Fetal Inactivation of the Nuclear Interacting SET Domain Protein 1 Impairs Terminal Erythroid Maturation and Results in Acute Erythroleukemia in Mice

The nuclear receptor binding SET domain protein 1 (NSD1) histone (H3K36) methyltransferase is target of recurrent genetic alterations in human hematological malignancies and certain solid cancers. To study its role in normal hematopoiesis, we inactivated the Nsd1 gene in the blood forming system of the mouse. Vav1-iCre mediated cleavage of floxed exon 5 resulted in almost complete loss of Nsd1 expression at the mRNA and protein level. After a latency of 7-17 weeks, all Vav1-iCre;Nsd1^fl/fl mice developed signs of disease, whereas heterozygous littermates expressed normal Nsd1 levels and remained healthy. Symptomatic mice presented anemia, reticulocytosis and thrombocytopenia with presence of erythroblasts on peripheral blood smears. Diseased mice had significant splenomegaly and infiltration of erythroblasts in spleen, liver and lung. Bone marrow (BM) transplantation from symptomatic mice was able to fully propagate the disease in wild type recipients, alone or in competition with normal cells. The majority of the BM and spleen cells of diseased mice expressed modest levels of the transferrin receptor (CD71^dim) and variable amounts of c-Kit and FcγR-II/III, but no TER119, CD34, B220 or Sca-1. The cells formed abnormal BFU-like dense reddish and partially hemoglobinized colonies in erythropoietin (EPO) containing methylcellulose that could be replated up to 4 rounds. In Vav1-iCre;Nsd1^fl/fl fetal livers, we also observed accumulation of abnormal erythroid progenitors, deficient for Nsd1, suggesting a prenatal origin of the phenotype. RNA sequencing of lineage marker-negative, Sca1⁺, c-Kit⁺ (LSK) cells revealed aberrant regulation of genes associated with/functioning in erythroid differentiation. Indeed, in vitro differentiation of Vav1-iCre;Nsd1^fl/fl erythroblasts was significantly impaired. Retroviral expression of Nsd1 was able to partially rescue the erythroid differentiation block. Interestingly, Vav1-iCre;Nsd1^fl/fl erythroblasts constitutively expressed significantly increased protein levels of the erythroid master transcriptional regulator GATA1 independent whether they were expanded as immature cells or induced for terminal maturation. Impaired terminal maturation of Vav1-iCre;Nsd1^fl/fl erythroblasts was associated with reduced transactivation of GATA1 positively-regulated targets (HbbA, HbbB, Gpa, Bcl2l1, p21), while expression of GATA1-repressed target genes (Gata2, c-kit, Spi1) was not affected. Strikingly, exogenous expression of Gata1 was able to overcome the differentiation block of Vav1-iCre;Nsd1^fl/fl erythroblasts depending on the integrity of the N- and C-zinc fingers, as seen in colony forming assays and liquid cultures. Chromatin immunoprecipitation (ChIP) revealed that binding of GATA1 to HbbA1 locus and to a lesser extent to regulatory elements in the Gata1 promoter region (G1-HE site) was impaired in Vav1-iCre;Nsd1^fl/fl erythroblasts, but restored upon overexpression of exogenous Gata1 . However, we found no significant changes of global H3K36me¹ and H3K36me² marks in wild type and Vav1-iCre;Nsd1^fl/fl erythroblasts. In addition, published ChIP-seq data revealed no enrichment for H3K36me^1/2 marks near critical GATA1 binding sites in HbbA1 or Gata1 suggesting that NSD1 most likely regulates GATA1 activity by direct modification or by interfering with GATA1 interacting partners necessary for efficient transactivation of critical mediators of terminal erythroid maturation. Taken together, our work unraveled NSD1 as a critical regulator of terminal erythroid differentiation.