Figure 2

Identification of SUV420H2 and HP1 interacting domains by in vitro GST-pull down. (A) The heterochromatic targeting module of SUV420H2 interacts with HP1 proteins. GST-tagged truncation SUV420H2 [347–435], as well as control GST-GFP fusion were bound to glutathione-Sepharose and incubated with nuclear extracts from HEK293 cells expressing TAP-tagged HP1α, HP1β, or HP1γ as indicated. After extensive washings, proteins were separated on SDS-PAGE and Western blot probed to reveal TAP-tagged proteins. (B) The chromoshadow domain of HP1γ is required for its interaction with SUV420H2. GST-SUV420H2 [347–435], as well as control GST-HP1γ fusions were bound to glutathione-Sepharose and incubated with nuclear extracts from HEK293 cells expressing TAP-HP1γ truncations or TAP-GFP control. HP1γ [ΔKKK] harbours a deletion from lysine 105 to lysine 107 within the hinge region, whereas HP1γ [ΔCSD] contains the 114 N-terminus amino-acids of HP1γ but not the chromoshadow domain. Loss of the chromoshadow domain abolishes the interaction with the heterochromatic targeting module of SUV420H2 and HP1γ oligomerization. (C) The chromoshadow domain (CSD) of HP1γ interacts with the SUV420H2 [347–435] region. GST-HP1γ [CSD] consisting of the HP1γ chromoshadow domain (AA 115–183), as well as control GST-HP1γ and GST-GFP fusions were bound to glutathione-Sepharose and incubated with in vitro translated TAP-SUV420H2 [347–435] fusion protein or TAP-GFP control. Bound TAP-tagged proteins are revealed by Western blotting using the peroxidase-anti-peroxidase (PAP) antibody (Sigma) which recognizes the protein A moiety of the TAP tag.