Fig 2: N-α-SUMOylation of CFL1 is reversible.The SUMOylation of CFL1 was inhibited by the knockdown of SAE1 (a), SAE2 (b), and Ubc9 (c), but enhanced by the knockdown of SENP1 (d). HEK-293T cells were transiently transfected as indicated. Cell lysates were used for De-IP by anti-CFL1 antibody followed by IB using anti-SUMO1 and anti-CFL1 antibodies. The original lysates were analyzed by IB using anti-CFL1, anti-SAE1 (a), anti-SAE2 (b), anti-Ubc9 (c), and anti-SENP1 (d) antibodies for input, as well as anti-GAPDH antibody for loading control. e CFL1 is physically associated with SAE1, SAE2, Ubc9 and SENP1. Lysates from HEK-293T cells transiently transfected with the vector control (−), CFL1WT-HA, CFL125KR-HA, HA-DUSP6, and His-SUMO1 as indicated for 24 h were subjected to IP with anti-HA antibody, followed by IB with anti-SAE1, anti-SAE2, anti-Ubc9, anti-SENP1 and anti-HA antibodies. The original lysates were analyzed by IB using anti-HA, anti-SAE1, anti-SAE2, anti-Ubc9 and anti-SENP1 antibodies for input, and anti-GAPDH antibody for loading control. f Similar to (e) but with expression of the vector control (−), CFL1WT-HA, CFL12KR-HA, CFL12KQ-HA, and His-SUMO1 as indicated. g–i Quantification of Western blots of (a)–(f). g Summary of SUMOylated CFL1 to total CFL1 presents the mean ± SEM of three independent experiments in (a)–(d); h Summary of coimmunoprecipitated (Co-IPed) SAE1, SAE2, Ubc9 and SENP1 normalized to total CFL1 or DUSP (HA) presents the mean ± SEM of three independent experiments in (e); i Summary of coimmunoprecipitated (Co-IPed) SAE1, SAE2, Ubc9 and SENP1 normalized to total CFL1 (HA) presents the mean ± SEM of three independent experiments in (f); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by one-way ANOVA with Tukey’s multiple comparison test. All blots represent ≥3 independent experiments. The exact p value and source data are provided as Source Data file.

Fig 3: SUMOylation regulates H3K27Ac enhancer marks and TFAP2C binding to enhancers. A) Histone marks at enhancers at a representative locus. B) Western blot analysis of Uba2 protein in whole cell extract. GAPDH was used as a loading control. C) ChIP-seq data of SUMO1 and SUMO2/3 peaks with and without UBA2 KD by adding Dox. D) Enhancer peaks responsive to UBA2 KD and most of these enhancer peaks were down regulated upon UBA2 KD.

Fig 4: NPM-ALK is SUMOylated at Lys24 and Lys32 residues. The mutated NPM-ALK recombinant proteins NPM-ALKK24R and NPM-ALKK32R were generated using the TnT coupled rabbit reticulocyte kit. In the upper panel, WB was performed to confirm the expression of recombinant NPM-ALK proteins. Wild-type NPM-ALK recombinant protein is present in lane 1, NPM-ALKK24R recombinant protein in lane 2, and NPM-ALKK32R recombinant protein in lane 3. In the lower panel, wild-type and mutated recombinant NPM-ALK proteins were subjected to an in vitro SUMOylation assay. SUMOylation was abolished with the K24R mutation for SUMO-1 and SUMO-3 and with the K32R mutation for SUMO-3. The two mutations failed to de-SUMOylate SUMO-2.

Fig 5: α-tubulin is SUMOylated at K96, K166, and K304 and deSUMOylated by SENP1. (A) Alignment of lysines (in red color) and surrounding sequences of α-tubulin isotypes in the mouse. (B) Immunoprecipitates with Flag M2 beads from HEK293T cells expressing HA-SUMO1 with Flag-tagged isotypes of α-tubulin as indicated were probed with SUMO1 and Flag Abs. (C) Flag-tagged wild-type (WT) and various α1A mutant at MS-predicted SUMOylation sites were expressed with HA-SUMO1 in CHO-K1 cells and then immunoprecipitated. Immunoprecipitates were detected by SUMO1 and Flag Abs. (D) List of putative SUMOylation sites on α1A isotype predicted by different softwares. (E) Flag-tagged WT and combined mutant α1A at bioinformatics-predicted SUMOylation sites were expressed with HA-SUMO1 in CHO-K1 cells and then immunoprecipitated. Immunoprecipitates were detected by SUMO1 and Flag Abs. (F) Schematic representation of SUMOylation sites on the structure of α-tubulin. K96, K166, and K304 are shown as red spheres. (G) Location of SUMOylation sites and the interface for lateral contact between two α-tubulins in one MT. K96, K166, and K304 are shown as red spheres, and H2-S3 loop and M-loop are in blue. (H) Flag-α1A was overexpressed with HA-SUMO1 or HA-SUMO1-K7,16,17R mutant in HEK293 cells. Immunoprecipitates using Flag M2 beads were probed with HA and Flag Abs. (I) Immunoprecipitates with Flag M2 beads from HEK293 cells expressing Flag-SENP1 or Flag-SENP2 were probed with Flag and α-tubulin Abs. (J) Immunoprecipitates with α-tubulin from HEK293 cells expressing Flag-SENP1 or Flag-SENP2 with Flag-SUMO1 were probed with SUMO1 and α-tubulin Abs. (K) Immunoprecipitates from E13.5 brain of SENP1+/+ and SENP1–/– mice were probed with SUMO1 and α-tubulin Abs. The experiments were repeated three times.