CRISPR mediated targeting of DUX4 distal regulatory element represses DUX4 target genes dysregulated in Facioscapulohumeral muscular dystrophy.
CRISPR mediated targeting of DUX4 distal regulatory element represses DUX4 target genes dysregulated in Facioscapulohumeral muscular dystrophy.
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DOI：
10.1038/s41598-021-92096-0
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发表时间：
2021-06-15
影响因子：
4.6
通讯作者：
Chadwick BP
中科院分区：
文献类型：
--
作者：
Das S;Chadwick BP
面肩肱骨肌营养不良症（FSHD）是一种使人衰弱的肌肉疾病，目前尚无有效的治疗方法。DUX4是一种在体细胞组织中被表观遗传沉默的胚胎基因，它的异常再激活是FSHD的原因。DUX4的疾病特异性再激活具有两个共同特征：DUX4的外显子3内存在非规范多聚腺苷化序列，该序列稳定致病性转录物；以及编码DUX4的大卫星重复序列D4Z4的抑制性染色质修饰缺失。我们使用CRISPR/Cas9通过两种独立的方法沉默DUX4。我们删除了DUX4致病性多腺苷化信号，导致致病性DUX4-fl转录下调。在另一种方法中，我们通过在外显子3内使用dCas9-KRAB平台播种异染色质来转录抑制DUX4。这些靶向DUX4实验的可行性最初在我们之前描述的非肌源性癌细胞系中进行了测试。随后，在一个永生化的患者成肌细胞系中，我们证明了通过任何一种方法靶向DUX4不仅导致致病性DUX4转录物的大幅下调，而且还导致其靶基因子集（已知的FSHD生物标志物）的大幅下调。这些发现为沉默聚腺苷酸化序列对致病性DUX4表达的影响提供了概念证明。
Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating muscle disease that currently does not have an effective cure or therapy. The abnormal reactivation of DUX4, an embryonic gene that is epigenetically silenced in somatic tissues, is causal to FSHD. Disease-specific reactivation of DUX4 has two common characteristics, the presence of a non-canonical polyadenylation sequence within exon 3 of DUX4 that stabilizes pathogenic transcripts, and the loss of repressive chromatin modifications at D4Z4, the macrosatellite repeat which encodes DUX4. We used CRISPR/Cas9 to silence DUX4 using two independent approaches. We deleted the DUX4 pathogenic polyadenylation signal, which resulted in downregulation of pathogenic DUX4-fl transcripts. In another approach, we transcriptionally repressed DUX4 by seeding heterochromatin using the dCas9-KRAB platform within exon 3. These feasibility of targeting DUX4 experiments were initially tested in a non-myogenic carcinoma cell line that we have previously characterized. Subsequently, in an immortalized patient myoblast cell line, we demonstrated that targeting DUX4 by either approach led to substantial downregulation of not only pathogenic DUX4 transcripts, but also a subset of its target genes that are known biomarkers of FSHD. These findings offer proof-of-concept of the effect of silencing the polyadenylation sequence on pathogenic DUX4 expression.
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影响因子：
4.8
作者：
Ansari, Amir Mehdi;Ahmed, A. Karim;Matsangos, Aerielle E.;Lay, Frank;Born, Louis J.;Marti, Guy;Harmon, John W.;Sun, Zhaoli
通讯作者：
Sun, Zhaoli
影响因子：
3.7
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Das S;Chadwick BP
通讯作者：
Chadwick BP
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影响因子：
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作者：
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通讯作者：
Buratowski, S
影响因子：
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作者：
DeSimone, Alec M.;Pakula, Anna;Emerson, Charles P., Jr.
通讯作者：
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