Fig. 3.

Distinct enzymes used in MTeC to vary the degree of H4K20 methylation demonstrate that full-length tandem tudor domain-containing proteins bind all three H4K20 methylated forms in vivo. (A) Schematic representation of MTeC bait fusion proteins. FLAG affinity tag followed by the first 44 aa of the WT H4 N-terminal tail (where K20 is WT or mutated to alanine) followed by the catalytic SET domain of PR-Set7 or Suv4-20h2. (B) Western blot analysis of the FLAG-immunoprecipitated MTeC bait fusion proteins from HEK 293 nuclear lysates by using FLAG or H4K20 methyl-specific antibodies. An HA-tagged JMJD2A tandem tudor domain only (C) or full-length (D) plasmid were cotransfected in HEK 293 cells with the histone H4 MTeC bait plasmids. Western blot analysis of the HA-immunoprecipitates from nuclear lysates were preformed by using FLAG or HA antibodies. Similar experiments were performed by using an HA-tagged 53BP1 tandem tudor domain only (E) of full-length plasmid (F).