Somatic mutations in U2AF1 have been identified in ~11% cases of MDS. U2AF1 is involved in the recognition of the 3' splice site required for the recruitment of the U2 snRNP during pre-mRNA splicing. Most U2AF1 mutations are found in two hotspots (S34 and Q157) within the first and second zinc finger domains. Transgenic and knock-in mice expressing U2AF1 S34F mutant exhibit impaired hematopoiesis. However, the role of wild-type U2AF1 in regulating hematopoietic stem cell (HSC) function and normal hematopoiesis has remained unknown.
To determine the role of U2AF1 in normal hematopoiesis, we generated a new conditional U2af1 knockout (floxed) mouse. We crossed U2af1 floxed mouse with Mx1-Cre mouse and the expression of Cre recombinase was induced with polyinosine-polycytosine (pI-pC) injection at 5 to 6 weeks after birth. We observed that deletion of U2af1 significantly reduced white blood cell, neutrophil, red blood cell and platelet counts in their peripheral blood compared with control animals within 10-14 days after pI-pC injection. Histopathologic analysis of the BM sections from U2af1-deficient mice showed severe BM aplasia. Flow cytometric analyses revealed a marked decrease in myeloid, erythroid and megakaryocytic precursors in the BM of U2af1-deficient mice compared with control animals. We also observed a marked decrease in Lin-Sca-1+c-kit+(LSK) and long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC), and multipotential progenitors (MPP) as well as common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and megakaryocyte-erythroid progenitors (MEP) in the BM of U2af1-deleted mice. Hematopoietic progenitor colony assays showed a significant decrease in myeloid (CFU-GM), erythroid (BFU-E), and megakaryocytic (CFU-Mk) colonies in the BM of U2af1-deficient mice.Together, these data suggest that loss of U2af1 causes severe defects in hematopoiesis.
We performed both non-competitive and competitive BM transplantation assays using U2af1-deficient BM to determine the role of U2af1 in HSC function. There was marked reduction of HSC, progenitors and all types of blood and BM cell precursors upon U2af1 deletion (by pI-pC administration) in the transplanted animals. Also, U2af1-deficient HSCs were unable to compete with WT HSCs and there was rapid loss of hematopoietic progenitors/precursors derived from the U2af1-deficient HSCs.
Since U2af1 deletion resulted in rapid decrease of hematopoietic progenitors in the BM, we asked whether deletion of U2af1 insulted the genome and induced apoptosis to hematopoietic cells in the BM. We observed significantly increased apoptosis in the total BM as wells as in c-kit+, Gr1+, Ter119+and CD41+cells suggesting that hematopoietic progenitors and precursors of multiple cell lineages underwent apoptosis upon U2af1 deletion. We also performed gamma-H2AX assay using imaging flow cytometry to evaluate DNA damage in total BM, Gr1+(myeloid) and CD71+(erythroid) cells in control and U2af1-deleted mice. We observed markedly elevated gamma-H2AX in total BM, Gr1+and CD71+cells from U2af1-deficient mice compared with control mice.In addition, we observed increased Chk1 phosphorylation (ser345), a hallmark for activation of the ATR pathway, and increased histone H2A K119 ubiquitination (H2AK119Ub), a marker for DNA damage response, in the BM of U2af1-deficient mice. Thus, depletion of U2af1 causes insult to the genome and induces DNA damage and increased cell death.
To gain insights into severe hematopoietic defects observed in U2af1-deficient mice, we performed transcriptome profiling of sorted LSK cells from U2af1 wild type (control) and U2af1-deleted mice. GSEA analysis of RNA sequencing data revealed significant downregulation of genes related to HSC maintenance in U2af1-deficient LSK. GSEA also revealed enrichment for cell cycle and DNA damage response-related genes, consistent with decreased proliferation and increased DNA damage and apoptosis observed in U2af1-deficient hematopoietic progenitors. We also determined the effects of U2af1 deletion on RNA splicing. Interestingly, we observed significant changes in gene expression as well as splicing alterations in several genes important for HSC survival and function. In conclusion, our results suggest a crucial role for U2af1 in the survival and function of HSC.
Disclosures
Mohi: Tolero Pharmaceuticals Inc.: Research Funding.
Arginine methylation of hnRNPK negatively modulates apoptosis upon DNA damage through local regulation of phosphorylation