Fig. 3: IRX3 represses SUMOylation.

IRX3 ChIP-seq, ATAC-seq, RNA-seq, and immunoblots were used to assess IRX3-dependent regulation of SUMOylation during adipocyte differentiation. a IRX3 ChIP-seq signal near TSS (\(\pm\)1.5 kb) of SUMO pathway genes in iWAT (n \(=\) 2) and gWAT (n \(=\) 1) preadipocytes before and after induction of differentiation (top); average ATAC-seq profiles at the same loci in ME3 control and IRX3-KO cells on day 1 (n \(=\) 3) (middle); log₂ fold changes in IRX3-KO vs. control ME3 cells on day 1 (n \(=\) 6) (bottom). *p_adj \(=\,\)0.01, **p_adj \(=\,\)0.001, ***p_adj \( < \) 0.001; multiple unpaired, two-sided t-tests with Holm–Sidak correction. b Immunoblot of SUMO pathway proteins from a in ME3 control and IRX3-KO cells on day 1 (n \(=\) 3). Representative of two experiments. c Quantification of b, normalized to GAPDH. *p \( < \) 0.05, **p \(=\) 0.009; unpaired, two-sided t-test. d RNA-seq of SUMO ligases and proteases not bound by IRX3 in WAT (n \(=\) 6)¹⁴; expression (left) and log₂ fold changes (right). *p_adj \(=\) 0.03, ***p_adj \( < \) 0.001; multiple unpaired, two-sided t-tests with Holm-Sidak correction. e–h Immunoblots and quantifications of global SUMO2/3 (e–f) and SUMO1 (g–h) conjugation in control and IRX3-KO ME3 cells on days 0 and 1 (n \(=\) 3). Representative of four (e-f) and two (g-h) experiments. *p_adj \(=\) 0.03, ***p_adj \( < \) 0.001; two-way ANOVA with Holm–Sidak correction.IRX3 rescue in IRX3-KO cells reduces SUMO2/3 and SUMO1 conjugation on day 1. Immunoblots (i, k) and quantifications (j, l) shown (n \(=\) 3). Representative of three (i, j) and two (k, l) experiments. *p_adj \( < \) 0.05, **p_adj \(=\) 0.002. m Schematic of the SUMOylation cycle. IRX3-bound genes in red; arrows indicate RNA (grey) and protein (orange) changes. Created in BioRender. Bjune, J. (2025) https://BioRender.com/1m0hzx6. Box plots show medians and interquartile ranges; bar graphs show means \(\pm \,\)SD. Source data are provided in the Source Data file.