Supplementary MaterialsSupplementary information 41598_2019_44720_MOESM1_ESM. Ash2l PF-00562271 is Rabbit polyclonal to KCNV2 essential for balanced gene expression and for hematopoietic stem and multi-potent progenitor cell physiology. is embryonically lethal, whereas the genes are deregulated in and KO cells. Loss of Mll3/KMT2C and Mll4/KMT2D results in death around birth and day E9.5, respectively14. Set1A and B (KMT2F and G, respectively) are also essential, the former during gastrulation, while the KO embryos survive until day E11.515. These findings suggest that each of the 6 KMT2 complexes is required for defined aspects of development and thus PF-00562271 are at least in part functionally distinct. For catalytic activity and for recruitment to chromatin KMT2 enzymes require the interaction with the WRAD complex, composed of WDR5, RBBP5, ASH2L, and two copies of DPY3010,11,23. Additional subunits are associated with distinct KMT2 complexes (aka COMPASS), further increasing diversity of these multi-protein cofactors10,24. WRAD components are essential as far as studied. Ash2l is required for early mouse development25 and for liver homeostasis26. Moreover, Dpy30 is essential during embryogenesis and critical for hematopoietic stem and progenitor cell differentiation27C29. In these studies, the heterozygous animals revealed no phenotype, suggesting that neither Ash2l nor Dpy30 is haploinsufficient. In summary, KMT2 complexes exert critical functions in mouse development and in organ homeostasis11,23,30. Epigenetic modifications of DNA and core histones play prominent roles in the development of hematopoietic malignancies, such as myeloid leukemia and aggressive lymphomas, and the corresponding writers, readers and erasers are considered as drug targets30C32. The association of KMT2 complexes with cancer has been well documented and is particularly evident for as translocations of this gene are associated with acute leukemias33. Other KMT2 methyltransferases have been linked to other malignancies (see e.g.34C37). An involvement of ASH2L in tumorigenesis has also been suggested. We have identified ASH2L as an 86?kDa interaction partner of the oncoprotein c-MYC38. Subsequently, ASH2L was found to cooperate with Ha-RAS in the transformation of rat embryo fibroblasts39. MYC is deregulated in the majority of hematopoietic malignancies40, and, together with ASH2L and other cofactors such as CBP/p300, regulates chromatin and gene transcription41C43. Furthermore, ASH2L interacts with MLK1 (megakaryocytic leukemia-1), a transcription factor originally identified in acute megakaryocytic leukemia and subsequently shown to affect megakaryocytic, monocytic, and granulocytic differentiation and function44C46. Moreover, low expression of ASH2L has been correlated with increased survival of patients with acute myeloid leukemia47. Beyond hematopoiesis, ASH2L is overexpressed in the majority of human tumors and its knockdown interferes with H3K4 methylation and tumor cell proliferation39,48C50. Together, these data suggest an important role of ASH2L for the differentiation and proliferation of hematopoietic cells both under physiologic conditions as well as during malignant transformation. To understand the function of Ash2l in the hematopoietic system in more detail, we generated conditional KO mice using the Mx1-Cre/loxP system. The loss of Ash2l protein expression in the hematopoietic system led to a differentiation block of early hematopoietic progenitor cells. This block was associated with a late cell cycle arrest. Consistent with this phenotype, genes encoding factors associated with G2/M-phase progression were down-regulated upon loss of Ash2l. The consequence of this differentiation block is severe pancytopenia with subsequent death of the animals. Results Mx1-Cre-dependent knockout of is lethal and prevents differentiation of hematopoietic cells We generated mice with alleles of harboring a floxed exon 4 and an Mx1-Cre transgene whose expression was stimulated by the intraperitoneal injection of the synthetic RNA analog polyinosinic-polycytidylic acid (pIC) (Fig.?1a)51. animals were affected starting at day 8 upon pIC treatment and had to be sacrificed subsequently (Fig.?1b). In the following experiments, we analyzed animals and cells at day PF-00562271 10. Activation of Cre led to efficient recombination of the floxed sequences (Fig.?1c). Histological examination of the bone marrow (BM) in the sternum by hematoxylin&eosin (H&E) staining revealed a reduced cellularity in the KO mice (Fig.?1d). The BM was populated less than half in KO vs. control mice (Fig.?1e). We observed that all lineages of blood-forming cells were affected with the appearance of dysmorphic megakaryocytes, showing lobulated nuclei and reduced amounts of cytoplasm (Fig.?1d, circles). In granulopoesis, a higher number of ring-like myelocytes (band granulocytes) and metamyelocytes was visible.