Targeted Reactivation of FMR1 Transcription in Fragile X Syndrome Embryonic Stem Cells
Targeted Reactivation of FMR1 Transcription in Fragile X Syndrome Embryonic Stem Cells
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DOI：
10.3389/fnmol.2018.00282
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发表时间：
2018-08-15
影响因子：
4.8
通讯作者：
Todd, Peter K.
中科院分区：
文献类型：
--
作者：
Haenfler, Jill M.;Skariah, Geena;Todd, Peter K.
脆性X综合征（FXS）是智力残疾和自闭症最常见的遗传原因。它由FMR 1的5'非翻译区（UTR）中CGG核苷酸重复序列的扩增引起。大的扩增引起重复序列和启动子超甲基化、异染色质形成、FMR 1转录沉默和脆性X蛋白FMRP的丢失。迄今为止，旨在纠正FMRP丧失所导致的后遗症的努力被证明是不够的，这可能是因为FMRP的多效性功能。由于重复序列不破坏FMRP编码序列，内源性FMR 1基因表达的再激活可以纠正FXS发病机制中的近端事件。在这里，我们利用重复的规则间隔回文重复序列/缺陷型CRISPR相关蛋白9（CRISPR/dCas 9）系统来选择性地重新激活沉默的FMR 1基因座的转录。当直接靶向人细胞中的CGG重复序列时，转录激活因子VP 192与dCas 9的融合强烈增强FMR 1转录并增加FMRP水平。使用先前未表征的FXS人胚胎干细胞（hESC）系获得转录沉默与连续传代，我们实现了基因座特异性转录激活FMR 1信使RNA（mRNA）的表达，尽管启动子和重复甲基化。然而，这些在转录水平上的变化并不伴随FXS细胞中FMRP蛋白表达的显著升高。这些研究表明，将转录激活因子导向CGG重复序列足以选择性地重新激活脆性X患者干细胞中的FMR 1 mRNA表达。
Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and autism. It results from expansion of a CGG nucleotide repeat in the 5' untranslated region (UTR) of FMR1. Large expansions elicit repeat and promoter hyper-methylation, heterochromatin formation, FMR1 transcriptional silencing and loss of the Fragile X protein, FMRP. Efforts aimed at correcting the sequelae resultant from FMRP loss have thus far proven insufficient, perhaps because of FMRP's pleiotropic functions. As the repeats do not disrupt the FMRP coding sequence, reactivation of endogenous FMR1 gene expression could correct the proximal event in FXS pathogenesis. Here we utilize the Clustered Regularly Interspaced Palindromic Repeats/deficient CRISPR associated protein 9 (CRISPR/dCas9) system to selectively re-activate transcription from the silenced FMR1 locus. Fusion of the transcriptional activator VP192 to dCas9 robustly enhances FMR1 transcription and increases FMRP levels when targeted directly to the CGG repeat in human cells. Using a previously uncharacterized FXS human embryonic stem cell (hESC) line which acquires transcriptional silencing with serial passaging, we achieved locus-specific transcriptional re-activation of FMR1 messenger RNA (mRNA) expression despite promoter and repeat methylation. However, these changes at the transcript level were not coupled with a significant elevation in FMRP protein expression in FXS cells. These studies demonstrate that directing a transcriptional activator to CGG repeats is sufficient to selectively reactivate FMR1 mRNA expression in Fragile X patient stem cells.