Cornelis Murre - US grants

DNA binding protein; protein structure function; gene expression; cell differentiation; immunoglobulin genes; B lymphocyte; virus protein; protein sequence; protein purification; intermolecular interaction; genetic transcription; crosslink; monoclonal antibody; tissue /cell culture; radiotracer; genetic manipulation; laboratory mouse; electroporation; transfection; affinity chromatography; ultraviolet radiation;

Although large series of genes have been identified that are translocated in human leukemic cells, the mechanism how they are involved in the promotion of leukemias is poorly understood. The objective of this proposal is focused on pbx-1, a recently identified DNA binding protein that contains a homeobox. The gene encoding pbx-1 is consistently rearranged in pediatric pre-B acute lymphoblastoid leukemia (ALL) due to a t(1;19) chromosomal translocation. Specifically, in pre-B ALL, the pbx-1 N-terminal domain has been replaced with a transactivation domain derived from E2A, normally a helix-loop-helix protein. The consistent presence of a translocated pbx gene in pre-B ALL makes it likely that the E2A/pbx fusion protein contributes to the malignant transforation of human pre-B cells. We propose in this research application to understand the mechanism by which E2A/pbx-1 promotes the aberrant growth of human pre-B cells. We have recently found that pbx binds DNA either as a homo- or as a heterodimer with homeobox proteins. We propose to continue these studies. First, we will determine the ability of pbx proteins to regulate gene expression in lymphoid cells. Next we propose to map the pbx domains and residues important for regulation of critical pbx target genes in lymphoid cells. We propose to characterize pbx proteins in normal and transformed B lymphocytes, in particular in cells derived from patients with pre-B All. The oncogenic potential of both wild-type and mutant-pbx proteins will be tested using transformation assays. To determine how E2A/pbx promotes lymphoid malignancies we will test the ability of wild-type and mutant E2A/pbx molecules to induce leukemias in mice. These studies should provide important information of how pbx molecules contribute to childhood leukemias and may suggest new avenues for the treatment of pre-B cell ALL.

DESCRIPTION (Adapted from the Applicant's Abstract): The overall objective of the research proposed in this application is to understand the molecular mechanisms that drive immunologic processes. It has become clear that the events that control lymphoid differentiation are a result of changing patterns of gene expression. The studies proposed here are focused on a class of transcription factors, helix-loop-helix proteins, that are in part responsible for the alterations in gene expression during B cell development. One particular class of helix- loop-helix (HLH) proteins are the E-proteins, that include E12, E47, E2-2 and HEB. E12 and E47 are encoded by one gene, E2A, and arise through differential splicing. E2-2 and HEB are encoded by different genes. The applicant showed previously that E2A gene products are required for proper B cell development. Differentiation of B cells in E2A mutant mice is blocked at a stage prior to immunoglobulin (Ig) gene rearrangement. Some B cell specific transcripts including B29 and Ig germline transcript - are expressed. Others, including RAG-1, RAG-2, mb-1, l5, VpreB and CD19 are absent. In addition, two transcription factors, including Pax -5 and EBF, implicated in the regulation of CD19 and mb-1 respectively, are absent in E2A (-/-) mice. These data suggested that E2A is a central regulator in early B cell development. It is proposed to continue these studies. The specific aims of the are focused on a further understanding of the role of E-proteins in B cell development. Specifically, the applicant plans to examine the individual roles of E12 and E47 in B cell differentiation. He will examine whether E12 or E47 regulate PAX-5 and EBF expression. The individual roles of Pax-5 and EBF in generating the E2A deficient phenotype will be studied. The role of E2-2 in lymphoid development will be investigated using gene targeting. It is planned to use transgenic mice to further dissect the contributions and redundancies of E-proteins in B cell development. Finally, the applicant will perform a genetic analysis of transcription factors in B cell development, including E-proteins, Oct-2 and PU.1.

Although large series of genes have been identified that are translocated in human leukemic cells, the mechanism how they are involved in the promotion of leukemias is poorly understood. The objective of this proposal is focused on pbx-1, a recently identified DNA binding protein that contains a homeobox. The gene encoding pbx-1 is consistently rearranged in pediatric pre-B acute lymphoblastoid leukemia (ALL) due to a t(1;19) chromosomal translocation. Specifically, in pre-B ALL, the pbx-1 N-terminal domain has been replaced with a transactivation domain derived from E2A, normally a helix-loop-helix protein. The consistent presence of a translocated pbx gene in pre-B ALL makes it likely that the E2A/pbx fusion protein contributes to the malignant transforation of human pre-B cells. We propose in this research application to understand the mechanism by which E2A/pbx-1 promotes the aberrant growth of human pre-B cells. We have recently found that pbx binds DNA either as a homo- or as a heterodimer with homeobox proteins. We propose to continue these studies. First, we will determine the ability of pbx proteins to regulate gene expression in lymphoid cells. Next we propose to map the pbx domains and residues important for regulation of critical pbx target genes in lymphoid cells. We propose to characterize pbx proteins in normal and transformed B lymphocytes, in particular in cells derived from patients with pre-B All. The oncogenic potential of both wild-type and mutant-pbx proteins will be tested using transformation assays. To determine how E2A/pbx promotes lymphoid malignancies we will test the ability of wild-type and mutant E2A/pbx molecules to induce leukemias in mice. These studies should provide important information of how pbx molecules contribute to childhood leukemias and may suggest new avenues for the treatment of pre-B cell ALL.

[unreadable] DESCRIPTION (provided by applicant): The overall objective of the research proposed in this grant application is to examine the regulation and function of E-proteins in T cell development and lymphomagenesis. E2A and HEB are helix-loop-helix proteins that play important roles throughout T-lineage maturation. Additionally, E2A and HEB function as tumor suppressors. In developing T cells the E2A proteins activate RAG2 gene expression, induce TCR beta gene rearrangement and regulate cell cycle progression. During the pre-TCR and alpha/beta TCR checkpoints, E2A/HEB DNA binding activity is controlled by the ras-Erk MAP kinase pathway. The regulation of E2A DNA binding activity is mediated, at least in part, through the induction of Id3 gene expression and by modulation of E2A protein synthesis. Here we propose to continue these studies. We would investigate the individual roles of E12, E47 and HEB in T-lineage development, lymphomagenesis and cell cycle progression. We would identify E12 and E47 target genes required to promote developmental maturation and cellular proliferation. We would examine mechanistically how E2A proteins regulate cell cycle progression both in vitro and in vivo. We would further define the pathway leading to the activation of Id3 expression. We would examine whether ras mediated transformation requires Id3 gene expression. Furthermore, we would examine how E2A protein synthesis is modulated during T-lineage development. Overall the dissection of the roles of E-proteins and Id3 in T-lineage development should help to clarify the mechanism of how HLH proteins regulate T cell homeostasis and lymphomagenesis. [unreadable] [unreadable]

Although large series of genes have been identified that are translocated in human leukemic cells, the mechanism how they are involved in the promotion of leukemias is poorly understood. The objective of this propose is focused on a class of homeodomain proteins, designated as TALE proteins. The TALE proteins include Pbx, Meis and Prep-1. The gene encoding pbx-1 is consistently rearranged in pediatric pre-B acute lymphoblastoid leukemia (ALL) due to a t (1;19) chromosomal translocation Specifically, in pre-B ALL, the pbx-1 N-terminal domain has been replaced with a transactivation domain derived from E2A, normally a helix-loop-helix protein. The consistent presence of a translocated pbx gene in pre-B ALL makes it likely that the E2A/pbx fusion protein contributes to the malignant transformation of human pre- B cells. We have recently identified a down-stream target gene of E2A/Pbx-1 encoding a growth factor, designated Wnt-16. We propose in this research application to understand the mechanism by which E2A/pbx-1 promotes the aberrant growth of human pre-B cells and how Wnt-16 contributes to the development of the leukemia. First, we would characterize Pbx and Prep-1 in normal. We would examine how Wnt-16 is regulated by E2A/Pbx-1. The oncogenic potential of Wnt-16 would be tested in cell lines employing dominant-negative LEF and Wnt-gene products. To determine how Wnt-16 promotes lymphoid malignancies we would test the ability of Wnt-16 to induce leukemias in mice. These studies should provide important information of how pbx molecules contribute to childhood leukemias and may suggest new avenues for the treatment of pre-B cell ALL.

EXCEED THE SPACE PROVIDED. The overall objective of the research proposed in this grant application is to continue our analysis the molecular pathways that regulate B lineage commitment and maturation. Our studies would be focused on the regulation and function of a subset of helix-loop-helix proteins, named E-proteins, in B-lineage development. E-proteins, E12,E47, HEB and E2-2, function at multiple stages thorughout B cell development. Specifically, E-proteins act:(1) to activateB- lineage specific gene expression, (2)to control Ig light chain gene rearrangement, (3)toregulate receptor editing, (4) to enforce the developmental checkpoint at the immature-B cell stage, (5)to regulate marginal zone versus follicular zone B cell development and (6) to induce AID gene expression. During the next grant cycle we would continue these studies. In particular we would investigate the relationship involving E2A, EBF and Pax-5 in early B lineage development. We would examine how E2Aproteins activateand repress transcription and how they regulate Ig light chain gene rearrangement. We have recently demonstrated that theE2A proteins have the ability to repress transcription by recruitment of a transcriptional co-repressor, named ETO-2. We would examine the function of ETO-2 during B cell development and how the activity of ETO-2 is regulated. We have also recently determined using mass spectrometry two serine residues in E12and E47that are modifiedby phosphorylation and one lysine residue modified by methylation. We would introduce mutations in these residues and examine their impact on the activity of E2A both in vitro and in the mouse germ-line. We would further characterize the role of E-proteins in the regulation AID transcription. Overall the dissection of the roles of E2A in B cell development should help to clarify the mechanism by how E-proteins regulate Blineage maturation.

DESCRIPTION (provided by applicant): The overall objective of the research proposed in this grant application is to determine the molecular pathways that act to initiate and complete T cell commitment. Recently we have isolated E2A-deficient hematopoietic progenitor cells that can be grown long term in vitro. Reconstitution of lethally irradiated mice with E2A-deficient hematopoietic progenitor cells leads to the development of T, NK, dendritic, myeloid and erythroid cell lineages, indicating that these cells are pluripotent. E2A deficient hematopoietic progenitor cells simultaneously express low levels of transcripts that encode for transcriptional regulators that contribute to the development of various hematopoietic cell lineages. These include EBF, Pax-5, TCF-1 andGATA-3. Enforced expression of E47 into E2A deficient hematopoietic progenitor cells leads to the activation of B-lineage specific gene expression whereas the transcription of factors involved in the commitment and development of alternative lineages is repressed. To determine how Notch signaling and E-protein activity regulate T lineage commitment, we have begun to examine how the combined activities of E-proteins and Notch signaling regulate down-stream target gene expression. Our preliminary studies show that both E47 and Notch-ICN directly activate HES1 gene expression in uncommitted E2A-deficient hematopoietic progenitor cells. Additionally, GATA-3 and TCF-1 gene expression is, albeit indirectly, regulated by Notch-ICN. In this grant application, we propose to continue these studies. We would determine how Notch signaling and E-proteins act to promote T-lineage commitment. We would identify target genes and enhancer elements that are regulated by Notch-ICN and E-proteins. We would examine how E-proteins and Notch mediated signaling act in concert to induce HES1 gene expression. We would examine the contributions of HES-1 and GATA-3 in promoting T-lineage maturation. We would employ both in vitro and in vivo approaches to clarify the role of Notch-ICN and E-proteins in T-lineage commitment. Overall the dissection of the roles of Notch signaling and transcriptional regulators should clarify the mechanisms by which T cell commitment is initiated and completed.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The E2A proteins E12 and E47 are expressed throughout B-cell development to activate B-cell specific gene expression and immunoglobulin gene rearrangement. Here we propose to examine the cellular localization of E12 and E47 during distinct stages of B-cell development utilizing biarsenical-tetracysteine labeling. E12 and E47 are transcriptional regulators that control developmental progression, cellular expansion, survival and gene rearrangement in developing lymphoid cells. How E12 and E47 regulate this diverse set of biological activities is unknown. Additionally it is unclear where E12 and E47 are localized during the distinct stages of lymphocyte development. In this application we propose to tag E12 and E47 utilizing the biarsenical-tetracysteine epitope. We would examine localization by fluorescence as well as EM studies. Ultimately we would like to combine electron microscopy and 3D-FISH to examine the localization of these transcription factors in the immunoglobulin locus. Data obtains from the studies may obtain insights into the mono-allelic activation of immunoglobulin gene rearrangement.

DESCRIPTION (provided by applicant): The E2A gene products belong to a class of helix-loop-helix (HLH) proteins, also named E-proteins. Members of the E-proteins include E12, E47, E2-2 and HEB. The DNA binding activities of the E- proteins are regulated by a distinct class of antagonistic HLH proteins, named Id gene products. The E2A proteins act at multiple stages during B cell development. In the lymphoid-primed multipotent progenitor and common lymphoid compartments, the E2A gene products act together with PU.1, EBF and Pax5 to specify a B cell fate. They maintain the expression of EBF and Pax5 in pro-B cells. In pre-B cells, the E2A proteins activate Ig: VJ gene rearrangement. In the immature-B cell compartment the E2A proteins induce receptor editing. E2A protein levels decrease upon innocuous BCR expression whereas E2A levels remain high in response to self-antigen. E2A abundance at this stage is regulated through a post-transcriptional mechanism that involves differences in protein stability. At the mature-B cell stage E2A levels are low but are elevated in response to BCR or TLR- mediated signaling, to promote class switch recombination (CSR). Here we propose to further analyze the activities of the E2A proteins at the immature- and mature-B cell stage. We would measure E12 and E47 turn-over rates in immature-B cells, expressing either an innocuous or self-reactive receptor. We would determine the individual roles of E12 and E47 at the immature-B cell stage. We would examine how the E2A proteins modulate receptor revision by identifying E2A targets in B cells expressing either an innocuous or self-reactive receptor. We would complement this analysis by performing a genome-wide screen for E2A binding sites. We would determine how the E2A proteins act with other families of transcriptional regulators to modulate down-stream target gene expression using a bioinformatics approach. We would examine the regulation and role of E-proteins in CSR. We would identify E2A binding sites, using a genome-wide screen, in naive and activated B cells. Our ultimate goal would be to identify the cis- regulatory codes that underpin the response to either tonic or auto-reactive or non-self reactive BCR- mediated signaling. PUBLIC HEALTH RELEVANCE: It is well known that white blood cells develop in the bone marrow from stem cells. The helix-loop- helix proteins, E12 and E47, program white blood cells to make antibodies. In the studies proposed here, we aim to determine how the helix-loop-helix proteins function in the suppression of autoimmunity.

DESCRIPTION (provided by applicant): The long-range goals of the research proposed in this application are to determine at high resolution the 3D-structure of the immunoglobulin heavy chain (Igh) locus. The Igh locus is organized into distinct regions that contain multiple variable (VH), diversity (DH), joining (JH) and constant (CH) coding elements. To probe the topography of the Igh locus, we have recently determined the spatial distance distributions using 12 genomic markers that spanned the entire locus. These spatial distance distributions were compared to computer simulations of alternative chromatin arrangements. This analysis predicted that the Igh locus is organized into compartments containing clusters of loops separated by linkers. We then used computational geometry to determine the mean relative 3D-positions of the VH, DH, JH and CH elements. Briefly, the data showed that during early B cell development, the entire repertoire of VH regions (2.5 Mbp) is merged and juxtaposed to the DH elements, allowing the VH regions to encounter DHJH elements with relatively high and similar frequencies. Here we propose to continue these studies. We would describe at high resolution the average Igh locus trajectories in interphase and mitotic chromatin. We would determine the spectrum of conformations adopted by the Igh locus fiber. We would use structured illumination microscopy to visualize the Igh chromatin territories. We would characterize compartments using physical approaches. We would identify loop bases using both physical and molecular biological approaches. We would use spatial distance measurements and computational geometry to determine whether the Igh locus structure is a general feature of antigen receptor loci and eukaryotic interphase chromatin. Taken together, these studies would provide a statistical description of Igh locus structure and provide mechanistic insight into how antibody diversity is generated. PUBLIC HEALTH RELEVANCE: In previous studies we have used geometry to show how a genetic locus is organized in 3D space. The studies proposed in this application should provide insight into how the structure of the genome permits the generation of an immune response to a wide variety of invading pathogens.

DESCRIPTION (provided by applicant): The E2A gene products belong to a class of helix-loop-helix (HLH) proteins, also named E-proteins. Members of the E-proteins include E12, E47, E2-2 and HEB. The DNA binding activities of the E- proteins are regulated by a distinct class of antagonistic HLH proteins, named Id gene products. Four Id genes are present in the mammalian genome, designated as Id1-4. E47 levels are high in T cell progenitors where they act to promote TCR2 gene rearrangement, induce the expression of genes involved in pre-TCR signaling and suppress proliferation. Once a pre-TCR has been generated, E47 levels decrease to permit developmental progression and cellular expansion. E47 expression also prevents the development of lymphoma in thymocyte progenitors. In this grant application we would examine how E- and Id protein activities are regulated to control developmental progression and suppress the development of lymphoma. PUBLIC HEALTH RELEVANCE: During previous studies we have demonstrated that a class of DNA binding proteins, named E- proteins are important for the development of white blood cells. We have also shown that the E- proteins make sure that white blood cells do not multiply inappropriately in healthy individuals. But mutations can make the E-proteins inactive. Such mutations can ultimately result in the development of lymphoid malignancies. The proposed studies would provide insight into how mutations in the genome of white blood cells lead to the development of lymphoma and should provide novel strategies for the treatment of human T cell leukemias.

Recent studies have provided substantial evidence that the genome consists not merely of linear structures but rather it is folded into elaborate patterns that permits interactions between genomic elements separated by large genomic distances. The precise folding and the spectrum of topologies of the genome remain to be determined. Recent studies have provided insight into the 3D-architectures of the immunoglobulin heavy chain (Igh) and ¿-globin loci. These data have revealed that these loci are organized into multiple compartments that are characterized by bundles of loops, consistent with the Multiple-Loop-Subcompartment (MLS) Model. We are now faced with the question as to whether the entire genome is folded as proposed by the MLS model. To address this question, we have recently employed a formaldehyde cross-linking approach that permits the identification of a genome-wide network of interacting genomic elements in pro-B cells. Our preliminary data indicates that the pro-B cell genome indeed is organized into clusters of loops that are connected by linkers as predicted by the MLS model. Here we would propose to continue these studies. We would perform an in depth analysis of individual compartments and examine how they are organized and how they relate to B-lineage specific programs of gene expression. We would identify and characterize intra-chromosomal interactions. Insulators and bridging factors would be identified and characterized both from a biochemical and functional perspective. Finally, we propose to determine the structure of mitotic chromosomes in pre-pro-B and pro-B cells. The overall goal would be to decipher the spectrum of conformations and trajectories formed by the pre-pro-B and pro-B cell chromatin fibers and how chromatin structure relates to function.

DESCRIPTION (provided by applicant): The specific aim of this proposal is to obtain partial support for a Summer Research Conference (SRC) sponsored by the Federation of American Societies for Experimental Biology (FASEB) on the subject of Molecular Mechanisms of Immune Cell Development and Function. This conference, originally titled Lymphocytes and the Immune System: Molecular, Cellular, and Interactive Mechanisms, has been chosen for sponsorship by FASEB after a competitive re-evaluation every other year since its inception in summer 2003. The distinctive features of this conference are the following. (1) The meeting places strong emphasis on molecular biology of gene regulation in lymphocyte development, including new insights emerging from genome-wide approaches. (2) Molecular mechanisms guiding immune receptor gene mutagenesis are an emphasis, explaining the basis of recognition specificity and also establishing precedents for long-range genomic control mechanism. (3) The program deliberately juxtaposes research on different classes of immune cells, so that mechanisms underlying developmental choices and larger patterns of shared use of particular molecular mechanisms can both be seen. (4) Many presenters have an explicit focus on gene network and signaling network connections rather than one gene at a time studies, to provide the most comprehensive available explanations of biological regulation. (5) The schedule integrates presentations from scientists at many levels, from internationally renowned experts to junior postdoctoral and graduate student speakers, in an intensive, all-plenary program. As a result, young trainees have uncommonly easy access to experienced senior figures, who can gain an appreciation for the young people's individual ideas. This meeting has received enthusiastic feedback from conference attendees of the previous meetings and a strong mandate for long-term continuation. The present application would provide needed support for the conference in the summer of 2013, which is the 6th conference in this highly successful series. At this time when funding is tight, many potential attendees, especially junior faculty, simply cannot afford to come unless they can receive some kind of travel or registration support, no matter how exciting their results may be. The funds requested would supplement limited funds provided by FASEB, as well as whatever contributions we can solicit from private industry. This support is crucial to make it possible to cover the costs of registration for invited speakers and to offer travel awards for a select group of junior scientist presenters.

The objectives of the research proposals in this application are to determine at a global scale the mechanisms that underpin the ?? versus ?? lineage choice. Many of the experiments proposed in the application involve genome-wide analyses and interpretation of the data obtained from such analyses. The purpose of the Genomics Core is to provide the laboratories with the following services that will permit the proposed studies to be completed. The Genomics Core would perform HiC, ChlP-Seq and RNA-Seq analyses, followed by bioinformatics analysis. Thus the entire spectrum of genome-wide analyses that concerns this POl would be performed at the UCSD Genomics Core Facility. Why a centralized Genomics Core? There are three reasons: (1) Except for the Murre laboratory, none of the participating laboratories have expertise in ChlP-Seq and RNA-Seq. (2) The Zhuang, Wiest and Zuniga-Pflucker laboratories do not have access to the bioinformatic infrastructure and analytical tools that have been established at UCSD. (3) The genomics core would permit us to standardize the ChlP-Seq and RNA-Seq analysis. This means using the same antibodies, same lot number and the same approach thereby allowing a careful and consistent comparison of the different binding patterns that will be derived from the various studies. (4) The Genomics Core will provide training to visiting research fellows. Thus, we envision that graduate students and postdoctoral fellows from the other laboratories would visit UCSD in order to become familiar with global approaches and analyses. Accordingly, an integral part of our approach is to provide personnel from different institutions access to these global analysis tools and provide training.

The long-range goals of our studies are to understand the mechanisms that enforce the ??T and pre-TCR checkpoints. We would like to describe these checkpoints in terms of global networks involving transcriptional regulators, signaling components and survival factors. Previously, we as well as others have demonstrated that E- and Id-proteins play critical roles in enforcing the ??, pre-TCR and TCR checkpoints. Most prominent among the E-proteins are the E2A gene products, E12 and E47. Two additional members that are closely related to E2A, named E2-2 and HEB, also belong to the E-protein family. The DNA binding activities of E-proteins are attenuated by a subset of helix-loop-helix (HLH) proteins, named ld1-4. E-proteins levels are high in T cell progenitors where they initiate TCRy, ? as well ? locus rearrangement, activate the expression of genes encoding for proteins involved in Notch- and pre-TCR signaling arid antagonize proliferation. Once a ?? or pre-TCR complex is assembled, IdS levels are elevated to suppress E2A DNA binding. Here we propose to continue these studies. We would use functional studies, genome-wide analyses and computational approaches to determine the mechanisms that underpin ?? versus ? selection. We would identify factors that cooperate with E-proteins to enforce the pre-TCR checkpoints. We would examine whether gradients of E-protein activity modulate DNA binding site preferences as well as enhancer repertoire selection beyond the pre-TCR checkpoint. We would examine how Notch- and pre-TCR signaling act in concert to modulate binding site as well as enhancer selection. We would describe ?? T cell development in terms of global networks of enhancer repertoires, interacting transcription factors, signaling components and survival factors. We would examine how differences in signaling by the pre- TCR and ?? TCR affect E2A occupancy and binding site selection. Finally, we would examine how these networks change during developmental progression and how such changes relate to enforcement of the ?? and pre-TCR checkpoints. RELEVANCE (See instructions): It has been established that an important population of cells, named ?? T cells, play critical roles in preserving epithelial structures that function as barriers. The proposal described here is aimed to understand the molecular mechanisms that promote their developmental progression. These studies may permit novel avenues for the treatment of immune diseases and interference with the development of malignancies.

DESCRIPTION (provided by applicant): It is now well established that in common lymphoid progenitors (CLPs), the E2A proteins act to induce the expression of EBF1 to establish B cell fate. However, hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) also express high levels of E2A, yet the EBF1 locus remains transcriptionally silent. These observations have raised the question as to why EBF1 expression is not activated by the E2A proteins in multipotent progenitors. Recent High-Throughput Chromosome Conformation Capture (Hi-C) studies have provided unexpected insights into this question. These studies showed that in multipotent progenitor cells the EBF1 locus is sequestered at the nuclear lamina. However, upon developing into pro- B cells the EBF1 locus relocates from the nuclear lamina to the transcriptionally permissive compartment in pro-B cells. Thus, we are now faced with the question as to how the EBF1 locus is sequestered at the nuclear lamina and how their release from the heterochromatin is regulated during the progression of developing hematopoietic progenitors. Factors that control the nuclear location of these key developmental regulators are the key to understanding how multipotency is enforced and how B and T lineage development is initiated. Here we propose to examine how sequestration of the EBF1 locus to the nuclear lamina relates to the enforcement of multipotency. We would describe in mechanistic terms how the EBF1 locus relocates from the lamina to the euchromatic compartment to orchestrate B cell fate.

? DESCRIPTION: The complete sequencing of the human genome has provided an unprecedented opportunity for the study of the structure and function of the human genome. While our genome has historically been viewed as a linear sequence of bases, it has progressively become clear that this is an inadequate way to represent our genetic information. Notably, research over the last 30 years has begun to shed light on the fact that the higher-order, 3-dimensional organization of our genome plays a critical role in the interpretation of the genetic information encoded in our genome. The structure of our genome in the nucleus has been clearly demonstrated to play influential roles in diverse nuclear processes including DNA replication and gene expression. Despite this, our understanding of the structure of our genome within the nucleus remains incomplete. The reasons for this include limitations in the resolution and throughput of existing tools in chromatin topology mapping, a scarcity of the analytical tools for studying genome structure datasets, and the difficulty to relate the nuclear structure to function. Due to recent advancements in molecular methods based on high-throughput DNA sequencing, single cell analytical approaches, and high-resolution microscopy, the time for breaking through these previous limitations has come. We will establish a highly collaborative, innovative team in order to develop the tools necessary to transform our understanding of chromatin architecture and function in mammalian cells. We will begin by developing datasets that establish gold standards for the study of nuclear structure and function using genetic, biochemical and imaging approaches. We will optimize current existing technologies for mapping genome wide chromatin interactions, while also developing novel, complementary approaches for studying chromatin structure. We will also develop innovative analytical methods to interpret the chromatin structural data, unraveling principles of structural- and temporal- chromatin organization. Our highly collaborative team will draw on the diverse experiences of its members to provide a synergistic environment to push the limits of our understanding of nuclear structure. We expect that the tools and datasets generated through the proposed research will dramatically advance our understanding of the chromatin structure and function in human cells.

PROJECT SUMMARY/ABSTRACT The development of technologies for genome wide mapping of chromatin interactions has yielded important insights into the organization of chromosomes. These studies have demonstrated that chromosomes are organized into topological domains, corresponding to genomic regions with high local interaction frequencies. Furthermore, studies of factors that contribute to higher order chromatin structure have indicated that certain regulatory elements and factors play key roles in establishing higher-order chromatin structure. What remains unclear at this time is how these structures, inferred from population averages of static chromatin interactions, are arranged in both space and time in live cell. We have recently developed live cell imaging and biophyiscal approaches to study the motion of individual loci. We will extend and use this strategy to validate finding of the structural models predicted by genome wide chromatin interaction experiments. In aim 1, we will develop methods for validating interactions between specific regulatory loci. Specifically, we will develop new experimental tools that enable tracking of multiple genomic loci with multi-color live cell imaging. We will used these tools to determine how the distributions and means of spatial distances in vivo relate to static chromatin interaction data, and identify the principles of chromatin organization. In aim 2, we will carry out experiments with multi-color live cell imaging to characterize the topological domains and individual elements using biophysical modeling. We will further use genome-editing technology to perturb the genome and determine the role of specific DNA elements in regulating chromatin dynamics and organization. The data and tools generated in this component will likely provide new insight into chromatin topology and dynamics in live cells.

PROJECT SUMMARY/ABSTRACT It is now well established that the onset of T cell development is initiated by the induction of Notch expression. During the previous grant cycle, we found that this critical step is controlled by the combined activities of E2A and HEB. Once Notch1 expression is activated, Notch signaling acts in concert with RUNX1, TCF1, E2A and GATA-3 to activate Bcl11b expression. Bcl11b next acts in concert with E2A to activate a T-lineage specific gene program. We (with Project 1) also demonstrated that E2A activates the expression of a non-coding transcript, named ThymoD, to reposition the Bcl11b enhancer from the nuclear lamina to the nuclear interior. These studies linked E-protein occupancy, non-coding transcription and Bcl11b expression into a common pathway that orchestrates the ?? versus ?? T cell fate decision. We now aim to describe, in physical and kinetic terms, Bcl11b locus movement relative to the onset of T cell development. To track genomic interactions in live lymphoid cells, we have developed a novel strategy. Specifically, we tracked the motion of DNA elements to visualize VDJ recombination in live B cells using tandem arrays of WT-TET and MUT-TET repressor binding sites. We propose to use the same strategy to describe in live T cells the trajectories of Bcl11b enhancer- promoter communication. Specifically, we will examine how Bcl11b enhancer-promoter communication is suppressed when sequestered at the nuclear lamina prior to commitment to the T cell lineage and after these loci return to the lamina upon adoption of the ?? T cell fate (with Projects 1 and 3). We will also examine how E-proteins and Notch signaling modulate Bcl11b enhancer-promoter communication to establish and maintain ?? T cell identity (with Projects 2 and 3) and how E-proteins, Notch signaling, non-coding transcription and nuclear repositioning are linked in four-dimensional space to orchestrate ?? T cell fate. Collectively these studies proposed here would be a first step towards describing the ?? versus ?? T cell fate choice in four- dimensional space and would not be possible outside of this integrated programmatic effort.

Antibodies are generated by somatic recombination involving variable (V), diversity (D) and joining (J) gene segments. During the previous grant cycle, we initiated studies aimed to visualize VH-DHJH interactions in live cells. Using single color labeling we were able to track the trajectories adopted by VH and DHJH elements. We found that VH and DHJH elements were subjected to fractional Langevin motion, in which VH and DHJH elements bounce back and forth in a spring-like fashion. However, since only a single genomic region was marked, this approach did not permit us to directly monitor VH-DHJH interactions. To directly visualize VH- DHJH encounters we developed a novel approach. We generated BCR-ABL transformed pro- B cell lines that carried tandem arrays of wild-type TET-operator and mutant TET-operator binding sites in the VH and DHJH regions, respectively. To mark the VH and DHJH regions these cells were transduced with virus expressing WT-TET GFP and MUT-TET SNAP-TAG. The results were surprising. VH-DHJH motion was severely sub-diffusive. We found that VH regions were trapped in distinct chromatin configurations that were remarkably stable (<60 minutes). Only VH regions located nearby DHJH regions had a chance for a VH-DHJH encounter. Comparison of simulated and experimental data suggested that such severely sub-diffusive motion was imposed by geometric confinement (loop domains) and phase separation/gelation (reversible cross-links within VH-DHJH and DH-JH loop domains). A caveat of these studies is that they were performed using BCR-ABL transformed pro-B cells. Here we propose to perform and extend these studies using primary pre-pro-B and pro-B cells. Specifically, we would track VH-DHJH motion but now in primary B cell progenitors. We would describe VH-DHJH motion in physical terms, including diffusion coefficients, scaling exponents, velocities and spatial confinement. We would image across long-time scales to examine how long VH and DHJH regions are trapped in distinct configurations and determine when (timing) and how (speed) such configurations change. We would measure first-encounter times as well as pairing times. We would examine whether transcriptional regulators and chromatin remodelers modulate VH-DHJH motion and first-encounter times. We would identify critical residues in RAG1 that would dictate pairing times. We would visualize VDJ recombination in live cells. Data obtained from these experiments would reveal whether and how space and time intersect to modulate the motion of paired coding and regulatory elements.