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TABLE S1: Suppression of PEV and TPE by Chromatin-associated Proteins
GeneAlleleMutationPEVTPEHIS-CDeficiencyDf(2L)38C7-10; 39D3-E1++-DeficiencyDf(2L)38F5; 39E7-F1+-H3.3DeficiencyDf(2L)25A2-D5-+H2AvDH2AvD810Deficiencya--Orc2Orc21Point mutationb+-Orc22Point mutationb++-Orc3lat1Point mutation-+lat6Point mutation-++Orc5Orc52Point mutation--AsxAsx1Point mutation--AsxxF23Point mutation--EzEz60Point mutationc--PcPc1Point mutation--phph-d401Deficiencyc-+++ph-p409Deficiencyc--PscPsc1Point mutation--Psc1.d19Point mutationd-+Psc1.d20Point mutation-++Psce22Point mutation-++Psch27Point mutation--BEAF-32DeficiencyDf(2R)51B5-11; 51D7-E2--Bj1DeficiencyDf(3L)64E1-13; 65C1-D6--CG6678DeficiencyDf(3R)93C3-6; 93F14-94A1-+dpadpa1Point mutation--Mcm7DeficiencyDf(3L)66E1-6; 66F1-6--mlemle9Point mutation--Mt2DeficiencyDf(3L)32F1-33F2-++spt4DeficiencyDf(2R)49A4-13; 49E7-F1--touDeficiencyDf(2R)48A-B-- a gift from S. Elgin
b gift from R. Kelly
c gift from H. Brock
d gift from T. Wu
Rationales
Histone and histone variants: The majority of histone genes in Drosophila are clustered in a block of tandem repeats in the 39D-E region ADDIN EN.CITE Ner2002555517Ner, S. S.Harrington, M. J.Grigliatti, T. A.Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada. ner@zoology.ubc.caA role for the Drosophila SU(VAR)3-9 protein in chromatin organization at the histone gene cluster and in suppression of position-effect variegationGenetics1763-741624AnimalsBase SequenceChromatin/*geneticsDNA/geneticsDrosophila/*genetics/growth & development/metabolismDrosophila Proteins/*geneticsFemaleGene Expression Regulation, DevelopmentalGenes, InsectHistones/*geneticsMaleMethyltransferases/*geneticsMultigene FamilyMutationRNA, Messenger/genetics/metabolismRepressor Proteins/*geneticsSupport, Non-U.S. Gov'tSuppression, Genetic2002Dec12524347http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12524347Samal19811045104517Samal, B.Worcel, A.Louis, C.Schedl, P.Chromatin structure of the histone genes of D. melanogasterCell401-9232Animals*Bacterial ProteinsChemistryChromatin/*analysisDNA Restriction EnzymesDeoxyribonuclease BamHIDeoxyribonuclease IDeoxyribonucleases*Deoxyribonucleases, Type II Site-SpecificDrosophila melanogasterEmbryo/analysisEndonucleases*GenesHistones/*geneticsMicrococcal NucleaseNucleosomes/analysisResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.Transcription, Genetic1981Feb6258802http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=6258802[31,91]. This region is known as the histone gene cluster, or HIS-C. We have previously demonstrated that removal of one copy of the HIS-C suppresses PEV in Drosophila ADDIN EN.CITE Moore1979525217Moore, G. D.Procunier, J. D.Cross, D. P.Grigliatti, T. A.Histone gene deficiencies and position--effect variegation in DrosophilaNature312-42825736AllelesAnimalsChromosome DeletionDrosophila melanogaster/*geneticsGenes, StructuralHeterochromatinHeterozygoteHistones/*geneticsLinkage (Genetics)1979Nov116133http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=116133Ner2002555517Ner, S. S.Harrington, M. J.Grigliatti, T. A.Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada. ner@zoology.ubc.caA role for the Drosophila SU(VAR)3-9 protein in chromatin organization at the histone gene cluster and in suppression of position-effect variegationGenetics1763-741624AnimalsBase SequenceChromatin/*geneticsDNA/geneticsDrosophila/*genetics/growth & development/metabolismDrosophila Proteins/*geneticsFemaleGene Expression Regulation, DevelopmentalGenes, InsectHistones/*geneticsMaleMethyltransferases/*geneticsMultigene FamilyMutationRNA, Messenger/genetics/metabolismRepressor Proteins/*geneticsSupport, Non-U.S. Gov'tSuppression, Genetic2002Dec12524347http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12524347[31,92], and we have confirmed this result here. Deletions of histone genes have also been shown to suppress TPE in budding yeast ADDIN EN.CITE Kaufman19982538253817Kaufman, P. D.Cohen, J. L.Osley, M. A.Lawrence Berkeley National Laboratory and Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3206, USA. pdkaufman@1bl.govHir proteins are required for position-dependent gene silencing in Saccharomyces cerevisiae in the absence of chromatin assembly factor IMol Cell Biol4793-806188Amino Acid SequenceChromatin*Chromosomal Proteins, Non-HistoneDNA-Binding Proteins/genetics/*physiologyFungal Proteins/genetics/*metabolismGene Dosage*Gene Expression Regulation, FungalHistones/geneticsHumansMolecular Sequence DataMutagenesisNuclear Proteins/genetics/*metabolismRepetitive Sequences, Nucleic AcidRepressor Proteins/genetics/*metabolismResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.Saccharomyces cerevisiae/*genetics*Saccharomyces cerevisiae ProteinsSequence Homology, Amino AcidTelomere1998Aug9671489http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9671489Norris19883049304917Norris, D.Dunn, B.Osley, M. A.Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA.The effect of histone gene deletions on chromatin structure in Saccharomyces cerevisiaeScience759-612424879Centromere/ultrastructureChromatin/physiology/*ultrastructureChromosome DeletionDNA Transposable ElementsGalactoseGene Expression RegulationGenes, FungalHistidineHistones/*geneticsMutationPhenotypeRNA, Messenger/geneticsRepetitive Sequences, Nucleic AcidResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.Saccharomyces cerevisiae/genetics/*ultrastructureTranscription, Genetic1988Nov 42847314http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=2847314[93,94]. However, haplo-deficiencies of the histone genes have no effect on TPE in Drosophila.
In addition to the main histone genes, which occur in multiple copies in all higher organisms, all eukaryotes have a variety of single copy histone variants. The functions of the variants are unknown and are currently the subject of intense study, however it appears very likely they are involved in regulating transcription. We tested two histone variants, H3.3 and H2AvD. A small intragenic null mutation in H2AvD did not suppress PEV or TPE.
A point mutation for H3.3 does not exist in the public databases and therefore we tested the effects of a small deficiency that removes H3.3 ADDIN EN.CITE Ahmad20021422142217Ahmad, K.Henikoff, S.Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A1-162, Seattle, WA 98109, USA.Histone H3 variants specify modes of chromatin assemblyProc Natl Acad Sci U S A16477-8499 Suppl 4CentromereChromatin/*metabolismDNA ReplicationHistones/chemistry/*metabolismResearch Support, Non-U.S. Gov't2002Dec 1012177448http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12177448[87]. Hemizygosity for H3.3 had a slight suppressing effect on TPE, but no effect on PEV.
Origin of Replication Complex Proteins: We tested three members of the Origin recognition complex (Orc): Orc2, Orc3 (also known as latheo) and Orc5. Mutations in Orc2 were previously known to be weak suppressors of PEV ADDIN EN.CITE Pak1997575717Pak, D. T.Pflumm, M.Chesnokov, I.Huang, D. W.Kellum, R.Marr, J.Romanowski, P.Botchan, M. R.Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotesCell311-23913Amino Acid SequenceAnimalsBase SequenceChromosomal Proteins, Non-Histone/*metabolismCloning, OrganismDNA-Binding Proteins/*metabolismDrosophila melanogasterHeterochromatin/*metabolismMolecular Sequence DataProtein BindingSupport, U.S. Gov't, P.H.S.Xenopus1997Oct 319363940http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9363940[61]. We confirm this result here, but found that Orc2 mutations have no effect on TPE. Orc5 does not suppress either PEV or TPE. Point mutations in Orc3 are weak to moderate Su(TPE), but have no effect on PEV. Orc3 is one of the genes that is removed by the Su(z)25 deficiency and therefore contributes to the strong suppressor effect of this deficiency.
Polycomb Group Genes: One of the first identified Su(TPE), Psc, is a member of the PcG of proteins. We confirmed that mutations in Psc suppress TPE, however, as appears to be the case with other Su(TPE), the effect is allele specific. Of the four alleles we tested, two are moderate Su(TPE)s, one is a weak Su(TPE) and one has no effect. We also tested mutations in six other PcG genes, but none suppressed TPE with the possible exception of ph-d (see Results).
Chromatin Proteins: We tested twelve other chromatin-associated proteins thought to be involved in the modulation of chromatin structure. Hemizygosity for the DNA methyltransferase protein Mt2, and a deficiency removing the putative gene CG6678, which contains a regulator of chromatin condensation (RCC1) domain suppressed TPE, but had no effect on PEV.
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