The role of E2A in hematopoietic development : establishment of a multipotent progenitor cell line to identify genetic and epigenetic regulatory programs in hematopoiesis

Over 55,000 bone marrow transplants are performed every year to treat patients suffering from bone and blood cancers, anemia, and auto-immune disorders. The success of these transplants is highly correlated to a high number of hematopoietic stem cells (HSCs) and/or early progenitor cells injected into the patient. Obtaining high numbers of these early progenitors from donors is extremely difficult, making ex vivo expansion of these cells an attractive option. Currently, however, there is still much uncertainty as to how to expand these cells while maintaining their pluripotency. In our lab, we have shown that mouse E47 knockout progenitor cells can be expanded almost indefinitely while retaining their pluripotency when cultured on S17 stromal cells in the presence of IL-7, SCF and Flt-3. Here, we overexpress Id2 under the control of a tetracycline-responsive promoter to expand multipotent progenitors that retain differentiating ability both in vitro and in vivo. This is a novel and robust method for ex vivo expansion of HSCs. We then exploited the in vitro growth capacity and multipotent differentiation potential of these Id2-HPCs to study both the genetic and epigenetic regulation of B and myeloid development in mice. We analyzed gene expression patterns and genome-wide binding of histone modifications, namely H3K4me1 and H3K4me3, to generate a predictive model of cis -regulatory elements at active enhancers in multipotent Id2-HPCs, B cells, or myeloid cells. We also utilized the Id2-HPCs to study the temporal changes in gene expression, epigenetic modifications and transcription factor binding in the pre-pro-B to pro-B cell transition. This analysis has confirmed the role of E2A to directly bind to nearly half of the regulated genes in B-cell development and, in conjunction with H3K4me1 binding patterns, has allowed the prediction of novel enhancers that are activated by specific combinations of transcription factors in a temporal fashio