Fig. 4

Targeting of Daxx and H3.3 at centromeres is SUMO-2 dependent. a SUMO-2 is required for Daxx association with centromeres. Representative images of HEp2 cells treated with MG132 (upper row) and FLAG-HA-CENP-B expressing HEp2 cells (untreated, middle row and MG132 treated, bottom row). SUMO-1 depletion (middle column) did not affect Daxx (green) accumulation at centromeres (CREST, red) compared to scramble siRNA transfection (left column), while SUMO-2 depletion (right column) abolished Daxx accumulation at centromeres in all three experimental conditions. Quantitation of the colocalization is presented in Additional file 3: Fig. S3. Graphs in the bottom panel show fluorescence intensity of Daxx (green) and CREST (red) signal along the ROI. b ChIP analysis of Daxx association with CEN and periCEN in HEp2 cells expressing FLAG-HA-Daxx. Depletion of SUMO-2, but not SUMO-1 abolished Daxx association with CEN and periCEN. c ChIP assay of H3.3 enrichment at CEN and periCEN in HEp2 cells stably expressing FLAG-HA-H3.3. Depletion of SUMO-2, but not SUMO-1, reduced H3.3 enrichment at CEN and periCEN. Bars represent the mean ± SD (n = 3). Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001; ***P < 0.001; **P < 0. 01; *P < 0.1; NS, nonsignificant