The acetyltransferase p300 is recruited in trans to multiple enhancer sites by lncSmad7. [ChIP-seq]. The acetyltransferase p300 is recruited in trans to multiple enhancer sites by lncSmad7. [ChIP-seq]

The histone acetyltransferase p300 (also known as KAT3B) is a general transcriptional coactivator that introduces the H3K27ac mark on enhancers triggering their activation and gene transcription. Genome-wide screenings demonstrated that a large fraction of long non-coding RNAs (lncRNAs) plays a role in cellular processes and organ development although the underlying molecular mechanisms remain largely unclear. We found 122 lncRNAs that interacts directly with p300. In depth analysis of one of these, lncSmad7, is required to maintain ESC self-renewal and it interacts to the C-terminal domain of p300. lncSmad7 also contains predicted RNA-DNA Hoogsteen forming base pairing. Combined Chromatin Isolation by RNA precipitation followed by sequencing (ChIRP-seq) together with CRISPR/Cas9 mutagenesis of the target sites demonstrate that lncSmad7 binds and recruits p300 to enhancers in trans, to trigger enhancer acetylation and transcriptional activation of its target genes. Thus, these results unveil a new mechanism by which p300 is recruited to the genome. Overall design: ChIP-seq of H3K27ac in ES cells, 6 samples total: 2 wild type, 2 lncSMAD7-/-, 2 Input DNA as control samples.

组蛋白乙酰转移酶p300（histone acetyltransferase p300，亦称KAT3B）是一类通用转录辅激活因子，可在增强子区域引入H3K27ac修饰以触发增强子活化及基因转录。全基因组筛选研究表明，尽管其背后的分子机制尚未完全阐明，但大部分长链非编码RNA（long non-coding RNAs，lncRNAs）均参与细胞进程与器官发育。本研究共鉴定出122种可与p300直接结合的lncRNAs。对其中一员lncSmad7的深入分析显示，该分子对维持胚胎干细胞（ESC）自我更新至关重要，且可与p300的C端结构域相互作用；lncSmad7还含有可形成RNA-DNA Hoogsteen碱基配对的预测序列。结合通过RNA沉淀进行染色质分离并后续测序的技术（Chromatin Isolation by RNA precipitation followed by sequencing，ChIRP-seq）与靶位点CRISPR/Cas9诱变实验结果证实，lncSmad7可通过反式作用结合并招募p300至增强子区域，进而触发增强子乙酰化及靶基因的转录激活。上述研究结果揭示了一种全新的p300基因组招募机制。整体实验设计：对胚胎干细胞中的H3K27ac进行染色质免疫共沉淀测序（ChIP-seq），共设置6个样本：2份野生型样本、2份lncSMAD7纯合敲除样本，以及2份输入DNA作为对照样本。