MMSET induces global changes in histone methylation. (A) Schematic representation of MMSET main isoforms showing the regions where the shRNA were designed. (Right) Cells containing the inducible shRNA were grown in the presence of doxycycline for 7 days. After 7 days, cell were washed and incubated for 7 more days without doxycycline to restore MMSET expression. Nuclear extracts were immunoblotted with anti-MMSET antibody. (B-C) Nuclear extracts from the knockdown and knockout systems were immunoblotted with the indicated antibodies. MMSET depletion induces methylation of H3K27me2/3 and decreases methylation of H3K36me2/3. (D) MMSET repletion induces global changes in chromatin. The MMSET knockout cell line was infected with retrovirus containing a vector control (lane 1), wild-type MMSET II (lane 2), or 2 different SET domain mutants (lanes 3-4). Nuclear extracts were immunoblotted with the indicated antibodies. Restoration of wild-type and active mutant of MMSET decreases methylation of H3K27me2/3 and increases methylation of H3K36me2/3. Repletion with the enzymatically dead mutant form of MMSET shows the same methylation pattern as the vector control. (E) MMSET expression affects chromatin structure. MNase assay in knockdown system. Lane 1: molecular weight marker; lanes 2-3: negative control with no enzyme. The presence of MMSET correlates with a more open chromatin state (lane 4). When MMSET is blocked, the chromatin closes, and it is not accessible for the digestion by MNase (lane 5). Re-expression of MMSET upon doxycycline removal opens the chromatin (lane 6).