Cerebellar gene expression profiles of mouse models for Rett Syndrome reveal MeCP2 targets

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. A genetic modification design type is where an organism(s) has had genetic material removed, rearranged, mutagenized or added, such as knock out. Keywords: genetic_modification_design Computed背景：MeCP2（甲基CpG结合蛋白2，methyl-CpG-binding protein 2）可结合CpG二核苷酸位点的甲基化胞嘧啶，同时也能结合未甲基化DNA，并参与调控染色质浓缩过程。女性体内MECP2基因突变会引发雷特综合征（Rett syndrome），这是一类以发育停滞与倒退、有目的的手部活动与言语能力丧失、刻板性手部动作、脑生长速度减缓、自主神经功能障碍以及癫痫发作为特征的神经发育障碍性疾病。多数突变为精子发生过程中产生的新发突变。MECP2基因定位于Xq28，受X染色体失活调控，因此患病女性为遗传嵌合体。罕见的半合子男性患者会罹患严重的先天性脑病。 方法：为明确MeCP2缺失所失调的生物学通路，研究人员对Mecp2突变小鼠的小脑组织开展了基于微阵列的全基因组基因表达分析。我们对比了两种不同MeCP2缺陷小鼠模型的雄性同窝突变体与野生型个体在2周、4周和8周龄时的转录本水平。对于上调的转录本，通过实时定量逆转录聚合酶链反应（real-time quantitative RT-PCR）进行验证。采用染色质免疫沉淀（chromatin immunoprecipitation）实验，验证MeCP2在体内与候选靶基因启动子区域的结合情况。 结果：在突变小鼠中，数百个基因的表达水平出现异常。上调基因的数量为下调基因的两倍，且仅有27个基因在多个时间点呈现差异表达。相较于携带大片段缺失的小鼠（Mecp2tm1.1Bird），外显子3缺失型小鼠（Mecp2tm1.1Jae）的失调基因数量降低了30%。在两个突变模型之间，每个时间点仅有极少的失调基因存在重叠。实时定量RT-PCR验证了4个基因的转录本水平上调：Irak1（白细胞介素-1受体相关激酶1，interleukin-1 receptor-associated kinase 1）；Fxyd1（磷酸溶血蛋白，phospholemman），可与钠钾ATP酶结合发挥功能；Reln，编码reelin蛋白，该蛋白是神经元分层与突触可塑性所必需的细胞外信号分子；以及Gtl2/Meg3，一种母本印记表达的非翻译RNA，可作为C/D框小核仁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与Gtl2的启动子区域。 结论：对小脑组织的转录组分析并未在Mecp2突变小鼠中检测到显著的全局表达变化。Irak1、Fxyd1、Reln与Gtl2的转录本水平上调，可能参与了MeCP2缺陷小鼠以及雷特综合征患者的神经元功能障碍进程。本研究的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科学假说。遗传修饰设计类型指对生物体的遗传物质进行删除、重排、诱变或添加的操作，例如基因敲除（knock out）。关键词：genetic_modification_design 计算型（Computed）