Gene Regulatory Networks and Chromatin Dynamics Regulating Cellular Differentiation lymphopoiesis

B-lymphocytes develop from pluripotent hematopoietic stem cells by a complex process involving several intermediate cell-stages. These stages are characterized on the basis of expression of cell surface markers and rearrangement of immunoglobulin heavy and light chain (IgH and IgL) genes. Our initial studies have demonstrated that the activity of the κE3’- enhancer, which is essential for rearrangement of the Ig kappa light chain (Igκ) gene, is regulated by a combination of various transcription factors, notably PU.1 and IRF4. Binding of these factors is particularly intriguing in that PU.1 bind to the κE3’-enhancer and recruits IRF4 to its adjacent binding site through phosphorylation dependent protein-protein interactions. Using a variety of biochemical approaches, we elaborated a structural model for assembly of PU.1/IRF4/DNA ternary complex. Importantly, mutation of PU.1 results in a profound block to B lymphopoiesis with an absence of B lineage progenitors. Gene expression analysis accompanied with complementation of PU.1-/- progenitors led to the discovery that PU.1 is required for the developmental induction of the transcription factor EBF. Ectopic expression of EBF rescued the development of B cells in PU.1-/- progenitors. Loss-of-function and gain-offunction analyses demonstrated that EBF promotes B cell fate choice of hematopoietic multipotent progenitors (MPPs) at the expense of myeloid lineages. By conducting genomewide expression analysis in combination with cross-validation of binding sites by surveying the genome, we have determined EBF specific gene signature. EBF alters gene expression program in part by regulating H3K4me1, a histone mark enriched at enhancer elements. These studies facilitated an assembly of an extended EBF-centered gene regulatory module that accounts for B cell fate choice of MPPs. Unlike mutation of PU.1, targeted disruption of IRF4 and its related factor, IRF8, results in a precise developmental arrest at the cycling pre-B cell stage. IRF-4,8-/- pre-B cells respond to IL- 7 and proliferate extensively in vitro but fail to express kappa and lambda light chain genes, which is critical for pre-B to B cell transition. Using IRF-4,8-/- pre-B cells, we have proceeded to demonstrate that two pathways converge to synergistically drive light-chain rearrangement. One pathway is directly dependent on IRF-4, whose expression is elevated by pre-BCR signaling,that acts on the κE3′ and λ enhancers to increase locus accessibility and positions a kappa allele away from pericentromeric heterochromatin. The other pathway is triggered by attenuation of IL-7 signaling and results in activation of the κ intronic enhancer. Based on these findings, we propose that stage-specific activation of light-chain recombination during B cell development is ensured by a combination of pre-BCR dependent activation of IRF4 and attenuated IL-7 signaling. Genome-wide expression analysis of IRF4 revealed a specific gene expression signature. Thus activation of IRF4 establishes a distinct gene regulatory module that drives Igκ light-recombination, which is necessary for pre-B to B cell transition. Based on these studies, we propose that developmental progression of B cells is contingent upon sequential distinct gene regulatory modules, which appear to be functionally separable and composed of transient signaling inputs, positive and negative feedback relationships.