The Condensin II Subunit, NcapH2, Is Required for Proper Embryonic Erythroid Terminal Maturation and Embryonic Viability in a Primary Mouse Model

Abstract

The process of nuclear condensation is an integral part of erythroid differentiation. We have previously shown that loss of the histone methyltransferase, Setd8, results in severe embryonic (primitive) erythroid anemia due to loss of chromatin condensation and proper erythroid terminal maturation (Malik 2017). Setd8 mediated H4K20 mono-methylation is an important mediator of higher order chromatin condensation and can recruit members of the Condensin II complex (Lau 2015). ondensin I and II complexes are five protein, large complexes responsible for DNA organization and packaging during mitosis. In eukaryotes, the complexes are composed of conserved SMC components, with each Condensin having a unique kleisin bridge protein and two HEAT subunits. Subunits of Condensin II are highly expressed in erythroid cells, suggesting an erythroid specific function. Overexpression of the Condensin II HEAT subunit, Cap-G2, accelerates erythroid differentiation in MEL cells (Xu 2006). To interrogate the role of Condensin II in a primary model, we mated endogenous EpoR driven cre expressing mice with mice containing a floxed allele around Condensin II kleisin subunit, NcapH2. Embryos homozygous for EpoR specific excision of NcapH2 suffered from severe primitive erythroid anemia with visible anemia beginning at embryonic day (E11.5) and lethality occurring at approximately E13.5. Peripheral blood analysis at E11.5 was significant for erythroblasts with enlarged cell size and nuclear size, as well erythroblasts with dysplastic appearing nuclei. Knockout embryos at E12.5 exhibited peripheral blood anemia, with an increase in the number of dysplastic appearing cells, and decreased fetal liver coloration suggesting a failure of both primitive and fetal definitive erythropoiesis. Failure of the embryonic erythroid lineage to expand was confirmed in a primary cell culture system in which loss of proliferation was limited to mid to late stage precursors, suggesting that Condensin II function is critical beyond the proerythroblast stage of maturation when nuclear condensation is occurring and mass spectrometry demonstrates that H4K20 mono-methylation accumulates. Characterization of primitive erythroblasts from knockout embryos showed increased cell size, increased scatter in the size of the nucleus, and a high frequency of cells with punctate nuclear staining. In addition, the subset of erythroid cells with punctate nuclei did not appear to have a nuclear envelope, suggesting a pre-mitotic error, likely during G2 of the cell cycle. Indeed, knockout cells showed a buildup of cells in G0-G1 of the cell cycle, suggesting arrest following a defective cell division. Together, our data suggests that Condensin II is required during erythroid maturation for proper cell cycle progression and chromatin organization, and that chromatin modifications, such H4K20me1, may be a required mechanism to control the higher-level organization of chromatin. This control is required in red cell precursors, as they undergo rapid expansion, maturation, and nuclear condensation simultaneously. Understanding these mechanisms will give valuable insight into the unique sensitivity of red cells to chromatin perturbations observed in bone marrow failure and myelodysplastic syndromes. Disclosures No relevant conflicts of interest to declare.