Transcription Elongation and Tissue-Specific Somatic CAG Instability

The expansion of CAG/CTG repeats is responsible for many diseases, including Huntington's disease (HD) and myotonic dystrophy 1. CAG/CTG expansions are unstable in selective somatic tissues, which accelerates disease progression. The mechanisms underlying repeat instability are complex, and it remains unclear whether chromatin structure and/or transcription contribute to somatic CAG/CTG instability in vivo. To address these issues, we investigated the relationship between CAG instability, chromatin structure, and transcription at the HD locus using the R6/1 and R6/2 HD transgenic mouse lines. These mice express a similar transgene, albeit integrated at a different site, and recapitulate HD tissue-specific instability. We show that instability rates are increased in R6/2 tissues as compared to R6/1 matched-samples. High transgene expression levels and chromatin accessibility correlated with the increased CAG instability of R6/2 mice. Transgene mRNA and H3K4 trimethylation at the HD locus were increased, whereas H3K9 dimethylation was reduced in R6/2 tissues relative to R6/1 matched-tissues. However, the levels of transgene expression and these specific histone marks were similar in the striatum and cerebellum, two tissues showing very different CAG instability levels, irrespective of mouse line. Interestingly, the levels of elongating RNA Pol II at the HD locus, but not the initiating form of RNA Pol II, were tissue-specific and correlated with CAG instability levels. Similarly, H3K36 trimethylation, a mark associated with transcription elongation, was specifically increased at the HD locus in the striatum and not in the cerebellum. Together, our data support the view that transcription modulates somatic CAG instability in vivo. More specifically, our results suggest for the first time that transcription elongation is regulated in a tissue-dependent manner, contributing to tissue-selective CAG instability.CAG/CTG重复序列（CAG/CTG repeats）的扩增与多种疾病密切相关，包括亨廷顿舞蹈症（Huntington's disease, HD）和1型肌强直性营养不良。CAG/CTG扩增在特定体细胞组织中不稳定，这会加速疾病进展。重复序列不稳定性的潜在机制十分复杂，目前仍不清楚染色质结构（chromatin structure）和/或转录（transcription）是否在体内影响体细胞CAG/CTG序列的不稳定性。为解决这一问题，我们利用R6/1与R6/2亨廷顿病转基因小鼠品系（R6/1 and R6/2 HD transgenic mouse lines），在亨廷顿病基因座上研究了CAG序列不稳定性、染色质结构与转录之间的关联。这两种小鼠携带相似的转基因，尽管整合位点不同，但均可重现亨廷顿病的组织特异性不稳定性。我们发现，相较于R6/1的匹配样本，R6/2组织中的不稳定性发生率更高。R6/2小鼠CAG序列不稳定性的升高与转基因高表达水平及染色质可及性（chromatin accessibility）相关。与R6/1的匹配组织相比，R6/2组织中亨廷顿病基因座的转基因mRNA水平与H3K4三甲基化（H3K4 trimethylation）水平均有所升高，而H3K9二甲基化（H3K9 dimethylation）水平则有所降低。不过，无论属于哪种小鼠品系，纹状体（striatum）与小脑（cerebellum）这两种CAG序列不稳定性水平差异显著的组织中，转基因表达水平与上述特定组蛋白修饰标记的水平均较为相似。值得注意的是，亨廷顿病基因座上的延伸型RNA聚合酶II（elongating RNA Pol II）水平（而非起始型RNA聚合酶II）具有组织特异性，且与CAG序列不稳定性水平相关。同样，与转录延伸相关的修饰标记H3K36三甲基化（H3K36 trimethylation），在纹状体的亨廷顿病基因座上显著升高，而在小脑中则无此现象。综上，我们的数据支持转录在体内调控体细胞CAG序列不稳定性这一观点。更具体地说，我们的研究首次表明，转录延伸以组织依赖的方式受到调控，进而促成了组织选择性的CAG序列不稳定性。