Distinct cytoplasmic and nuclear fractions of Drosophila heterochromatin protein 1: their phosphorylation levels and associations with origin recognition complex proteins

Abstract

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae.

Figure 1

HP1 fractionates as multiple oligomeric species from a cytosolic extract from early Drosophila embryos. Immunoblot analyses of (A) fractions from a Superose 6 gel filtration column (HP1 oligomers: large, fractions 13–15; medium, fractions 18–20; small, fractions 22–24) and (B) fractions from sucrose density sedimentation (HP1 oligomers: large, fractions 17–20; medium, fractions 8–14; small, fractions 1–5). The peak fractions for molecular weight standards: (A) thyroglobulin, 670 kD; gamma globulin, 158 kD; ovalbumin, 44 kD; and (B) thyroglobulin, 19.5 S; catalase, 11.2 S; aldolase, 7.3 S; and ovalbumin, 3.5 S are indicated.

Figure 2

The cytoplasmic oligomers of HP1 and differentially salt-extracted nuclear fractions contain different HP1 phosphoisoforms. Immunoblot analyses of phosphoisoforms in (A) small, medium, and large HP1 oligomers, and a mixture of small and large oligomers. (B) Immunoblot analyses of HP1 in salt- extracted fractions (0–0.06 M, 0.06–0.5 M, and 0.5–1.0 M KCl) from cycle 14 interphase nuclei.

Figure 3

DmORC subunits coimmunoaffinity purify with large and medium HP1 oligomers. (A) Immunoblot analyses of fractions from Superose 6 gel filtration column with antibodies that recognize DmORC2 and HP1. (B) Immunoblot analyses of unfractionated cytoplasmic extract with antibody prepared against HP1 peptide for immunoaffinity purifications. (C) Immunoblot analyses of immunoaffinity-purified small, medium, and large HP1 oligomers with antibodies that recognize DmORC2, DmORC6, and HP1. Each gel is loaded as follows: lane 1, total cytoplasmic extract, 200 μg; lane 2, pooled gel filtration fractions applied to anti-HP1 resin, containing small, medium, and large oligomers, respectively, 2% total; lane 3, flowthrough from each anti-HP1 column, 2% total; lane 4, 0.5 M KCl elution; lane 5, 1.0 M KCl elution; lane 6, 100 mM glycine, pH 2.0 elution (∼2 μg protein) from each anti-HP1 column; and lane 7, total protein eluted from control IgG column. (D) Coomassie-stained profile of glycine eluate from control and anti-HP1 columns loaded with gel filtration fractions containing small (lanes 1 and 2), medium (lanes 3 and 4), and large (lanes 5 and 6) oligomers, respectively. Polypeptides corresponding to immunoblot signals for DmORC2, DmORC6 (ORC2 and ORC6), HP1, and three novel polypeptides (p55, p40, and p35) are indicated.

Figure 4

Distinct nuclear fractions of HP1 and their associations with DmORC proteins. (A) Immunoblot analyses of immunoaffinity-purified fractions from salt-extracted interphase nuclei (60 mM, 0.5 M, and 1.0 M) with antibodies that recognize HP1, DmORC2, and DmORC6. Each lane contains: (1) 4 μg (1%) of each salt-extracted nuclear fraction applied to anti-HP1 resin; (2) 0.5 M KCl elution; and (3) 100 mM glycine, pH 2.0, elution from anti-HP1 column. (B) Quantitation of HP1 in salt-extracted nuclear fractions: (1) 0–60 mM KCl; (2) 60 mM–0.5 M KCl; and (3) 0.5–1 M KCl from pre-cycle 14 and cycle 14 embryos.

Figure 5

Heterochromatic localization of HP1 in diploid nuclei is perturbed in mutants for ORC2 gene. (A) Interphase nuclei from (a) wild-type or heterozygous k43 larvae; (b) homozygous k43 ¹ larvae; and (c) homozygous k43 ^γ4e larvae, stained with DAPI (left panels) and immunostained with antibodies that recognize HP1 (right panels). (B) Metaphase chromosomes from (a) wild-type or heterozygous k43 larvae; (b) homozygous k43 ¹ larvae; and (c) homozygous k43 ^γ4e larvae, stained with DAPI (left panels) and immunostained with antibodies that recognize HP1 (center panels); pseudo-color merged images (right panels), HP1-immunostaining (red) and DAPI-staining (green). Individual chromosomes are indicated (2, 3, 4, X, and Y).

Figure 5

Heterochromatic localization of HP1 in diploid nuclei is perturbed in mutants for ORC2 gene. (A) Interphase nuclei from (a) wild-type or heterozygous k43 larvae; (b) homozygous k43 ¹ larvae; and (c) homozygous k43 ^γ4e larvae, stained with DAPI (left panels) and immunostained with antibodies that recognize HP1 (right panels). (B) Metaphase chromosomes from (a) wild-type or heterozygous k43 larvae; (b) homozygous k43 ¹ larvae; and (c) homozygous k43 ^γ4e larvae, stained with DAPI (left panels) and immunostained with antibodies that recognize HP1 (center panels); pseudo-color merged images (right panels), HP1-immunostaining (red) and DAPI-staining (green). Individual chromosomes are indicated (2, 3, 4, X, and Y).

Figure 6

ORC mutations have minimal effect on GAGA localization into heterochromatin. Metaphase chromosomes from (A) wild-type or heterozygous k43 larvae; (B) homozygous k43 ¹ larvae (representative Y chromosome exhibiting: normal [inset] and abnormal [arrow] GAGA localization); (C) homozygous k43 ^γ4e larvae stained with DAPI (left panels) and immunostained with antibodies that recognize GAGA (center panels); pseudo-color merged images (right panels), GAGA-immunostaining (red) and DAPI-staining (green). Individual chromosomes are indicated (2, 3, 4, X, and Y).

Figure 7

Polytenization of chromosomes and HP1 enrichment at the chromocenter of polytene chromosomes is affected by ORC2 mutations. HP1 immunolocalization (right panel) on DAPI-stained polytene chromosomes (left panel) from: (A) wild-type larvae (arrowhead indicates the chromocenter); (B) k43 ¹-homozygous mutant larvae; and (C) k43 ^γ4e homozygous mutant larvae (enhanced exposure of HP1 immunostaining in far right panel, showing reduced HP1 signal at euchromatic sites of HP1 localization also).