Depletion of L3MBTL1, a PcG Gene Deleted in Patients with 20q-Associated with Hematologic Malignancies, Accelerates Erythroid Differentiation of Hematopoietic Progenitor Cells

L3MBTL1 is a polycomb gene located in 20q12, within the common deleted region identified in patients with 20q deletion associated with polycythaemia vera (PV), myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). L3MBTL1 is expressed within CD34+ haematopoietic progenitor cells from which myeloid malignancies arise and its Drosophila homologue encodes a tumour suppressor protein. L3MBTL1 represents then a candidate target gene in 20q deletion patients. To gain insight into the role of L3MBTL1 in hematopoiesis we knocked down the level of L3MBTL1 mRNA through lentiviral expression of short hairpin RNAs in CD34+ hematopoietic stem/progenitor cells isolated from human cord blood (CB) cells. We achieved an approximately 80% drop in endogenous L3MBTL1 mRNA level, as determined using quantitative RT-PCR. Transduced and sorted CD34+ GFP+ CB cells were plated in liquid cultures and induced to cytokine-driven differentiation. The effect of L3MBTL1-knock down was assessed by colony assays and by fluorescence-activated cell sorting (FACS), using lineage–specific cell surface markers. We demonstrate that the knock down (KD) of L3MBTL1 remarkably accelerates the differentiation of hematopoietic CD34+ cells into erythrocytes. The relative percentage of mature erythroid precursors cells, defined as CD71+ and Glycophorin A+ cells, consistently increased in the L3MBTL1-KD population, as the CD34+ cells reached this advanced stage of erythroid differentiation in fewer days than control cells. As confirmation, Giemsa staining after cytospin preparations of L3MBTL1-KD cells showed more mature morphology compared to the control cells, and benzidine staining revealed many more Hb containing-positive cells in the L3MBTL1-depleted cell population compared to the control. Monitoring of globin gene expression demonstrated that L3MBT-Knock down is involved in the regulation of only a subset of these genes, primarily the expression of the epsilon and zeta globin genes, the embryonic globin genes belonging to the beta and alpha globin gene clusters respectively. In addition to these effects a significant slowing of proliferation was seen, which likely reflects the increased differentiation of these cells. We have previously identified a role for L3MBT in binding histones H1 and H4 that contain monomethylated and dimethylated lysine residues H1k2b and H4k20 (Kalakonda et al. 2008) and compacting chromatin (Trojer et al. Cell 2007). Precisely how this feature of L3MBTL1 function in regulating the erythroid differentiation of hematopoietic cells is unknown. We however link lack of L3MBTL1 with the possible pathogenesis of PV associated with 20q deletion. We will present additional data that attempt to define the kinetics of alpha-like and beta-like globin gene expression during the erythroid maturation of L3MBTL1-KD cells in culture and to investigate a supposed synergism with the known molecular pathways of normal and malignant erythroid differentiation.