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酶在内的表观遗传调节因子有助于正常的造血。在小鼠的条件突变中，我们和其他人已经证明了位于X染色体上的组蛋白3赖氨酸27(H3K27)去甲基酶UTX(KDM6A)是成年雌性小鼠胚胎发育和造血所必需的。UTX是少数逃脱X染色体失活的基因之一，因此女性同时表达两种UTX等位基因。值得注意的是，在Y染色体上存在一对UTX，UTY，因此男性表达半合子对UTX/UTY。值得注意的是，据报道，UTY的脱甲基酶活性明显低于UTX，这可能会导致性别差异。UTX和UTY之间的其他差异包括癌症中UTX突变频率显著更高。我们还观察到了UTX突变/UTY-wt(雄性)胚胎中的杂合子表型，这在UTX-突变/UTX-wt(雌性)或UTY-突变/UTX-wt(雄性)胚胎中并不明显，这表明在小鼠的发育过程中，UTY并不能替代所有的UTX功能。脱甲基酶活性的差异可以解释这些UTX-UTY的差异，然而UTX不需要去甲基化的功能已经被报道。为了解决UTX和UTY的差异，我们将通过对UTX和UTY loxP等位基因的条件突变，结合移植实验、转录组分析和靶基因鉴定，来研究它们在小鼠HSPC动态平衡中的作用。为了评估去甲基酶依赖和独立功能的贡献，我们将使用BAC转基因突变或不突变的BAC转基因体外培养条件下的UTX和UTY间充质干细胞进行造血分化试验，以检测去甲基酶活性，这可能得到永生化HSPC的类似实验的支持。根据最近的预测，UTX/UTY蛋白复合体是二聚体，我们还将评估男性ESCs是否包含三种类型的复合体(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。这项研究提案将对UTX/UTY的表观遗传调控，特别是小鼠HSPC的动态平衡提供新的见解。它将建立在我们正在进行的对组蛋白3赖氨酸甲基化回路的分析的基础上，并对其进行补充，并可能揭示性别差异的分子基础。

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.