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, 3 (11), e209

POF and HP1 Bind Expressed Exons, Suggesting a Balancing Mechanism for Gene Regulation

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POF and HP1 Bind Expressed Exons, Suggesting a Balancing Mechanism for Gene Regulation

Anna-Mia Johansson et al. PLoS Genet.

Abstract

Two specific chromosome-targeting and gene regulatory systems are present in Drosophila melanogaster. The male X chromosome is targeted by the male-specific lethal complex believed to mediate the 2-fold up-regulation of the X-linked genes, and the highly heterochromatic fourth chromosome is specifically targeted by the Painting of Fourth (POF) protein, which, together with heterochromatin protein 1 (HP1), modulates the expression level of genes on the fourth chromosome. Here we use chromatin immunoprecipitation and tiling microarray analysis to map POF and HP1 on the fourth chromosome in S2 cells and salivary glands at high resolution. The enrichment profiles were complemented by transcript profiles to examine the link between binding and transcripts. The results show that POF specifically binds to genes, with a strong preference for exons, and the HP1 binding profile is a mirror image of POF, although HP1 displays an additional "peak" in the promoter regions of bound genes. HP1 binding within genes is much higher than the basal HP1 enrichment on Chromosome 4. Our results suggest a balancing mechanism for the regulation of the fourth chromosome where POF and HP1 competitively bind at increasing levels with increased transcriptional activity. In addition, our results contradict transposable elements as a major nucleation site for HP1 on the fourth chromosome.

Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. High Resolution ChIP-chip Analysis of POF and HP1 Binding to Chromosomes 4 and 2L in S2 Cells and Salivary Glands
The smoothed data for one of the three biological replicates of each experiment are shown. Figure S1 shows a comparison of the replicates and the effect of the smoothing process. A typical 80-kb region from the fourth chromosome (A) is compared to a typical 80-kb control region from Chromosome 2L (B). The scale bars show the genomic positions of the two regions in kb. HP1 enrichment in salivary glands is shown in dark blue (HSG), HP1 in S2 cells (HS2) in light blue, POF in S2 cells (PS2) in orange, and POF in salivary glands (PSG) in red. The enrichments are the log2 ratios between specific IP/input. Genes expressed from left to right are shown above the horizontal line in each panel, and the genes expressed in the opposite direction are shown below the line. Regions that are repeat-masked and therefore not represented by probes on the arrays are shaded. Note that the fourth chromosome is enriched in repeated regions.
Figure 2
Figure 2. POF Binds Preferentially within Genes with a Preference for Exons
(A) The amounts of sequences with binding divided into 100 binding classes. In S2 cells, 509 kb out of 860 kb qualified as binding compared to 377 kb in salivary glands. The amounts of intergenic compared to genic sequences (B), exons compared to introns (C), and UTR compared to coding sequences (D) plotted against increasing (stronger) binding classes. S2 cells are shown to the left of salivary glands. The black line corresponds to expectations if the binding was randomly distributed, and the shaded areas represent the situation when only 5% of the probes are left, ranging from 6.5 kb (CDS in salivary glands) to 20 kb (genes in S2 cells). These areas will therefore be sensitive to random effects and annotation inaccuracies. (E) The average binding profile of POF to bound genes in S2 cells and salivary glands. Exons from genes of different lengths were scaled to align 5′ and 3′ ends and divided into ten regions. The mean binding of each region of the gene is represented by black lines and the standard deviations by gray lines.
Figure 3
Figure 3. POF and HP1 Colocalize within Genes while HP1 Binding Shows an Additional “Promoter Peak”
(A) POF and HP1 enrichment profiles at the Thd1 Pur-alpha gene pair locus. POF and HP1 binding at the Ank locus (B) and the Crk locus (C). HP1 binding in salivary glands is shown in dark blue, HP1 in S2 cells in light blue, POF in S2 cells in orange, and POF in salivary glands in red. Genes expressed from left to right are represented by rectangles above the horizontal line in each panel and the genes expressed in the opposite direction are shown below the line. Exons are indicated in black and introns in gray. Note the peak of HP1 enrichment (arrowheads) in the region upstream of the transcription start points of Ank and Crk, present in both S2 cells and salivary glands. All enrichment values for all single probes on the fourth chromosome plotted as HP1 enrichment on the y-axis and POF enrichment on the x-axis for S2 cells (D) and salivary glands (E). Probes in the putative promoter region (−500 to +200 bp from the transcription start point) are shown in black.
Figure 4
Figure 4. Correlations of POF and HP1 Binding Strength with Each Other and with Transcription Levels
(A) Transcript profiling (light gray), POF enrichment (orange), and H3K9ac enrichment (green) at the eIF-4G locus (transcribed from right to left) in S2 cells. Note that the transcript profile allows predictions of exon usage and transcript levels of unannotated exons (exemplified by blue boxes). The H3K9ac is typically enriched at the 5′ end of transcribed genes, as seen for eIF-4G. The combination of transcript profiling and the H3K9ac profile also allows prediction of novel genes, e.g., a novel gene can be predicted downstream of eIF-4G (red box) transcribed from left to right. (B) POF binding in S2 cells (PS2) plotted against relative transcription values in S2 cells (TP_S2). (C) POF binding in salivary glands (PSG) versus transcription values in salivary glands (TP_SG). (D) HP1 binding in S2 cells (HS2) versus relative transcription values in S2 cells. (E) HP1 binding in salivary glands (HSG) versus relative transcription values in salivary glands. (F) POF binding in S2 cells versus HP1 binding in S2 cells, and (G) POF binding in salivary glands versus HP1 binding in salivary glands. Note the more even distribution of different transcription levels in salivary glands compared to S2 cells.
Figure 5
Figure 5. Cell-Specific Binding of POF and HP1 Is Linked to Levels of Transcription
Relative binding values for POF (A) and HP1 (B) for Chromosome 4 genes in S2 cells versus salivary glands. The zfh2 gene is indicated as a black dot. (C) POF and HP1 enrichments at the zfh2 locus in S2 cells and salivary glands. The transcript profiles on a log2 scale in S2 cells (light gray) and salivary glands (dark gray). The shaded boxes indicate the calculated expression values for zfh2 in the two cell types. (D) The log2 differences in POF binding and differences in expression between salivary gland cells and S2 cells plotted with respect to the positions of the genes along the fourth chromosome. Genes with higher binding levels in salivary glands are shown as red bars above the horizontal line while genes with higher binding levels in S2 cells are shown as orange bars below the line. The widths of the bars reflect the gene length. The relative change in expression is shown below the gene annotation map (the zfh2 gene is indicated). Genes that are more expressed in salivary glands are shown as dark gray bars above the horizontal line, and genes that are more expressed in S2 cells are shown as light gray bars below the horizontal line. Note that the POF binding differences are reflected by the differences in expression.
Figure 6
Figure 6. Silenced Trangene Insertions Correspond to Sites with High HP1 Enrichment but Not with Low Expression Regions
(A) HP1 enrichment profiles and transcript profiles surrounding the insertion of the silenced transgene 39C-24 and the nonsilenced transgene 4-M1030 (B). (C) Both the HP1 binding and the transcript levels are lower in the regions near (±500 bp) nonsilenced insertions (non-PEV) compared to regions near silenced insertions (PEV) in S2 cells and salivary glands (D). The silenced (n = 17) and nonsilenced (n = 6) transgenes are from Sun et al. [35].
Figure 7
Figure 7. Mean Enrichment Profiles of HP1 and POF in Salivary Glands, Calculated from Enrichment Values for All Bound (by POF) and All Unbound Chromosome 4 Genes (Merged Exons)
The promoter regions of bound genes (500 bp upstream of the transcription start point) were divided into five 100-bp fragments, and the mean enrichment in each fragment was calculated and is indicated. The mean enrichments within intergenic regions (IG), surrounding (±200 bp) repetitive regions (Masked), and 1360 elements are indicated. The enrichment of HP1 and POF is shown in blue and red, respectively.

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