Regulation of Notch Signaling By O-Glycans during Lymphopoiesis and Myelopoiesis

Abstract

IntroductionNotch signaling is essential for the optimal generation of T, B and myeloid cells. Epidermal growth factor-like (EGF) repeats in the extracellular domain of Notch receptors are modified by O-fucose and O-GlcNAc glycans transferred by protein O-fucosyltransferase 1 (POFUT1) and EGF O-GlcNAc-transferase (EOGT), respectively. EOGT promotes Notch ligand binding and Notch signaling in the developing retina in mice, and its loss is the basis of Adam's Oliver Syndrome 4 (AOS4) in humans. The loss of POFUT1 in humans leads to the Dowling Degos Disease 2 (DDD2). Thus, O-fucose and O-GlcNAc glycans may have different functions in Notch signaling, reflected in different requirements for T, B or myeloid cell development.Methods:Eogt control, Eogt null, Pofut1 floxed and Vav1-iCre transgenic mice were used to generate single and compound mutant mice with inactive Eogt, Pofut1 or both Eogt and Pofut1. Antibody markers of hematopoietic stem progenitor cell (HSPC), lymphoid and myeloid subsets were used to identify different T, B and myeloid cell subsets by flow cytometry using the Cytek TM Aurora Flow Cytometer. FCS files were analyzed using FlowJo software (BD). CD45.1+ (B6.SJL-Ptprc a Pepc b/BoyJ #002014) congenic mice were used for bone marrow transfer. Expression of NOTCH1 and binding of soluble Notch ligands to DN T cells was analyzed by flow cytometry. Expression of different Notch target genes was determined using qRT-PCR anlaysis.Results:Eogt null mice exhibited altered production of certain T cell subsets in thymus and B cell subsets in spleen, most similar to alterations observed in mice lacking LFNG, MFNG and RFNG. This phenotype was cell-autonomous as Eogt null bone marrow cells transferred defective T and B cell development to irradiated recipient mice. More severe defects in hematopoiesis were observed in mice conditionally lacking Pofut1 in hematopoietic stem cells (HSC) via Vav1-iCre. However , deletion of both O-fucose and O-GlcNAc glycans together in Eogt:Pofut1 double knockout HSC, led to the most severe decrease in common lymphoid and myeloid progenitors in bone marrow. In thymus, the dramatic reduction in T cell subsets observed in Pofut1 cKO mice was reduced further (in DN2 subsets) in Eogt:Pofut1 dKO mice. In spleen, there was a significantly greater decrease in follicular B and other B cell populations, and a greater increase in CD11b/c+ and Gr1+ myeloid cells in Eogt:Pofut1 dKO mice. Splenomegaly occurred in both Pofut1 cKO and Eogt:Pofut1 dKO mice, with a greater increase in extramedullary hematopoiesis in the dKO spleen. Binding of soluble DLL4-Fc was >90% reduced in Pofut1 cKO DN T cells, and not further reduced in dKO cells, while NOTCH1 expression at the cell surface was only slightly reduced. The relative expression of Notch target genes including Hes1, CD25, cMyc, and Deltex1 was similarly reduced in Pofut1 cKO and Eogt:Pofut1 dKO DN T cell progenitors.Conclsuion:The combined data suggest that O-fucose and O-GlcNAc glycans act in an additive fashion to optimize Notch signaling in lymphoid and myeloid differentiation.Keywords: Notch Signaling, O-Glycans, Protein O-fucosyltransferase 1 (POFUT1), O-GlcNAc transferase (EOGT), Lymphopoiesis, Myelopoiesis. DisclosuresStanley: Aviceda Therapeutics, Inc.: Consultancy, Current holder of stock options in a privately-held company.