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• W2065134033 abstract "Association of the highly conserved heterochromatin protein, HP1, with the specialized chromatin of centromeres and telomeres requires binding to a specific histone H3 modification of methylation on lysine 9. This modification is catalyzed by the Drosophila Su(var)3-9 gene product and its homologues. Specific DNA binding activities are also likely to be required for targeting this activity along with HP1 to specific chromosomal regions. The Drosophila HOAP protein is a DNA-binding protein that was identified as a component of a multiprotein complex of HP1 containing Drosophila origin recognition complex (ORC) subunits in the early Drosophila embryo. Here we show direct physical interactions between the HOAP protein and HP1 and specific ORC subunits. Two additional HP1-like proteins (HP1b and HP1c) were recently identified in Drosophila, and the unique chromosomal distribution of each isoform is determined by two independently acting HP1 domains (hinge and chromoshadow domain) (47Smothers J.F. Henikoff S. Mol. Cell Biol. 2001; 21: 2555-2569Crossref PubMed Scopus (135) Google Scholar). We find heterochromatin protein 1/origin recognition complex-associated protein (HOAP) to interact specifically with the originally described predominantly heterochromatic HP1a protein. Both the hinge and chromoshadow domains of HP1a are required for its interaction with HOAP, and a novel peptide repeat located in the carboxyl terminus of the HOAP protein is required for the interaction with the HP1 hinge domain. Peptides that interfere with HP1a/HOAP interactions in co-precipitation experiments also displace HP1 from the heterochromatic chromocenter of polytene chromosomes in larval salivary glands. A mutant for the HOAP protein also suppresses centric heterochromatin-induced silencing, supporting a role for HOAP in centric heterochromatin. Association of the highly conserved heterochromatin protein, HP1, with the specialized chromatin of centromeres and telomeres requires binding to a specific histone H3 modification of methylation on lysine 9. This modification is catalyzed by the Drosophila Su(var)3-9 gene product and its homologues. Specific DNA binding activities are also likely to be required for targeting this activity along with HP1 to specific chromosomal regions. The Drosophila HOAP protein is a DNA-binding protein that was identified as a component of a multiprotein complex of HP1 containing Drosophila origin recognition complex (ORC) subunits in the early Drosophila embryo. Here we show direct physical interactions between the HOAP protein and HP1 and specific ORC subunits. Two additional HP1-like proteins (HP1b and HP1c) were recently identified in Drosophila, and the unique chromosomal distribution of each isoform is determined by two independently acting HP1 domains (hinge and chromoshadow domain) (47Smothers J.F. Henikoff S. Mol. Cell Biol. 2001; 21: 2555-2569Crossref PubMed Scopus (135) Google Scholar). We find heterochromatin protein 1/origin recognition complex-associated protein (HOAP) to interact specifically with the originally described predominantly heterochromatic HP1a protein. Both the hinge and chromoshadow domains of HP1a are required for its interaction with HOAP, and a novel peptide repeat located in the carboxyl terminus of the HOAP protein is required for the interaction with the HP1 hinge domain. Peptides that interfere with HP1a/HOAP interactions in co-precipitation experiments also displace HP1 from the heterochromatic chromocenter of polytene chromosomes in larval salivary glands. A mutant for the HOAP protein also suppresses centric heterochromatin-induced silencing, supporting a role for HOAP in centric heterochromatin. Early microscopic studies revealed the eukaryotic nucleus to have a heterogeneous morphology. The bulk of eukaryotic chromatin has a decondensed amorphous appearance during interphase. However, certain chromosomal regions retain the compact appearance of metaphase chromatin throughout the cell cycle and, according, have been termed “heterochromatin” (1Heitz E. Jahrb. Wissensch Bot. 1928; 69: 762-818Google Scholar). The distinct cytological properties of heterochromatin translate into distinct functional properties as well; for example, it is typically replicated later in S phase and is transcriptionally inert relative to the more typical “euchromatin” (2Lima de Faria A. Jaworska H. Nature. 1968; 217: 138-142Crossref PubMed Scopus (203) Google Scholar, 3Ris H. Korenberg R.D. The Structure and Replication of Genetic Material. Academic Press, Inc., New York1979: 268-361Google Scholar). In Drosophila, heterochromatin has been found to induce silencing of euchromatic genes that become juxtaposed to it by a chromosomal rearrangement, a phenomenon known as position effect variegation (4Muller H.J. J. Genet. 1930; 22: 299-334Crossref Scopus (304) Google Scholar). Ironically, the relatively few genes that normally reside within heterochromatin suffer a similar fate when translocated to a euchromatic chromosomal region (5Hearn M.G. Hedrick A. Grigliatti T.A. Wakimoto B.T. Genetics. 1991; 128: 785-797Crossref PubMed Google Scholar, 6Eberl D.F. Duyf B.J. Hilliker A.J. Genetics. 1993; 134: 277-292Crossref PubMed Google Scholar, 7Lu B.Y. Emtage P.C. Duyf B.J. Hilliker A.J. Eissenberg J.C. Genetics. 2000; 155: 699-708Crossref PubMed Google Scholar). These distinct cytological and functional properties of heterochromatin are thought to reflect its unique nucleoprotein composition. For example, euchromatic genes that have undergone heterochromatin-induced silencing adopt a more highly ordered nucleosomal array (8Wallrath L.L. Elgin S.C.R. Genes Dev. 1995; 9: 1263-1277Crossref PubMed Scopus (408) Google Scholar). In some cases, the silenced gene is also recruited into the heterochromatin compartment of the nucleus, where it is thought to be sequestered from the enzymatic machinery for a variety of DNA metabolic processes including transcription. One conserved feature of heterochromatin is its non-coding repetitive DNA sequence content, although little conservation is observed between these repeats at the primary sequence level (9Kit S. J. Mol. Biol. 1961; 3: 711-716Crossref PubMed Scopus (170) Google Scholar, 10Rae P.M. Proc. Natl. Acad. Sci. U. S. A. 1970; 67: 1018-1025Crossref PubMed Scopus (90) Google Scholar, 11Pardue M.L. Gall J.G. Science. 1970; 168: 1356-1358Crossref PubMed Scopus (819) Google Scholar). Another conserved feature of heterochromatin is the heterochromatin-associated protein, heterochromatin protein 1 (HP1). 1The abbreviations used are: HP1, heterochromatin protein 1; Su(var), Suppressor of variegation; ORC, origin recognition complex; DmORC, Drosophila DNA replication ORC; HOAP, HP1/ORC-associated protein; di-MeK9, di-methylated lysine 9 containing histone H3; DAPI, 4,6-diamidino-2-phenylindole. This protein, first described in Drosophila, is enriched in the heterochromatin of species ranging from fission yeast to humans (12James T.C. Elgin S.C.R. Mol. Cell. Biol. 1986; 6: 3862-3872Crossref PubMed Scopus (480) Google Scholar, 13James T.C. Eissenberg J.C. Craig C. Dietrich V. Hobson A. Elgin S.C. Eur. J. Cell Biol. 1989; 50: 170-180PubMed Google Scholar, 14Eissenberg J.C. Elgin S.C.R. Curr. Opin. Genet. Dev. 2000; 10: 204-210Crossref PubMed Scopus (395) Google Scholar). The Drosophila protein (HP1a) is encoded by the Suppressor of variegation (Su(var)) 2-5 gene, which belongs to a group of Su(var) genes with mutant phenotypes of reversing the heterochromatin-induced silencing of euchromatic genes (15Eissenberg J.C. Tharappel C.J. Foster-Hartnett D.M. Hartnett T. Ngan V. Elgin S.C.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9923-9927Crossref PubMed Scopus (449) Google Scholar, 16Eissenberg J.C. Hartnett T. Mol. Gen. Genet. 1993; 240: 333-338Crossref PubMed Scopus (39) Google Scholar, 17Sinclair D.A.R. Mottus R.C. Grigliatti T.A. Mol. Gen. Genet. 1983; 191: 326-333Crossref Scopus (99) Google Scholar, 18Wustmann G. Szidonya. J. Taubert. H. Reuter G. Mol. Gen. Genet. 1989; 217: 520-527Crossref PubMed Scopus (130) Google Scholar). Because early studies failed to demonstrate DNA binding activity for HP1, the mechanism for its association with heterochromatin has remained a mystery until the recent discovery that a specific covalent modification (methylation on lysine 9) of histone H3 provides a chromatin binding site for it in species ranging from fission yeast to humans (19Bannister A.J. Zegerman P. Partridge J.F. Miska E.A. Thomas J.O. Allshire R.C. Kouzarides T. Nature. 2001; 410: 120-124Crossref PubMed Scopus (2184) Google Scholar, 20Lachner M. O'Carroll D. Rea S. Mechtler K. Jenuwein T. Nature. 2001; 410: 116-120Crossref PubMed Scopus (2177) Google Scholar, 21Jacobs S.A. Taverna S.D. Zhang Y. Briggs S.D. Li J. Eissenberg J.C. Allis C.D. Khorasanizadeh S. EMBO J. 2001; 20: 5232-5241Crossref PubMed Scopus (330) Google Scholar, 22Cowell I.G. Aucott R. Mahadevaiah S.K. Burgoyne P.S. Huskisson N. Bongiorni S. Prantera G. Fanti L. Pimpinelli S. Wu R. Gilbert D.M. Shi W. Fundele R. Morrison H. Jeppesen P. Singh P.B. Chromosoma (Berl.). 2002; 111: 22-36Crossref PubMed Scopus (232) Google Scholar, 23Schotta G. Ebert A. Krauss V. Fisher A. Hoffman J. Rea S. Jenuwein T. Dorn R. Reuter G. EMBO J. 2002; 21: 1121-1131Crossref PubMed Scopus (484) Google Scholar). Interestingly, this histone modification is catalyzed by the product of the Drosophila Su(var)3-9 gene and its homologues in other species, and recognition of this histone binding site by HP1 requires the conserved chromodomain of HP1. The mechanism used by the cell to target HP1 and the Su(var)3-9 H3 methyltransferase activity specifically to heterochromatin is not as well understood. A number of HP1-interacting proteins have been identified that directly or indirectly bind DNA. Some of these (e.g. TIF1 proteins and SP100) are capable of acting as HP1-dependent transcriptional co-repressors when tethered to heterologous DNA binding domains (24Le Douarin B. Nielsen A. Garnier J.-M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (468) Google Scholar, 25Seeler J.S. Marchio A. Sitterlin D. Transy C. Deejan A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7316-7321Crossref PubMed Scopus (229) Google Scholar, 26Lehming N. Le Saux A. Schuller J. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7322-7326Crossref PubMed Scopus (149) Google Scholar, 27Nielsen A.L. Ortiz J.A. You J. Oulad-Abdelghani M. Khechumian R. Gansmuller A. Chambon P. Losson R. EMBO J. 1999; 18: 6385-6395Crossref PubMed Scopus (294) Google Scholar, 28Lechner M.S. Begg G.E. Speicher D.W. Rauscher III, F.J. Mol. Cell. Biol. 2000; 20: 6449-6465Crossref PubMed Scopus (169) Google Scholar). The DNA binding activities of TIF1-interacting zinc finger Kruppel repressor proteins (KRAB-ZFPs) are thought to target the transcriptional repressing activity of HP1 through TIF1β (29Ryan R.F. Schultz D.C. Ayyanathan K. Singh P.B. Friedman J.R. Fredericks W.J. Rauscher III, F.J. Mol. Cell. Biol. 1999; 19: 4366-4378Crossref PubMed Scopus (315) Google Scholar, 30Matsuda E. Agata Y. Sagai M. Katakai T. Gonda H. Shimizu A. J. Biol. Chem. 2001; 276: 14222-14229Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 31Schultz D. Ayyanathan K. Negorev D. Maul G.G. Rauscher III, F.J. Genes Dev. 2002; 16: 919-932Crossref PubMed Scopus (903) Google Scholar). These proteins are found in complexes with lysine 9 histone H3 methyltransferase activities (31Schultz D. Ayyanathan K. Negorev D. Maul G.G. Rauscher III, F.J. Genes Dev. 2002; 16: 919-932Crossref PubMed Scopus (903) Google Scholar). The retinoblastoma protein targets both HP1 and the mammalian Su(var)3-9 homologue (SUV39H) to the mammalian cyclin E promoter, presumably through the DNA binding activity of the E2F transcription factor (32Nielsen S.J. Schneider R. Bauer U.M. Bannister A.J. Morrison A. O'Carroll D. Firestein R. Cleary M. Jenuwein T. Herrera R.E. Kouzarides T. Nature. 2001; 412: 561-565Crossref PubMed Scopus (744) Google Scholar, 33Ogawa H. Ishiguro K.-i. Gaubatz S. Livingston D.M. Nakatani Y. Science. 2002; 296: 1132-1136Crossref PubMed Scopus (629) Google Scholar). These data support a role for specific DNA binding activities in recruiting HP1- and histone-modifying activities to mammalian euchromatic genes. Mammals contain three different isoforms of HP1 that differ in their euchromatic and heterochromatic localizations, and it has even been proposed that HP1 heterodimers may play a role in recruiting euchromatic genes to the heterochromatin compartment (30Matsuda E. Agata Y. Sagai M. Katakai T. Gonda H. Shimizu A. J. Biol. Chem. 2001; 276: 14222-14229Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 34Minc E. Allory Y. Worman H.J. Courvalin J.-C. Buendia B. Chromosoma (Berl.). 1999; 108: 220-234Crossref PubMed Scopus (280) Google Scholar, 35Nielsen A.L. Oulad-Abdelghani M. Ortiz J.A. Remboutsika E. Chambon P. Losson R. Mol. Cell. 2001; 7: 729-739Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). DNA binding activities may similarly play a role in recruiting HP1 to heterochromatic regions. For example, fission yeast mutants for homologues of the mammalian α-satellite sequence binding centromere protein B (CENP-B) have reduced levels of lysine 9-methylated histone H3 and the HP1 homologue, Swi 6, at centromeres (36Nakagawa H. Lee J.-K. Hurwitz J. Allshire R.C. Nakayama J.-i. Grewal S.I.S. Tanaka K. Murakami Y. Genes Dev. 2002; 16: 1766-1778Crossref PubMed Scopus (84) Google Scholar). The double-stranded interference RNA-processing machinery has also been found to have a role in Swi 6 targeting to centromeres and the silent mating type loci of Schizosaccharomyces pombe (37Volpe T.A. Kidner C. Hall I.M. Teng G. Grewal S.I. Martienssen R.A. Science. 2002; 297: 1833-1837Crossref PubMed Scopus (1635) Google Scholar). In this paper, we examine the role of a DNA binding activity that we identified as a component of a maternally loaded complex of HP1 in the early Drosophila embryo (38Huang D.W. Fanti L. Pak D.T.S. Botchan M.R. Pimpinelli S. Kellum R. J. Cell Biol. 1998; 142: 307-318Crossref PubMed Scopus (103) Google Scholar, 39Shareef M.M. King C. Damaj M. Badugu R.-K. Huang D.W. Kellum R. Mol. Biol. Cell. 2001; 12: 1671-1685Crossref PubMed Scopus (114) Google Scholar). This complex also contained subunits of the Drosophila DNA replication origin recognition complex (DmORC); thus, the unidentified component was designated as HP1/ORC-Associated Protein (HOAP). The DmORC2 subunit is enriched in centric heterochromatin of early embryos, and mutants for this protein also suppress heterochromatin-induced silencing and display defects in HP1 localization in centric heterochromatin of diploid nuclei (38Huang D.W. Fanti L. Pak D.T.S. Botchan M.R. Pimpinelli S. Kellum R. J. Cell Biol. 1998; 142: 307-318Crossref PubMed Scopus (103) Google Scholar, 40Pak D.T.S. Pflumm M. Chesnokov I. Huang D.W. Kellum R. Marr J. Romanowski P. Botchan M. Cell. 1997; 91: 311-323Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). These data support a role for the ORC in localizing HP1 to heterochromatin. The amino terminus of the HOAP protein contains similarity to the HMG box of sequence-specific HMG proteins and binds specific double-stranded AT-rich satellite sequences of Drosophila melanogaster in vitro (39Shareef M.M. King C. Damaj M. Badugu R.-K. Huang D.W. Kellum R. Mol. Biol. Cell. 2001; 12: 1671-1685Crossref PubMed Scopus (114) Google Scholar). It is also localized in heterochromatin, predominantly at telomeres, but weaker enrichment is also observed in pericentric heterochromatin (39Shareef M.M. King C. Damaj M. Badugu R.-K. Huang D.W. Kellum R. Mol. Biol. Cell. 2001; 12: 1671-1685Crossref PubMed Scopus (114) Google Scholar). Mutants for the HOAP-encoding gene, caravaggio, display a telomere fusion phenotype that is associated with a defect in HP1 localization at telomeres (41Cenci G. Siriaco G. Raffa G.D. Kellum R. Gatti M. Nat. Cell Biol. 2003; 5: 82-84Crossref PubMed Scopus (139) Google Scholar). Here we report the molecular parameters that specify interactions between HOAP and the predominantly heterochromatin-localized HP1a isoform (commonly known as Drosophila HP1). We also present evidence that these interactions play a role in the association of this HP1 protein with pericentric heterochromatin. Protein Expression—Hexahistidine-tagged recombinant HOAP proteins were expressed from a cDNA of the anon fe 1G5 gene cloned into the pET20b expression vector (39Shareef M.M. King C. Damaj M. Badugu R.-K. Huang D.W. Kellum R. Mol. Biol. Cell. 2001; 12: 1671-1685Crossref PubMed Scopus (114) Google Scholar). FLAG-tagged recombinant HP1 proteins were expressed from a cDNA of the HP1a gene cloned into the PQE30 (Qiagen) expression vector. The HP1a cDNA was obtained by PCR amplification using EST clone LD10408 and a forward primer-containing DNA sequence encoding the FLAG tag (5′-GCGCGCGGAATTCATGGACTATAAAGACGATGACAAAGGCAAGAAAATCGACAACCCT-3′) and reverse primer (5′-GCGCGCGTCTAGAATCTTCATTATCAGAGTACCAGGATAG-3′). Truncated HOAP and HP1 proteins were expressed from deletion derivatives of these expression vectors created by the introduction of restriction sites into the HOAP- and HP1-coding sequences by site-directed mutagenesis (Stratagene QuikChange site-directed mutagenesis kit, catalog number 200518). All cDNA clones used to express recombinant proteins were sequenced before their use for expressing proteins in BL21 (DE 3) Escherichia coli strain. Hexahistidine-tagged proteins were purified by nickel nitrilotriacetic acid-agarose (Qiagen catalog number 30210) chromatography using imidazole for protein elution, and FLAG-tagged proteins were purified by FLAG M2 (Sigma A-1205) chromatography using FLAG peptide (Sigma F3290) for protein elution. 35S-Labeled ORC, HP1a, HP1b, and HP1c proteins were synthesized in vitro using a coupled transcription/translation reaction system (TNT Quick Coupled Transcription/Translation System, Promega, catalog number L1170) and cDNA clones LD11626 (ORC1), GH 13824 (ORC2), GM 14657 (ORC3), LD 43280 (ORC4), LP 12153 (ORC5), RE 52740 (ORC6), LD 10408 (HP1a), RE 72354 (HP1b), and RE 28447 (HP1c) produced by the Berkeley Drosophila Genome Project and distributed by Research Genetics (Invitrogen). Immunoprecipitation Experiments—Immunoprecipitation experiments were carried out as described by Huang et al. (38Huang D.W. Fanti L. Pak D.T.S. Botchan M.R. Pimpinelli S. Kellum R. J. Cell Biol. 1998; 142: 307-318Crossref PubMed Scopus (103) Google Scholar). An anti-HOAP immunoaffinity resin (39Shareef M.M. King C. Damaj M. Badugu R.-K. Huang D.W. Kellum R. Mol. Biol. Cell. 2001; 12: 1671-1685Crossref PubMed Scopus (114) Google Scholar) was used to immunoprecipitate recombinant FLAG-tagged HP1 proteins, 35S-labeled ORC subunits, or HP1a, HP1b, HP1c proteins with recombinant hexahistidine-tagged HOAP. M2 resin (Sigma A-1205) was used in co-precipitation experiments of hexahistidine-tagged HOAP proteins with recombinant FLAG-tagged HP1 proteins. All immunoprecipitation reactions were carried out with equimolar concentrations of co-precipitating proteins. Peptide competition experiments were carried out with a 10- and 100-fold molar excess of competing PRMVI, PETEMNE, PGETEMNE, GETEMNE, HP1a hinge (KSKRTTDAEZDTIPVSGST), HP1b hinge (RSKRKSFLEDDTEEQKKLI), and HP1c hinge (KKRGEKKPKCEEIQKLR) peptide synthesized by Research Genetics (Invitrogen). Immunoprecipitation reactions were incubated with rotation for 1 h at 4 °C in Buffer A (50 mm Hepes, pH 7.6, 10% glycerol (w/v), 1 mm sodium metabisulfite, 100 mm phenylmethylsulfonyl fluoride, 200 mm benzamidine, and a 1:100 dilution of protease inhibitor mixture (1.6 mg/ml benzamidine and 1.0 mg/ml each phenanthroline, aprotinin, leupeptin, and pepstatin)) containing 100 mm KCl. Immunoprecipitations were washed 3 times (15 min each) in the same buffer followed by 1 wash in Buffer A containing 0.5 m KCl and 1 wash in Buffer A containing 1.0 m KCl before elution of the bound protein with 100 mm glycine, pH 2.0. HP1 and HOAP immunoblotting was performed on 2% of the input sample, 2% of the unbound supernatant fraction, and 25% of the bound pellet fraction. Immunoblotting signals were detected by enhanced chemiluminescence detection or by autoradiography of 35S-labeled in vitro translated protein. Adobe Photoshop 7.0 software was used to scan and process all digital images. Gel Filtration—Purified recombinant HOAP and HP1 proteins were combined at stoichiometries of 1:1, 1:2, and 1:4 and loaded onto a Sephacryl S-200 gel filtration column that had been pre-equilibrated in Buffer A. Trichloroacetic acid precipitates of 1-ml fractions from the column were analyzed by Coomassie staining on SDS-polyacrylamide gels. Native protein molecular weight standards from Bio-Rad (catalog number 151-1901) were used. Peptide Challenge Assays—The effect of a peptide on HP1 association with the insoluble chromatin fraction was determined by incubating five salivary glands in 50 μl of Cohen's permeabilization buffer (42Alfageme C.R. Rudkin G.T. Cohen L.H. Chromosoma (Berl.). 1980; 78: 1-31Crossref PubMed Scopus (75) Google Scholar) (10 min) containing a 6 μm concentration of the challenging peptide (di-MeK9hisH3 (Upstate Biotechnology catalog number 12-430), PETEMNE, GETEMNE, PGTEMNE, PRMVI, or HP1a, -b, and -c hinge peptide, as described above) in a 500-μl microcentrifuge tube. The glands were then pelleted by centrifugation for 2 min at 8,000 × g. HP1 and HOAP immunoblotting was then performed on a trichloroacetic acid precipitate of the entire solubilized supernatant fraction and the entire solubilized pellet fraction from each set of treated glands. Equivalent loading of protein samples was monitored by Ponceau S staining of protein transferred to nitrocellulose before immunoblotting. Polytene chromosome immunostaining was used to determine the effect of each peptide on HP1 association with the chromocenter. After peptide extraction as described above, salivary glands were fixed in formaldehyde and used for polytene chromosome squash preparations. Chromosome squash preparations and immunostaining were carried out as previously described (39Shareef M.M. King C. Damaj M. Badugu R.-K. Huang D.W. Kellum R. Mol. Biol. Cell. 2001; 12: 1671-1685Crossref PubMed Scopus (114) Google Scholar) using rat anti-HOAP (1:200) and rabbit anti-HP1 (1:1000) primary antibodies and fluorescein-labeled anti-rabbit IgG and rhodamine-labeled anti-rat IgG secondary antibodies (1:1000). The effect of overexpressing HOAP on HP1 chromocenter displacement by peptide treatment was determined using salivary glands dissected from a heat shock-inducible HOAP transgenic line (39Shareef M.M. King C. Damaj M. Badugu R.-K. Huang D.W. Kellum R. Mol. Biol. Cell. 2001; 12: 1671-1685Crossref PubMed Scopus (114) Google Scholar) that had been subjected to 37 °C heat shock treatment for 30 min followed by a 30-min recovery at room temperature. Similar treatments of control animals of the same genotype lacking the HOAP transgene were carried out in parallel. A Photometrics CoolSnap cooled high speed digital color camera and MetaView imaging software were used to acquire images at equivalent exposure settings for all specimens. Position Effect Variegation Modifier Assays—The wm4 and BL1-hsLacZ reporter genes were used to assay a mutant for HOAP (cav) (41Cenci G. Siriaco G. Raffa G.D. Kellum R. Gatti M. Nat. Cell Biol. 2003; 5: 82-84Crossref PubMed Scopus (139) Google Scholar) for phenotypes associated with modifying pericentric heterochromatin-induced silencing. Crosses were carried out between w1 /Y;cav/TM3Sb males and wm4 females and between w1 ;cav/TM3Sb males and BL1-hsLacZ/TM3Sb females. Eye variegation phenotypes of progeny of the cross between w1 ;cav/TM3Sb and wm4 were visually scored as described in Table I. β-Galactosidase activity was quantitated in protein extracts prepared from female progeny of the cross between cav/TM3Sb and BL1-hsLacZ/TM3Sb after a 30-min 37 °C heat shock followed by 15 min recovery using chlorophenol red-β-d-galactopyranoside as substrate (43Simon J.A. Lis J.T. Nucleic Acids Res. 1987; 15: 2971-2988Crossref PubMed Scopus (108) Google Scholar).Table IMutant for HOAP protein suppresses heterochromatin-induced silencing of two different reporter geneswm4/wm4 female × w1 /Y;cav/TM3Sb maleVariegationaVariegation is scored as % pigmented cells: <20% = strong; ≈50% = moderate; >80% = weak.wm4/w1 ; cav %wm4/w1 ; TM3SbStrong727Moderate4835Weak4538BL1/TM3Sb female × cav/TM3Sb maleBL1/cavBL1/TM3Sbβ-Galactoside activitybβ-Galactosidase activity from expression of hsLacZ reporter is given in units/mg of protein.42.529.6a Variegation is scored as % pigmented cells: <20% = strong; ≈50% = moderate; >80% = weak.b β-Galactosidase activity from expression of hsLacZ reporter is given in units/mg of protein. Open table in a new tab HOAP Interacts Directly with HP1—Co-precipitation experiments were carried out with bacterially expressed recombinant HOAP and HP1 proteins to determine whether HOAP is capable of directly interacting with HP1 (Fig. 1). An anti-FLAG M2 resin was used to precipitate hexahistidine-tagged HOAP protein with FLAG-tagged HP1. The full-length HOAP protein (full) co-precipitated with HP1 in these experiments (Fig. 1A). The domain of HOAP that is responsible for this binding to HP1 was then mapped to the carboxyl-terminal half of the protein. Truncated versions of the FLAG-tagged HP1 protein were then used to map the HP1 domain responsible for binding the HOAP carboxyl terminus. An anti-HOAP immunoresin was used to precipitate carboxyl-terminal HOAP protein and associated HP1 fragments (Fig. 1B). The carboxyl-terminal chromoshadow domain (CSD) and the hinge (H) domain located between the conserved chromo and chromoshadow domains of HP1 were each found to independently bind the HOAP carboxyl terminus. The amino-terminal HP1 chromodomain (CD) failed to bind the HOAP carboxyl terminus. A pentapeptide motif (PRMVI) located in the HP1 chromoshadow domain and also in a number of HP1-interacting proteins (p150 subunit of CAF-1, TIF1 proteins, and the Su-(var)3-7 protein) has been shown or implicated to mediate the association of these proteins with HP1 (44Brasher S.V. Smith B.O. Rasmus R.H. Nietlispach D. Thiru A. Nielsen P.R. Broadhurst R.W. Ball L.J. Murzina N.V. Laue E.D. EMBO J. 2000; 19: 1587-1597Crossref PubMed Scopus (239) Google Scholar, 45Cowieson N.P. Partridge J.F. Allshire R.C. McLaughlin P.J. Curr. Biol. 2000; 10: 517-525Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 46Smothers J.F. Henikoff S. Curr. Biol. 2000; 10: 27-30Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Mutations in the pentapeptide motif of mouse HP1β (mMOD1) also prevent it from forming homodimers (44Brasher S.V. Smith B.O. Rasmus R.H. Nietlispach D. Thiru A. Nielsen P.R. Broadhurst R.W. Ball L.J. Murzina N.V. Laue E.D. EMBO J. 2000; 19: 1587-1597Crossref PubMed Scopus (239) Google Scholar). Although HOAP lacks a canonical pentapeptide motif, its interaction with the HP1 chromoshadow domain prompted us to determine whether this motif is required for the HOAP/HP1 interaction. We used a molar excess of a competing PRMVI peptide in the co-precipitation assay to examine the role of this motif in this interaction (Fig. 1C, +PRMVI). A 10-fold molar excess of this peptide was found to interfere with the interaction between the HOAP carboxyl terminus and the HP1 chromoshadow domain. In contrast, the interaction of the HOAP carboxyl terminus with the HP1 hinge domain was not even inhibited by a 100-fold molar excess of the peptide. A mutant form of the HP1 protein containing a substitution of glutamic acid for valine at position 190 of the PRMVI motif also failed to bind the HOAP carboxyl terminus (Fig. 1D, V190E). The carboxyl terminus of the HOAP protein contains three copies of a novel proline-containing repeat (PETEM/INE) that could also have a role in HP1 binding (39Shareef M.M. King C. Damaj M. Badugu R.-K. Huang D.W. Kellum R. Mol. Biol. Cell. 2001; 12: 1671-1685Crossref PubMed Scopus (114) Google Scholar). A synthetic peptide for this sequence was also used in competitive binding experiments with the HOAP carboxyl terminus and each of its HP1 interaction domains (Fig. 1C, +PETEMNE). A 100-fold molar excess of the peptide was able to inhibit the interaction between the HOAP carboxyl terminus and the HP1 hinge domain but had no effect on the interaction between the HOAP carboxyl terminus and the HP1 chromoshadow domain. A 10-fold molar excess of PETEMNE peptide also partially interfered with the interaction between the HOAP carboxyl terminus and the HP1 hinge domain (data not shown). Mutant forms of the peptide (Fig. 1C, +PGETEMNE and +GETEMNE) did not inhibit binding of the HP1 hinge domain to the HOAP carboxyl terminus. Also, a P290E substitution in the third PETEMNE motif of the HOAP carboxyl terminus prevented it from binding the HP1 hinge domain but had no effect on its binding to the chromoshadow domain (Fig. 1D, P290E). HOAP Interacts with a Dimer of HP1—The ability of the PRMVI peptide to interfere with binding of the HOAP carboxyl terminus to the HP1 chromoshadow domain suggests some role for this motif in the HOAP/HP1 interaction. The peptide interference could result from direct competition by the peptide for a site of interaction between carboxyl-terminal HOAP and the PRMVI motif of HP1. Alternatively, the interference could be an indirect consequence of the peptide impeding HP1 homodimerization. The peptide might then indirectly interfere with an HP1/HOAP interaction that requires HP1 in dimeric form. To address this possibility, gel filtration exp" @default.
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• W2065134033 title "Novel Drosophila Heterochromatin Protein 1 (HP1)/Origin Recognition Complex-associated Protein (HOAP) Repeat Motif in HP1/HOAP Interactions and Chromocenter Associations" @default.
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