Abstract
Erythropoiesis requires dramatic changes in gene expression in a cell that is rapidly proliferating and undergoing progressive nuclear condensation in anticipation of enucleation. Disruption of this process is associated with myelodysplastic syndromes and congenital anemias. Our lab has demonstrated that Setd8, the sole histone methyltransferase that can generate H4K20me1, plays an essential role in this process (Malik 2019). H4K20me1 accumulates during terminal erythroid maturation (Murphy Blood 2021) and can regulate chromatin structure and gene expression through interaction with multiple partners, including the Condensin II Complex. The Condensin II complex is a ring-like structure composed of two conserved SMC components (SMC2 and SMC4), two HEAT subunits (NCAPG2 and NCAPD3), and a kleisin subunit NCAPH2. The Condensin II complex plays an important role in chromatin condensation during mitosis, and establishing higher-order chromatin interactions in interphase cells, with some studies suggesting it also regulates gene expression (Yuen Science Adv 2017; Iwasaki Nature Comm 2019). Similar to Setd8, many subunits of the Condensin II complex are highly expressed in erythroid cells compared to most other cell types (biogps.org). We hypothesized that the Condensin II complex, in conjunction with Setd8 and H4K20me1, is important for establishing appropriate patterns of chromatin architecture and gene expression in maturing erythroblasts.
To address this hypothesis, we deleted the NCAPH2 subunit in erythroid cells by crossing mice with floxed alleles of NCAPH2 with mice expressing cre-recombinase under the direction of the Erythropoietin receptor promotor (EpoRCre). Homozygous disruption of NCAPH2 was embryonic lethal by E13.5. NCAPH2 mutant embryos were similar in appearance to littermate controls until E12.5 when they developed notable pallor and a dramatic decline in the number of benzidine positive cells. Cell cycle analyses demonstrated that an accumulation of cells in G2/M preceded the dramatic decline in erythroblast numbers at E12.5. In contrast to cells from littermate controls, the NCAPH2 mutant cells were very heterogenous in cell and nuclear size and morphology. Surpisingly, most NCAPH2 mutant cells appeared to be hemoglobinized, suggesting sufficient iron accumulation and heme synthesis. In vitro cultures derived from primitive erythroid progenitors replicated in vivo findings, including normal early erythropoeisis, with significant abnormalities during mid- to late- maturation. Western blot in cycloheximide treated primitive erythroid cultures revealed that NCAPH2 has a long half-life, which likely contributes to the relative normalicy of early primitive erythoproesis. NCAPH2 mutant embryos also had a dramatic failure of definitive erythropoiesis, as evidenced by loss of mature erythroblasts in the fetal liver at E13.5. To gain insights into the mechanisms underlying these findings, we performed RNA-seq of NCAPH2 mutant, NCAPH2 het, and NCAPH2 WT erythroblasts from E11.5 embryos. Comparing NCAPH2 mutant and NCAPH2 WT erythroblasts there were 1121 down regulated genes and 743 upregulated genes (adj p-value <0.05). As expected, the downregulated genes were enriched for pathways related to cell cycle, such as Mitotic Spindle Organization (adj pvalue 5e-42). The upregulated genes were enriched for a variety of pathways including p53 transcriptional network (adj pvalue 4e-10), neutrophil mediated immunity (2e-9), DNA-binding transcription factor (adj pvalue 7e-5), and regulation of erythrocyte differentiation (adj pvalue 5e-4). Intriguingly, 91/340 genes differentially expressed in Setd8 mutant erythroblats were also differentially expressed in NCAPH2 mutant cells, including genes typically repressed early in erythroid commitment, such as GATA2 and SPI1. Cut&Tag in CD34+ derived erythroblasts demonstrated occupancy of H4K20me1 at these loci. Mass spectrometry of proteins isolated via mono-mehtylated H4K20 peptides in erythroid extracts identified Condensin II components, supporting a model where the Condensin II complex directly interacts with H4K20me1. Together, these results demonstrate that the Condensin II complex is essential for erythropoiesis, and may work in conjunction with Setd8 and H4K20me1 to establish appropriate patterns of gene expression in maturing erythroblasts.
No relevant conflicts of interest to declare.
