Abstract
BackgroundStroke 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 (E2). 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.MethodsTo 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.ResultsKdm5c 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.ConclusionsThe 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.
Highlights
Microglia from aged females had significantly higher levels of kdm5c compared to male microglia (Fig. 1a). kdm6a mRNA showed the same pattern (Fig. 1b)
Effects of Lysine-specific demethylase 6A (KDM6A) manipulation on microglial cytokine production As interferon regulatory factor 4 (IRF4)/IRF5 regulate gene expression of anti-/pro-inflammatory cytokines respectively in microglia [22], we examined the levels of cytokines in the homogenates of cultured neonatal microglia by RT-PCR after kdm5c or kdm6a manipulation. kdm6a siRNA treatment led to significant decreases in TNFα mRNA levels in both male and female microglia (Fig. 7a, b), while lenti-kdm6a significantly increased TNFα mRNA in microglia of both sexes (Fig. 7c, d). kdm6a knockdown by siRNA induced a significant decrease in iNOS mRNA in males with a decrease trend in females, and kdm6a overexpression induced a significant increase in pro-inflammatory iNOS in females with an increase trend in males (Fig. 7e–h)
It is likely that the baseline sex difference in microglial expression of kdm6a/kdm5c is masked by their global expression in the brain; these X chromosome inactivation (XCI) escapees in microglia may be susceptible to the ischemic insult, and once a stroke occurs, the baseline sex difference is amplified leading to a global sex difference in the aged ischemic brains
Summary
There are 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. Stroke is a sexually dimorphic disease [3, 4]. Young women have lower stroke incidence than young men thought to be due, in part, to the protective effect of estrogen [5, 6]. Stroke sensitivity is mediated primarily by gonadal hormones in young animals, regardless of their chromosome complement, but as animals age, chromosomal sex drives ischemic outcomes [9]. Because the number of X chromosomes correlates with susceptibility to ischemic disease, we asked if specific X genes contribute to this phenotype
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