title: Loss of heterochromatin protein 1 (HP1) chromodomain function in mammalian cells by intracellular antibodies causes cell death.
abstract: The chromodomain (CD) is a highly conserved motif present in a variety of animal and plant proteins, and its probable role is to assemble a variety of macromolecular complexes in chromatin. The importance of the CD to the survival of mammalian cells has been tested. Accordingly, we have ablated CD function using two single-chain intracellular Fv (scFv) fragments directed against non-overlapping epitopes within the HP1 CD motif. The scFv fragments can recognize both CD motifs of HP1 and Polycomb (Pc) in vitro and, when expressed intracellularly, interact with and dislodge the HP1 protein(s) from their heterochromatin localization in vivo. Mouse and human fibroblasts expressing anti-chromodomain scFv fragments show a cell-lethal phenotype and an apoptotic morphology becomes apparent soon after transfection. The mechanism of cell death appears to be p53 independent, and the cells are only partly rescued by incubation with the wide spectrum caspase inhibitor Z-VAD fmk. We conclude that expression of anti-chromodomain intracellular antibodies is sufficient to trigger a p53-independent apoptotic pathway that is only partly dependent on the known Z-VAD-inhibitable caspases, suggesting that CD function is essential for cell survival.
authors.affiliation: Department of Neuroscience, University of Roma Tor Vergata, Via Montpellier 1, 00133 Rome, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Filesi
authors.forename: Alessio
authors.initials: A
authors.lastname: Cardinale
authors.forename: Sjaak
authors.initials: S
authors.lastname: van der Sar
authors.forename: Ian G
authors.initials: IG
authors.lastname: Cowell
authors.forename: Prim B
authors.initials: PB
authors.lastname: Singh
authors.forename: Silvia
authors.initials: S
authors.lastname: Biocca
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism / Q000494:pharmacology; D019556:COS Cells; D016923:Cell Death / Q000235:genetics* / Q000276:immunology; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism*; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000172:deficiency* / Q000235:genetics; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D005057:Eukaryotic Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D008322:Mammals / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D010641:Phenotype; D011233:Precipitin Tests; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
chemical_list: D017304:Annexin A5; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004791:Enzyme Inhibitors; D000939:Epitopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D016159:Tumor Suppressor Protein p53; D000090266:Chromobox Protein Homolog 5