Gene expression patterns vary in clonal cell cultures from Rett syndrome females with eight different MECP2 mutations.
Gene expression patterns vary in clonal cell cultures from Rett syndrome females with eight different MECP2 mutations.
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DOI：
10.1186/1471-2350-3-12
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发表时间：
2002-11-05
影响因子：
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通讯作者：
Francke, Uta
中科院分区：
文献类型：
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作者：
Traynor, Jeff;Agarwal, Priyanka;Francke, Uta
背景技术背景：患有神经系统疾病Rett综合征的女性是X连锁MECP 2突变的杂合子，MECP 2编码甲基CpG结合蛋白2（MeCP 2），被认为是转录抑制因子。为了鉴定MeCP 2调节的靶基因，我们研究了具有四种常见突变的单细胞来源的野生型和突变型MECP 2表达成纤维细胞克隆中的整体基因表达（R106 W、R306 C、705 delG、1155 del 32）和包括四种突变MeCP 2的类淋巴母细胞系（LCL）中的细胞毒性。（T158 M、803 delG、R168 X和1159 del 28）表达的细胞系和5个（1159 del 28、R106 W、R255 X、803 delG、803 delG）野生型MeCP 2表达细胞系。表达研究用寡核苷酸芯片（Affyestival U95）进行，并验证了实时定量RT-PCR使用Sybr绿色。结果：49个转录本的表达增加，21个转录本的表达减少，在至少3/4突变/野生型成纤维细胞的比较。通过定量RT-PCR测定的11个基因的转录水平与微阵列数据高度相关。因此，仅通过RT-PCR测试来自两个Rett个体的多个额外克隆。在突变体和野生型MeCP 2表达克隆中发现了显著的表达差异。比较淋巴母细胞系的表达谱产生了16个差异表达的基因。结论：MeCP 2缺乏不会导致全球基因表达失调。MeCP 2的体内功能不涉及广泛的转录抑制，或者其功能在也表达其他甲基-CpG结合蛋白的细胞类型中是多余的。我们的数据表明，克隆成纤维细胞株可能会显示出很大的株间变异，使它们成为全基因组表达谱研究的困难和不稳定的资源。
BACKGROUND: Females with the neurological disorder Rett syndrome are heterozygous for mutations in X-linked MECP2 that encodes methyl-CpG binding protein 2 (MeCP2) thought to act as a transcriptional repressor. To identify target genes for MeCP2 modulation, we studied global gene expression in single cell-derived wild-type and mutant MECP2 expressing fibroblast clones with four common mutations (R106W, R306C, 705delG, 1155del32) and in lymphoblastoid cell lines (LCLs) that included four mutant MeCP2 (T158M, 803delG, R168X and 1159del28) expressing, and five (1159del28, R106W, R255X, 803delG, 803delG) wild-type MeCP2 expressing lines.METHODS: Clonality and mutation status were verified by androgen receptor methylation assays for X-inactivation and by sequencing MECP2 transcripts. Expression studies were done with oligonucleotide microarrays (Affymetrix U95) and verified with real-time quantitative RT-PCR using Sybr Green.RESULTS: Expression of 49 transcripts was increased, and expression of 21 transcripts was decreased, in at least 3 of 4 mutant/wild-type fibroblast comparisons. Transcript levels of 11 genes, determined by quantitative RT-PCR, were highly correlated with the microarray data. Therefore, multiple additional clones from two Rett individuals were tested by RT-PCR only. Striking expression differences were found in both mutant and wildtype MeCP2 expressing clones. Comparing expression profiles of lymphoblastoid cell lines yielded 16 differentially expressed genes.CONCLUSIONS: MeCP2 deficiency does not lead to global deregulation of gene expression. Either MeCP2's in vivo function does not involve widespread transcriptional repression, or its function is redundant in cell types that also express other methyl-CpG binding proteins. Our data suggest that clonal fibroblast strains may show substantial inter-strain variation, making them a difficult and unstable resource for genome-wide expression profiling studies.