Fig. 6: Molecular mechanism of the activation of Nr1h2 intrinsic program.

a A dual-Glo luciferase assay depicted to test SINE-B1 enhancer activity in ESC and NrESC, as well as siNr1h2 and siNT NrESC samples. Created in BioRender.com. b Genome browser view of Nr1h2-FLAG ChIP-seq (turquoise), H3K27ac ChIP-seq (orange, ESC; purple, NrESC) and RNA-seq signal profile (red) surrounding NrESC up-DEGs (Scd1, Abcg1 and Scd2). Red-colored boxes and dashed lines indicate the candidate SINE-B1 enhancer regions being tested. c Quantification of the normalized luminescence (normalized to Renilla signal and ESC signal) in three constructs testing for SINE-B1 candidates nearby Scd1, Abcg1 and Scd2 in ESC and NrESC (top), or siNT and siNr1h2 NrESC (bottom). Two-tailed Welch’s t-test. Data are represented as mean ± s.d.; n = 3 independent assays. d Western blot analysis showing that Kdm1a is only associated with Nr1h2-FLAG in T09-treated Nr1h2-FLAG and Kdm1a-HA overexpressing sample (Nr-FLAG and Kd-HA), compared to GFP control sample (G-FLAG and G-HA). 3 independent experiments were repeated with similar results. e Heatmap of ChIP-seq signal on Nr1h2-FLAG ChIP-seq peaks. The ChIP-seq of Kdm1a¹³³ was obtained from the GEO database. f Genome browser view of Nr1h2-FLAG ChIP-seq (turquoise), Kdm1a ChIP-seq (blue) and RNA-seq signal profile (red) surrounding NrESC markers (Grn and Scd1). g Expression of Grn, Scd1 and Krt8 in ESC overexpressing EGFP-FLAG or Nr1h2-FLAG under T09 treatment. Expression is relative to Gapdh and normalized to EGFP-FLAG ESC expression. Data are represented as mean ± s.d.; n = 3 technical replicates. h Immunofluorescence of Scd1 in ESC and NrESC. Scale bar, 20 µm. 3 independent experiments were repeated with similar results. i Sequential ChIP-qPCR indicating the increased co-binding of Kdm1a and Nr1h2-FLAG under T09 treatment at Abca1, Hdac2, Grn and Scd1, normalized to EGFP-FLAG control. Regions not bound by Kdm1a and Nr1h2-FLAG were used as negative control. Data are represented as mean ± s.d.; n = 3 technical replicates. j Quantification of blastoid formation efficiency from NrESC treated with recombinant Grn, Igg, Anti-Grn, Igg + T09, or Anti-Grn + T09. One-way ANOVA with Bonferroni correction for multiple comparisons. Data are represented as mean ± s.d.; n = 3 independent assays. k Quantification of blastoid formation efficiency from NrESC treated with T09 and/ or Scd1 inhibitor. One-way ANOVA with Bonferroni correction for multiple comparisons. Data are represented as mean ± s.d.; n = 3 independent assays. l Model schematic showing multifaceted regulatory roles of Nr1h2-specific intrinsic program in NrESC. Created in BioRender.com. Source data are provided as a Source Data file.