Calibrated ChIP-seq for Xist-mediated polycomb recruitment. Calibrated ChIP-seq for Xist-mediated polycomb recruitment

Calibrated ChIP-seq was employed to quantitatively address the questions of how and where Xist recruits polycomb modifications (H2AK119ub and H3K27me3) to the inactive chromosome. 'Dox' samples were performed after 24 hours of induced Xist expression. Suz12 is the core enzymatic component of PRC2 while Pcgf3/5 are non-canonical PRC1 components which initiate the polycomb cascade in Xist-mediated chromosome inactivation. Xist PID region, encompassing the B-repeat and part of C-repeat, was characterized to bind hnRNPK directly, then recruit the Pcgf3/5. Notably, Suz12 KO does not affect Xist-dependent H2AK119ub deposition too much in a 24hour Xist induction. Overall design: H3K27me3 and H2AK119ub calibrated ChIP-seq for Xist and XistDelPID-induced chromosome inactivation [endogenous Xist model]. H3K27me3 and H2AK119ub calibrated ChIP-seq for WT, Pcgf3/5 acute double knockout, and Suz12KO [transgenic Xist model].

本研究采用校准型染色质免疫共沉淀测序（calibrated ChIP-seq），定量解析X染色体失活特异性转录本（Xist）如何、在何处将多梳蛋白修饰（polycomb modifications）——即组蛋白H2A赖氨酸119位泛素化（H2AK119ub）与组蛋白H3赖氨酸27位三甲基化（H3K27me3）——招募至失活染色体。其中，"Dox"样本为经多西环素诱导Xist表达24小时后制备的实验样品。Suz12是多梳抑制复合体2（PRC2）的核心酶活组分，而Pcgf3/5属于非经典多梳抑制复合体1（PRC1）组分，可启动Xist介导的染色体失活过程中的多梳级联反应。Xist的PID区域包含B重复序列与部分C重复序列，经实验证实可直接结合异质性核糖核蛋白K（hnRNPK），进而招募Pcgf3/5。值得注意的是，在24小时Xist诱导条件下，Suz12敲除（KO）对Xist依赖的H2AK119ub沉积无显著影响。整体实验设计如下：针对内源性Xist模型，开展Xist诱导与XistDelPID诱导的染色体失活相关的H3K27me3、H2AK119ub校准型染色质免疫共沉淀测序检测；针对野生型（WT）、Pcgf3/5急性双敲除以及Suz12KO的转基因Xist模型，同样完成上述两类组蛋白修饰的校准型染色质免疫共沉淀测序检测。