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In Drosophila melanogaster, the two chromosomal proteins HP1 and HP2 colocalize on heterochromatic and euchromatic sites in polytene chromosomes. Mutations in the HP2 gene act as dominant suppressors of position effect variegation, demonstrating a role for HP2 in the formation or maintenance of heterochromatin. In this paper, we investigated whether a putative homolog of the D. melanogaster HP2 is involved in the facultative heterochromatinization process in mealybugs. Using an antibody raised against the Drosophila HP2, we identified in the mealybug Planococcus citri a cross-reactive epitope, which we refer to as HP2-like. We investigated the HP2-like pattern during the male embryo development where the entire paternal haploid chromosome set becomes heterochromatic. The HP2 antibody heavily decorates the chromocenters, where it localizes with HP1, and marks the chromatin before it acquires the full cytological characteristics of the male-specific heterochromatin. In euchromatic chromosomes, HP2-like is mainly concentrated at telomeric sites. The interplay between HP2-like and HP1-like was studied by dsRNA interference experiments. Extinguishing HP1-like expression by RNAi does not prevent the association of HP2-like with facultative heterochromatin, implying that HP2-like binds to chromatin in a HP1-independent manner. Our results confirm and extend the structural and functional conservation of proteins involved in heterochromatin assembly.

We show that methylated lysine 9 of histone H3 (Me9H3) is a marker of heterochromatin in divergent animal species. It localises to both constitutive and facultative heterochromatin and replicates late in S-phase of the cell cycle. Significantly, Me9H3 is enriched in the inactive mammalian X chromosome (Xi) in female cells, as well as in the XY body during meiosis in the male, and forms a G-band pattern along the arms of the autosomes. Me9H3 is a constituent of imprinted chromosomes that are repressed. The paternal and maternal pronuclei in one-cell mouse embryos show a striking non-equivalence in Me9H3: the paternal pronucleus contains no immunocytologically detectable Me9H3. The levels of Me9H3 on the parental chromosomes only become equivalent after the two-cell stage. Finally, we provide evidence that Me9H3 is neither necessary nor sufficient for localisation of heterochromatin protein 1 (HP1) to chromosomal DNA.

The behavior of chromosomes during development of the mealybug Planococcus citri provides one of the most dramatic examples of facultative heterochromatization. In male embryos, the entire haploid paternal chromosome set becomes heterochromatic at mid-cleavage. Male mealybugs are thus functionally haploid, owing to heterochromatization (parahaploidy). To understand the mechanisms underlying facultative heterochromatization in male mealybugs, we have investigated the possible involvement of an HP-1-like protein in this process. HP-1 is a conserved, nonhistone chromosomal protein with a proposed role in heterochromatinization in other species. It was first identified in Drosophila melanogaster as a protein enriched in the constitutive heterochromatin of polytene chromosome. Using a monoclonal antibody raised against the Drosophila HP-1 in immunoblot and immunocytological experiments, we provide evidence for the presence of an HP-1-like in Planococcus citri males and females. In males, the HP-1-like protein is preferentially associated with the male-specific heterochromatin. In the developing male embryos, its appearance precedes the onset of heterochromatization. In females, the HP-1-like protein displays a scattered but reproducible localization pattern along chromosomes. The results indicate a role for an HP-1-like protein in the facultative heterochromatization process.