Drosophila heterochromatin protein 1 (HP1)/origin recognition complex (ORC) protein is associated with HP1 and ORC and functions in heterochromatin-induced silencing

Abstract

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

Figure 1

The anon fe 1G5 gene product contains sequence similarity to sequence specific HMG proteins. (A) The amino-terminal region of the anon fe 1G5 gene product contains a conserved amino acid sequence motif of sequence-specific–binding HMG proteins. (B) The carboxyl-terminal region of the anon fe 1G5 gene product contains 3 copies of a novel repeated sequence. Identical amino acids are shown in light gray; similar amino acids are shown in dark gray.

Figure 2

HOAP is associated with cytoplasmic complexes containing HP1 and ORC subunits. Antibodies were increased against an anon fe 1G5 fusion protein expressed in bacteria in 2 different rabbits. Lanes 1–3, immunoblotting of the bacterially expressed anon fe 1G5 fusion protein and the endogenous anon fe 1G5 gene product in the embryo cytoplasmic extract (lane 2) with the use of antibodies produced by 1 rabbit and the endogenous anon fe 1G5 gene product in the embryo cytoplasmic extract with the use of antibodies produced by a second rabbit (lane 3). The antibody increased against the DmORC 2 peptide was used to immunoblot the endogenous DmORC 2 protein in the embryo cytoplasmic extract (lane 4). (B) Antibodies that recognize HP1, HOAP (anon fe 1G5 gene product), and DmORC 2 were used to immunoblot gel filtration fractions of the embryo cytoplasmic extract. (C) The immunoprecipitate of HP1 from the embryo cytoplasm extract was immunoblotted with antibodies that recognize the anon fe 1G5 gene product (HOAP) and HP1. Lane 1, input protein (5% total); lane 2, protein not retained on α-HP1 beads (5% total); lane 3, protein not retained on nonimmune IgG beads (5% total); lane 4, protein retained on α-HP1 beads (100% total); lane 5, protein retained on nonimmune IgG beads. (D) Immunoprecipitates of the endogenous anon fe 1G5 gene product (HOAP) from fractions of the cytoplasmic extract containing a large HP1 complex were immunoblotted with antibodies that recognize DmORC 6, HP1, the anon fe 1G5 gene product (HOAP), and DmORC 2. Lane 1, input protein (5% total); lane 2, protein not retained on α-anon fe 1G5 beads (5% total); lane 3, protein not retained on nonimmune IgG beads (5% total); lane 4, protein retained on α-anon fe 1G5 beads (100% total); lane 5, protein retained on nonimmune IgG beads.

Figure 3

HOAP is in a tightly bound interphase chromatin fraction containing HP1 and DmORC subunits. (A) Equal volumes of protein that were sequentially extracted from interphase nuclei with 60 mM and 0.5 and 1.0 M KCl and in the remaining pellet fraction were immunoblotted with antibodies that recognize ORC 2, HOAP, HP1, and ORC 6. (B) Formaldehyde cross-linked fractions of salt-resistant chromatin were immunoprecipitated with α-HOAP and α-HP1 antibodies and immunoblotted with antibodies that recognize DmORC 2, HOAP, HP1, DmORC 6, and DmLamin B. Lane 1, input chromatin (10% total); lane 2, chromatin retained on α-HOAP beads (50% total); lane 3, chromatin retained on α-HP1 beads (50% total); lane 4, chromatin retained on nonimmune IgG beads (50% total). The input chromatin in lanes 5–8 was treated with MNase I before immunoprecipitation with α-HP1 antibodies. Immunoprecipitated chromatin fractions were immunoblotted with antibodies that recognize DmORC 2, HOAP, and HP1. Lane 5, input chromatin treated with MNase I for 10 min (10% total); lanes 6–8, chromatin treated with MNase I for 0 (lane 6), 1 (lane 7), and 10 (lane 8) min that was retained on α-HP1 beads (50% total each).

Figure 4

HOAP colocalizes with subfractions of HP1 and DmORC2 in interphase nuclei. (A) Immunolocalization of HOAP (green), HP1 (red), and HOAP (green) and HP1 (red) merged in DAPI-stained interphase nuclei (blue) of a cycle 14 Drosophila embryo (side view). (B) Immunolocalization of HOAP (green), HP1 (red), and HOAP (green) and HP1 (red) merged in DAPI-stained interphase nuclei (blue) of a larval brain squash. (C) Immunolocalization of HOAP (green), DmORC2 subunit (red), and HOAP (green) and DmORC2 (red) merged in DAPI-stained interphase nuclei (blue) of a larval brain squash. (D) Immunolocalization of HOAP [HOAP (Def)] in DAPI-stained interphase nuclei [DAPI (Def)] of a brain squash from homozygous Df(3R)crb-F89-4 larvae and HOAP [HOAP (mitotic)] in DAPI-stained metaphase chromosomes [DAPI (mitotic)] of a wild-type larval brain squash.

Figure 5

HOAP is localized predominantly at telomeres and weakly throughout regions of pericentric heterochromatin of polytene chromosomes. Shown are immunostaining of HOAP (green; A), HP1 (red; B), and HOAP (green) and HP1 (red) merged (C) and an enlargement of HOAP (green) and HP1 (red) merged at the chromocenter (D). The HP1 signal appears faded when superimposed over the bright signal from the intensely DAPI-stained chromocenter. (B) Inset, HP1 signal alone.

Figure 6

Punctate localization of HOAP in heterochromatin is conserved in closely related Drosophila species that contain an anon fe 1G5 gene and similar satellite sequence compositions. Larval brain squashes from D. melanogaster (A), D. simulans (B), D. mauritiana (C), and D. virilis (D) were stained with DAPI and α-HP1 and α-HOAP antibodies.

Figure 7

HOAP binds D. melanogaster satellite and TAS in vitro. (A) Coomassie brilliant blue staining of the bacterially expressed hexahistidine-tagged recombinant HOAP protein used in the electrophoretic mobility shift assays. (B) Electrophoretic mobility shift assays were carried out with the recombinant HOAP protein and Drosophila satellite sequences AATAT (lanes 1 and 2), AATAG (lanes 3 and 4), AATAC (lanes 5 and 6), AAGAC (lanes 7 and 8), AAGAG (lanes 9 and 10), AACAA (lanes 11 and 12), AATAAAC (lanes 13 and 14), AATAGAC (lanes 15 and 16), AAGAGAG (lanes 17 and 18), and AATACATAG (lanes 19 and 20). For each pair of lanes, the odd-numbered lane contains a given satellite sequence free probe, and the even-numbered lane contains that same probe in the presence of HOAP protein. (C) Competitive binding studies with recombinant HOAP protein, satellite sequences AATAT, AATAG, and AATAACATAG, and TAS element. For each labeled probe (AATAT, AATAG, AATAACATAG, or TAS, as indicated on the left) lanes are as follows: lane 1, free labeled probe; lane 2, labeled probe in the presence of HOAP protein; lanes 3–22, binding of HOAP protein to the probe indicated to the left competed with an increasing (50, 100, 200, and 400×) molar excess of cold AATAT competitor (lanes 3–6), AATAG competitor (lanes 7–10), AATAACATAG competitor (lanes 11–14), TAS competitor (100, 200, and 300× molar excess; lanes 15–18), and nonspecific DNA competitor (scs sequence; lanes 19–22). (D) Electrophoretic mobility shift assays were carried out with the labeled D. melanogaster satellite sequence AATAACATAG (lanes 1 and 2) and D. virilis satellite sequences ACAAACT (lanes 3 and 4), ATAAACT (lanes 5 and 6), and ACAAATT (lanes 7 and 8) in the absence and presence of HOAP protein, respectively.

Figure 8

The anon fe 1G5 gene displays a reciprocal modifier of variegation phenotypes. The variegated phenotype of white^m4 was observed in a wild-type genetic background (A) and a Df(3R)F89-4/TM3Ser genetic background (B). Variegated expression of the In(3L)BL hs-LacZ reporter was observed in a wild-type genetic background (C), a Df(3R)F89-4/TM3Ser genetic background (D), and the presence of 2 copies of a heat shock–induced hs-anon fe 1G5 transgene (E).