Contributions of the H3K27 demethylases UTX (KDM6A) and UTY (KDM6C) to hematopoiesis and sex specific differences in gene expression

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Accumulating evidence indicates that epigenetic regulators including histone methyltransferases and demethylases contribute to normal hematopoiesis. Using conditional mutagenesis in mice we and others have shown that the histone 3 lysine 27 (H3K27) demethylase UTX (KDM6A), located on the X-chromosome, is required for embryonic development and hematopoiesis in adult female mice. Utx is one of the few genes that escape X-chromosome inactivation and so females express both Utx alleles. Notably, a Utx paralog, Uty, is present on the Y-chromosome so males express the hemizygous pair Utx/Uty. Notably, it has been reported that UTY has significantly less demethylase activity than UTX, which has potential implications for sex-specific differences. Other differences between UTX and UTY include significantly higher UTX mutation frequencies in cancers. We have also observed a heterozygous phenotype in Utx-mutant/Uty-wt (male) embryos that is not apparent in Utx-mutant/Utx-wt (female) or Uty-mutant/Utx-wt (male) embryos, indicating that UTY does not substitute for all UTX functions during mouse development. The difference in demethylase activities could account for these UTX-UTY differences, however UTX functions that do not require demethylation have been reported. To address the differences between UTX and UTY, we will investigate their roles in murine HSPC homeostasis using conditional mutagenesis of Utx and Uty loxP alleles combined with transplantation experiments, transcriptome analyses and target gene identification. To evaluate the contributions of demethylase dependent versus independent functions, we will employ an in vitro hematopoietic differentiation assay from conditional Utx and Uty mESCs using BAC transgenes point mutated or not for demethylase activity, potentially supported by similar experiments with immortalized HSPCs. Following the recent prediction that the UTX/UTY protein complexes are dimeric, we will also evaluate whether male ESCs contain three types of complex (UTY/UTY; UTY/UTX and UTX/UTX) as opposed to one type in female ESCs (UTX/UTX) to determine if this could also be a source of sex specific differences. This research proposal will deliver new insights into UTX/UTY epigenetic regulation in general and murine HSPC homeostasis in particular. It will build upon and complement our ongoing analyses of the histone 3 lysine methylation circuitry in hematopoiesis and may uncover a molecular basis for sex specific differences.