X 染色体逃逸基因通过炎症信号的表观遗传修饰参与缺血性性别二态性。
X chromosome escapee genes are involved in ischemic sexual dimorphism through epigenetic modification of inflammatory signals.
机构信息
Department of Neurology, McGovern Medical School, The University of Texas Health Science Center at Houston, 6431 Fannin Street, Houston, TX, 77030, USA.
Department of Anesthesiology, Center for Shock, Trauma and Anesthesiology Research (STAR) Center, University of Maryland School of Medicine, Baltimore, MD, USA.
出版信息
J Neuroinflammation. 2021 Mar 12;18(1):70. doi: 10.1186/s12974-021-02120-3.
BACKGROUND
Stroke is a sexually dimorphic disease. Previous studies have found that young females are protected against ischemia compared to males, partially due to the protective effect of ovarian hormones, particularly estrogen (E). However, there are also genetic and epigenetic effects of X chromosome dosage that contribute to stroke sensitivity and neuroinflammation after injury, especially in the aged. Genes that escape from X chromosome inactivation (XCI) contribute to sex-specific phenotypes in many disorders. Kdm5c and kdm6a are X escapee genes that demethylate H3K4me3 and H3K27me3, respectively. We hypothesized that the two demethylases play critical roles in mediating the stroke sensitivity.
METHODS
To identify the X escapee genes involved in stroke, we performed RNA-seq in flow-sorted microglia from aged male and female wild type (WT) mice subjected to middle cerebral artery occlusion (MCAO). The expression of these genes (kdm5c/kdm6a) were confirmed in four core genotypes (FCG) mice and in post-mortem human stroke brains by immunohistochemistry (IHC), Western blot, and RT-PCR. Chromatin immunoprecipitation (ChIP) assays were conducted to detect DNA levels of inflammatory interferon regulatory factor (IRF) 4/5 precipitated by histone H3K4 and H3K27 antibodies. Manipulation of kdm5c/kdm6a expression with siRNA or lentivirus was performed in microglial culture, to determine downstream pathways and examine the regulatory roles in inflammatory cytokine production.
RESULTS
Kdm5c and kdm6a mRNA levels were significantly higher in aged WT female vs. male microglia, and the sex difference also existed in ischemic brains from FCG mice and human stroke patients. The ChIP assay showed the IRF 4/5 had higher binding levels to demethylated H3K4 or H3K27, respectively, in female vs. male ischemic microglia. Knockdown or over expression of kdm5c/kdm6a with siRNA or lentivirus altered the methylation of H3K4 or H3K27 at the IRF4/5 genes, which in turn, impacted the production of inflammatory cytokines.
CONCLUSIONS
The KDM-Histone-IRF pathways are suggested to mediate sex differences in cerebral ischemia. Epigenetic modification of stroke-related genes constitutes an important mechanism underlying the ischemic sexual dimorphism.
背景
中风是一种存在性别差异的疾病。先前的研究发现，年轻女性相较于男性对缺血具有保护作用，这部分归因于卵巢激素（尤其是雌激素）的保护作用。然而，X 染色体剂量也存在遗传和表观遗传效应，这会导致损伤后中风的敏感性和神经炎症增加，尤其是在老年人群中。逃逸 X 染色体失活（XCI）的基因在许多疾病中导致性别特异性表型。Kdm5c 和 kdm6a 是分别使 H3K4me3 和 H3K27me3 去甲基化的 X 逃逸基因。我们假设这两种去甲基酶在介导中风易感性方面发挥着关键作用。
方法
为了确定参与中风的 X 逃逸基因，我们对经历大脑中动脉闭塞（MCAO）的老年雄性和雌性野生型（WT）小鼠中经流式分选的小胶质细胞进行了 RNA-seq 分析。通过免疫组织化学（IHC）、Western blot 和 RT-PCR，在四种核心基因型（FCG）小鼠和死后中风大脑中对这些基因（kdm5c/kdm6a）的表达进行了确认。进行染色质免疫沉淀（ChIP）检测，以检测由组蛋白 H3K4 和 H3K27 抗体沉淀的炎症性干扰素调节因子（IRF）4/5 的 DNA 水平。通过 siRNA 或慢病毒对 kdm5c/kdm6a 的表达进行操作，在小胶质细胞培养物中确定下游途径，并研究其在炎症细胞因子产生中的调控作用。
结果
在老年 WT 雌性小胶质细胞中，kdm5c 和 kdm6a mRNA 水平明显高于雄性小胶质细胞，在 FCG 小鼠和人类中风患者的缺血性大脑中也存在这种性别差异。ChIP 检测表明，IRF 4/5 与去甲基化的 H3K4 或 H3K27 的结合水平在雌性缺血性小胶质细胞中高于雄性。用 siRNA 或慢病毒敲低或过表达 kdm5c/kdm6a 会改变 IRF4/5 基因的 H3K4 或 H3K27 甲基化，进而影响炎症细胞因子的产生。
结论
KDM-组蛋白-IRF 途径被认为介导了脑缺血的性别差异。与中风相关的基因的表观遗传修饰构成了缺血性性别二态性的重要机制。
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