Critical roles for the transcription factor PU.1 in early lymphopoiesis in adult mice

© 2013 Dr. Swee Heng PangPU.1 is a central regulator of foetal haematopoiesis. Conditional knockout of PU.1 in adult mice demonstrated disrupted myeloid and lymphoid developments. While this suggests a role of PU.1 in early haematopoietic progenitors, the progenitor stage at which PU.1 is important remain unknown. Moreover, the source of immature myeloid cells that contribute to the development of acute myeloid leukaemia (AML) in PU.1fl/flMxCre+ mice is still poorly defined. With the use of PU.1fl/-MxCre+RAG1+/gfp and PU.1fl/flRosa26CreERT2 mice that allows the inducible inactivation in stem cells and early lymphoid progenitors, I identify that PU.1 plays a critical role in lymphoidprimed multipotent progenitors (LMPPs), and deficiency in PU.1 results in a developmental block at the transition from LMPPs to common lymphoid progenitors. In addition, I demonstrate that the aberrant myeloid cells that eventually develop into AML originate from the PU.1 deficient Lin-Sca-1+c-Kit+ (LSK) cells upon transplantation. Results from PU.1fl/-RAG1cre mice suggest that PU.1 deficient LMPPs within the LSK cells are the most likely candidate that forms these abnormal cells. Gene expression studies using PU.1 deficient LSK cells revealed distinct difference in total transcriptomes when compared to the wild-type LSK controls. Earlier studies reported that specific deletion of PU.1 in B cells revealed its dispensable role in B cell development in the bone marrow. I demonstrate similar findings by using PU.1fl/-RAG1cre mice that delete PU.1 in an earlier stage of B cell progenitors. I further investigate the importance of PU.1 in interacting with IRF4 and IRF8. Deficiencies in both PU.1 and IRF4/8 revealed distinct consequences for B cell development. Strikingly, the pre-B cells from PU.1/IRF4 deficient mice are hyperproliferative to IL-7, and express elevated level of pre-BCR. Both PU.1 and IRF4/8 deficient mice develop pre-B acute lymphoblastic leukaemia, from which isolated cells can be maintained in culture supplemented with IL-7. I also demonstrate that there is a dose dependent pattern observed from a range of PU.1/IRF4 deficient mice in which lack of one copy of both would result in leukaemia. These leukaemic cells expressed low levels of Spib, Izkf1 (Ikaros) and Blnk, and by using ChIPseq, my collaborators and I show that Spib and Ikaros are transcriptionally activated by PU.1 and IRF4/8. Restoration of Spi-B and Ikaros expression in these leukaemic cells reduce their IL-7 dependent growth in culture. In conclusion, these findings demonstrate that PU.1 is functionally important in LMPPs, and together with IRF4/8, they are essential transcriptional regulators as well as tumour suppressors in early B cell development