Lsh regulates LTR retrotransposon repression independent of Dnmt3b function (RNA-Seq)

Background: Global DNA methylation contributes to genomic integrity by supressing repeat associated transposition events. Several chromatin factors are required in addition to DNA methyltransferases to maintain DNA methylation at intergenic and satellite repeats. Embryos lacking Lsh, a member of the SNF2 superfamily of chromatin helicases, are hypomethylated. The interaction of Lsh with the de novo methyltransferase, Dnmt3b, facilitates the deposition of DNA methylation at stem cell genes. We wished to determine if a similar targeting mechanism operates to maintain DNA methylation at repetitive sequences. Results: We used HELP-seq to map genome wide DNA methylation patterns in Lsh-/- and Dnmt3b-/- somatic cells. DNA methylation is predominantly lost from specific genomic repeats in Lsh-/- cells: LTR-retrotransposons, LINE-1 repeats and mouse satellites. RNA-seq experiments demonstrate that specific IAP (Intracisternal A-type particle) LTRs and satellites, but not LINE-1 elements, are aberrantly transcribed inLsh-/- cells. LTR hypomethylation in Dnmt3b-/- cells is moderate and hypomethylated repetitive elements (IAP, LINE-1 and satellite) are silent. Chromatin immunoprecipitation (ChIP) indicates that repressed LINE-1 elements gain H3K4me3, but H3K9me3 levels are unaltered in Lsh-/- cells, indicating that DNA hypomethylation alone is not permissive for their transcriptional activation. Mis-expressed IAPs and satellites lose H3K9me3 and gain H3K4me3 in Lsh-/- cells. Conclusions: Our study emphasizes that regulation of repetitive elements by DNA methylation is selective and context dependent. We propose a model where Lsh is specifically required at a precise developmental window to target de novo methylation to repeat sequences, which is subsequently maintained by Dnmt1 in somatic cells to enforce repeat silencing thus contributing to genomic integrity. Two pairs of RNA samples compared: WT and Lsh-/- RNA isolations from tail-tip fibroblasts; WT and Lsh-/- RNA isolations from E13.5 mouse embryos.研究背景：全基因组DNA甲基化（global DNA methylation）通过抑制重复序列相关的转座事件维持基因组稳定性。除DNA甲基转移酶外，还需多种染色质因子以维持基因间区及卫星重复序列的DNA甲基化水平。缺失Lsh（染色质解旋酶SNF2超家族成员）的胚胎会出现低甲基化表型。Lsh与从头甲基转移酶Dnmt3b的相互作用，可促进DNA甲基化在干细胞基因区域的沉积。本研究旨在探究是否存在类似的靶向机制，以维持重复序列区域的DNA甲基化。 研究结果：本研究利用HELP-seq技术，在Lsh敲除（Lsh-/-）及Dnmt3b敲除（Dnmt3b-/-）体细胞中绘制全基因组DNA甲基化图谱。在Lsh-/-体细胞中，DNA甲基化主要从特定基因组重复序列中丢失：包括长末端重复序列逆转录转座子（LTR-retrotransposons）、LINE-1重复序列及小鼠卫星序列。RNA测序（RNA-seq）实验显示，特定的IAP（Intracisternal A-type particle）LTR序列及卫星序列在Lsh-/-细胞中出现异常转录，但LINE-1元件并无此现象。在Dnmt3b-/-细胞中，LTR区域的低甲基化程度较为温和，且低甲基化的重复序列元件（IAP、LINE-1及卫星序列）保持沉默。染色质免疫共沉淀（Chromatin immunoprecipitation，ChIP）实验结果表明，在Lsh-/-细胞中，受抑制的LINE-1元件获得了H3K4me3组蛋白修饰，但H3K9me3修饰水平未发生改变，这提示仅DNA低甲基化并不足以使其转录激活。在Lsh-/-细胞中，异常表达的IAP及卫星序列会丢失H3K9me3修饰并获得H3K4me3修饰。 研究结论：本研究证实，DNA甲基化对重复序列元件的调控具有选择性且依赖于具体语境。我们提出如下调控模型：Lsh需在特定发育窗口期发挥特异性作用，将从头甲基化靶向引导至重复序列区域；后续在体细胞中，Dnmt1可维持该区域的甲基化水平，从而强化重复序列的沉默状态，最终维持基因组稳定性。 本研究共设置两组对照RNA样本：分别为尾尖成纤维细胞的野生型（wild type）与Lsh-/- RNA提取样本，以及E13.5期小鼠胚胎的野生型与Lsh-/- RNA提取样本。