Fig. 6.

MLCK localizes in the nucleus, at the vicinity of WDR5 and euchromatin regions, and regulates WDR5-mediated-H3K4 methylation. (A) Jurkat cells were cultured in suspension, 2D, and 3D conditions. Then, cells were lysed and cytoplasmic, nuclear, and chromatin-bound fractions analyzed by western blotting. RhoGDI, Lamin B1 and Histone H3 were used as internal control of cytoplasmic, nuclear/chromatin and chromatin fractions. (B) Midsection of a representative nucleus from isolated nuclei sedimented on poly-Lysine coated coverslips and stained to visualize MLCK, the chromatin, and WDR5 or H3K4me3. (C and D) Jurkat cells were incubated with Blebbistatin, for 30 min before culturing them in 3D. Then, WDR5 was immunoprecipitated (C), and its H3K4 methyltransferase activity (D) determined. Mean n = 4 ± SD. ***P < 0.001. (E) Jurkat cells were incubated with Blebbistatin or ML-7 inhibitors for 30 min before culturing them in 3D. Then WDR5 was immunoprecipitated and its interactions with ASH2L and RbBP5 were determined.