Fig. 6

Hira and Ubn2 regulate TE expression. a, b ChIP-qPCR analysis of Hira (a) and Ubn2 (b) binding on ERV1 family member MMERGLN_LTR, ERV2 family members (IAPLTR2b and RLTR10D) and ERV3 family members (MERVL and MT2). Biological triplicate data (n = 3 extracts) are presented as mean ± s.e.m. ns: non-significant, **p < 0.01; ***p < 0.001 in Student’s t test. c Hira and Ubn2 binding profile around the center of MT2 locus (MERVL-LTR) respectively. The ChIP-seq signal was calculated as the log2 ratio of the normalized number of reads relative to the input. d GSEA analysis of upregulated genes after Ubn2 knockdown for the enrichment of 2C genes. Red, upregulated genes; blue, downregulated genes; NES, normalized enrichment scores; FDR, false discovery rate. The Kolmogorov–Smirnov statistic was used for the calculation of the P value. e Flow cytometry analysis of the 2C⁺ population in 2C::tdTomato reporter ESCs after depletion of Ubn2 by shRNA. Data are represented as mean ± s.e.m. (n = 3 independent experiments). **p < 0.01 in Student’s t test. f, g The expression level of pluripotent gene Oct4 and trophectoderm markers (Eomes, Cdx2, and Fgfr2) in Ubn2 (f) and Hira (g)-depleted ESCs cultured under ESC or TSC medium for 3 days, as measured by RT-qPCR and normalized to Gapdh levels. Data are represented as mean ± s.e.m. (n = 3 independent experiments). ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001 in Student’s t test. h qPCR analysis of Hira, Ubn2, MERVL, and ERV2 family member (RLTR12H and IAPLTR2b) after Hira depletion in Ubn2 overexpression ESCs. The results were normalized to Gapdh. Data are represented as mean ± s.e.m. (n = 3 independent experiments). ns: non-significant, *p < 0.05; ***p < 0.001 in Student’s t test