James R Hagman - US grants

DESCRIPTION (provided by applicant): Early B cell Factor (EBF) is an essential regulator of genes in B lymphocytes. EBF drives B cell development from early progenitors to mature B cells by activating genes encoding the pre-B and mature B cell receptors (pre-BCR and BCR). In mice lacking EBF, B cell development is arrested, immunoglobulins are not produced and EBF target genes are not activated. In the absence of EBF mb-1 (Ig-a) genes remain in hypermethylated, inaccessible chromatin. Recently, we demonstrated that EBF is required for, and indeed, initiates CpG demethylation and remodeling of nucleosomes at the B cell-specific mb-1 promoter. Remodeling of chromatin by EBF facilitates binding of the promoter by the B lineage commitment factor Pax5. Our studies identified a new 'functional'hierarchy of transcription factors that acts in addition to the 'genetic'hierarchy identified previously (EBF regulates expression of pax5). We have proposed that B lineage determination by EBF is largely a function of its essential role as a chromatin remodeling 'pioneer factor'. To understand how EBF drives B lineage determination, we propose to 1) identify gene targets of EBF that are important in B lineage specification, 2) determine how EBF initiates demethylation of the mb-1 promoter and 3) determine the molecular basis of EBF-dependent chromatin remodeling.

DESCRIPTION (provided by applicant): The Pax family of DNA-binding proteins includes essential regulators of tissue-specific gene expression in humans and other higher eukaryotes. Pax proteins are essential for the formation of differentiated cells and tissues, however, increasing levels of DNA binding activity results in neoplastic transformation and tumorigenesis. For example, chromosomal abnormalities and gene rearrangements resulting in overexpression of Pax-5 are associated with B lineage lymphomas in humans. Mechanisms contributing to Pax-5-mediated lymphomagenesis are not understood, but multiple lines of evidence suggest that the dosage of Pax-5 is exquisitely regulated in normal B cells by transcriptional and post-translational mechanisms. As one mechanism contributing to post-translational regulation of Pax-5, we propose that Pax- 5 DNA binding is regulated, in part, by the redox status of highly conserved cysteine residues in its paired DNA-binding domain. Thus, DNA binding by Pax-5 (and other Pax family members) may be reduced in response to oxidative stress. To date, this hypothesis has only been tested using limited in vitro model systems that do not adequately reflect the complexity of homeostatic mechanisms governing transcriptional activity in vivo. Moreover, it has not been determined whether S-thiolation (glutathionylation) of Pax-5 is an important mechanism for controlling its activity in vivo. In this application, we propose a genetic approach that bypasses previously encountered deficiencies associated with transfection assays and other in vitro experimental protocols. Our experiments will address a relatively unexplored area of molecular biology with profound implications for understanding how cells maintain precise levels of key regulatory factors. Our studies will eventually aid in devising new therapeutics for treating Pax-related cancers (lymphoma, astrocytoma, and rhabdomyosarcoma).

DESCRIPTION (provided by applicant): The Pax family of DNA-binding proteins includes essential regulators of tissue-specific gene expression in humans and other higher eukaryotes. Pax proteins are essential for the formation of differentiated cells and tissues, however, increasing levels of DNA binding activity results in neoplastic transformation and tumorigenesis. For example, chromosomal abnormalities and gene rearrangements resulting in overexpression of Pax-5 are associated with B lineage lymphomas in humans. Mechanisms contributing to Pax-5-mediated lymphomagenesis are not understood, but multiple lines of evidence suggest that the dosage of Pax-5 is exquisitely regulated in normal B cells by transcriptional and post-translational mechanisms. As one mechanism contributing to post-translational regulation of Pax-5, we propose that Pax- 5 DNA binding is regulated, in part, by the redox status of highly conserved cysteine residues in its paired DNA-binding domain. Thus, DNA binding by Pax-5 (and other Pax family members) may be reduced in response to oxidative stress. To date, this hypothesis has only been tested using limited in vitro model systems that do not adequately reflect the complexity of homeostatic mechanisms governing transcriptional activity in vivo. Moreover, it has not been determined whether S-thiolation (glutathionylation) of Pax-5 is an important mechanism for controlling its activity in vivo. In this application, we propose a genetic approach that bypasses previously encountered deficiencies associated with transfection assays and other in vitro experimental protocols. Our experiments will address a relatively unexplored area of molecular biology with profound implications for understanding how cells maintain precise levels of key regulatory factors. Our studies will eventually aid in devising new therapeutics for treating Pax-related cancers (lymphoma, astrocytoma, and rhabdomyosarcoma).

DESCRIPTION (provided by applicant): EBF1 is a crucial regulator of B lymphocyte lineage specification and commitment. In mice lacking EBF1 (encoded by the Ebf1 gene), B cell development is arrested and immunoglobulins (Ig) are not produced. Recently, we determined that mice with a single wild type Ebf1 allele (Ehet mice) exhibit defects in B cell development in the bone marrow. B cell development is further impaired in Ehet mice that also possess a single functional Runx1 (Rhet) allele. Runx1 encodes the Runt domain transcription factor Runx1 (CBF12/PEBP21), a functional partner of EBF1. In single Ehet and double (ERhet) haploinsufficient mice, we observed 1) substantially decreased numbers of CD19+ cells in the bone marrow, 2) delayed activation of pre-B cell markers, 3) reduced levels of Ig;light (L) chain rearrangements and 4) the loss of B cell identity, as evidenced by the mixing of B cell and NK cell phenotypes. The loss of B cell identity occurred in the presence of Pax5, a defined mediator of B cell commitment. We will address the central hypothesis that EBF1 is a primary regulator of B cell lineage specification and commitment. We propose that 1) the appropriate dosage of EBF1 is critical for establishing B cell identity and progression, and 2) this role of EBF1 is dependent on its functions as a transcriptional repressor, which is an undefined mechanism. We will continue our studies by identifying signaling defects in pro-B/pre-B cells that express reduced levels of EBF1. In the last aim, we will address whether EBF1 is required for the maintenance of B cell identity by utilizing new Ebf1 conditional knockout (Ecko) mice developed in our laboratory. Together, these studies will result in important new insights concerning functions of EBF1 in the regulation of B cell lineage specification, commitment and progression. PUBLIC HEALTH RELEVANCE: Early B cell Factor (EBF1) is essential for the production of B lymphocytes, which produce antibodies in response to foreign pathogens. Here, we will determine how EBF1 regulates the development of these cells in bone marrow. We will also address how EBF prevents the expression of genes of other types of hematopoietic cells, resulting in the commitment of progenitors to become B cells. Our studies are important for understanding how EBF controls these processes in normal cells, autoimmune diseases and cancer.

DESCRIPTION (provided by applicant): Summary B cell development in the bone marrow proceeds in a stepwise process from multipotent hematopoietic stem cells (HSCs) to committed immature B lymphocytes. Research has elucidated a B cell-specific regulatory network of transcription factors necessary for the development of early B cells. These proteins include Early B cell Factor 1 (EBF1), which drives B lineage specification and commitment. Although much is known about EBF1, there are many unanswered questions concerning its regulation in B cells and its potential for protein- protein interactions. Several lines of evidence point to Zfp521 (Evi3/ZNF521/EHZF) as a co-regulator of gene transcription with EBF1. Zfp521 interacts physically with EBF1 and can repress its activity in vitro. Zfp521 is co- expressed with EBF1 in developing B cells. However, little is known concerning functions of Zfp521 in B cell development. Specifically, the mechanisms by which Zfp521 modulates activities of EBF1 and the basis and consequences of physical interactions between Zfp521 and EBF1 are unknown. We have demonstrated that early B cell development is perturbed greatly in the absence of Zfp521. This is evidenced by a lack of expression of markers of late pro-B cells and pre-B cells and decreased immature B cells in bone marrow of Zfp521-deficient mice. As a mechanism that integrates the activities of both Zfp521 and EBF1, recent studies in our laboratory using newly developed antibody reagents detected Zfp521 binding to promoters that also bind EBF1. Co-occupancy of Zfp521 and EBF1 on these genes suggests a new model for co-regulation of B lymphopoiesis. In this manner, EBF1, the B lineage determination factor, is regulated by Zfp521 in developing B cells. Hypotheses: Zfp521 is critically important for B cell lymphopoiesis and function. We propose that it regulates the B cell program via EBF1-dependent and -independent mechanisms. Zfp521 interacts with EBF1 to limit its DNA binding, but Zfp521 also assembles complexes with EBF1 on target gene DNA. Determining the basis of interactions between EBF1 and Zfp521 at the molecular level is critical for understanding how the B cell regulatory network generates mature B cells capable of secreting antibodies in humoral immune responses. Therefore, we will: Determine how Zfp521 regulates gene transcription in developing B cells. Determine how Zfp521 functions in the B cell regulatory network by identifying its binding sites in pro- B cells. Define structural requirements and functional consequences of Zfp521 interactions with EBF1.

DESCRIPTION (provided by applicant): V(D)J recombination is the hallmark of adaptive immunity. In T and B cells, a series of highly regulated somatic gene rearrangement events produce functional genes from gene segments. This process results in the expression of functional antigen receptors. Immunoglobulin (Ig) genes encode B cell receptors, while T cell receptors are encoded by Tcr loci. The central mechanisms necessary for the assembly of these genes are cleavage and ligation of variable (V), diversity (D, at a subset of antigen receptor loci), and Joining (J) segments by V(D)J recombination. V(D)J recombinase complexes comprise multiple proteins including Recombination activating genes 1 and 2 (Rag1 and Rag2), which recognize and cleave recombination signal sequences (RSS) when RSS are in an 'accessible' state. Here, we will determine the roles of Protein Arginine Methyltransferase 5 (PRMT5), methylated arginine, and its removal by epigenetic 'erasers' in this process. Accessibility of RSS for recombination is largely controlled by epigenetic mechanisms including DNA methylation and post-translational modifications of histones. The importance of each of these mechanisms has been documented in V(D)J recombination. However, far less is understood concerning the importance of arginine methylation and the identities of enzymes that add or remove methyl groups from arginine during lymphocyte development. We hypothesize that V(D)J recombination of T cell receptor (Tcra) loci is regulated by PRMT5, which dimethylates arginine symmetrically at multiple residues of histones H3 and H4, as well as some non-histone proteins. Provocatively, Prmt5 transcripts are expressed in developing thymocytes (and B cells), but not at stages that express the Rag1 gene and feature ongoing V(D)J recombination. These observations suggest that arginine methylation by PRMT5 inhibits V(D)J recombination. Indeed, depletion of Prmt5 mRNA in a pre-T cell line greatly increases mature TCR? expression on the plasma membrane. We predict that this mechanism regulates antigen receptor assembly in both T and B cells. Therefore, we will address functions of arginine methylation and PRMT5 in T and B cell lines that undergo efficient V(D)J recombination in vitro. Our evidence suggests that the loss of arginine methylation is an active process involving one or more epigenetic erasers, which are likely members of the Jumonji family of protein dioxigenases. We will use biochemical methods to identify candidate arginine demethylases in lymphocyte progenitors. Together, our experiments will address novel epigenetic mechanisms that control antigen receptor assembly during lymphocyte development, but are also important for gene regulation in a wide variety of contexts including cancer.

ABSTRACT Genetic variation between men and women predisposes differences in their responses to infection. For example, women respond much more vigorously to agents and vaccines including influenza, toxoplasmosis, and legionella, and are more prone to developing autoimmune disorders including Grave's disease, rheumatoid arthritis, and systemic lupus erythematosis (SLE). The molecular bases of sex differences in immunity and diseases of the immune system are unknown, although the presence of key immune modulatory genes on the X chromosome suggests a basis for sex-specific responses. The importance of sex-specific factors has been recognized by the National Institutes of Health, which changed its policies to require investigators to address and report differences between the sexes in pre-clinical studies. In this regard, we have observed that mature mIg+ B cells of mice express high levels of Serine/Arginine-specific Protein Kinase 3, a PKC-like kinase encoded by an X-linked gene. We propose that SRPK3 regulates humoral immunity in humans and mice. Using mice with floxed Srpk3 genes that delete specifically in B cells (using Cd79a-Cre), we observed that SRPK3 is necessary for efficient T-independent (TI) type 2 antibody responses against the model antigen NP-Ficoll. Preliminary experiments also suggest graded antibody responses in female mice with different gene dosage of Srpk3 (Srpk3+/+, Srpk3+/?, and Srpk3?/?). We hypothesize that SRPK3 is required for appropriate antibody responses, which are disrupted in its absence due to aberrant pre-mRNA splicing and the lack of SRPK3-mediated phosphorylation. To address this hypothesis, we will determine requirements for SRPK3 in responses to model antigens and identify targets of the kinase in pre-mRNA splicing and the phosphorylated proteome in marginal zone B cells. We conclude that the loss of SRPK3 function results in significant immunodeficiencies in males and females.

PROJECT SUMMARY The generation of B and T lymphocytes follows their progression from uncommitted progenitors through a series of defined stages of development. At each stage, cells express a distinct set of genes, or transcriptome. These patterns of gene expression are controlled, in part, by tissue- and stage-specific epigenetic factors that limit the accessibility of chromatin. We have demonstrated that Nucleosome Remodeling and Deacetylase (NuRD) complexes, which include the Chromodomain Helicase and DNA binding 3 (CHD3; also known as Mi-2?) or the related protein CHD4 (Mi-2?) proteins, are gatekeepers of gene transcription. CHD4 has been studied extensively in B and T cells, while very little is known concerning its closely related paralog CHD3. The literature suggests that normal lymphocyte development likely requires both CHD proteins due to their distinct functions, but this has not been proven in an in vivo model system. Here, we will generate gene-targeted mice for the conditional knockout of Chd3 genes in lymphocytes of mice. These mice are currently unavailable commercially and from other research laboratories. Thus, we will generate a valuable resource that can be shared with other investigators with interests in lymphocyte development, epigenetics, and specific functions of NuRD complexes.