Genomic mapping of Suppressor of Hairy-wing binding sites in Drosophila

Abstract

Background: Insulator elements are proposed to play a key role in the organization of the regulatory architecture of the genome. In Drosophila, one of the best studied is the gypsy retrotransposon insulator, which is bound by the Suppressor of Hairy-wing (Su [Hw]) transcriptional regulator. Immunolocalization studies suggest that there are several hundred Su(Hw) sites in the genome, but few of these endogenous Su(Hw) binding sites have been identified.

Results: We used chromatin immunopurification with genomic microarray analysis to identify in vivo Su(Hw) binding sites across the 3 megabase Adh region. We find 60 sites, and these enabled the construction of a robust new Su(Hw) binding site consensus. In contrast to the gypsy insulator, which contains tightly clustered Su(Hw) binding sites, endogenous sites generally occur as isolated sites. These endogenous sites have three key features. In contrast to most analyses of DNA-binding protein specificity, we find that strong matches to the binding consensus are good predictors of binding site occupancy. Examination of occupancy in different tissues and developmental stages reveals that most Su(Hw) sites, if not all, are constitutively occupied, and these isolated Su(Hw) sites are generally highly conserved. Analysis of transcript levels in su(Hw) mutants indicate widespread and general changes in gene expression. Importantly, the vast majority of genes with altered expression are not associated with clustering of Su(Hw) binding sites, emphasizing the functional relevance of isolated sites.

Conclusion: Taken together, our in vivo binding and gene expression data support a role for the Su(Hw) protein in maintaining a constant genomic architecture.

Figure 1

Su(Hw) binding profile across 3 Mb Adh region. Schematic of enrichment profiles for embryo, brain, and wing imaginal disc are shown as a plot of enrichment of array fragments against genomic coordinates. Light gray vertical lines on the plots indicate fragments with enrichment greater than 1.7-fold. The positions of high scoring Patser matches to the new Suppressor of Hairy-wing (Su [Hw]) binding consensus are indicated below the enrichment plots. The upper line indicates positions of matches with P < e^-15, and the lower line indicates positions of matches with P between e^-12 and e^-15 and having enrichment >1.7-fold in at least one of the chromatin sources. Annotation tracks are provided in Additional data file 9. kb, kilobases; Mb, megabases.

Figure 2

Correlation of ChIP enrichment using either anti-Su(Hw) on wild-type chromatin or anti-GFP on chromatin from Su(Hw)-GFP transgenic. The enrichment values are plotted as the arsinh transformation (approximately equivalent to the log2 scale) of the ratio of specific versus control ChIP. Correlation coefficient is 0.66. ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; Su(Hw), Suppressor of Hairy-wing.

Figure 3

Enhanced Su(Hw) binding site consensus derived from in vivo ChIP. (a) WebLogo of the gypsy consensus. (b) WebLogo of the new consensus. (c) Aligned stack of the motif identified by MEME; 42 sites contained in 41 array fragments. The box indicates the 20 base pair sequences corresponding to the WebLogo in panel b. ChIP, chromatin immunopurification; Su(Hw), Suppressor of Hairy-wing.

Figure 4

Closeness of match to the Su(Hw) binding site consensus is associated with in vivo binding. The Patser P value for each Patser match is plotted against the enrichment (arsinh transformation; approximately equal to log₂ ratio) of the fragment containing the matching sequence. The enrichment value is the highest mean value from the three chromatin sources. The vertical line indicates the Patser P = e^-15; for matches with P < e^-15, 63% show enrichment greater than 0.5 (1.4-fold) and 53% show enrichment greater than 0.8 (1.7-fold). Su(Hw), Suppressor of Hairy-wing.

Figure 5

Conservation of Su(Hw)and Su(Hw) binding sites. (a) Example of a conserved Suppressor of Hairy-wing (Su [Hw]) binding site in an intron of the cyclin E gene. Although the overall conservation of the intron is variable, the binding site itself is a conserved entity. (b) PhastCons scores across all 2,281 predicted genomic Su(Hw) binding sites with a Patser P value < e^-15. The binding sites are centred over position 0 and 100 base pairs left and right of the site are shown. The blue line indicates the median PhastCons score for a given position, and the black bar shows the 25th and 75th percentiles of the scores. It is evident that Su(Hw) binding sites are generally highly conserved, whereas their genomic context is not.

Figure 6

Selected genomic Su(Hw) binding sites. (a) Intronic sites in CG31814. (b) Sites separating genes transcribed from the same strand (CG18095 and CG31771). (c) Suppressor of Hairy-wing (Su [Hw]) site in the cyclin E (CycE) gene. Gene models are from the FlyBase genome browser [55]; dark gray bars represent enriched 1 kilobase fragments from the tiling array and asterisks represent the location of Patser sites.

Figure 7

Expression changes in the 3 Mb Adh region with respect to Su(Hw) binding sites. Expression changes (as absolute fold change according to the scale bar) are indicated by the bars above the gene models, with upregulated genes in orange and downregulated genes in blue. The bars mark the 5' end of each gene. The location of Su(Hw) binding sites are plotted on the three rows All sites, Cluster 1, and Cluster 2. Cluster 2 indicates the two sites within 100 base pairs of each other. Cluster 1 indicates the six pairs of sites within 1 kilobase of each other. All sites plots the locations of the remaining 83 sites in the region. The maps are plotted and rendered using the Affymetrix Integrated Genome Browser.

Figure 8

The Su(Hw) binding site has a pronounced DNA flexibility profile. Higher stacking free energy values are associated with DNA flexibility [38]. Blue indicates the stacking free energy profile for 100 best matches to Suppressor of Hairy-wing (Su [Hw]) consensus based on Patser P value; black indicates the profile for 100 random sequences; and red indicates the profile for the 12 Su(Hw) sites in the gypsy element. A representative sequence is given at the top. The gray zone marks the region between the highly conserved G nucleotides at positions 5 and 17 in the new Su(Hw) binding consensus.

Figure 9

Genes with expression changes in su(Hw) mutant larvae (L3) and wing discs. A cluster diagram showing changes in gene expression in a su(Hw) null condition compared with changes in the heterozygous controls (fold change ≥ 1.7, P ≤ 10^-3 for the mutants and approximately half the fold change at P ≤ 10^-2 for the heterozygotes). The table lists those genes with greater than 1.7-fold expression change that have predicted Suppressor of Hairy-wing (Su [Hw]) binding sites within 30 kilobases (kb). The Expression column shows the absolute fold change for each gene. The Distance column indicates the distance between the gene model and Su(Hw) sites(s); for those genes with predicted sites within the gene model, the number of sites are indicated. If there is more than one site, the distance between them is given. The Location column indicates where the predicted sites lie with respect to the gene models. UTR, untranslated region.