A tight regulation of transcription factor activity is critical for proper development. For instance, modifications of RUNX transcription factors dosage are associated with several diseases, including hematopoietic malignancies. In Drosophila, Myeloid Leukemia Factor (MLF) has been shown to control blood cell development by stabilizing the RUNX transcription factor Lozenge (Lz). However, the mechanism of action of this conserved family of proteins involved in leukemia remains largely unknown. Here we further characterized MLF’s mode of action in Drosophila blood cells using proteomic, transcriptomic and genetic approaches. Our results show that MLF and the Hsp40 co-chaperone family member DnaJ-1 interact through conserved domains and we demonstrate that both proteins bind and stabilize Lz in cell culture, suggesting that MLF and DnaJ-1 form a chaperone complex that directly regulates Lz activity. Importantly, dnaj-1 loss causes an increase in Lz+ blood cell number and size similarly as in mlf mutant larvae. Moreover we find that dnaj-1 genetically interacts with mlf to control Lz level and Lz+ blood cell development in vivo. In addition, we show that mlf and dnaj-1 loss alters Lz+ cell differentiation and that the increase in Lz+ blood cell number and size observed in these mutants is caused by an overactivation of the Notch signaling pathway. Finally, using different conditions to manipulate Lz activity, we show that high levels of Lz are required to repress Notch transcription and signaling. All together, our data indicate that the MLF/DnaJ-1-dependent increase in Lz level allows the repression of Notch expression and signaling to prevent aberrant blood cell development. Thus our findings establish a functional link between MLF and the co-chaperone DnaJ-1 to control RUNX transcription factor activity and Notch signaling during blood cell development in vivo.

The hematopoietic system plays a critical role in establishing the proper response against invading pathogens or in removing cancerous cells. Furthermore, deregulations of the hematopoietic differentiation program are at the origin of numerous diseases including leukemia. Importantly, many aspects of blood cell development have been conserved from human to Drosophila. Hence, Drosophila has emerged as a potent genetic model to study blood cell development and leukemia in vivo. In this chapter, we give a brief overview of the Drosophila hematopoietic system, and we provide a protocol for the dissection and the immunostaining of the larval lymph gland, the most studied hematopoietic organ in Drosophila. We then focus on the various paradigms that have been used in fly to investigate how conserved genes implicated in leukemogenesis control blood cell development. Specific examples of Drosophila models for leukemia are presented, with particular attention to the most translational ones. Finally, we discuss some limitations and potential improvements of Drosophila models for studying blood cell cancer.