Study of MED1 acetylation in ER positive breast cancer cells [RNA-Seq III]

Chromatin occupancy of MED1 and other Mediator components, as well as Pol II, were studied in MCF7 cells. MED1 acetylation affects chromatin occupancy of its related factors, which was analyzed with CUT&RUN-seq, ChIP-seq and sequential ChIP-seq assay (by pan-acetyl-lysine antibody) with MED1 KR mutant (HA tag) where acetylation residues were replaced. Combined with RNA seq analysis for gene expression in MCF7 cells with MED1 KO and KR/KQ MED1 ectopic expression, the role of MED1 acetylation in gene transcription control was investigated. Overall design: ChIP-seq and/or CUT&RUN-seq for MED1 and other Mediator compoments, as well as Pol II, were conducted in MCF7 cells. In addition to normal MCF7 cells, the cells with depletion of endogenous MED1 and ectopic expression of WT or KR mutant MED1 (HA tag) were also tested. Sequential ChIP-seq by FLAG (for MED1) antibody and pan-acetyl-lysine antibody were conducted. RNA seq (for mature mRNA) was conducted for MCF7 cells with MED1 KO (vs control), and 6KR/Q MED1 ectopic expression (Vs WT MED1 expression).

本研究在MCF7细胞中探究了MED1及其他中介体（Mediator）组分、RNA聚合酶II（Pol II）的染色质占据情况。MED1的乙酰化可影响其相关因子的染色质结合特征，本研究通过CUT&RUN测序（CUT&RUN-seq）、染色质免疫沉淀测序（ChIP-seq），以及针对乙酰化残基已被替换的带HA标签（HA tag）的MED1 KR突变体、以泛乙酰化赖氨酸抗体介导的连续染色质免疫沉淀测序（sequential ChIP-seq）实验，对该现象展开了分析。结合对MED1敲除（MED1 KO）以及过表达野生型（WT）或KR/KQ突变型MED1的MCF7细胞的RNA测序（RNA-seq）基因表达分析，本研究进一步探究了MED1乙酰化在基因转录调控中的作用。实验整体设计：在MCF7细胞中针对MED1及其他中介体组分、Pol II开展了ChIP-seq和/或CUT&RUN-seq实验。除正常MCF7细胞外，本研究还检测了内源MED1敲除且分别过表达带HA标签的野生型或KR突变型MED1的细胞组。此外还开展了两组连续ChIP-seq实验：分别以FLAG抗体（靶向MED1）和泛乙酰化赖氨酸抗体进行富集。针对MCF7细胞的RNA-seq（用于检测成熟mRNA）实验包含两个对比组：一是MED1敲除细胞与对照细胞的对比，二是过表达6KR/Q突变型MED1的细胞与过表达野生型MED1的细胞的对比。