifications in the bim regulators dependent upon negative selection of signals, ultimately mediated by PKCci. Finally we will complete the construction of the signals, ultimately by PKCa . Finally Positive Selection Indicator mice that will be essential for the genetic strategies Positive Selection will be essential the strategies described in the original application. Our revised studies will set the ground to define the Our revised studies will set the ground to define the described in original elements in a pathway carrying signals from the antigen receptor and PKCcx to the carrying signals from the antigen receptor and PKCa to the proteins controlling BIM activation and negative selection. Precisely defining the full proteins controlling BIM activation and negative selection. 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DESCRIPTION (provided by applicant): Although the human genome encodes about 30 different ATP-dependent chromatin remodeling complexes, their mechanism[s] are largely unknown. In part, this is due to the lack of rapidly acting small molecule inhibitors that could permit precise temporal analysis needed for mechanistic studies. One family of these complexes based on the Brg and Brm ATPases (BAF or mSWI/SNF complexes) are both necessary and in some contexts sufficient to induce the pluripotent state. BAF complex subunits are also tumor suppressors and undergo loss of heterozygosity in certain human malignancies. Finally, they have been shown to be necessary for explicative senescence and their deletion increases the proliferative life of human fibroblasts. Proteomic analysis indicates that the diverse functions of BAF complexes are the result of combinatorial assembly of the complexes from gene families encoding the subunits. Genome wide analysis of the occupancy of BAF complexes in ES cells has revealed that they most commonly suppress their target genes from a distance, suggesting an unanticipated mechanism of action. To understand the poorly defined mechanisms used by SWI/SNF-like BAF complexes to promote pluripotency and suppress tumors, this project aims to identify a comprehensive set of rapidly acting small- molecule inhibitors using a knock-in ESC reporter line. This line contains luciferase inserted into the Bmi1 locus and undergoes rapid activation upon genetic deletion of the ATPase subunit of the BAF complex. The 2 MDa complex contains 13 subunits and has an expected surface area of 0.5 to 2.5 million E2. The extraordinarily large surface of the complex is hypothesized to carry out a sequential series of reactions that can be ordered and probed with small molecules. Once identified, we will define the binding sites of the small molecule inhibitors using established embryonic stem cell lines with null mutations in subunits of the complex. Because the inhibitors could also block regulatory mechanisms impinging upon BAF complexes, we will characterize their effect upon defined post-translational modifications of the complex. The time-of-action of each of the inhibitors in both the repression of Polycomb genes and the activation of important pluripotency targets such as Fgf4 and Bmp4 will help to further define the level of function of each of the rapidly acting inhibitors. Specifically, their effects will be determined on higher-order chromatin structures, long-range interactions, chromatin accessibility, nucleosome positioning, and transcription factor occupancy. This comprehensive and systematic investigation should lead to deeper understanding of the actions of this chromatin-remodeling complex, which plays an essential role in pluripotency, human tumor suppression and cellular senescence. In addition, this valuable toolbox of small molecule probes will be invaluable for other researchers studying chromatin regulation. PUBLIC HEALTH RELEVANCE: We intend to find small molecules that inhibit the activities of a complex of proteins that control the packaging of DNA into the nucleus of a cell. This protein complex plays a critical role in human embryonic stem cells and human tumor suppression. Inhibitors of this complex could lead to improved stem cell therapies and new cancer treatments.

DESCRIPTION (provided by applicant): Recent screens for driving mutations in human malignancy have repeatedly identified subunits of mammalian SWI/SNF-like BAF complexes as tumor suppressors. Biochemical studies indicate that the identified subunits: BAF250a, Brg (BAF190), BAF155, BAF60b, BAF53a, and BAF47 (hSNF5) are dedicated to these complexes and not found as individual proteins or as parts of other complexes. In addition, we have recently found that SS18 (mutated in Synovial Sarcoma), Bcl7 and Bcl11 appear to be dedicated, stable subunits of BAF complexes. These studies suggest that SWI/SNF-like BAF complexes might be one of the most commonly mutated chromatin regulators in human cancer. BAF complexes regulate chromatin structure and are composed of about 14 subunits that are combinatorially assembled from the products of gene families encoding the subunits. The mechanisms underlying their frequent mutation in cancer are unclear. We have found that conditional deletion or depletion of the oncogenic subunits leads to stalling in mitosis and anaphase bridge formation, strongly implicating a failure to decatenate DNA during M phase. Importantly, Topoisomerase IIa (Topo IIa), which resolves catenated DNA at M phase associates with BAF complexes and Brg is essential for chromatin binding by Topo IIa. Furthermore, purified BAF complexes are required for optimal decatenation by purified Topo IIa in vitro. These observations suggest that a failure of decatenation by Topo IIa contributes to the genesis of human cancers, which is the central hypothesis of this application. This hypothesis is supported by the high frequency of concurrent mutations in other genes or aneuploidy in tumors bearing apparent initiating mutations in BAF subunits. To study these oncogenic BAF mutations, the SS18 translocation to SSX is particularly useful since it produces a sterotypic in-frame fusion of the SS18 BAF subunit to a member of the SSX gene family located on the X chromosome. This precise translocation attaches 78aa of SSX to SS18 and is almost certainly the driving event in synovial sarcoma (SS), which account for about 8% of sarcomas. Remarkably, the translocation of one allele leads to partial dissolution of BAF complexes. We will begin our studies by determining whether the SS18-SSX fusion causes a loss or gain of function for BAF complexes. We will define the consequences of this translocation for BAF complex binding over the genome to determine if target genes are lost or gained. We will then determine why the wildtype SS18 allele is repressed and leads to the formation of little protein compared to the translocated allele. We will examine the potentially oncogenic role of Topo IIa's dependence upon BAF complexes. Finally, we will attempt to understand how activating mutations in a- catenin and PI3K apparently cooperate with loss-of-function mutations in BAF subunits to lead to cancer. At the conclusion of our work we expect to have gained insight into the mechanism of transformation by mutations of the subunits of BAF complexes, which are emerging as major contributors to human cancer.

DESCRIPTION (provided by applicant): The development of the vertebrate nervous system requires switching of polymorphic ATP-dependent chromatin remodeling complexes from a neural progenitor complex (npBAF) to a neuron-specific complex (nBAF) at mitotic exit. This switch is in part directed by two microRNAs, miR-124 and miR-9, which when expressed in fibroblasts can convert them into functional neurons. Recent exome sequencing studies have found frequent mutations of BAF complex subunits in non-syndromic mental retardation, microcephally, schizophrenia and less frequent BAF complex mutations in sporadic autism and sporadic ALS. The apparently genetically dominant nature of these mutations is consistent with the instructive role of subunit switching during neurogenesis and suggests that these chromatin regulators may have rate-limiting functions. Relatively little is known of the underlying mechanisms directing subunit switching, or how subunit switching relates to other more well-defined programs of neural development. Also, the mechanisms underlying the contribution of subunit mutations to a diverse range of neurologic diseases in humans are unknown. Indeed most of the mutations are in subunits not required for conventional activities such as nucleosome remodeling. Our preliminary data suggest that repression of three progenitor subunits and their substitution with neuron-specific subunits leads to mitotic exit of neural progenitors (NPGs) and functional differentiation. We will test this hypothesis using genetic approaches and define the genetic circuitry involved in repression of the npBAF subunits and the activation of the nBAF subunits. We will also define the proteins that bind to the neuron- specific surfaces of the complexes that mediate their role in dendritic morphogenesis, dendritic targeting and neural fate determination. Using chromatin immunoprecipitation and sequencing we will define the genome- wide consequences, which result from the normal exchange of three subunits within these 2 mega dalton chromatin remodeling complexes. Our studies and those of others indicate that an important function of BAF complexes is to oppose Polycomb complexes; however the mechanisms underlying this opposition are not understood. Thus, we will use a method recently devised in our lab to understand the underlying mechanism of BAF-Polycomb opposition. At the conclusion of our studies we hope to have an understanding of the fundamental genetic circuitry and mechanisms critical for this epigenetic switch and to gain insight into the roles of polymorphic BAF complexes in diverse human neurologic diseases.

ABSTRACT Recent exome sequencing studies have found that over 20% of human cancers have deleterious mutations in the genes encoding the subunits of mSWI/SNF (BAF) complexes. A much larger number of tumors have amplifications or deletions of these genes. These complexes are polymorphic assemblies of 15 subunits encoded by 28 genes giving rise to remarkable combinatorial specificity. This biologic specificity is reflected in the highly selective pattern of oncogenic mutations in specific subunits in specific cancers. Cancer mutations generally have the characteristics of tumor suppressors and are generally heterozygous, implying that they play a genetically dominant role in suppressing tumor formation. Work in our lab and others has led to the conclusion that the ability of these complexes to oppose polycomb-mediated repression contributes to their roles in both development and oncogenesis. Hence, we will focus our work on the mechanisms underlying the opposition between BAF and polycomb and its therapeutic consequences. First we will use a novel, newly designed in vivo chromatin remodeling assay to fully characterize the nature of the opposition on a minute-by- minute basis. Secondly, we will define the energetic requirements for BAF-polycomb opposition and the essential role of ATP in regulating binding and release of PRC1 from BAF. Third, we will fully characterize the direct interaction between BAF and polycomb repressive complex 1 (PRC1) in terms of subunits and domains that are essential for this interaction. Fourth, we will explore the consequences of disruption of BAF-polycomb opposition for repair, recombination and transcription over the genome. Finally, we will define the therapeutic potential of a group of small molecule BAF inhibitors that we identified in earlier screens. At the conclusion of these studies we should have a mechanistic understanding of BAF-polycomb opposition and have explored at least two potential paths for the production of cancer-specific drugs. 

PROJECT SUMMARY Methods for regulating cellular processes within distinct populations of neurons are needed to elucidate relationships between molecular mechanisms, circuits, and behavior; and to develop cell type- or circuit- selective treatments for neurological disorders. We propose a novel, non-genetic, small molecule method? transcription factor-chemically induced proximity (TF-CiP)?that harnesses the cell type- and circuit-specificity of endogenous transcription factors to regulate gene expression in subsets of neurons. TF-CiP utilizes a bifunctional small molecule to heterodimerize an ?anchor? transcription factor, which naturally binds to a target gene, with a ?hijacked? transcription factor, which enhances or represses transcription of the target gene. Cell specificity is determined from the intersection of expression of each transcription factor. TF-CiP can theoretically be adapted for any organism and because transcription factors are well conserved, it is possible that the same TF-CiP small molecule can be used to modulate neuronal processes across animal species. We will develop TF-CiP to regulate the expression of the rate-limiting enzyme for brain serotonin synthesis, TPH2, as a means to tune serotonin levels in subsets of serotonergic neurons. Although the population of central serotonergic neurons is relatively small, these neurons send projections throughout the brain and serve important roles in regulating mood, anxiety, sleep, and social behavior. Achieving circuit-specificity for a serotonin-modulatory small molecule would be an improvement over current therapies, which can have undesirable side-effects due to indiscriminate targeting of serotonin signaling in the central and peripheral nervous systems. TPH2 transcription is regulated by stress, sex hormones, and several transcription factors. We leverage this knowledge along with single-cell RNA-sequencing and projection mapping data in serotonergic neurons as a resource for candidate TF-CiP transcription factors. In Aim 1, we will screen for transcription factors that regulate TPH2 transcription cooperatively upon chemically-induced heterodimerization of their FRB and FKBP tags. This screen will employ a Tph2-Venus reporter and orthophthalaldehyde- mediated serotonin visualization as readouts in serotonergic cells. In Aim 2, we will work with structural biologists and chemists to synthesize bifunctional TF-CiP molecules for TPH2 transcriptional regulation. This will involve virtual binding screens of over 8 million small molecules, binding validation using surface plasmon resonance and isothermal titration calorimetry, small molecule functionalization, and chemical linkage of two transcription factor-binding molecules. In Aim 3, TF-CiP molecules will be screened for cytotoxicity, selectivity, and efficacy at regulating TPH2 transcription and serotonin synthesis in cells. In Aim 4, we will test TF-CiP molecules for efficacy and selectivity in vivo using murine social behavior assays and brain immunostaining, respectively. Successful completion of this study will provide the first neuropharmaceutic that is capable of targeting specific subsets of neurons or circuits without the aid of exogenous genetically-encoded proteins.