Genome-wide Analysis Reveals Mecp2-dependent Regulation of MicroRNAs in a Mouse Model of Rett Syndrome (mm8 promoter arrays)

MicroRNAs (miRNAs) are a class of small non-coding RNAs that function as post-transcriptional regulators of gene expression. Many miRNAs are expressed in the developing brain and regulate multiple aspects of neural development including neurogenesis, dendritogenesis and synapse formation. Rett syndrome (RTT) is a progressive neurodevelopmental disorder caused by mutations in the gene encoding Methyl-CpG binding protein 2 (MECP2). While Mecp2 is known to act as a global transcriptional regulator, miRNAs that are directly regulated by Mecp2 in the brain are not known. Using massively parallel sequencing methods, we have identified miRNAs whose expression is altered in cerebella of Mecp2-null mice before and after the onset of severe neurological symptoms. In vivo genome-wide analyses indicate that promoter regions of a significant fraction of dys-regulated miRNA transcripts, including a large polycistronic cluster of brain-specific miRNAs, are DNA methylated and directly bound by Mecp2. Functional analysis demonstrates that the 3' untranslated region (UTR) of messenger RNA encoding Brain-derived neurotrophic factor (Bdnf) can be targeted by multiple miRNAs aberrantly up-regulated in absence of Mecp2. Taken together, these results suggest that dys-regulation of miRNAs may contribute to RTT pathoetiology, and also provide a valuable resource to further investigate the role of miRNAs in RTT. Chromatin extracted from postnatal 6-8 week old cerebellar (CB) tissues of wild-type (WT) or Mecp2-null (KO) male mice was immunoprecipitated with indicated antibodies and analyzed by NimbleGen custom mouse 385K promoter tiling microarrays (a 2-array set covering the promoter regions of all Refseq protein-coding genes and miRNA transcripts with predicted transcription start sites). Whole cell extract (WCE) was used as input controls in all experiments. DNA methylation profiles in WT CB were also analyzed by methylated DNA immunoprecipitation (MeDIP) followed by hybridization to the same promoter tiling microarrays (MeDIP-chip).微小RNA（microRNAs，miRNAs）是一类小型非编码RNA，可作为基因表达的转录后调控因子发挥功能。诸多miRNA在发育中的大脑内表达，并调控神经发生、树突发生与突触形成等多个神经发育过程。 雷特综合征（Rett syndrome，RTT）是一种由编码甲基CpG结合蛋白2（Methyl-CpG binding protein 2，MECP2）的基因突变引发的进行性神经发育障碍。尽管已知Mecp2可作为全局转录调控因子发挥作用，但大脑中直接受Mecp2调控的miRNA目前尚不明确。 本研究借助大规模并行测序技术，鉴定出了Mecp2基因敲除小鼠小脑在严重神经症状发作前后表达量发生改变的miRNA。全基因组体内分析显示，存在异常表达的miRNA转录本（包括一个大型脑特异性miRNA多顺反子簇）的启动子区域存在DNA甲基化修饰，且可被Mecp2直接结合。功能实验证实，编码脑源性神经营养因子（Brain-derived neurotrophic factor，Bdnf）的信使RNA（messenger RNA，mRNA）的3'非翻译区（UTR）可被Mecp2缺失时异常上调的多种miRNA靶向结合。 综上，上述结果表明miRNA的异常调控可能参与雷特综合征的发病机制，同时也为进一步探究miRNA在雷特综合征中的作用提供了宝贵的研究资源。 本研究使用出生后6-8周龄野生型（wild-type，WT）或Mecp2基因敲除（Mecp2-null，KO）雄性小鼠的小脑（CB）组织提取染色质，通过指定抗体进行免疫沉淀，随后使用NimbleGen定制小鼠385K启动子平铺式微阵列（一套覆盖所有RefSeq编码蛋白基因及带有预测转录起始位点的miRNA转录本启动子区域的2张阵列）进行分析。所有实验均以全细胞提取物（WCE）作为输入对照。 本研究还通过甲基化DNA免疫沉淀（methylated DNA immunoprecipitation，MeDIP）后与上述启动子平铺式微阵列杂交的技术（MeDIP-chip），分析了野生型小鼠小脑的DNA甲基化谱。