Formation of an active epigenetic mark and its recycling is mediated by cell cycle-specific RNAs [RIP-seq]

The mechanisms by which epigenetic modifications are established in gene regulatory regions of active genes remain elusive. The data presented show that the establishment and recycling of a major epigenetic mark, the acetylated form of the replacement histone H2A.Z, is regulated by cell cycle-specific long noncoding RNAs encoded in regions adjacent to the promoters of active genes. These transcripts, termed SPEARs (S Phase EArly RNAs), are induced in early S phase: their expression precedes that of the downstream genes on which they exert their regulatory action. SPEARs drive the modification and deposition of the acetylated form of histone H2A.Z by bringing together the replacement histone and the histone acetyl transferase TIP60. This widespread bimodal pathway constitutes a novel RNA-mediated mechanism for the establishment of epigenetic marks and cell-specific epigenetic profiles, thereby providing a unifying explanation for the accuracy and persistence of epigenetic marks on chromatin. All samples were done using the cell line HL-60. 1 replicate each. RIP-Sequencing targetting acH2A.Z, H2A.Z and TIP60 with corresponding input. RNA-Sequencing on nuclear extract, nuclear-extracts synchronized at S-Phase and nascent nuclear-extracts synchronized at S-Phase. ChIP-Seq was performed targeting the acH2A.Z and H2A.Z histone marks with cells treated with DMSO, ACTD and DRB with appropriate controls.

活跃基因的基因调控区域中，表观遗传修饰的建立机制仍未明确。本研究呈现的数据表明，主要表观遗传标记之一——替换型组蛋白H2A.Z（replacement histone H2A.Z）的乙酰化形式的建立与循环利用，受到活跃基因启动子邻近区域编码的细胞周期特异性长链非编码RNA（long noncoding RNA, lncRNA）的调控。这类转录本被命名为SPEARs（S期早期RNA，S Phase EArly RNAs），在S期早期被诱导表达，其表达先于其所调控的下游基因。SPEARs通过将替换型组蛋白H2A.Z与组蛋白乙酰转移酶TIP60（histone acetyl transferase TIP60）相结合，介导乙酰化替换型组蛋白H2A.Z的修饰与沉积过程。这一广泛存在的双模态通路，为表观遗传标记的建立以及细胞特异性表观遗传图谱的形成提供了一种全新的RNA介导机制，由此统一解释了染色质上表观遗传标记的精准性与持久性。所有样本均采用HL-60细胞系构建，每组设置1次生物学重复。实验采用靶向乙酰化组蛋白H2A.Z（acH2A.Z）、替换型组蛋白H2A.Z及TIP60的RNA免疫沉淀测序（RIP-Sequencing），并设置相应的输入对照；同时对核提取物、S期同步核提取物以及新生S期同步核提取物开展RNA测序（RNA-Sequencing）；此外，针对经二甲基亚砜（DMSO）、放线菌素D（ACTD）以及DRB处理的细胞，以乙酰化组蛋白H2A.Z与替换型组蛋白H2A.Z为靶点进行染色质免疫共沉淀测序（ChIP-Seq），并设置恰当的对照样本。