Intrabody-mediated diverting of HP1β to the cytoplasm induces co-aggregation of H3–H4 histones and lamin-B receptor

Journal Article · Thu Oct 15 04:00:00 EDT 2015 · Experimental Cell Research

DOI:https://doi.org/10.1016/J.YEXCR.2015.09.006· OSTI ID:22746368

Cardinale, Alessio ^[1]; Filesi, Ilaria ^[2]; Singh, Prim B. ^[3]; Biocca, Silvia ^[2]

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1. Laboratory of Molecular and Cellular Neurobiology, IRCCS San Raffaele Pisana, Via di Val Cannuta 247, 00166 Rome (Italy)
2. Department of Systems Medicine, University of Roma Tor Vergata, Via Montpellier 1, 00133 Roma (Italy)
3. Department of Natural Sciences and Psychology, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF (United Kingdom)

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. - Highlights: • Intrabodies against heterochromatin protein 1 β (HP1β) inhibit its traffic to the nucleus. • Anti-HP1β intrabodies sequester HP1β in cytoplasmic aggregates. • Anti-HP1β scFv causes co-aggregation of LBR and H3–H4 histones in the cytoplasm. • Methylated histone H3 at K9 (Me9H3) is not affected by anti-HP1β scFv expression. • Anti-HP1β scFv induces altered nuclear morphology and apoptosis.

• OSTI ID: 22746368
• Journal Information: Experimental Cell Research, Journal Name: Experimental Cell Research Journal Issue: 1 Vol. 338; ISSN 0014-4827; ISSN ECREAL
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
AGGLOMERATION
ALBUMINS
APOPTOSIS
CATTLE
CENTROMERES
CHAIN REACTIONS
CYTOPLASM
DNA
DNA REPAIR
FLUORESCENCE
FLUORIDES
HETEROCHROMATIN
HISTONES
HYDROGEN 4
MEMBRANES
METHYLATION
MICE
MORPHOLOGY
MUTANTS
PHENOTYPE
PHOSPHATES
POLYMERASE CHAIN REACTION
POLYMERASES
RECEPTORS
SODIUM