Fig. 1: LSH tethering induces transcriptional repression.

a Schematic graph to illustrate recruitment of FRB-tagged LSH by rapamycin to the CiA: Oct4 locus in the CIP system. Rapamycin dimerizes with FRB and FKBP. The CiA: Oct4 mouse ES cells contain one modified Oct4 allele harboring one array of DNA binding sites (12 × ZFHD1) in the promoter region upstream of an in-frame eGFP reporter inserted in exon 1 of the Oct4 gene. Scale bar, 40 μm. b Western blot analysis for detection of LSH (wild type)-FRB-V5, LSH (K 237 A)-FRB-V5 (ATP mutant site), and ZnF-FKBP-HA fusion proteins in the engineered mouse ES cells using V5 and HA antibodies. Cells expressing both fusion proteins were used to establish the CIP system, and cells lacking LSH fusion protein served as controls. c ChIP-qPCR analysis using V5 antibody for detection of wild-type and ATP mutant LSH (K 237A) protein recruitment to the Oct4 locus site within 60 min after rapamycin treatment in the CIP system (n = 3 independent experiments). Cells that lacked LSH fusion protein served as controls. Data are represented as mean ± SD. d Flow cytometry was used to measure reporter-GFP expression after recruitment of wild-type or ATP mutant LSH (K 237A)-FRB-V5 fusion protein at indicated time points in the CIP system. Cells that lacked LSH fusion protein served as controls. e Determination of GFP and OCT4 protein level by WB after rapamycin treatment at indicated time points in the wild-type LSH CIP system. f ChIP-qPCR analysis to determine Pol II enrichment at the ZFHD binding site and GFP reporter site at indicated time points in the wild-type and ATP mutant LSH (K 237A) CIP system after rapamycin treatment (n = 4 independent experiments). ChIPs using IgG served as the controls. Data are represented as mean ± SD. Significance assessed using one-way ANOVA with Tukey’s multiple comparison test (**adjusted p = 0.0022, ***adjusted p < 0.0001, n.s. means not significant). Source data are provided as a Source Data file.