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

越来越多的证据表明，包括组蛋白甲基转移酶和去甲基化酶在内的表观遗传调节因子有助于正常的造血。通过对小鼠进行条件诱变，我们和其他人已经证明，位于x染色体上的组蛋白3赖氨酸27 （H3K27）去甲基化酶UTX （KDM6A）是成年雌性小鼠胚胎发育和造血所必需的。Utx是为数不多的逃避x染色体失活的基因之一，因此雌性同时表达两个Utx等位基因。值得注意的是，在y染色体上存在Utx平行体Uty，因此雄性表达半合子对Utx/Uty。值得注意的是，有报道称UTY的去甲基酶活性明显低于UTX，这可能与性别特异性差异有关。UTX和UTY之间的其他差异包括UTX在癌症中的突变频率明显更高。我们还在UTX -mutant/ UTX -wt（雄性）胚胎中观察到杂合表型，而在UTX -mutant/ UTX -wt（雌性）或UTX -mutant/ UTX -wt（雄性）胚胎中则不明显，这表明在小鼠发育过程中，UTY并不能替代所有UTX的功能。去甲基化酶活性的差异可以解释这些UTX- uty差异，然而，已经报道了不需要去甲基化的UTX功能。为了解决UTX和UTY之间的差异，我们将利用UTX和UTY loxP等位基因的条件诱变，结合移植实验、转录组分析和靶基因鉴定，研究它们在小鼠HSPC稳态中的作用。为了评估去甲基化酶依赖与独立功能的贡献，我们将对使用BAC转基因点突变或未突变的条件Utx和Uty mESCs进行体外造血分化试验，可能会得到永生化HSPCs的类似实验的支持。根据最近的预测，UTX/UTY蛋白复合物是二聚体，我们还将评估男性ESCs是否含有三种类型的复合物（UTY/UTY； UTY/UTX和UTX/UTX），而不是女性ESCs中的一种类型（UTX/UTX），以确定这是否也可能是性别特异性差异的来源。这项研究计划将为一般的UTX/UTY表观遗传调控，特别是小鼠HSPC稳态提供新的见解。它将建立并补充我们正在进行的造血过程中组蛋白3赖氨酸甲基化电路的分析，并可能揭示性别特异性差异的分子基础。

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.