Michael Atchison - US grants

The immunoglobulin Kappa locus is transcriptionally controlled by two developmental stage-specific enhancers. In addition to transcription, these enhancers have been implicated in the processes of somatic rearrangement and somatic mutation, necessary for proper B cell development. Therefore, understanding the mechanisms of kappa locus enhancer activity is important. The intron enhancer lies within the intron separating the joining from constant region exons and is largely controlled by the availability of the active form of NF-kappa B. The 3 prime enhancer lies 8.5 kilobases downstream of the constant region and is controlled by the interplay between positive-and negative-acting sequences. We have identified many of the proteins that bind to the 3 prime enhancer to control its activity. The transcription factors PU.1, PIP, E2A, c-fos, c-jun, ATF1, and CREM bind to the central 132 basepair core of the enhancer. These proteins can assemble on enhancer DNA sequences as a higher order complex (the enhanceosome). We have identified some of the protein contacts between the various enhancer binding proteins. Some of these contacts are necessary for enhanceosome formation and for transcriptional activity. In experiments proposed here, we will determine the mechanism of specific protein interactions between various enhancer binding proteins. Specifically, interactions between PU.1, PIP, c-fos, c-jun, ATF1, CREM, and E2A will be characterized. Mutants of the PU.1 protein will be assayed for their ability to physically interact with other enhancer binding proteins. The consequences of mutations that disrupt protein interactions for enhanceosome formation and for enhancer activity will be determined. We will also study various physical properties of the enhanceosome complex. We will also study the role of protein interactions by PU.1 on hematopoietic development. Mice deficient in PU.1 lack B cells, T cells, and myeloid cells. We will insert various Pu.1 mutants with altered abilities to make various protein contacts into embryonic stem (ES) cells deficient in PU.1. The 'corrected' ES cells will be used to prepare chimeric mice and their contribution to various hematopoietic lineages will be determined. Finally, we will study the role of various positive-acting, inducible, and negative-acting enhancer DNA sequences on the developmental control of enhancer activity in transgenic mice. Many of the proteins that bind to the 3 prime enhancer are encoded by proto-oncogenes. Therefore, these studies will also be useful for understanding the roles of these proteins in oncogenesis.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Veterinary students are trained in comparison of multiple animal species and application of knowledge across species boundaries. Veterinarians are thus, attuned to the identification of animal systems that might serve as models of human disease. Recruitment of veterinarians into biomedical research careers should have a positive impact on human health. However, a relatively small number of veterinarians are actively involved in biomedical research. Exposure of veterinary students early in their training to research, is likely to result in more veterinarians pursuing biomedical research careers. The short-term training program at the UP, SVM, enables veterinary students to participate in biomedical research and thus, become familiar with career opportunities. Program students receive training in the development of research ideas, writing of research proposals, performance of biomedical research, and the presentation of data in written and oral formats. The program is entering its 13th year overall and fifth year of National Institutes of Health (NIH) funding. The program has supported 136 different students, 89 of whom have graduated from the veterinary school. Data indicate that program participants are far more likely to pursue post-graduate education than non-participants. Those that pursue post-graduate education tend to enter careers at academic institutions, or the pharmaceutical industry rather than clinical private practice. Program applicants, with the help of an advisory committee, identify faculty sponsors at the UP and write a research proposal that is well defined and addresses an interesting problem in biomedical research. Applications are reviewed with respect to the credentials of the student, merit of the research proposal, and training environment of the sponsor's laboratory. Students perform research in the mentor's laboratory during June, July, and August and participate in weekly seminars that develop skills needed for research careers and explore various career options. Ethics and the responsible conduct of research are also addressed. Students present their research orally and submit their work in the form of a written scientific manuscript. Students also participate in a school-wide research day held each Spring. Future goals include further expansion of funded slots and development of additional strategies for recruitment of veterinary students into research careers. [unreadable] [unreadable]

The POU transcription factor Oct4 is a key regulator of mammalian germline development and pluripotency of embryonic stem cells and is thus one of the most important factors in controlling the expression of genes associated with mammalian reproduction. Subtle changes in Oct4 protein levels determine differentiation of these earliest stem cells along three different lineages. The recently described plasticity of adult stem cells has sparked new and further interest in Oct4, as it may also be involved in the process by which these cells regain pluripotency. Dimer interactions are often crucial in transducing inter- and intracellular signals into biological function. Different POU dimer conformations exhibit distinct activities. The objective of the proposed work is to understand the role of Oct4 dimers in mouse germline development and embryonic stem cell potency. Studying aberrant conformations of Oct4 provide insight into embryo loss and infertility. Essentially, the proposed research will establish a firm structure - function relationship for two different Oct4 dimers. The Oct4 protein exhibits incredible diversity in the recognition of cognate DNA elements. Its DNA-binding domain, the POU domain, imparts this feature through its two structurally independent subdomains connected by a flexible linker region. To modulate Oct4 function in a precise manner during germline development, the definitive protein-protein interactions necessary for both Oct4 dimer conformations will be defined based on the molecular structure of both conformations in complex with DNA (aim 1). The role of Oct4 dimer formation in germline development and stem cell potency will be established by introducing point mutations into the endogenous Oct4 gene and by analyzing the resulting phenotypes in the developing mouse embryo (aim 2). To fully assess the function of both dimers the manner by which Oct4 POU dimer activity is regulated will be unraveled. These include intra- and inter-molecular interactions of Oct4 (aim 3). Using a powerful in vitro system recently developed in the laboratory to derive germ cells from embryonic stem cells, the role of both dimers in germ cell formation will be completely characterized, (aim 4). By this, the cellular mechanisms causing pluripotency and germline determination can be explored in defined culture conditions. These studies will allow stem cell potency and the germ cell lineage to be manipulated both in vitro and in vivo. The anticipated results will have relevant applicability to stem cell and germline biology and lead to a better understanding of human reproduction.

[unreadable] DESCRIPTION (provided by applicant): YY1 is a developmentally important transcription factor that regulates expression of numerous genes. The Drosophila Polycomb group (PcG) protein, Pleiohomeotic (PHO), bears sequence homology with YY1. We found that YY1 can functionally replace PHO as a PcG protein in vivo. YY1 expression in Drosophila results in PcG-dependent transcriptional repression and rescue of pho mutant flies. DNA binding by YY1 causes recruitment of PcG proteins and modification of histones by deacetylation and methylation. We found that YY1 sequences 201-226, when linked to the GAL4 DNA binding domain are necessary and sufficient for transcriptional repression. These YY1 residues physically interact with CtBP, PCNA and SUMO-1. We will address the following questions concerning YY1 PcG function: 1) What is the mechanism of PcG transcriptional repression by YY1? By ChIP assay we will determine the YY1 sequences needed for PcG recruitment to DNA and histone modification. In vivo rescue experiments will determine the sequences needed for organismal development. Genetic and ChIP approaches will determine the importance of PCNA, CtBP and SUMO-1 in YY1 medicated PcG repression. We will also explore the temporal requirements of YY1 DNA binding, PcG recruitment, and histone modification. 2) What is the mechanism of CtBP function in YY1 DNA binding and PcG repression? CtBP mutation results in reduced YY1 DNA binding and PcG recruitment in vivo. We will determine the effect of CtBP mutation on YY1 stability, DNA binding ability, intracellular location, and possible sequestration into a complex. EMSA and GST pull-down studies will explore the importance of CtBP sumoylation for interaction with YY1. We will use ChIP assays to characterize PcG binding to numerous PREs that bind to YY1. 3) How does YY1 function in mammalian PcG repression systems? A number of candidate mammalian PRE sequences have been identified that bind YY1 and other PcG proteins. We will use ChIP studies to determine whether CtBP, PCNA, or sumoylated proteins bind to mammalian PREs. We will use overexpression and RNAi knock-down of YY1, CtBP, SUMO-1 and PCNA to explore the effects on PcG recruitment, histone modification, and gene expression. PcG proteins are also implicated in muscle differentiation. Therefore, we will test the roles of YY1, CtBP, SUMO-1, and PCNA in differentation of myblasts into myotubes. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We propose a series of 5 annual National Veterinary Scholar Symposia with the theme: Veterinarians in Biomedical Research: Building Capacity. The goals of the Symposia are: 1) To provide national forums for veterinary students and post-graduate veterinarians engaged in biomedical research. This will provide opportunities for veterinary scientists-in-training to present their data, and to learn about state-of-the-art interdisciplinary biomedical research from renowned scientists. 2) To cultivate connections among veterinary students, fellows, faculty, government, and industrial scientists performing comparative biomedical research. The meeting will provide both formal (plenary and poster sessions) and informal opportunities for networking. 3) To engage veterinary students, fellows, faculty, and nationally recognized academic scientists, government scientists, and pharmaceutical industry scientists in a constructive dialog about the future directions of veterinary research. Lecturers presenting cutting edge biomedical research will also initiate discussions about the potential roles of veterinarian-scientists in future biomedical research teams consistent with the goals of the NIH Roadmap. 4) To provide opportunities for connections between veterinary students performing research and Directors of T32, and other post-graduate programs. There is a significant shortage of veterinary graduates applying for positions in postdoctoral research programs designed for veterinarian-scientists, and this conference will enable connections between students and Directors of training programs. These symposia will bring together hundreds of veterinary students interested in research, with these directors. We will also make concerted efforts to recruit and retain women and students from under-represented minority groups and to invite veterinarian-scientists as speakers from such groups. The above goals address our long-term objective of promoting contributions from veterinarians with advanced training in the biomedical sciences, to the study of mechanisms of disease in both animals and humans. The unique perspectives offered by veterinarian-scientists are likely to promote innovation in diagnosis and treatment, and to stimulate the development of multidisciplinary research teams consistent with the goals of the NIH Roadmap.

DESCRIPTION (provided by applicant): Nationwide there is a shortage of veterinarian-scientists involved in biomedical or public health research. Three recent reports by the National Research Council have argued for increased efforts to expand the number of veterinarians in research careers. Veterinary students receive extensive training in the comparison of multiple animal species and the application of knowledge across species boundaries. As such, veterinarians are well attuned to the identification of animal models that serve as models of human disease. Recruitment of veterinarians into biomedical research careers would have a positive impact on human health and is consistent with the NIH Roadmap. However, a relatively small number of veterinarians are actively involved in biomedical research. Exposure of veterinary students, early in their training, to biomedical research has been shown to increase the numbers of veterinarians who pursue research careers. For the past 17 years (10 years with NIH T35 support) the University of Pennsylvania School of Veterinary Medicine has administered a short-term summer research program for first and second year veterinary students to participate in research training. This program enabled 222 different veterinary students to perform biomedical research with 110 different faculty members at the University of Pennsylvania. Tracking data indicate that program graduates are far more likely to pursue post-graduate education and are nearly 8 times more likely to pursue PhD studies. In this program, veterinary students, with the help of an advisory committee, identify faculty sponsors at the University. A core of 34 well funded and experienced faculty serve as trainers, but other qualified faculty at Penn are permissible for training if they meet criteria defined by the program executive committee. With the help of their mentors, students write a short research proposal that addresses an interesting problem in biomedical research. Applications are reviewed with respect to the credentials of the student, merit of the research proposal, and training environment of the sponsor's laboratory. Students receiving funding perform research in the mentor's laboratory during the months of June, July, and August. Students also participate in weekly seminars that provide training in grant writing, data presentation in written and oral formats, and information on research training opportunities and career options. Students are required to present their research in an oral presentation and must submit their work in the form of a written scientific manuscript. Students also present their work in either poster or oral format at a school-wide research day held each Spring and present posters at the annual Merck-Merial National Conference. The program provides training in all aspects of biomedical research including development of research ideas, preparation of a grant proposal, performance of biomedical research, and presentation of results in written, poster, and oral formats.

DESCRIPTION (provided by applicant): There is an urgent need to increase the number of veterinarian-scientists with expertise in infectious disease research. Over 60% of all infectious diseases of animals can also affect humans, and incidences of new, emerging zoonotic infectious diseases are increasing. Moreover there continues to be a threat of bioterrorism, with the risk of the deliberate introduction of pathogens into the U.S. food supply. A recent report by the National Research Council points out that there is an unprecedented decline in interest in public health and biomedical research by veterinarians, and argues that society's need to protect against these threats is outgrowing the veterinary knowledge base. Our program seeks to directly address this national need through our VMD-PhD training program in infectious disease-related research. This program is contained within the umbrella of our existing VMD-PhD program which has a 38 year track record of success. Our training program includes infectious disease-related programatic structures such as Infectious disease discussion groups, infectious disease-related seminars, the annual infectious-disease retreat, a global health overview course, and externships at government public health agencies. This is coupled with infectious disease-related veterinary and graduate didactic education, rigorous biomedical PhD thesis research, and veterinary clinical training. The program is further supported by synergistic activities provided by the larger VMD-PhD and MD-PhD programs. Students receive VMD training at the Penn School of Veterinary Medicine, and PhD training within one of the Penn Biomedical Graduate Groups devoted to research in infectious disease-related research (1) Microbiology, Virology, and Parasitology, 2) Immunology, or 3) Epidemiology and Biostatistics). This application brings together 43 faculty trainers with established research programs in the above disciplines. These faculty have a rich history of predoctoral and postdoctoral training and have trained nearly 700 individuals. Throughout the program, VMD and PhD curricula are interdigitated and programs are in place to bridge the two training programs to provide maximal synergy. Extensive oversight and advising systems are also in place to provide an efficient and well structured program. In summary, we seek to address a pressing national need for more veterinarian-scientists through our VMD-PhD program in infectious disease-related research.

? DESCRIPTION (provided by applicant): B cell development requires the ordered V(D)J rearrangement of immunoglobulin (Ig) genes. Defects in Ig rearrangement lead to severe immunodeficiencies while aberrant chromosomal translocations result in leukemia and lymphoma. During development, Ig rearrangement is tightly regulated with heavy chain locus (IgH) accessibility and rearrangement occurring at the pro-B cell stage and light chain kappa locus (Ig?) accessibility and rearrangement at the pre-B cell stage. As the heavy and light chain loci are huge (up to 3.4 mb), distal V(D)J rearrangement requires an additional developmentally controlled physical contraction process to bring distal V region genes into the proximity of D and J gene segments. While it is clear that accessibility and locus contraction are developmentally regulated, the mechanistic basis for these effects is currently unclear. During the past funding period, we have identified YY1 as a critical regulator of Ig locus contraction. We demonstrated that YY1 localizes at over 20 sites spanning the Ig? locus in cells poised to undergo Ig? rearrangement, and that YY1 co-localizes at these sites with components of the condensin, cohesin, and Polycomb Group (PcG) complexes, proteins involved in large-scale chromosomal interactions. We recently also found that YY1 binds to the Ig? Cer DNA element that regulates Ig? locus contraction and recruits condensin proteins in a YY1-dependent manner. Based on our cumulative data, we hypothesize that YY1 binds to Ig loci, and that developmentally regulated interactions of DNA-bound YY1 with various components of the condensin, cohesin, and PcG complexes, as well as with CTCF, a YY1-interacting protein known to impact Ig locus contraction, regulates the temporal rearrangement of the Ig loci. By ChIP-seq approaches we will determine (1) whether developmentally regulated YY1-dependent recruitment of PcG, condensin, cohesin, and CTCF proteins is required for differential recombination of the IgH and Ig? loci during B cell maturation. Using proteomic, co-IP, and modification-specific antibodies we will (2) determine the mechanistic basis for developmentally regulated YY1 interactions with condensin, cohesin, PcG, and CTCF proteins. We hypothesize that (3) the YY1, condensin, cohesin, and PcG protein co-localization sites physically interact with each other, and with the Cer regulatory sequence to mediate locus contraction and Ig? rearrangement. We will test this using 3C assays in pro-B, pre-B, and YY1 knock-out backgrounds, and will define the mechanism of YY1 function in Ig locus contraction by expressing YY1 mutants with defined functions in YY1-null cells followed by 3D-FISH to measure locus contraction. We anticipate that our studies will provide foundational insight into the mechanisms of YY1-mediated Ig locus contraction and will result in a tremendous advances in our understanding of B cell development and immune function, as well as mechanisms resulting in leukemia and lymphoma.

DESCRIPTION (provided by applicant): The earliest developmentally regulated events that mark a gene for lineage-specific expression is still poorly understood. However, these early epigenetic and DNA occupancy events are key for regulating gene expression patterns that subsequently regulate developmental processes. The B cell developmental system is an excellent and tractable system to explore epigenetic regulatory mechanisms, as extensive analysis has led to the phenotypic and functional characterization of specific developmental stages that can be readily identified and isolated by cell surface markers. The immunoglobulin (Ig) genes are very well characterized genes that are differentially controlled during B cell development, and that utilize large scale, global regulatory mechanisms (somatic rearrangement and Ig locus contraction), as well as more localized regulatory mechanisms (enhancer activation, inducible transcription). Therefore, they are outstanding model genes to explore the molecular events regulating gene expression. We will utilize the Ig system to determine the early epigenetic events that initially target the Ig kappa locus during B cell development. We will utilize embryonic stem (ES) cell as well as complementary in vivo and ex vivo approaches to define the earliest epigenetic events that lead to large-scale Ig locus contraction, transcriptional activation, and Ig gene rearrangement during B cell development. Early events in B cell development are initiated by, and are critically dependent upon, the transcription factor PU.1. We developed a PU.1-null ES cell system expressing various PU.1 mutants that will enable us, in conjunction with conditional PU.1 knockout systems, to determine the PU.1- dependent functions important for changes in chromatin structure at the Ig kappa locus (Aim 2) and for B cell development (Aim 3). Finally, despite utilizing the same recombination machinery, the IgH and IgL loci are differentially accessible to the recombination machinery during early B cell development. Our preliminary results suggest that an important mechanism for controlling inaccessibility of the Ig kappa locus at the pro-B cell stage is binding of transcription factor STAT5 to a site that overlaps the central PU.1 binding site in the Ig kappa 3' enhancer as well as to sites flanking the intron enhancer. We will assess the consequences of STAT5 binding on enhancer activity, kappa locus transcription, and somatic rearrangement and will determine if competitive displacement represents a novel mechanism for developmental regulation of Ig gene function (Aim 4). We anticipate our studies will specifically elucidate novel regulatory mechanisms at the Ig: locus that control its developmental expression, thereby regulating the consequent progression of B cell development. On a more global level, we predict that these studies will reveal new paradigms for developmental control mechanisms mediated through a single enhancer regulatory element, thereby providing insights into the developmental disorders and/or malignancies caused by aberrant expression of lineage-specific or developmentally restricted genes. PUBLIC HEALTH RELEVANCE: Disruptions in the developmental control of gene expression result in numerous diseases. Defects in the developmental processes studied here can result in either severe immune defects, or in the development of malignancies caused by defective transcription factor function. Understanding these processes therefore directly relates to public health.

DESCRIPTION (provided by applicant): Three reports by the National Research Council of the National Academies of Science have argued for increased efforts to expand the number of veterinarians in research careers. Veterinary students receive extensive training in the comparison of multiple animal species and the application of knowledge across species boundaries. As such, veterinarians are well attuned to the identification of animal models that might serve as models of human disease. However, a relatively small number of veterinarians are actively involved in biomedical research. Exposure of veterinary students, early in their training, to biomedical research has been shown to increase the numbers of veterinarians who pursue biomedical research careers. For the past 22 years the University of Pennsylvania School of Veterinary Medicine, has administered a short-term summer research program for first and second year veterinary students to participate in research training. This program has enabled 317 different veterinary students to perform biomedical research with 134 different faculty members at Penn. Veterinary students, with the help of an executive committee, identify faculty sponsors at Penn. A core of 33 well-funded and experienced faculty serve as training mentors. Students write a research proposal that is well defined and addresses an interesting problem in biomedical research. Applications are reviewed with respect to the credentials of the student, merit of the research proposal, and training environment of the sponsor's laboratory. Students perform research in the mentor's laboratory during the months of June, July, and August and participate in weekly seminars that provide training in grant writing, data presentation in written, poster and oral formats, and information on career opportunities in academia, industry, and government. Students also participate in trips to NIH, the Philadelphia Science Center, and the Merial-NIH Veterinary Scholars Symposium. Students present their research oraly and in poster format, and must submit their work in the form of a written scientific manuscript. Thus students receive training in all aspects of biomedical research. Our outcomes data indicate that Program graduates are much more likely to pursue further education and graduate studies, and are much less likely to pursue private practice clinical careers.

DESCRIPTION: Long-distance DNA interactions are critically involved in developmental regulation of gene expression, stem cell biology, and immune functions. The immunoglobulin (Ig) genes best exemplify the importance of long- distance DNA interactions as somatic rearrangement of these genes requires linkage of DNA sequences separated by as much as 3 megabases. Similarly, Ig class switch recombination (CSR) requires formation of both constitutive and cytokine-inducible loops within switch region DNA sequences, and interactions between enhancer sequences (E? and 3'RR enhancers) that are separated by 200 kb. Although many long-distance loops have been identified at the Ig loci, the molecular mechanisms that control their formation remain unclear. We recently published the novel observation that the transcription factor YY1 controls antibody repertoires at the Ig? locus and physically interacts with condensin, cohesin, and Polycomb Group (PcG) proteins, all of which are involved in long-distance DNA loop formation. Moreover, co-localization of YY1 with these proteins within the Ig? locus suggests that YY1 controls long-distance DNA interactions. In further support, recent evidence shows that YY1 plays critical roles in long-distance DNA interactions at the IgH, Ig?, and the Th2 cytokine loci. Notably, our preliminary data demonstrate that YY1 conditional knock-out reduces CSR and dramatically ablates long-distance DNA loops required for CSR. Based on our combined data, we hypothesize that YY1 developmentally regulates CSR by both constitutive and cytokine inducible binding to DNA, and subsequent recruitment of proteins (condensin, cohesin, PcG proteins, etc.) required for long-distance DNA loop formation. We are uniquely poised to test this hypothesis in our recently developed powerful and innovative YY1 conditional knockout/reconstitution primary ex vivo splenoctye system. As YY1 is a ubiquitous protein, its role in DNA loop formation must be regulated tissue-specifically and B cell stage-specifically by either post-translational modification, or by developmentally regulated protein-protein interactions. Therefore, in addition to determining how YY1 contributes to long-distance DNA loop formation, we will identify the YY1- interacting proteins required for loop formation, and characterize the developmental and inducible mechanisms governing this process. Defining the role of YY1 in CSR will provide foundational insights into humoral immune mechanisms, and may lead to new paradigms of long-distance DNA interactions, and the translocations that drive lymphomagenesis. We have over two decades experience with YY1 function and have developed numerous unique molecular clones and genetically modified mouse lines to address YY1 function in DNA loop formation.

Hematopoietic development is an ordered process in which stem cells give rise to multiple lineages. While early progenitors can be multipotent, lineage-specific progenitors reach a stage where they become exclusively committed to that lineage. For example, B and T cell lineages differentiate from lymphoid-primed progenitors produced in the bone marrow, and exclusive commitment to the B cell lineage occurs as cells transition from the pre-pro-B to the pro-B cell stage. Despite the commitment of pro-B cells to the B lineage, we have made the surprising discovery that conditional knock-out of the ubiquitous multi-functional transcription factor YY1 in pro-B cells, results in the loss of B lineage commitment and the consequent ability to develop into the T cell lineage both in vitro and in vivo. To understand the mechanistic basis for this surprising lineage plasticity, we have developed a new lineage tracing mouse line that will enable us to determine how YY1-null pro-B cells develop into T lineage cells (de-differentiation to more primitive progenitors, or trans-differentiation), assess the potential for YY1- null pro-B cells to develop into other hematopoietic lineages, and determine if YY1-null T cells also exhibit lineage plasticity (Aim 1). Mechanistically, lineage-specific transcription factors bind to DNA and regulate gene expression prior to subsequent large-scale alterations in chromatin structure needed for lineage commitment. Rigorous studies by our laboratory as well as others indicate that despite its ubiquitous expression pattern, YY1 controls long-range chromatin interactions (LRCIs) in a lineage- specific fashion. Our findings support the hypothesis that DNA binding by lineage-specific transcription factors enables YY1 recruitment to distinct genomic loci, thereby enabling YY1 to both generate LRCIs that stabilize lineage-appropriate gene expression, and to generate repressive chromatin marks (H3K27me3) at lineage-inappropriate genes. We will thus, compare the molecular genetic phenotype (gene expression patterns, chromatin accessibility, epigenetic structure, and chromatin folding) of YY1- null pro-B cells developed into DN1, DN2a, DN2b, DN3, DP, CD4+, and CD8+ T cells, compared to wild- type T lineage cells, as well as YY1 conditional knockout T lineage cells (Aim 2). We hypothesize that in the absence of YY1, T lineage development can proceed, but LRCIs needed to stably maintain lineage- specific gene expression, and heterochromatin needed for repression of alternative lineages will fail to fully develop, potentially enabling continuing lineage plasticity. Our experiments may reveal a common mechanism for controlling lineage plasticity, vastly expanding potential applicability of directing YY1-null cells into multiple lineages.

During B cell lineage commitment, a dynamic shift of genes between transcriptionally restricted and transcriptionally permissive compartments at the pre-pro-B to pro-B cell transition results in activation of the B lineage program and repression of alternative lineage programs. While B lineage commitment is generally believed to be driven by lineage-specific transcription factors, we have made the surprising discovery that conditional knock-out of the ubiquitous transcription factor YY1 results in loss of B lineage commitment, allowing subsequent development into the T cell lineage both in vitro and in vivo. Pioneer transcription factors such as Ebf1 promote transcription of B lineages genes and repress expression of alternative lineage genes to initiate B lineage commitment, but stable commitment requires changes in chromatin structures at the pro-B cell stage. As YY1 is a key factor controlling lineage-specific gene regulatory long-range chromatin interactions (LRCIs), we hypothesize that YY1 knock-out in pro-B cells results in loss of chromatin LRCIs that stably maintain B lineage-specific gene expression. Consistent with this, we found reduction of B lineage transcripts after YY1 knock-out. YY1 can also mediate Polycomb Group (PcG) repression, and we found that YY1 knock-out resulted in increased expression of alternative lineage genes, suggesting that YY1 loss abrogates repressive chromatin structures needed to prevent expression of these genes. Thus, we hypothesize that YY1 knock-out in pro-B cells results in lost chromatin structures that stably maintain lineage-specific gene expression, as well as loss of repressive chromatin structures needed to prevent alternative lineage gene expression, thus leading to lost B lineage commitment. To test this, we will determine chromatin folding patterns, nuclear localization of key genes, chromatin accessibility, and epigenetic structures in wild-type and YY1-null pro-B cells to define the genomic structures regulated by YY1 during B lineage commitment. To determine if analogous effects of YY1 are operative in the T lineage, we will determine if YY1 loss promotes lineage plasticity of YY1-null DN3 cells. YY1 is also necessary in pro-B cells for Igk locus contraction required for rearrangement of distal Vk genes. It has been suggested that YY1 plays a structural role in regulating chromatin structures, but it is unclear if this requires the YY1 transcriptional activation, PcG, or self-association functions. We will utilize an established panel of YY1 mutants that are compromised in these functions to assess in parallel, the mechanisms of YY1 regulation of chromatin structures needed for B lineage commitment, and those needed for Igk locus contraction and Jk-Vk rearrangement. As YY1 is involved in embryogenesis and development of multiple tissue types, determining how YY1 controls genomic structures to specify B lineage commitment will provide a new paradigm for the function of a ubiquitous factor in lineage-specific development.