Transcriptional Control of Early Lymphopoiesis

The hematopoietic stem cell (HSC) gives rise to all lymphoid cell types by differentiating to the common lymphoid progenitor (CLP), which then undergoes commitment to and differentiation along the B, T or NK cell lineage. Several transcription factors control the earliest phase of lymphopoiesis. The three regulators E2A, EBF and Pax5 are essential for differentiation of the CLP into B-lymphocytes, whereas Id2 and Notch1 are required for NK and T cell development, respectively. Within the B-lymphoid lineage, E2A and EBF are thought to coordinately activate the B cell gene expression program, while Pax5 restricts the different developmental options of lymphoid progenitors to the B cell pathway. Notch1, similar to Id2, appears to block the primary B-lymphoid fate of the CLP by inactivating the earliest B cell factor E2A and by simultaneously activating the T-lymphoid gene expression program. To date, little is known about the molecular mechanisms by which E2A, EBF and Notch1 control early lymphopoiesis. The main reason for this is the fact that inactivation of the E2A, EBF or Notch1 gene leads to the loss of the earliest B- or T-lymphoid precursor cells, respectively. In contrast, the Pax5-deficient pro-B cells with their broad developmental potential can be cultured ex vivo, which has facilitated a detailed analysis of the Pax5 function in early B-lymphopoiesis. In this grant application, we propose to investigate the function of E2A, EBF and Notch1 at the molecular level by a combination of gain-of-function and conditional loss-of-function experiments. We will investigate whether precocious expression of E2A and EBF under the control of the Ikaros locus will skew the developmental potential of the HSC towards the B cell pathway. A posttranslational Id1-ER induction system and a floxed EBF allele will be generated for conditional inactivation of E2A and EBF in early pro-B cells. E2A- and EBF-deficient pro-B cells will be compared with wild-type pro-B cells by gene expression profiling to identify E2A and EBF target genes. Likewise, Notch1 target genes will be identified by microarray screening of Pax5-deficient pro-B cells, which have been exposed to Delta or Jagged ligands to activate Notch1 signaling. Finally, the function of selected target genes will be analyzed by siRNA knock-down approaches. Together, these experiments will provide novel insight into the molecular mechanisms controlling early lymphocyte development.

The hematopoietic stem cell (HSC) gives rise to all lymphoid cell types by differentiating to the common lymphoid progenitor (CLP), which then undergoes commitment to and differentiation along the B, T or NK cell lineage. Several transcription factors control the earliest phase of lymphopoiesis. The three regulators E2A, EBF and Pax5 are essential for differentiation of the CLP into B-lymphocytes, whereas Id2 and Notch1 are required for NK and T cell development, respectively. Within the B-lymphoid lineage, E2A and EBF are thought to coordinately activate the B cell gene expression program, while Pax5 restricts the different developmental options of lymphoid progenitors to the B cell pathway. Notch1, similar to Id2, appears to block the primary B-lymphoid fate of the CLP by inactivating the earliest B cell factor E2A and by simultaneously activating the T-lymphoid gene expression program. To date, little is known about the molecular mechanisms by which E2A, EBF and Notch1 control early lymphopoiesis. The main reason for this is the fact that inactivation of the E2A, EBF or Notch1 gene leads to the loss of the earliest B- or T-lymphoid precursor cells, respectively. In contrast, the Pax5-deficient pro-B cells with their broad developmental potential can be cultured ex vivo, which has facilitated a detailed analysis of the Pax5 function in early B-lymphopoiesis. In this grant application, we propose to investigate the function of E2A, EBF and Notch1 at the molecular level by a combination of gain-of-function and conditional loss-of-function experiments. We will investigate whether precocious expression of E2A and EBF under the control of the Ikaros locus will skew the developmental potential of the HSC towards the B cell pathway. A posttranslational Id1-ER induction system and a floxed EBF allele will be generated for conditional inactivation of E2A and EBF in early pro-B cells. E2A- and EBF-deficient pro-B cells will be compared with wild-type pro-B cells by gene expression profiling to identify E2A and EBF target genes. Likewise, Notch1 target genes will be identified by microarray screening of Pax5-deficient pro-B cells, which have been exposed to Delta or Jagged ligands to activate Notch1 signaling. Finally, the function of selected target genes will be analyzed by siRNA knock-down approaches. Together, these experiments will provide novel insight into the molecular mechanisms controlling early lymphocyte development.