Fig. 6: EGR1 was identified as a direct downstream target gene of IRX2 in cardiac fibroblasts that regulates the angiotensin II (Ang II)-induced fibrotic response.

A CFs were infected with an adenovirus carrying IRX2 and then subjected to Ang II stimulation for 24 h. ChIP-seq was performed with two anti-IRX2 antibodies (IRX2-1B7 and IRX2-1C1). Similar spatial distributions of IRX2 peaks were identified by the two anti-IRX2 antibodies. B IRX2-regulated genes identified in the ChIP-seq data (bottom) were overlaid with upregulated differentially expressed genes revealed by RNA-seq (top). With this strategy, 70 genes were identified. C Two IRX2 binding sites were identified in the ChIP-seq peak distributed in the promoter site of Egr1. D The Egr1 promoter was stimulated by IRX2 overexpression. Egr1 promoter activity was measured by a luciferase assay upon Irx2 overexpression in CFs isolated from wild-type mice (n = 5 for each group). E The mutant (Mut) promoters (pro) deleting IRX2 binding sites cannot be stimulated by Irx2 overexpression (n = 5 for each group). F Independent ChIP-PCR was performed with Irx2-overexpressing CFs to verify IRX2 binding to the promoter of Egr1 (n = 5). G–H Egr1 mRNA and EGR1 protein expression in CFs isolated from Irx2 cfKO mice and littermate controls with or without Ang II infusion for 12 weeks (n = 6). I Egr1 mRNA levels were detected in CFs with Irx2 deficiency caused by Cre expression (n = 5). J–K Egr1 mRNA and EGR1 protein expression in CFs isolated from Irx2 mfTg mice and littermate controls with or without Ang II infusion for 4 weeks (n = 6). L Egr1 mRNA level in Irx2-overexpressing human CFs after Ang II treatment for 24 h (n = 6). Data are shown as the mean ± SEM, and analysed using one-way ANOVA followed by Tukey post hoc test (H, I and K) or Tamhane’s T2 test (D–G, J and L). Source data are provided as a Source Data file.