Effect of conditional triple-knockout of Jmjd2a/Kdm4a, Jmjd2b/Kdm4b and Jmjd2c/Kdm4c or overexpression of KDM4C/JMJD2C mutants with different phase separation ability on gene expression in mouse embryonic stem cells. [RNA-Seq]

To investigate the functions of JMJD2/KDM4 family in mouse embryonic stem cells (mESCs), we treat the the conditional triple-knockout mESCs of Jmjd2a/Kdm4a, Jmjd2b/Kdm4b and Jmjd2c/Kdm4c with 4-hydroxytamoxifen (4-OHT) for 72h. Further, to investigate whether JMJD2/KDM4 phase separation affects 2-Cell-like cells (2CLCs) induction, we generated IDR-truncated JMJD2C/KDM4C (Mut) to disrupt its phase separation ability, and rescued it by by fusing two IDRs in other species (hIDR1 and hIDR2). We overexpressed those phase separation mutants in the Zscan4-GFP::Mervl-tdTomoto mESCs separately. Overall design: We then performed gene expression profiling analysis using data obtained from RNA-seq of 8 different cells (Ctrl (WT mESC), JMJD2 TKO, JMJD2C FL (Full length), JMJD2C Mut (IDR truncated), Mut-hIDR1 (rescued by hIDR1, from human HNRNPA), hIDR1, Mut-hIDR2 (rescued by hIDR2, from human FUS), hIDR2.

为探究JMJD2/KDM4家族在小鼠胚胎干细胞（mouse embryonic stem cells，mESCs）中的功能，我们对Jmjd2a/Kdm4a、Jmjd2b/Kdm4b及Jmjd2c/Kdm4c的条件性三敲除小鼠胚胎干细胞采用4-羟基他莫昔芬（4-hydroxytamoxifen，4-OHT）处理72小时。进一步，为探究JMJD2/KDM4相分离（phase separation）是否影响2-细胞样细胞（2-Cell-like cells，2CLCs）的诱导，我们构建了固有无序区域（intrinsically disordered region，IDR）截短型JMJD2C/KDM4C（突变体，Mut）以破坏其相分离能力，并通过融合来自其他物种的两个IDR结构域（hIDR1与hIDR2）对其进行功能挽救。我们将上述相分离突变体分别在Zscan4-GFP::Mervl-tdTomato小鼠胚胎干细胞中过表达。整体实验设计：我们对8组不同样本的RNA测序（RNA-seq）数据开展基因表达谱分析，各组分别为：野生型小鼠胚胎干细胞对照（Ctrl (WT mESC)）、JMJD2三敲除组（JMJD2 TKO）、全长JMJD2C过表达组（JMJD2C FL，Full length）、IDR截短型JMJD2C突变体组（JMJD2C Mut，IDR truncated）、人源HNRNPA来源的hIDR1挽救突变体组（Mut-hIDR1）、单独hIDR1过表达组（hIDR1）、人源FUS来源的hIDR2挽救突变体组（Mut-hIDR2）、单独hIDR2过表达组（hIDR2）。