Fig. 1.

Umbrea binds directly to Hip and HP1 in vitro and the three proteins are associated with one another in a protein complex in vivo. In addition, Umbrea is a protein interaction partner of HOAP. Umbrea uses the same binding modules in Hip as HP1 and HP1 interacts with Umbrea using its chromo shadow domain. Full-length Umbrea, Hip, HP1 and HOAP proteins (depicted in; a were expressed in bacteria and assayed for their ability to interact with each other in; b Note: Hip contains three HP1-binding interfaces (I, II and III; described in Fig. 3), whereas HOAP contains a HMG-like domain and three copies of a proline-containing repeat (RP1-3) (Shareef et al. 2001); b A GST-Hip, GST-HP1 and GST-HOAP fusion protein, or GST alone, were used in a GST pull-down assay and analysed for their ability to retain recombinantly expressed His-tagged full-length Umbrea. Eluted proteins were probed with an anti-His tag antibody. Umbrea interacts with Hip, HP1 and HOAP; c Anti-HP1 and anti-Hip western blot analysis after immunoprecipitation (IP). Nuclear extract from larval salivary glands was immunoprecipitated using a combination of both anti-Umbrea antibodies, or mock-precipitated using the corresponding preimmune sera. Mock precipitation with preimmune sera did not retain Hip and HP1, respectively, indicating that Umbrea interacts specificially with Hip and HP1 in vivo; d The Hip protein sequence contains numerous charged amino acid residues, such as K, R, E and D (bold type; positively and negatively charged amino acids are underlined and in italics, respectively). (Figure modified after Fig. 4 in Schwendemann et al. 2008.) As described in Schwendemann et al. (2008), Hip contains three HP1-binding interfaces depicted below (I, II and III). The Hip N- and C-terminal fragments used for the following experiments are indicated.; e As in (B), His-tagged full-length Umbrea was used as an input in GST pull-down assays with different Hip N-terminal (GST-N-Hip, GST-N1-Hip, GST-N2-Hip, GST-N3-Hip and GST-N4-Hip) or C-terminal (GST-C-Hip, GST-C1-Hip, and GST-C2-Hip) fragments. Western blot analysis with anti-His tag antibody revealed that fragments N-Hip, C-Hip, N1-Hip, N2-Hip and N4-Hip, but not N3-Hip, C1-Hip or C2-Hip, interact with Umbrea. Three sequences that are necessary for Umbrea binding are underlined; e. Note: these sequences are identical to the three HP1-binding interfaces of Hip (Schwendemann et al. 2008); f To characterize the interaction between Hip and Umbrea, His-tagged full-length Hip was used as an input in GST pull-down assays but Umbrea N-terminal (GST-N-Umbrea), C-terminal fragments (GST-C-Umbrea), or the chromo shadow domain (GST-cs-Umbrea) were immobilized on glutathione agarose beads. The Umbrea chromo shadow domain but not N- or C-terminal regions is sufficient to interact with Hip; g Same conditions as before, but this time full-length Umbrea was fused to GST (GST-Umbrea) and GST pull-down assays with truncated forms of His-tagged HP1 (HP1-cd-His, HP1-hinge-His or HP1-cs-His) were performed. The HP1 chromo shadow domain (HP1-cs) but not the HP1 chromo domain (HP1-cd) or the hinge region (HP1-hinge) is necessary to interact with Umbrea. Finally, as described above, but only the chromo shadow domain of Umbrea was fused to GST (GST-cs-Umbrea) and analysed for the ability to pull down the HP1 chromo shadow domain (HP1-cs-His). Taken together, the chromo shadow domains of both Umbrea and HP1 are sufficient to mediate a direct interaction between the two proteins