Ldb1, a ubiquitously expressed LIM domain binding protein, is essential in a number of tissues during development. It interacts with Gata1, Tal1, E2A and Lmo2 to form a transcription factor complex regulating late erythroid genes.
It has been identified by the host laboratory a number of novel Ldb1 interacting proteins in erythroleukemic cells, in particular the repressor protein Eto2 (and its family member Mtgr1) and the cyclin dependent kinase Cdk9. Morpholino mediated knockdowns in zebrafish show these f actors to be essential for definitive hematopoiesis. In accordance with the zebrafish results these factors are co-expressed in pre-hematopoietic cells of the early mouse embryo although the complexes were originally identified in late erythroid cells.
Based on the change in subcellular localisation of Eto2, we postulate that it plays a central role in the transition from the migration and expansion phase of the pre-heamatopoietic cells to the establishment of definitive hematopoietic stem cells. My project will focus on the understanding of the role of the two newly identified Ldb1 binding partners, (Eto2 and Cdk9) in the regulation of the formation of the blood system.
In order to achieve my goals, I will use a multidisciplinary approach involving affinity tagging of the proteins (allowing purification and mass spectrometry identification of interacting partners), functional testing of the novel partner proteins in zebrafish, and mouse genetic modification to obtain in vivo data through conditional knockouts.
The role of Eto2 and Cdk9 will be studied at the cellular and organism level. I will use mice and cells to identify their target genes and to gain insight into their role in the development of stem cells and erythroid differentiation in vivo.
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