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Figures
- Fig. 1.
lacI-HP1 fusion protein co-localizes with endogenous HP1. Polytene chromosomes from expressor stock LH2, expressing lacI-HP1, were squashed and stained with antibodies to HP1 (red) and antibodies to lacI (green). The chromocenter (C) and telomeres (T) are indicated. Complete co-localization is observed as yellow in the merged image.
- Fig. 2.
Tethered HP1 causes silencing at some, but not all, genomic sites. The left column shows the eye phenotype observed for stocks 157.1, J3.2 and S9.2 with and without tethered GFP-lacI. The middle column shows the eye phenotype with and without tethered lacI-HP1. The right column shows localization of the lacI-HP1 fusion protein on polytene chromosomes. The arrow denotes the location of lac repeats. Polytene chromosomes were stained with antibodies against lacI (green).
- Fig. 3.
Tethered lacI-HP1 causes ectopic associations. Third instar larvae of stocks 157.1, 157.4.112 and P2.5 expressing lacI-HP1 were stained with antibodies to lacI (green). Arrows indicate the locations of lac repeats. The percent of nuclei showing ectopic associations, out of 60 nuclei scored for each cytological position, are shown at the bottom of each panel. Associations were observed between the tethered site and other HP1-containing sites (A,B), but were also observed at sites not enriched with HP1 (C).
- Fig. 4.
HP1 functions downstream of SU(VAR)3-9. (A) The lac repeats show a low level of HP1 and H3 K9 methylation in the absence of lacI-HP1. Polytene chromosomes from stock 157.1 containing a reporter transgene with 256 copies of lac repeats were stained with antibodies to HP1 (green) and antibodies to H3 K9 di-methyl (red). Arrows indicate the locations of lac repeats. (B) Tethered HP1 does not intensify H3 K9 methylation. Strong staining is observed for the HP1 antibody, but not the H3 K9 di-methyl antibody, upon expression of lacI-HP1. (C) Expression of a Su(var)3-9-EGFP transgene leads to recruitment of SU(VAR)3-9-EGFP (upper row) and increased H3 K9 methylation (bottom row). (D) Silencing by tethered HP1 (stock 157.1) was unaffected in Su(var)3-906 hetero- and homozygotes as indicated by a white eye phenotype (middle and bottom rows). The homozygous Su(var)3-906 flies show a rough eye phenotype due to the roughest mutation used as a marker on the Su(var)3-906-bearing chromosome (bottom row). By contrast, Su(var)3-906 heterozygotes show suppression of white gene in In(1)wm4, a stock carrying an inversion placing the white gene next to centric heterochromatin, and 118E-10 a stock carrying a hsp70-white P-element inserted within centric heterochromatin.
Tables
- Table 1.
Summary of silencing assays
lacI repeat line lacI repeat location Copies of lacI repeats Eye color phenotype without lacI-HP1 Eye color phenotype with lacI-HP1 P-element insertion site 157.1 1E4 256 Orange White None 179.1 86D 256 Orange White 8.63 kb downstream of Sodh-2 157.4.112 61F3 256 Orange White 17 bp upstream of BtbII, P6.4 88E12-F1 256 Yellow White 1.69 kb downstream of ea P20.21 99A4-5 256 Orange White None P8.6 96B1 256 Orange White 25 bp upstream of BcDNA:GH12663 P11.3 96C1-2 256 Yellow White None P19.9 63C5 256 Orange White None P2.5 65D4-5 256 Orange White 6 bp upstream of sgl P23.9 87D9-11 256 Yellow White 4 bp downstream of CtBP J11.1 42C2-3 256 Yellow White 444 bp upstream of l(2) 01289 J3.2 93A7-B1 256 Red Red 62 bp upstream of Atpα F3.1.1 54B17-C1 256 Orange White None 107.1 40B-D 256 Orange White None P25.22 89B2-B3 256 Orange White 3.45 kb upstream of He189B J8.1 89B16-17 ∼58 Orange White 6.5 kb upstream of tara S4.5 73A1-73A2 ∼58 Dark orange Yellow None S11.7 61D1 ∼55 Dark orange Yellow None S9.2 73A2-73A4 ∼46 Red Yellow 48 bp downstream of argos S15.14 1E4 ∼33 Orange Yellow None CN2 50C20-22 32 Orange Light orange 3.84 kb upstream of Aats-phe CN10 63C5 32 Dark orange Yellow None P9.4 85A7-9 ∼20 Dark orange Yellow None J2.3 86E13-15 ∼15 Orange Light orange None 6.4P5 77B6-9 4 Dark orange Orange 166 bp upstream of trb1 64.AB 67A8-B1 4 Orange Light orange None -
Nearby genes listed are only those in which the transcription start site, or a predicted transcription start site, resides within 10 kb of the P-element insert according to FlyBase. `None' indicates absence of genes within 10 kb of the P-element insert site. `Upstream' and `downstream' denote the location of the P-element with respect to the gene. `∼' indicates lines in which the number of lac repeats were estimated based on the size of the lac repeat-containing fragment.
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