title: Intrabody-mediated diverting of HP1β to the cytoplasm induces co-aggregation of H3-H4 histones and lamin-B receptor.
abstract: Diverting a protein from its intracellular location is a unique property of intrabodies. To interfere with the intracellular traffic of heterochromatin protein 1β (HP1β) in living cells, we have generated a cytoplasmic targeted anti-HP1β intrabody, specifically directed against the C-terminal portion of the molecule. HP1β 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-HP1β intrabody sequesters HP1β 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-HP1β intrabody-expressing cells. Methylated histone H3 at K9 (Me9H3), a marker of constitutive heterochromatin, is not affected by the anti-HP1β intrabody expression. Hyper-acetylating conditions completely dislodge H3 from HP1β:LBR containing aggregates. The expression of anti-HP1β scFv fragments induces apoptosis, associated with an alteration of nuclear morphology. Both these phenotypes are specifically rescued either by overexpression of recombinant full length HP1β or by HP1β mutant containing the chromoshadow domain, but not by recombinant LBR protein. The HP1β-chromodomain mutant, on the other hand, does not rescue the phenotypes, but does compete with LBR for binding to HP1β. These findings provide new insights into the mode of action of cytoplasmic-targeted intrabodies and the interaction between HP1β and its binding partners involved in peripheral heterochromatin organisation.
authors.affiliation: Laboratory of Molecular and Cellular Neurobiology, IRCCS San Raffaele Pisana, Via di Val Cannuta 247, 00166 Rome, Italy.
authors.forename: Alessio
authors.initials: A
authors.lastname: Cardinale
authors.affiliation: Department of Systems Medicine, University of Roma Tor Vergata, Via Montpellier 1, 00133 Roma, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Filesi
authors.affiliation: Department of Natural Sciences and Psychology, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, United Kingdom.
authors.forename: Prim B
authors.initials: PB
authors.lastname: Singh
authors.affiliation: Department of Systems Medicine, University of Roma Tor Vergata, Via Montpellier 1, 00133 Roma, Italy. Electronic address: biocca@med.uniroma2.it.
authors.forename: Silvia
authors.initials: S
authors.lastname: Biocca
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053144:Cell Nucleus Shape; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D066329:Protein Aggregates; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000378:metabolism*; D000097682:Lamin B Receptor
chemical_list: D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D066329:Protein Aggregates; D018160:Receptors, Cytoplasmic and Nuclear; D057127:Single-Chain Antibodies; D000097682:Lamin B Receptor; C000715593:CBX1 protein, human
keywords: Chromoshadow domain; Heterochromatin proteins; Histone methylation; Protein aggregation; Single-chain Fv fragment