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. 2012 Jun;32(11):2121-34.
doi: 10.1128/MCB.06622-11. Epub 2012 Apr 2.

POF regulates the expression of genes on the fourth chromosome in Drosophila melanogaster by binding to nascent RNA

Anna-Mia Johansson  1 , Per Stenberg, Anders Allgardsson, Jan Larsson
Affiliations

POF regulates the expression of genes on the fourth chromosome in Drosophila melanogaster by binding to nascent RNA

Anna-Mia Johansson et al. Mol Cell Biol. 2012 Jun.

Abstract

In Drosophila, two chromosome-wide compensatory systems have been characterized: the dosage compensation system that acts on the male X chromosome and the chromosome-specific regulation of genes located on the heterochromatic fourth chromosome. Dosage compensation in Drosophila is accomplished by hypertranscription of the single male X chromosome mediated by the male-specific lethal (MSL) complex. The mechanism of this compensation is suggested to involve enhanced transcriptional elongation mediated by the MSL complex, while the mechanism of compensation mediated by the painting of fourth (POF) protein on the fourth chromosome has remained elusive. Here, we show that POF binds to nascent RNA, and this binding is associated with increased transcription output from chromosome 4. We also show that genes located in heterochromatic regions spend less time in transition from the site of transcription to the nuclear envelope. These results provide useful insights into the means by which genes in heterochromatic regions can overcome the repressive influence of their hostile environment.

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Figures

Fig 1
Fig 1
The time course for the release and reassociation of POF from its chromosomal targets closely resembles that for RNP2. POF (yellow) and RNP2 (green) are released from chromosomes after heat shock. Third-instar Drosophila larvae were heat shocked for 45 min and allowed to recover for 60, 90, or 140 min prior to dissection. After a recovery time of approximately 140 min, the distributions of both POF and RNP2 on the fourth chromosome (indicated by boxes) have been restored to their original patterns. DNA is stained with DAPI (blue).
Fig 2
Fig 2
POF binding to chromosome 4 genes depends on active transcription. Binding of POF on chromosome 4 genes is decreased upon blocking transcription by DRB treatment. (A) The binding of POF to three chromosome 4 genes (CG31998, Zyx102EF, and MED26) was analyzed by ChIP using antibodies against POF. The y axis indicates enrichment plotted as percentage of input. Results are shown for two biological replicates. DRB treatment causes release of POF from the main part of the gene body. Note that the genes are expressed from right to left, and the primers used are indicated according to reference . CG4016 represents a control gene located on chromosome 2R, i.e., not targeted by POF. (B) POF is reduced on the fourth chromosome after a 60- or 90-min DRB treatment of salivary glands. Note that in DRB-treated nuclei, RNP2 redistributes from a diffuse binding pattern to a more specific banded pattern, consistent with its release from the main part of the gene bodies. Arrowheads indicate distal ends of chromosome 4.
Fig 3
Fig 3
POF binds preferentially to chromosome 4 transcripts rather than transcripts originating from the other chromosome arms. (A) Schematic outline of the RIP method. The light gray indicates the period in the sample preparation where POF can be released from its target and reassociate with free RNA. Dark gray indicates the point at which the RNA molecules bound by POF are fixed. N1, N3, and N6 denote native nuclear extracts sonicated for 1, 3, or 6 min, respectively. FA denotes formaldehyde cross-linking. (B, C, D) Mean ratios for native samples of POF-RIP/Input (B), MSL2-RIP/Input (C) and MOF-RIP/Input (D) calculated for all probes within exons for each chromosome arm (log2 scale). Squares indicate the mean value, and whiskers indicate the 95% confidence interval. Sample N1 is indicated in red, N3 in green, and N6 in blue. (E, F, G) Mean ratios for cross-linked samples of POF-RIP/Input (E), MSL2-RIP/Input (F), and MOF-RIP/Input (G) calculated for all probes within exons for each chromosome arm (log2 scale). Squares indicate the mean value, and whiskers indicate the 95% confidence interval. FA is indicated in red and WCFA in green.
Fig 4
Fig 4
POF associates with chromatin via RNP2. The figure shows the ChIP-chip POF and RIP-chip POF binding profiles and the transcriptome profile. (A) POF binding profile for the entire fourth chromosome. The chromatin IP (ChIP-chip) profile is shown in orange. The RNA IP (RIP-chip) profiles are shown for the three independent replicates N1, N3, and N6 in red, and the transcriptome profile (TP) is shown in gray. Genes expressed from left to right are represented by rectangles above the horizontal line; genes expressed in the opposite direction are shown below the line. Exons are indicated in black and introns in gray. POF binding profiles and transcriptome profile at a 100-kb region (B) and at the MED26 locus (C). Regions that are repeat masked and therefore not represented on the arrays are shaded. Numbers on the x axis denote chromosomal positions along the fourth chromosome in kilobases.
Fig 5
Fig 5
POF binding to chromosome 4 is RNase resistant. Isolated salivary glands were treated with PBS ± RNase and double stained with MLE-POF or MSL2-POF. MLE (red) is completely released from the X chromosome after RNase treatment, while POF (green) remains unaffected. MSL2 (red; lower panel) is not affected by the RNase treatment. The rightmost column shows enlargements of the chromocenters and chromosome 4 regions.
Fig 6
Fig 6
POF binds to chromosome 4 transcripts with an exon bias. (A) POF-RIP enrichment at the eIF-4G locus. Genes expressed from left to right are represented by rectangles above the horizontal line, and genes expressed in the opposite direction are shown below the line. Exons are indicated in black and introns in gray. The POF-RIP profile is smoothed with a 90-bp bandwidth. Note that the previously detected unannotated exon and the novel gene are enriched (indicated by star and arrowhead, respectively) (27). The mean ratios of POF-RIP/Input (B) for all probes within genes, all probes within exons only (C), and all probes within introns only (D), sorted by chromosome arms. (E) Exon versus intron ratios for all chromosome arms. Squares indicate the mean values, and whiskers indicate the 95% confidence intervals.
Fig 7
Fig 7
The fourth chromosome shows a decrease in RNP2 pausing and a more pronounced decrease of RNP2 density over the gene body compared to the other autosomes. (A) The average RNP2 pausing index (PI) for genes grouped by chromosome arms; (B) the average RNP2 elongation density index (EdI) for genes grouped by chromosome arms. Squares indicate the mean values, and whiskers indicate the 95% confidence intervals. The PI and EdI values are from reference .
Fig 8
Fig 8
POF does not affect splicing nor does it protect RNA from degradation. (A) Mean expression of all probes within exons for each chromosome arm. The y axis shows the absolute levels of exon transcripts (log2 scale) in wild-type flies. Note the increased expression on chromosome 4 and chromosome X. (B) Transcriptome profile for wild-type (gray) and PofD119 mutant (red) adult female flies at the eIF-4G locus. The lower panel shows the ratio between the Pof mutant and wt. Genes expressed from left to right are represented by rectangles above the horizontal line, and genes expressed in the opposite direction are shown below the line. Exons are indicated in black and introns in gray. The transcriptome profiles are smoothed with a bandwidth of 90 bp. (C) Metagene transcriptome profiles for expressed genes are shown for chromosome 4 in the wild type (solid gray) and on chromosome 2L in the wild type (dashed gray) and compared with chromosome 4 in PofD119 mutants (solid red) and chromosome 2L in PofD119 (dashed red). To construct a metagene (schematically illustrated below the graph), all exons between the first annotated transcriptions start site and the last transcription stop site for each gene were fused. The genes were rescaled to the same relative length, and the transcripts were divided into 10 bins. The first and last bins were subjected to annotation artifacts and were excluded from the plot. (D) Ratios of PofD119/wild-type metagene profiles for chromosome 4 (solid line) and chromosome 2L (dashed line). Note that the decrease in transcript signals for chromosome 4 genes is uniform along the gene body. (E) Mean ratio between transcript profile for PofD119 and the wild type for each chromosome arm. Squares indicate the mean value and whiskers indicate 95% confidence intervals.
Fig 9
Fig 9
Transcripts encoded from chromosome 4 and pericentric heterochromatin are more efficiently exported relative to transcripts from other chromosomes. Mean relative ratio in log2 scale of RNA from whole-cell extract (WCFA) compared to RNA from nuclear extract (FA) for all genes on every chromosome arm (A) and for chromosome 2L and 4 compared to the heterochromatin-rich regions 2L:31 and pericentric heterochromatin from chromosome arms 2L, 2R, and 3L (B). Squares indicate the mean values, and whiskers indicate the 95% confidence intervals.
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