Transcription profiling of cerebellum from Mecp2 mutant mice to identify pathways mis-regulated by MeCP2 deficiency in Rett Syndrome

Background MeCP2, methyl-CpG-binding protein 2, binds to methylated cytosines at CpG dinucleotides, as well as to unmethylated DNA, and affects chromatin condensation. MECP2 mutations in females lead to Rett syndrome, a neurological disorder characterized by developmental stagnation and regression, loss of purposeful hand use and speech, stereotypic hand movements, deceleration of brain growth, autonomic dysfunction and seizures. Most mutations occur de novo during spermatogenesis. Located at Xq28, MECP2 is subject to X inactivation, and affected females are mosaic. Rare hemizygous males suffer from a severe congenital encephalopathy. Methods To identify pathways mis-regulated by MeCP2 deficiency, microarray-based global gene expression studies were carried out on cerebellum of Mecp2 mutant mice. We compared transcript levels in mutant/wildtype male sibs of two different MeCP2-deficient mouse models at 2, 4 and 8 weeks of age. Increased transcript levels were evaluated by real-time quantitative RT-PCR. Chromatin immunoprecipitation assays were used to document in vivo MeCP2 binding to promoter regions of candidate target genes. Results In the mutants, several hundred genes showed altered expression levels. Twice as many were increased than decreased, and only 27 genes were differentially expressed at more than one time point. The number of misregulated genes was 30% lower in mice with an exon 3 deletion (Mecp2tm1.1Jae) than in mice with a larger deletion (Mecp2tm1.1Bird). Between the mutants, few misregulated genes overlapped at each time point. Real-time quantitative RT-PCR assays validated increased transcript levels for four genes: Irak1, interleukin-1 receptor-associated kinase 1; Fxyd1, phospholemman, associated with Na, K-ATPase; Reln, encoding reelin, an extracellular signaling molecule essential for neuronal lamination and synaptic plasticity; and Gtl2/Meg3, an imprinted maternally expressed non-translated RNA that serves as a host gene for C/D box snoRNAs and microRNAs. Chromatin immunoprecipitation assays documented in vivo MeCP2 binding to promoter regions of Fxyd1, Reln, and Gtl2. Conclusions Transcriptional profiling of cerebellum failed to detect significant global changes in Mecp2-mutant mice. Increased transcript levels of Irak1, Fxyd1, Reln, and Gtl2 may contribute to the neuronal dysfunction in MeCP2-deficient mice and individuals with Rett syndrome. Our data provide testable hypotheses for future studies of the regulatory or signaling pathways that these genes act on.**背景**：甲基CpG结合蛋白2（methyl-CpG-binding protein 2, MeCP2）可结合CpG二核苷酸（CpG dinucleotides）上的甲基化胞嘧啶，同时也能结合非甲基化DNA，并参与调控染色质浓缩。MECP2基因突变在女性中可引发雷特综合征（Rett syndrome）——一种以发育停滞与倒退、有目的的手部使用及言语能力丧失、刻板样手部动作、脑生长减速、自主神经功能障碍及发作为特征的神经系统疾病。多数突变在精子发生过程中新发。MECP2基因定位于Xq28，受X染色体失活调控，受累女性为嵌合体个体。罕见的半合子男性患者会罹患严重的先天性脑病。 **方法**：为识别MeCP2缺失所导致的失调通路，我们对Mecp2突变小鼠的小脑组织开展了基于微阵列的全基因表达分析。我们比较了两种不同MeCP2缺陷小鼠模型的雄性同胞在2、4、8周龄时的突变型与野生型转录本水平。通过实时定量逆转录聚合酶链反应（real-time quantitative RT-PCR）验证了上调的转录本丰度。采用染色质免疫沉淀（chromatin immunoprecipitation）实验验证了MeCP2在体内与候选靶基因启动子区域的结合。 **结果**：突变小鼠体内有数百个基因的表达水平发生改变。上调基因的数量为下调基因的两倍，且仅27个基因在多个时间点呈现差异表达。携带外显子3缺失的小鼠（Mecp2tm1.1Jae）的失调基因数量比携带大片段缺失的小鼠（Mecp2tm1.1Bird）少30%。在每个时间点，两种突变模型的失调基因重叠度均较低。实时定量逆转录聚合酶链反应验证了4个基因的转录本水平上调：白细胞介素-1受体相关激酶1（interleukin-1 receptor-associated kinase 1, Irak1）、Fxyd1（编码与Na,K-ATP酶相关的磷酸膜蛋白（phospholemman））、Reln（编码reelin，一种对神经元分层及突触可塑性至关重要的细胞外信号分子），以及Gtl2/Meg3——一种印记母源表达的非翻译RNA，可作为C/D盒小核仁RNA（C/D box snoRNAs）与微小RNA（microRNAs）的宿主基因。染色质免疫沉淀实验证实，MeCP2在体内可结合Fxyd1、Reln及Gtl2的启动子区域。 **结论**：对小脑组织的转录组分析未检测到Mecp2突变小鼠存在显著的全局表达变化。Irak1、Fxyd1、Reln及Gtl2的转录本水平上调，可能参与了MeCP2缺陷小鼠及雷特综合征患者的神经元功能障碍。本研究数据为后续探索这些基因所作用的调控或信号通路提供了可验证的假说。