Effect of PEV modifiers on bw^D trans-inactivation. Su(var)3-7/+ is a very strong suppressor of bw^D inactivation, but Su(var)205 and Rpd3 are only mild suppressors while Su(var)3-9 does not suppress trans-inactivation at all. Known functions and products of the Su(var)s are in parentheses below the genotypes. The wild-type chromosomes do not carry balancers and the + chromosome in the v; +/bw^D control is from the Canton-S wild-type line.

Effect Su(var)3-7 on trans-inactivation. (A) An extra dose of Su(var)3-7 enhances trans-inactivation. (Left) The trans-inactivated P{hsp26-Pt} transgene in the presence of bw^D. (Right) The bw^D inactivated transgene with an extra copy of Su(var)3-7 carried in the transgene P{Suv3-7}T21a. (B) Bar graph with standard error of the nuclease accessibility of each genotype (n = 3). See Figure 1 for transposon and probe details. The value for ab28/+ is set at 100% and relative percentage cuts for the other genotypes are calculated. The P-value from a Student's t-test for pairs of interest is above the bracket. In the presence of an extra copy of Su(var)3-7, the restriction enzyme accessibility was significantly lower as compared to ab28/bw^D. 39C-2 is a positive control.

Chromatin structure of the mini-white regulatory region in trans to bw^D. (A) Fly eyes of the genotypes y w^67c23; P{lacW}chrw/+ (mw/+, left) and y w^67c23; P{lacW}chrw/bw^D; P{cos bw⁺}/+ (mw/bw^D, right). The photo is taken from Csink et al. (2002). See Csink et al. (2002) for a detailed explanation of the genotypes. (B) The P{lacW} transposon has a mini-white reporter gene and a lacZ enhancer trap. The lacZ has termination (T) sequences from hsp70. A detailed view of the mini-white regulatory region shows two sets of primers designed to amplify ChIP DNA products. Primer hsp70-mw utilizes the chimeric region of hsp70 and mini-white and amplifies 199 bases primarily of mini-white. Primer mw(reg) covers the transcription start (+1) and goes into the first exon of mini-white. (C) Bar charts with standard error of data from duplex PCR with di-AcH3 antibody-precipitated adult fly DNA (n = 3). Lower acetylation levels on the transposon opposite bw^D with both primer sets are found. (D) Bar charts with standard error of data from duplex PCR with HP1 antibody-precipitated adult fly DNA (n = 3). Both primers show significantly higher occupation of HP1 on the transposon in the presence of bw^D.

Analysis of sequences in cis to bw^D detects acetylation, methylation, and HP1 differences. (A) The P{lacW} transposon inserted near the chrw locus, 4.7 kbp from bw^D on the opposite chromosome. A distal flanking deletion on the wild-type chromosome removes 17 kbp of genomic sequences, including the bw gene, giving rise to the csc2 chromosome shown in the top line. Therefore, csc2/bw^D has the transposon and bw^D in the same location as in chrw/bw^D, but changes the distal cis sequences for the transposon. Three primers sets were designed to study cis changes in chromatin structure by the bw^D heterochromatic insert. The first primer, bw (exon), is 71 bp from the bw^D insertion. This primer set will most likely pick up the simple sequence satellite sequences in the heterochromatic insert. bw (UTR) is 10.3 kbp from the insertion, 12 bp from the transcription start site of the bw gene. The primer set cg30181 is 12 kbp from the bw^D insertion in the gene CG30181 next to bw, in its first exon, 400 bp from its transcription start site. (B–D) Bar charts with standard error of data from duplex PCR with (B) ²MeH3K9, (C) di-AcH3, and (D) HP1 antibody-precipitated larval DNA (n = 3). Methylation level is higher in the presence of bw^D with bw (exon) primer set but not farther away in the bw gene or in the gene CG30181 next to bw. HP1 levels are higher in the presence of bw^D with all three cis sequences amplifying primer sets. Higher HP1 levels are seen 12 kbp from the bw^D insertion.

Chromatin structure of the hsp26 regulatory region in trans to bw^D. (A) Detailed view of the hsp26-Plant region of the transposon showing the Plant primer set amplifying 367 bp of the coding region used in the ChIP assays. Another primer set, Plant(s), utilizes the same forward primer as Plant but uses a different reverse primer to amplify 293 bp of the coding region. (B) Phosphorimager scan of a polyacrylamide gel showing products of the duplex PCR with the Plant primer and an internal control primer using HP1-precipitated DNA from adult flies. The ratio of signal in the Plant band and the control band from precipitated chromatin, normalized to ratios from the input DNA, are plotted as bar charts with standard error, and P-values are calculated to determine significant differences as described in materials and methods. HP1 levels are significantly higher for all three genotypes, ab28/bw^D, ab28/E(bw^D)40, and 39C-2, compared to ab28/+ in the transcribed region of the transposon (n = 3). (C) Bar charts with standard error of data from duplex PCR with ²MeH3K9 antibody-precipitated third instar larval DNA (n = 3, where n is three independent chromatin extractions). Methylation level on the transcribed region is slightly higher for the transposon inserted in the centric heterochromatin as compared to ab28/+. No difference in the methyl modification levels is seen with ab28/bw^D or ab28/E(bw^D)40. (D) Bar charts with standard error of data from duplex PCR with HP1 antibody-precipitated third instar larval DNA (n = 3). HP1 is significantly enriched on 39C-2 in the coding region as compared to the ab28/+ control but no different for ab28/bw^D and ab28/E(bw^D)40.

Nucleosomal compaction and trans-inactivation. (A) The P{hsp26-Pt} transposon inserted near the chrw gene, 4.7 kbp proximal to the bw^D insertion site on the opposite chromosome. The chromosomal region around bw is depicted by colored boxes representing known and annotated genes drawn to scale. bw^D is not to scale. (B) Eye phenotypes of flies of the indicated genotype. All flies are also white⁻ at the endogenous locus on the X chromosome, so silencing is of the hsp70-white reporter gene in P{hsp26-Pt}. P{hsp26-Pt}ab28/+ is the negative control with wild-type eyes. P{hsp26-Pt}ab28/bw^D is trans-inactivated. P{hsp26-Pt}ab28/E(bw^D)40 is more inactivated than P{hsp26-Pt}ab28/bw^D. P{hsp26-Pt}39C-2 contains the transposon inserted in the centric heterochromatin and is silenced in cis (Wallrath and Elgin 1995). (C) The P{hsp26-Pt} transposon with the hsp26 promoter driving a unique plant cDNA and the hsp70 promoter driving the white gene. The details of the hsp26-Pt region show a positioned nucleosome flanked by two DNAse hypersensitivity sites (proximal, PDH; distal, DDH) containing XbaI restriction sites and two SalI sites, one each in the hsp26 regulatory region and the plant fragment, used to generate Plant probe (Pt probe) (Wallrath and Elgin 1995). (D) Southern blot showing restriction enzyme accessibility of the four genotypes P{hsp26-Pt}ab28/+ (ab28/+), P{hsp26-Pt}ab28/bw^D (ab28/bw^D), P{hsp26-Pt}ab28/E(bw^D)40 [ab28/E(bw^D)40], and P{hsp26-Pt}39C-2 (39C-2). The SalI cuts generate a 3-kbp parent fragment, the SalI–XbaI distal cuts generate a 1.2-kbp fragment, and the SalI–XbaI proximal cut gives rise to a 0.8-kbp fragment. On the Southern blot are chromatin digests from the four genotypes with XbaI and SalI. (E) The percentage accessibility of each genotype plotted as bar charts with standard error (n = 3). The value for ab28/+ is set at 100% and relative percentage cuts are calculated for the other three genotypes (materials and methods). The P-values from a one-tailed Student's t-test for pairs of interest are above the brackets. All three genotypes, ab28/bw^D, ab28/E(bw^D)40, and 39C-2, are significantly less accessible than ab28/+. The restriction enzyme sites are significantly more protected in the presence of E(bw^D)40 as compared to ab28/bw^D.