The phosphorylation by aurkb regulates transcriptional functions of KLF1 during erythropoiesis Free

Erythroid Krüppel-like factor (EKLF/KLF1) is a master transcriptional regulator of erythropoiesis, and when KLF1 is mutated, it leads to varied phenotypes ranging from mild symptoms such as increase in fetal hemoglobin to severe dyserythropoietic anemia and in extreme cases hydrops fetalis. Therefore, it is critical to understand how KLF1 is regulated and how this regulation contributes towards normal red blood cell development. In the current study, we have characterized a novel putative phosphorylation site for Aurora B Kinase (AURKB) on KLF1, and we have investigated the importance of this site for KLF1's transcriptional functions.

Firstly, to verify whether KLF1 and AURKB interact, we employed immunoprecipitation using Human Embryonic Kidney cells (HEK293T) cells transfected with FLAG-tagged mouse KLF1 and found that AURKB coimmunoprecipitates with KLF1. Further, by conducting an in vitro kinase assay using recombinant AURKB and KLF1 proteins and then running the reaction on an SDS-page gel, followed by staining the gel with a phospho-protein stain, we found that AURKB does phosphorylate KLF1.

To test how the putative AURKB phosphorylation site on KLF1 affects its functions, we used a cytosine base editor for mutating the site from a serine to a leucine, to generate a phosphorylation-dead site in Human Umbilical Derived Erythroid Progenitor cells (HUDEP2). Using these cells, we found that through western blotting, there was an increase in fetal gamma globin and a decrease in adult beta globin. We also saw an increase in Lin-28 homolog B (LIN28B) protein, which is important for fetal globin expression in fetal blood cells. There was no significant difference in the level of KLF1 protein when the mutant cells were compared to wild type. To look at how the hemoglobin tetramer profile is affected, we used High-Performance Liquid Chromatography (HPLC), and we found that the phosphorylation dead mutants contained more than 70% of fetal hemoglobin when compared to the wild type, indicating an almost complete reversal of hemoglobin switching that normally occurs at birth.

To investigate the impact of the phosphorylation site on KLF1's transcription ability, we did a reporter assay by nucleofecting the cells with the beta globin promoter attached to a firefly gene and then measured the chemiluminescence. We observed a significant decrease in KLF1's transcriptional activity in the HUDEP2 cells harboring phospho-dead mutant KLF1 when compared to the wild-type HUDEP2 cells.

Upon analyzing the progress of terminal differentiation, we saw a delay with the cell surface marker glycophorin A expression when doing flow cytometry, with the levels of glycophorin A increasing by day 4 of differentiation for the wild-type cells, while the mutant cells only showed an increase at day 6 of differentiation. We also observed that the mutant cells have a larger cell area than the wild-type cells and fail to decrease in size by day 4 of differentiation; the mutant cells only start to decrease in size around day 6 of differentiation compared to expanding cells. The mutant cells also have a delay in hemoglobinization, indicated by the paler cell pellet color and the amount of hemoglobin per million cells via HPLC. Interestingly, on day six of differentiation, there is a sharp increase in the amount of hemoglobin in these mutant cells that is more than the wild-type counterpart. Overall, these observations in HUDEP2 cells harboring phospho-dead mutant KLF1 are consistent with a fetal erythroid profile, similar to what is observed in erythroid cells with increased LIN28B levels.

Overall, we have discovered a novel a mechanism by which the function of the erythroid master regulator, KLF1, is regulated by its interaction with AURKB and by phosphorylation at its AURKB phosphorylation motif. Given that the mutation at the AURKB phosphorylation site on KLF1 leads to around 70% fetal hemoglobin in erythroid cells compared to wild type cells that express less than 1% fetal hemoglobin, these investigations could contribute towards developing treatments for beta hemoglobinopathies such as sickle cell anemia and beta thalassemia, where an increase in fetal hemoglobin can ameliorate the symptoms.