Influence of Repressive Histone and DNA Methylation upon D4Z4 Transcription in Non-Myogenic Cells.
Influence of Repressive Histone and DNA Methylation upon D4Z4 Transcription in Non-Myogenic Cells.
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DOI：
10.1371/journal.pone.0160022
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Chadwick BP
中科院分区：
文献类型：
--
作者：
Das S;Chadwick BP
我们研究了疾病相关的大卫星阵列D4 Z4，并专注于影响其疾病背景之外的染色质状态的表观遗传因素。我们使用了HCT 116细胞系，该细胞系含有稳定由D4 Z4编码的人双同源框基因DUX 4的体细胞转录物所需的非典型多聚腺苷酸化（poly-A）信号。在HCT 116中，D4 Z4被包装到组成型异染色质中，其特征是DNA甲基化和组蛋白H3在赖氨酸9（H3 K9 me 3）处的三甲基化，导致D4 Z4衍生转录本的基础水平较低。然而，DNA甲基转移酶基因DNMT 1和DNMT 3B的双敲除（DKO），但不是单独的，导致DNA和H3 K9甲基化的显著损失。这与来自阵列的转录物水平的上调相结合，包括DUX 4同种型（DUX 4-fl），其与DUX 4蛋白一起在疾病面肩肱型肌营养不良症（FSHD）中的体肌中异常表达，如通过DUX 4的bondafide靶标在DKO而不是HCT 116细胞中的上调间接指示的。在HCT 116中用组蛋白甲基化的化学抑制剂处理的结果表明，在不存在DNA低甲基化的情况下，单独的H3 K9 me 3损失足以促进DUX 4-fl转录。此外，来自具有非典型多聚-A信号和D4 Z4处DNA低甲基化的免疫缺陷、着丝粒不稳定和面部异常综合征1（ICF 1）患者的细胞系的表征显示，与对照相比，患者中DUX 4靶基因上调，尽管保留了H3 K9 me 3。总之，这些数据表明DNA甲基化和H3 K9 me 3都是D4 Z4沉默的决定因素。此外，我们表明，除了睾丸，有可观的表达剪接和多腺苷酸化D4 Z4衍生的转录本，包含完整的DUX 4开放阅读框（ORF）沿着DUX 4靶基因表达在胸腺中，这表明DUX 4可能提供正常的功能，在这个体细胞组织。
We looked at a disease-associated macrosatellite array D4Z4 and focused on epigenetic factors influencing its chromatin state outside of the disease-context. We used the HCT116 cell line that contains the non-canonical polyadenylation (poly-A) signal required to stabilize somatic transcripts of the human double homeobox gene DUX4, encoded from D4Z4. In HCT116, D4Z4 is packaged into constitutive heterochromatin, characterized by DNA methylation and histone H3 tri-methylation at lysine 9 (H3K9me3), resulting in low basal levels of D4Z4-derived transcripts. However, a double knockout (DKO) of DNA methyltransferase genes, DNMT1 and DNMT3B, but not either alone, results in significant loss of DNA and H3K9 methylation. This is coupled with upregulation of transcript levels from the array, including DUX4 isoforms (DUX4-fl) that are abnormally expressed in somatic muscle in the disease Facioscapulohumeral muscular dystrophy (FSHD) along with DUX4 protein, as indicated indirectly by upregulation of bondafide targets of DUX4 in DKO but not HCT116 cells. Results from treatment with a chemical inhibitor of histone methylation in HCT116 suggest that in the absence of DNA hypomethylation, H3K9me3 loss alone is sufficient to facilitate DUX4-fl transcription. Additionally, characterization of a cell line from a patient with Immunodeficiency, Centromeric instability and Facial anomalies syndrome 1 (ICF1) possessing a non-canonical poly-A signal and DNA hypomethylation at D4Z4 showed DUX4 target gene upregulation in the patient when compared to controls in spite of retention of H3K9me3. Taken together, these data suggest that both DNA methylation and H3K9me3 are determinants of D4Z4 silencing. Moreover, we show that in addition to testis, there is appreciable expression of spliced and polyadenylated D4Z4 derived transcripts that contain the complete DUX4 open reading frame (ORF) along with DUX4 target gene expression in the thymus, suggesting that DUX4 may provide normal function in this somatic tissue.