Independence of Repressive Histone Marks and Chromatin Compaction during Senescent Heterochromatic Layer Formation (ChIP-Seq). Homo sapiens

The expansion of repressive epigenetic marks has been implicated in heterochromatin formation during embryonic development, but the general applicability of this mechanism is unclear. Here we show that nuclear rearrangement of repressive histone marks H3K9me3 and H3K27me3 into non-overlapping structural layers characterizes senescence-associated heterochromatic foci (SAHF) formation in human fibroblasts. However, the global landscape of these repressive marks remains unchanged upon SAHF formation, suggesting that in somatic cells heterochromatin can be formed through the spatial repositioning of pre-existing repressively marked histones. This model is reinforced by the correlation of pre-senescent replication timing with both the subsequent layered structure of SAHFs and the global landscape of the repressive marks, allowing us to integrate microscopic and genomic information. Furthermore, modulation of SAHF structure does not affect the occupancy of these repressive marks nor vice versa. These experiments reveal that high-order heterochromatin formation and epigenetic remodeling of the genome can be discrete events. Overall design: ChIP-seq for different histone marks in both growing and Ras-induced senescent fibroblasts, in the presence or absence of certain sh-RNAs K9me3Grow2.bed (growing) Chip Seq Analysis of H3K9me3 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT K9me3Sen2.bed (senescent) Chip Seq Analysis of H3K9me3 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment K9me2Grow3.bed (growing) Chip Seq Analysis of H3K9me2 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment K9me2Sen3.bed (senescent) Chip Seq Analysis of H3K9me2 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT K27me3Sen3.bed (senescent) Chip Seq Analysis of H3K27me3 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT K27me3Grow2.bed (growing) Chip Seq Analysis of H3K27me3 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment K36me3Grow2.bed (growing) Chip Seq Analysis of H3K36me3 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment K36me3Sen2.bed (senescent) Chip Seq Analysis of H3K36me3 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT K4me3Grow2.bed (growing) Chip Seq Analysis of H3K4me3 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment K4me3Sen3.bed (senescent) Chip Seq Analysis of H3K4me3 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT

抑制性表观遗传标记的扩增已被证实与胚胎发育过程中的异染色质形成密切相关，但该机制的普遍适用性仍有待阐明。本研究发现，抑制性组蛋白标记H3K9me3与H3K27me3重排形成非重叠的结构层，这一特征是人类成纤维细胞中衰老相关异染色质焦点（senescence-associated heterochromatic foci，SAHF）形成的标志性事件。然而，SAHF形成后，这些抑制性标记的全基因组景观并未发生改变，这表明在体细胞中，异染色质可通过预先携带抑制性标记的组蛋白的空间重定位形成。该模型得到了以下相关性结果的支撑：衰老前细胞的复制时序与SAHF后续形成的层状结构以及抑制性标记的全基因组景观均存在显著关联，这使得我们能够整合显微成像与基因组学两类实验数据。此外，调控SAHF的结构既不会影响这些抑制性标记的染色质占据情况，反之亦然。本实验揭示了高阶异染色质形成与基因组表观重塑可作为两个相互独立的生物学过程。

总体实验设计：对增殖状态及Ras诱导衰老的成纤维细胞中不同组蛋白标记开展染色质免疫共沉淀测序（Chromatin Immunoprecipitation Sequencing，ChIP-seq）实验，实验分组包含存在或缺失特定短发夹RNA（short hairpin RNA，sh-RNA）的组别。具体样本信息如下：
- K9me3Grow2.bed（增殖组）：ER:Ras转染的IMR90人二倍体成纤维细胞经4-OHT处理第6天后的H3K9me3 ChIP-seq分析样本
- K9me3Sen2.bed（衰老组）：未进行4-OHT处理的ER:Ras转染IMR90人二倍体成纤维细胞的H3K9me3 ChIP-seq分析样本
- K9me2Grow3.bed（增殖组）：未进行4-OHT处理的ER:Ras转染IMR90人二倍体成纤维细胞的H3K9me2 ChIP-seq分析样本
- K9me2Sen3.bed（衰老组）：ER:Ras转染的IMR90人二倍体成纤维细胞经4-OHT处理第6天后的H3K9me2 ChIP-seq分析样本
- K27me3Sen3.bed（衰老组）：ER:Ras转染的IMR90人二倍体成纤维细胞经4-OHT处理第6天后的H3K27me3 ChIP-seq分析样本
- K27me3Grow2.bed（增殖组）：未进行4-OHT处理的ER:Ras转染IMR90人二倍体成纤维细胞的H3K27me3 ChIP-seq分析样本
- K36me3Grow2.bed（增殖组）：未进行4-OHT处理的ER:Ras转染IMR90人二倍体成纤维细胞的H3K36me3 ChIP-seq分析样本
- K36me3Sen2.bed（衰老组）：ER:Ras转染的IMR90人二倍体成纤维细胞经4-OHT处理第6天后的H3K36me3 ChIP-seq分析样本
- K4me3Grow2.bed（增殖组）：未进行4-OHT处理的ER:Ras转染IMR90人二倍体成纤维细胞的H3K4me3 ChIP-seq分析样本
- K4me3Sen3.bed（衰老组）：ER:Ras转染的IMR90人二倍体成纤维细胞经4-OHT处理第6天后的H3K4me3 ChIP-seq分析样本