Stephen Smale - US grants

The very general problem of solving non-linear systems of equations efficiently has many important applications, in virtually every area of science and engineering. Steven Smale and his students and collaborators will use methods of geometry, especially Newton's method and path following, to study their relevance to these problems, with the aim of eventually leading to substantial reduction in costs of computation.

Hematopoiesis is a complex developmental process through which pluripotent stem cells in mammalian fetal liver or adult bone marrow give rise to several types of terminally differentiated blood cells, including cells in this pathway are likely to play significant roles in the onset of leukemias and immunodeficiencies. To understand the basis for these defects, the regulatory mechanisms for hematopoiesis need to be understood at the molecular level. The primary objective of the research described in this application is to identify and characterize proteins that regulate gene expression during early stages of lymphocyte differentiation. Towards this end, the transcriptional control of the terminal deoxynucleotidyltransferase (TdT) gene is being investigated. This gene is expressed specifically in both early B and early T cells and its transcriptional control mechanisms appear unique when compared with the control mechanisms for the immunoglobulin genes. The analysis currently focuses on a DNA-binding protein, called LyF-1, that has been implicated in the regulation of the TdT gene and of several other lymphoid genes. LyF-1 has been purified from cultured lymphocytes. The purified protein will be used to characterize the biochemical properties of LyF-1 and to obtain LyF-1 amino acid sequence. The amino acid sequence will lead to isolation of the LyF-1 gene. The nucleotide sequence of the gene will be determined and its DNA-binding domain defined, potentially leading to the isolation of related genes. The regulation of LyF-1 expression will also be examined. Knowledge of the activation pathway for LyF-1 will further our understanding of the activation pathways for the TdT gene and for LyF-1 will further our understanding of the activation pathways for the TdT gene and for other lymphoid genes. In addition to the extensive analysis of LyF-1, two other potential regulators of TdT transcription will be characterized. The TdT-DBE element is located downstream of the TdT transcription start site. Through an interaction with a putative DNA-binding protein, NF-TdT-DBE, this element strongly activates TdT transcription in vitro. NF-TdT-X is a protein that has been shown to compete with LyF-1 for binding to the TdT promoter and, therefore, may be a negative regulator of TdT transcription. The proteins and DNA sequence elements responsible for these activities will be analyzed in more detail to determine their roles in TdT regulation. Eventually, this systemic analysis of TdT transcription should allow precise TdT regulation to be recapitulated with purified proteins in a cell-free transcription reaction. These experiments will help us reach our long-term goals, which are 1) to understand the regulation of early stages of lymphocyte differentiation at the molecular level, and 2) to elucidate defects in the immunodifferentiation program that lead to leukemogenesis and immunodeficiency.

9417022 Rankin The American Mathematical Society requests a grant in the amount of $110,688 for the twenty--fifth AMS-SIAM Summer Seminar in Applied Mathematics, to be held in the summer of 1995. Previous seminars were held annually or at two or three year intervals since 1957 for a total of 24 seminars. The 1995 topic, "Mathematics of Numerical Analysis", was selected by the 1993 AMS-SIAM Committee on Applied Mathematics. Members of the Organizing Committee for the 1995 Sumer Seminar are: Stephen Smale (chair), Gene Allgower, Lenore Blum, Alexandre Chorin, Phillippe Ciaret, Felipe Cucker, James Demmel, Ron DeVore, Gene Golub, Arieh Iserles, Bert Jongen, Herb Keller, J.L. Lions, Jim Renegar, Michael Shub, Gilbert Strang, Shmuel Winograd and Henryk Wozniakowski. The 1995 Summer Seminar will be held during a four-week period in July/August, 1995, at Park City, Utah. The Society will be responsible for making suitable arrangements for lecture and seminar rooms, and for handling administrative matters.

Smale The investigator studies algorithms based on Newton's method for finding zeros of a system of polynomials. The focus of the analysis is to obtain bounds on the number of Newton steps required. Such bounds will depend on the number of variables, the degrees of the polynomials, and the desired accuracy. The goal of the project is a complexity theory of nonlinear equation solving. Equation solving is at the heart of much of mathematics and the main theoretical problem associated to its modern theory of computation is its complexity theory. Equation solving, together with its computational aspects, is a main way that mathematics is used by engineering, physics, economics, and many other disciplines.

Learning theory deals with the design and analysis of algorithms that learn from data. The goal of this proposal is to extend the recent theory to reach out and cover new areas, thus enhancing its impact and power. There are four directions the PI's wish to pursue.

A first direction examines the question of "unsupervised" learning, i.e., learning in the absence of a teacher who provides labels and feedback. Problems of clustering, density estimation, information extraction etc. are instantiations that will be considered. Our approach makes contact with ideas in geometry and topology. A second direction considers "active learning" where input points of the data are determined in some manner other than random sampling. This direction makes contact with Shannon sampling theory, and parts of scientific computation. A third direction considers "population learning" where one considers a population of learning agents interacting with and learning from each other and studies the dynamic evolution of such populations. This framework contains natural models for the evolution of language in societies of linguistic agents and the evolution of financial markets in societies of economic agents. A fourth direction examines problems of large scale data analysis, classification, and retrieval in the context of speech, image, and text data. Many of the algorithms developed will be empirically
evaluated.

This proposal will stimulate cross fertilization between diverse fields such as linguistics, engineering, mathematics, economics, computer science, and statistics.

DESCRIPTION (provided by applicant): As the UCLA-Caltech Medical Scientist Training Program (MSTP) approaches its 25th anniversary, its mission remains the same as it was when the Program was established in 1983: to promote the education of outstanding physician-scientists. To fulfill this mission, our current goals are to 1) recruit exceptionally bright and accomplished students who exhibit an unusual degree of passion for scientific knowledge and a life-long commitment to research and leadership, 2) help guide admitted students toward outstanding training environments that encourage individual thinking and provide students with the tools needed to develop into accomplished physician-scientists, 3) provide a comprehensive support system to meet the trainees' needs and 4) play an increasingly prominent role in guiding the career development of undergraduate students within the UCLA community, especially those from under-represented ethnic groups and disadvantaged backgrounds. To accomplish these goals as effectively as possible, the UCLA-Caltech MSTP is now run by two equal Co-Directors and two Associate Directors, all of whom are deeply committed to the Program. The Program is structured for an average of eight years of study. A new integrated, problem-based medical school curriculum is particularly well-suited for MSTP students, due to increased time for independent exploration and increased emphasis on research advances that contributed to current knowledge of disease etiology, diagnosis, and treatment. For their Ph.D. research, students choose mentors from a wide array of science and engineering Ph.D. Programs, with current students obtaining Ph.D.s in fields ranging from Philosophy to Policy Analysis. The MSTP's commitment to excellence was perhaps most apparent when UCLA and Caltech entered into an affiliation agreement ten years ago. This affiliation, which provides an opportunity for two students per year to perform their thesis research at Caltech, not only has increased the number of outstanding mentors available to students, but also appears to have increased the Program's visibility and recruitment success. The MSTP derives great benefit from recent dramatic improvements in physical facilities at both UCLA and Caltech, from the financial health of the universities, and from the recruitment of a large number of outstanding new faculty members to UCLA and Caltech and to UCLA's new California Nanosciences Institute and Institute for Stem Cell Biology and Medicine.

[unreadable] DESCRIPTION (provided by applicant): Members of the NF-?B/Rel family of transcription factors are well-established as critical regulators of pro- inflammatory genes in cells of the innate immune system, and they play important roles in several other biological processes. The phenotypes of mice lacking individual NF-?B family members suggest that each protein targets unique sets of genes. However, although much has been learned about the signal transduction mechanisms that activate NF-?B dimers in response to various stimuli, the fundamental reasons different NF-?B dimers are capable of regulating different genes remain largely unexplored. Because some genes that require a specific NF-?B family member for expression play critical roles in dictating the type of immune response elicited by a stimulus, an understanding of the mechanistic basis of family member selectivity may lead to novel strategies for modulating immune responses with an unusually high degree of specificity, to more effectively combat infectious diseases and chronic inflammatory diseases. We became interested in the mechanisms of NF-?B family member selectivity when we found that the II12b gene, which encodes the p40 subunit of the pro-inflammatory cytokine IL-12, exhibits an unusually strong requirement for the c-Rel member of the NF-?B family. Because earlier studies had shown that the DNA- contacting residues of c-Rel are identical to those of another family member, p65, and that the DNA recognition sequences for c-Rel and p65 are similar, we asked why c-Rel, but not p65, is critical for Il12b activation. Our results provided compelling evidence that c-Rel is required because c-Rel homodimers can bind non-consensus NF-?B sequences with high affinity, whereas p65 homodimers bind with high affinity only to sequences that closely match the NF-?B consensus. Notably, 46 residues of c-Rel were found to be responsible for its unique DNA-binding properties. To further elucidate NF-?B selectivity mechanisms, we will first use a variety of interconnected strategies to advance our knowledge of gene activation by c-Rel. We will also prepare bacterial artificial chromosome (BAC) transgenic mice to examine more rigorously the hypothesis that the 46 residues of c-Rel are fully responsible for its unique functions. Finally, because of the success of our p65-c-Rel chimeric protein strategy for uncovering c-Rel selectively mechanisms, we will use this same strategy as a starting point toward understanding the mechanism of selective gene activation by p65. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A primary goal of this application is to elucidate the mechanisms by which pro-inflammatory genes are regulated in macrophages, with a long-term objective of uncovering strategies for modulating immune responses and inflammation in normal and diseased states. An equally important goal is to use a pro- inflammatory gene as a model for elucidating fundamental mechanisms of gene activation by mammalian RNA polymerase II in response to an acute stimulus. To achieve these goals, we will use as a model gene Il12b, which encodes the p40 subunit of the heterodimeric cytokine IL-12. II12b is representative of many pro- inflammatory genes, but it plays an unusually important role in bridging the innate and adaptive immune systems and is a key regulator of immune responses against tumors and infectious agents. Considerable insight into the regulation of inducible genes has been obtained over the past two decades, primarily through studies of transfected promoter-reporter plasmids. However, much less is known about gene regulation in an endogenous chromatin environment. The chromatin immunoprecipitation assay (ChIP) has made it possible to examine endogenous events, but functional strategies to compliment this descriptive technique have been limited. We hypothesize that important new insight into endogenous gene regulation mechanisms can be obtained by introducing a series of mutations directly into an endogenous locus. To accomplish our objectives, we will introduce mutations into promoter and enhancer elements at the endogenous Il12b locus. In addition to disrupting known control elements, we will disrupt DNA elements and regions that have been highly conserved through evolution, but did not contribute important functions in transfection assays. These latter mutations will test the hypothesis that highly conserved sequences are generally critical for transcription in an endogenous setting. ChIP and restriction enzyme accessibility will be used to monitor the effect of each mutation on the cascade of events leading to Il12b transcription. In the final aim, we will examine how the Il12b locus becomes assembled into a chromatin state poised for activation by exploring the intriguing observation that an inducible enhancer is already marked in embryonic stem (ES) cells. We will identify proteins that associate with the Il12b enhancer and other model enhancers in ES cells and ask whether these interactions are essential for transcription in differentiated cells. Public Health Relevance Statement: The aberrant expression of pro-inflammatory genes plays a major role in a number of common diseases, including cancer, atherosclerosis, and a number of inflammatory autoimmune disorders. The objective of the research proposed in this application is to increase our understanding of the molecular mechanisms regulating pro-inflammatory gene expression. A major deficiency in our current knowledge is that most studies of pro- inflammatory gene expression have relied, by necessity, on artificial experimental approaches that only lead to a partial view of key regulatory mechanisms. Using recent technological advances and knowledge gained from comparative genome analyses, we propose to study mechanisms regulating pro-inflammatory genes in their native genomic environment. The long-term goal of this research is to develop strategies for the selective modulation of pro-inflammatory genes in the context of human disease.

Summary The 15th Annual Meeting of the International Cytokine Society (ICS) will be held in San Francisco, California from October 26-30, 2007. This international conference, titled "Cytokines in Health and Disease," will bring together leading investigators in the fields of cytokine signaling and biology, immunology, cancer research, and infectious diseases. The meeting is organized by Drs. Stephen Smale, Richard Flavell, Laurie Glimcher, Christopher Hunter, and Abul Abbas, who study cytokines from a broad range of perspectives. A major goal of the meeting will be to promote interactions between scientists performing cutting-edge studies of the molecular mechanisms of cytokine function, signal transduction, and gene expression, and those working to translate this knowledge into novel therapies for human disease. The therapeutic potential of cytokines and of modulators of cytokine signaling and expression are now appreciated, raising the need for enhanced interactions between basic, translational, and clinical researchers in this exciting field. In addition to approximately 48 invited speakers scheduled for Plenary Sessions and Symposia, a number of abstracts submitted by registrants will be selected for oral presentations in Special Topics Sessions, with other registrants given the opportunity to discuss their work at poster sessions. Scientists from academic institutions as well as from the biotechnology and pharmaceutical industries have been invited and encouraged to participate. Outstanding junior investigators, postdoctoral researchers, and graduate/medical students will also be encouraged to participate, and awards will be given to three or four young researchers in each of these categories. A special effort will be made to support the career development of under-represented minorities and women. Although our overriding objective is to bring together researchers studying cytokines from diverse perspectives, one major theme of the meeting will be recent studies of the emerging field of T helper 17 (Th17) cell development and functions in normal physiology and a variety of diseases, with one Plenary Session devoted entirely to this important and exciting new research area. The titles of other Plenary Sessions and Symposia are "Innate Immune Recognition and Responses," "T-Cell Subsets," "Cytokines, Chemokines, and Inflammation," "Inhibitory Receptors and Cytokines," "Gene Regulation," "Inflammation and Cancer," "Signal Transduction," "Regulation of Macrophage Activities and Functions," and "Clinical Progress." It is the firm belief of the Organizing Committee and ICS Leadership that, by bringing together leaders, junior investigators, and trainees in these diverse areas of cytokine research, this meeting will inspire important new avenues of investigation and will be of great benefit to the career development of promising young investigators and trainees in the cytokine field.

DESCRIPTION (provided by applicant): Inflammation can be beneficial for a normal immune response to microbial pathogens. However, prolonged inflammation can promote tissue damage during infection and has been closely linked to a diverse range of diseases, including cancer, atherosclerosis, and several inflammatory autoimmune diseases. Although a number of anti-inflammatory drugs are available, none of them are considered to be ideal for a variety of reasons, including insufficient target specificity and limited potency. Therefore, new strategies are needed for the development of inhibitors of pro-inflammatory genes and proteins. One major limitation in pursuing pharmaceuticals that inhibit the transcription of specific pro-inflammatory genes is that our understanding of the molecular mechanisms responsible for selective gene regulation remains rudimentary. Transcription factors such as NF-?B and AP-1 contribute to the activation of many pro-inflammatory genes. However, because of their broad functions, these factors may not be appropriate targets for the inhibition of specific subsets of inducible genes. During the past few years, a number of signal transduction pathways activated by Toll-like receptors (TLRs) and other transmembrane receptors have been elucidated, resulting in great strides toward the identification of pathways that lead to selective gene regulation. However, it will be difficult to fully appreciate the mechanisms responsible for the differential regulation of pro-inflammatory genes without an understanding of the logical organization of the control regions and DNA sequence elements associated with these genes. Our laboratory has found that genes induced by lipopolysaccharide (LPS) in murine macrophages can be divided into six broad classes on the basis of several criteria, including their (1) requirement for new protein synthesis, (2) requirement for nucleosome remodeling by the SWI/SNF family of ATP-dependent nucleosome remodeling complexes, (3) requirement for the transcription factor IRF3, and (4) the presence of a CpG island promoter. The experiments proposed in this application focus on the four classes of primary response genes, which are defined as genes directly activated by LPS signaling pathways in the absence of new protein synthesis. The proposed experiments will examine a variety of hypotheses, including the hypothesis that the critical properties of primary response genes are stimulus-specific and the hypothesis that CpG islands are frequently associated with SWI/SNF-independent primary response genes because the high CpG-content is incompatible with the assembly of stable nucleosomes. We will also examine the role of IRF3 in the activation of a specific class of SWI/SNF-dependent primary response genes. Finally, we will make use of bacterial artificial chromosomes containing representative members of each gene class to initiate more detailed analyses of the diverse activation mechanisms. Together, these studies should greatly expand our knowledge of the selective regulation of pro-inflammatory genes in cells of the innate immune system. PUBLIC HEALTH RELEVANCE: The aberrant expression of specific pro-inflammatory genes plays a major role in a number of common diseases, including cancer, atherosclerosis, and a number of inflammatory autoimmune disorders. Significantly, recent studies have shown that enhanced expression of some inflammatory genes helps protect against disease, whereas other genes enhance disease progression. The objective of the proposed research is to better understand the molecular mechanisms regulating the differential expression of inflammatory genes in cells of the immune system. The long-term goal of this research is to develop pharmacologic strategies for the selective modulation of pro-inflammatory genes, leading to the enhanced expression of protective genes and reduced expression of detrimental genes.

The Gene Regulation Program Area is composed of 21 members, spanning 8 Departments within UCLA. In ;he past competing cycle, investigators from this Program authored 374 publications, of which 94 (25%) were nter-programmatic and 15 (4%) intra-programmatic. 155 (41%) were placed in high-impact journals. 15 members of this Program Area used 8 out of the 8 Shared Resources that are currently funded by the JCCC. During the current funding year, peer-reviewed funding totaled $12.2 million in total costs, including $1.6 million from the National Cancer Institute. As with other Program Areas, JCCC fosters a number of interactive activities and many of the Shared Resources that support investigators in the GR Program Area. During the current grant cycle, funds from the JCCC in the form of CCSG Developmental Funds, institutional support and philanthropic gifts to the GR Program Area total $832,185. These funds supported Interdisciplinary Grants, Seed Grants, recruitment/retention, Program Area Leadership support, funding for the use of emerging Shared Resources and trainees. Twelve of the Program Area Members were the recipients of JCCC support. The Gene Regulation Program Area is completing its first full CCSG cycle. The initial goal of the Program Area was to bring together the strong existing group of gene regulation researchers at UCLA and to develop a collective interest in the application of gene regulation studies to problems of cancer origin, development, and therapy. The original group of 12 has grown to 21 members, in large part by recruitment of a group of extraordinary young investigators as Assistant Professors. Substantial investment by the JCCC, the DGSoM, and the College of Letters and Science has benefited this Program Area enormously. During the current CCSG cycle, we have focused on four major objectives: (1) to promote among our members the study of fundamental mechanisms of gene regulation, using appropriate model organisms, mammalian cells, and animal models; (2) to study the molecular/transcriptional mechanisms that mediate cell differentiation, nflammation and viral latency and investigate how aberrations in these processes affect cancer initiation and progression; (3) to serve as a resource for the other Program Areas in the JCCC in the use of gene regulation concepts and methodologies to advance their research problems; and (4) to promote the translation of existing knowledge and new discoveries into translational and clinical applications. Interactions among members are fostered by a monthly meeting, a weekly journal club, and a seminar series that brings leaders in the field to UCLA.

DESCRIPTION (provided by applicant): Inflammation can be beneficial for a normal immune response to microbial pathogens. However, prolonged inflammation can promote tissue damage during infection and has been closely linked to a diverse range of diseases, including cancer, atherosclerosis, and several inflammatory autoimmune diseases. Although a number of anti-inflammatory drugs are available, none of them are considered to be ideal for a variety of reasons, including insufficient target specificity. Therefore, new strategies are needed for the development of selective inhibitors of pro-inflammatory genes and proteins. One major limitation in pursuing pharmaceuticals that inhibit the transcription of specific pro-inflammatory genes is that our understanding of the molecular mechanisms responsible for selective gene regulation is surprising limited. Signaling pathways such as the NF-?B and AP-1 pathways are known to contribute to the activation of many pro-inflammatory genes. However, because of their broad functions, these pathways are not appropriate targets for the selective modulation of individual genes. Because it has proved to be difficult to uncover the mechanisms of selective regulation through the use of conventional experimental strategies, we have begun to attack the selectivity question using a new strategy that should lead to a much broader appreciation of this issue, with the possibility of identifying therapeutic lead compounds. Specifically, we are generating macrophage cell lines from mice in which fluorescent protein reporter genes are regulated by cytokine gene control regions in their native chromatin environment. High-throughput screens will then be performed to identify small molecules that differentially alter the expression of key cytokine genes, including the genes encoding IL-12 p40, IL-12 p35, IL-23 p19, and IL-10. The rationale for inserting fluorescent protein reporter genes into a native chromatin environment is that our past studies have revealed that conventional promoter-reporter plasmids, often used for high-throughput screens, fail to assemble into physiologically relevant chromatin structures upon stable transfection. Furthermore, the fluorescent protein reporter assay is preferable to an ELISA assay that monitors endogenous cytokine secretion because the ELISA is susceptible to misleading effects on cytokine translation, processing, and secretion. By testing small-molecule libraries in which the molecular targets are known, we hope to gain unprecedented insight into the signaling pathways that contribute to selective gene regulation. The signaling pathways identified will then be examined in greater depth to elucidate selectivity mechanisms. Larger libraries of compounds whose targets are unknown will also be screened to gain further insight into the potential for selective regulation, with the possibility of identifying therapeutic lead compounds. PUBLIC HEALTH RELEVANCE The objective of the proposed research is to explore the feasibility of a novel high-throughput screening strategy that may lead to the discovery of small molecules capable of modulating the expression of proteins involved in inflammation. The small molecules identified will facilitate our ongoing studies of the signaling pathways that regulate the selective expression of inflammatory genes. Furthermore, the proposed screens may lead to the discovery of therapeutic lead compounds for the treatment of diseases associated with aberrant inflammation, including atherosclerosis, cancer, and a number of inflammatory autoimmune disorders.

DESCRIPTION (provided by applicant): Epigenetic properties responsible for the broad developmental potential of embryonic stem cells (ES cells), hematopoietic stem cells (HSCs), and other types of stem cells are of considerable interest because these cells are advantageous for a number of therapeutic strategies. Stem cells are of equal interest to cancer biologists because of evidence that many human cancers are caused by the aberrant expansion of cells with stem cell-like properties. Recent studies suggest that genes involved in early developmental decisions are poised for activation through their association with bivalent histone modification domains, consisting of both active and repressive epigenetic marks. However, these bivalent domains are not generally associated with typical tissue-specific genes expressed in differentiated cells. Instead, previous studies of the liver-specific Alb1 gene suggested that typical tissue-specific genes are assembled into inaccessible chromatin structures in pluripotent cells and that, during or after gastrulation, pioneer transcription factors initiate a cascade of events that promotes chromatin decondensation and ultimately leads to transcriptional activation. In contrast to this hypothesis, we recently demonstrated that well-characterized enhancers for three tissue-specific genes, Ptcra, Il12b, and Alb1, are selectively marked by unmethylated CpG dinucleotides in pluripotent ES cells. The unmethylated CpGs appear to result from the binding of transcription factors to the enhancers, even though the binding of these factors in ES cells does not lead to nuclease hypersensitivity and does not always promote histone modifications typically associated with active or silent genes. Preliminary functional studies suggest that the enhancer marks we have observed in ES cells may be critical for transcriptional activation of the tissue-specific genes in differentiated cells. In the absence of the enhancer marks, pre-methylated enhancer- promoter-reporter plasmids were resistant to transcriptional activation in differentiated cells, and were unable to establish unmethylated windows at their enhancers. These results lead to the hypothesis that enhancer marks must be established in early development because the more repressive chromatin environment found in differentiated cells is incompatible with transcriptional activation of unmarked genes. To further explore this hypothesis and better understand the establishment and maintenance of the enhancer marks in ES cells, we will continue our studies of the same three model genes used for our preliminary experiments, the thymocyte- specific Ptcra gene, macrophage/dendritic cell-specific Il12b gene, and liver-specific Alb1 gene. A major goal will be to identify the specific DNA elements required for establishment of the enhancer marks, as a first step toward rigorously analyzing the functional significance of the marks. The enhancer marks will also be evaluated in induced pluripotent stem cells (iPS), as a strategy for better evaluating the establishment and significance of the marks during epigenetic reprogramming. PUBLIC HEALTH RELEVANCE: The molecular features of embryonic stem cells, hematopoietic stem cells, and many other types of stem cells are of considerable interest because of the therapeutic potential of stem cell-derived tissues. This study will explore a recently discovered feature of mouse embryonic stem cells that may be critical for their unique properties.

DESCRIPTION (provided by applicant): Ikaros is the founding member of a small family of C2H2 zinc finger DNA-binding proteins and has been found to play critical roles in lymphocyte development and tumor suppression. Like many of the approximately 800 C2H2 zinc finger proteins encoded by mammalian genomes, Ikaros contains multiple tandem zinc fingers within its DNA-binding domain. Since the discovery of Ikaros in 1992, much has been learned about its biological functions from elegant studies of Ikaros mutant mice. However, its precise intracellular functions and mechanisms of action have remained elusive, largely because it does not appear to function as a typical transcription factor and because target genes responsible for its most important biological functions have been difficult to identify. We have come to realize that the only way we can fully understand its mechanisms of action, and the only way we can evaluate hypotheses that have emerged from our biochemical studies, is to study Ikaros domains and biochemical activities in a native physiological setting. Toward this end, we have introduced specific mutations into the endogenous Ikzf1 locus, which encodes Ikaros. Two mutant strains have been characterized to date. These two strains contain deletions of exons encoding the first and last zinc fingers of the DNA-binding domain. These mutant mice were created (1) to test a hypothesis that these two fingers regulate binding to distinct sets of target genes, (2) to facilitate the discovery of new Ikaros target genes, and (3) to contribute to the broader C2H2 zinc finger field by exploring the biological reason for the existence of tandem arrays of zinc fingers. Importantly, we have found that each mutant strain exhibits a select subset of the phenotypes previously described in Ikaros null mice, providing strong evidence that the two fingers regulate different target genes and biological functions. In Aim 1, we will further characterize selective phenotypes of the mutant strains to better understand how these two fingers differentially regulate previously described target genes. In Aim 2, an unbiased approach that takes advantage of the selective phenotypes of the mutant strains will be used to identify and characterize new Ikaros target genes. Finally, we will characterize a third mutant mouse strains that lacks a key residue involved in Ikaros multimerization, and we will begin to use a similar approach to examine the functional significance of two co-repressors that are known to interact with Ikaros.

Description Embryonic stem cells (ESC) and induced pluripotent stem cells (IPSC) hold great promise for the treatment and study of debilitating diseases. Stem cells are also of considerable interest to cancer biologists because of evidence that many human cancers are caused by the aberrant expansion of cells with stem cell-like properties. Although considerable knowledge has been acquired, much remains to be learned about the fundamental molecular properties of stem cells and of the pluripotent state, which is defined as the capacity to differentiate into nearly all cell lineages. From a gene regulation perspective, most studies of pluripotency have focused on, 1. master transcriptional regulators of pluripotency and the gene networks controlled by these regulators, 2. bivalent histone modification domains that characterize the promoters of genes involved in early developmental decisions, and 3. fundamental differences in chromatin structure that distinguish pluripotent cells from differentiated cells. Recently, evidence has emerged from our lab and others that the marking of enhancers for typical tissue-specific genes by pioneer transcription factors and unmethylated CpG dinucleotides may also be critical for establishing or maintaining the pluripotency state. It has been hypothesized that these enhancer marks provide competence for transcriptional activation in differentiated cell types. The goals of the proposed research are to better understand how tissue-specific enhancers are marked in ESC and to rigorously examine the functional significance of these enhancer marks. In Aim 1, bacterial artificial chromosomes (BACs) will be used to identify and characterize DNA motifs and transcription factors that positively and negatively regulate the establishment of unmethylated windows observed in ESC and IPSC at well-characterized tissue-specific enhancers. By working closely with the other three project Pis, we will gain further insight into the relevance of the enhancer marks by characterizing their conservation between human and mouse ESC and IPSC, by examining the timing of their establishment during reprogramming, and by evaluating their organization within the overall 3-dimensional nuclear architecture of pluripotent cells. In Aim 2, the functional relevance of the enhancer marks will be examined through the conditional recruitment of repressive chromatin complexes capable of erasing the marks in ESC. In these experiments, we will test the hypothesis that erasure of the marks in ESC compromises transcriptional activation of the tissue-specific genes following differentiation. Finally, using the same tools that are generated for these experiments, we will assist Dr. Zaret with the goals of his project (Project 2) by evaluating the ability of pluripotency factors to gain access to silent chromatin assembled in vivo through the action of different repressive chromatin complexes. There will be no human or animal experimentation with the proposed work.

The goals of this research are to elucidate transcriptional networks that participate in the immune response to infection by Mycobacterium leprae (mLEP) and to devise therapeutic strategies that enhance the immune response by manipulating these networks. Many mLEP patients develop a self-limiting tuberculoid form of the disease (T-lep), in which an effective immune response is dependent on transcriptional networks in macrophages activated by Toll-like receptor ligands. Type II interferon (IFN-gamma), and IL-15. These stimuli drive the transcriptional activation of genes encoding key antimicrobial peptides and cytokines that promote a robust T helper 1 response. Vitamin D plays a major role in enhancing this response, as demonstrated by the increased susceptibility of vitamin D-deficient individuals to bacterial infection. In other patients, disseminated lepromatous (L-lep) lesions develop that appear to be caused, in part, by immune suppression by the Type I IFN, IFN-Beta. Projects 1, 2, and 4 of this application will focus on microbial stimuli that drive the mLEP response, proteins induced by IFN-gamma and IFN-Beta that either enhance or suppress the antimicrobial response, and vitamin D metabolic pathways as they pertain to antimicrobial immunity. In this Project, we hypothesize that elucidation and dissection of transcriptional networks activated by stimuli that either catalyze or suppress the immune response to mLEP infection will lead to key regulatory nodes and mechanisms that can be targeted by novel therapeutics strategies. This hypothesis is based on emerging excitement about the potential of chromatin and epigenetic regulators as therapeutic targets. We propose to: 1) use high-throughput sequencing analysis of transcriptional networks (RNAseq) to understand how multiple stimuli help shape the effective and ineffective immune responses that characterize T-lep and L-lep lesions; 2) elucidate the mechanism by which vitamin D suppresses the expression of important cytokines like IL-12 while promoting the expression of key antimicrobial peptides, towards the goal of uncoupling these activities in a therapeutic setting; 3) elucidate the mechanisms that distinguish the Type I and Type II IFN responses, towards the translational goal of converting the suppressive Type I response into an effective Type II response. These studies have considerable potential to translate basic knowledge of gene regulation circuitry into disease therapies and will provide knowledge of transcriptional networks that will be of great value to the goals of the entire CORT team and to other basic and translational researchers studying antimicrobial immunity.

This project will significantly improve the learning environment for thousands of life science majors by converting introductory courses in biology and math to student-centered modes of instruction. Instructor teams will be trained to use interactive video technology to implement active learning pedagogies in core biology courses. The project team also will design a new math curriculum that incorporates biology-relevant content and computational applications, supplemental instruction opportunities for underrepresented racial minority students, and student-centered teaching methods demonstrated to positively impact students most at risk for dropping out of science. Expected outcomes include more students motivated and prepared to explore diverse science careers, fewer students under-performing in their introductory coursework, and a more diverse undergraduate pool completing science and math degrees. Project activities also will produce research faculty who are more engaged in assessing student learning as well as future faculty (teaching fellows) who can serve as advocates of student-centered teaching at other institutions. To augment these activities, a new position will be created within the UCLA Career Center that promotes student interest in career alternatives to medicine and awareness of emerging careers in science. The project evaluation plan will provide new knowledge about the impact of interactive videos on student learning, the effectiveness of student-centered teaching on knowledge retention and persistence in science majors, and the support structures necessary to engage research-focused faculty in curricular reform. All of these activities will incorporate faculty mentoring strategies and incentives aimed at surmounting institutional barriers that tend to inhibit educational reform.