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. [HiChIP]
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. Besides, to investigate whether JMJD2/KDM4 phase separation affects Enhancer-Promoter loops, 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). Further, to investigate whether the functions of JMJD2/KDM4 are catalytic-dependent, we also generated catalytic mutants of JMJD2B and JMJD2C. We overexpressed those phase separation or catalytic mutants in the aboved mESCs separately and TKO endogenous Jmjd2 by adding 4-OHT 72h (for phase separation mutants) or 36h (for catalytic mutants). Overall design: We then performed H3K27ac HiChIP analysis using data obtained from 9 different cells (Ctrl (WT mESC), JMJD2 TKO, JMJD2C Mut (IDR truncated), Mut-hIDR1 (rescued by hIDR1, from human HNRNPA), hIDR1, Mut-hIDR2 (rescued by hIDR2, from human FUS), hIDR2, CMuts (rescued by catalytic mutants of JMJD2B and JMJD2C) and EV (empty vector, negative control of CMuts).