Intrabody-mediated diverting of HP1ß to the cytoplasm induces co-aggregation of H3-H4 histones and lamin-B receptor.

Diverting a protein from its intracellular location is a unique property of intrabodies. To interfere with the intracellular traffic of heterochromatin protein 1ß (HP1ß) in living cells, we have generated a cytoplasmic targeted anti-HP1ß intrabody, specifically directed against the C-terminal portion of the molecule. HP1ß is a conserved component of mouse and human constitutive heterochromatin involved in diverse nuclear functions including gene silencing, DNA repair and nuclear membrane assembly. We found that the anti-HP1ß intrabody sequesters HP1ß into cytoplasmic aggregates, inhibiting its traffic to the nucleus. Lamin B receptor (LBR) and a subset of core histones (H3/H4) are also specifically co-sequestered in the cytoplasm of anti-HP1ß intrabody-expressing cells. Methylated histone H3 at K9 (Me9H3), a marker of constitutive heterochromatin, is not affected by the anti-HP1ß intrabody expression. Hyper-acetylating conditions completely dislodge H3 from HP1ß:LBR containing aggregates. The expression of anti-HP1ß scFv fragments induces apoptosis, associated with an alteration of nuclear morphology. Both these phenotypes are specifically rescued either by overexpression of recombinant full length HP1ß or by HP1ß mutant containing the chromoshadow domain, but not by recombinant LBR protein. The HP1ß-chromodomain mutant, on the other hand, does not rescue the phenotypes, but does compete with LBR for binding to HP1ß. These findings provide new insights into the mode of action of cytoplasmic-targeted intrabodies and the interaction between HP1ß and its binding partners involved in peripheral heterochromatin organisation.