Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos - PubMed (original) (raw)

Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos

Bernd Schuettengruber et al. PLoS Biol. 2009.

Abstract

Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In Drosophila, PcG and trxG factors bind to regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. Here, we used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, we mapped histone modifications associated with PcG-dependent silencing and TRX-mediated activation. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. Our results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N-bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation.

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Conflict of interest statement

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

Figures

Figure 1

Figure 1. Genomic Distribution of PcG and TrxG Proteins and associated Histone Modifications in a Segment of Chromosome 3R

The plots show the ratios (fold change) of specific IP versus mock IP assays along part of the chromosome 3R. Significantly enriched fragments (_p_-value < 1E−04) are shown in red. All the profiles generated are available for viewing in an interactive browser at

http://purl.oclc.org/NET/polycomb

. Position of genes (FlyBase annotation 4.3) is shown at the top of the figure. Transposons and previously predicted PREs (M. Rehmsmeier, personal communication; [24,25]) are indicated by gray bars. Note that PC and H3K27me3 are bound to large genomic regions, whereas the other profiles show sharp localized binding. PcG recruitment factors were bound at PREs as well as at many other promoter regions where no PcG binding is detected. The N-terminal fragment of TRX (TRX-N) shows only weak binding to PREs, but colocalizes with H3K4me3 and sequence-specific DNA binding proteins at many promoter regions. The C-terminal fragment of TRX (TRX-C) is only strongly bound at PcG binding sites. ANT-C, Antennapedia complex; ato, atonal; dsx, doublesex; grn, grain; hb, hunchback.

Figure 2

Figure 2. Venn Diagrams Showing Overlap between Bound Regions of Different Protein Profiles

All the bound regions taken for analysis were with _p_-value < 1E−04. For PC and H3K27me3, the unstitched regions (see Text S1) were analysed. PRC1 denotes the regions cobound by PC and PH. Recruiters are the regions cobound by PHO, DSP1, GAF, and PHOL. (A) PcG binding is highly correlated. Nearly all PH sites are bound by PC and H3K27me3. Minimal overlap is seen between H3K27me3 and TRX-N/H3K4me3 or TRX-C/H3K4me3. (B) Occurrence of PcG recruitment factors along with PRC1 and TRX-N. Note that a large proportion of each factor is bound with TRX-N. Interestingly, PHO co-occurs with nearly all the PRC1 (PC+PH). PHOL minimally colocalizes with PRC1 but colocalizes extensively with TRX-N. (C) Occurrence of PcG recruitment factors along with PRC1 and TRX-C. Note the high overlap of TRX-C with PRC1.

Figure 3

Figure 3. Genome-Wide Architecture of Polycomb and Trithorax Marks and Recruiters

(A) Spatial clusters. We dissected our multifactor genome-wide dataset into groups of loci with common factor and histone mark occupancy (spatial clusters). Clusters are probabilistically tied together to reflect a typical genomic organization (Figure S18). Our algorithm detected two superclusters, one representing H3K27me3-marked domains (left) and the other representing H3K4me3-marked domains (right), and further decomposed each supercluster into distinct genomic behaviors. Here, we depict each cluster as a block, where rows represent the 2 kb (−1 kb to +1 kb) around cluster centers, color-coded to reflect the binding intensity of nine marks and factors (yellow indicates strong binding, blue negative enrichment). (B) We also plotted the enrichment of clusters' locations relative to the TSS (_x_-axis, zero reflect the TSS itself), normalized by the genome-wide frequency of distances from the TSS. (C) Frequency of clusters in the genome. The relative abundance of the eight clusters is shown. About two-thirds of the genome is not associated with either of our two superclusters (i.e., lboth H3K4me3 and H3K27me3 are lacking). (D) Transcription factor (TF) peaks in three clusters. We show the number of peaks (over 1.5 chip enrichment) for the PH sites, K4me3-recruiter, and K4me3-TSS clusters. The vast majority of TF peaks is observed in these three clusters, with some exceptions for GAF and TRX (unpublished data).

Figure 4

Figure 4. Average Chromatin Profiles at PH Sites and Transcription Start Sites

(A) Shown are average fold changes of selected factors around PH local maxima (100-bp intervals in a 2.5-kb flanking region). Note the dip in values of H3K27me3 and PC at PH peaks and the stronger binding of PHO and TRX-C compared to other recruiters and TRX-N, respectively. (B and C) We classified annotated TSS (FlyBase 4.3) according to the existence of a nearby PH site (B) or H3K4me3 local maximum (C). Shown are the average fold changes for selected factors around such TSSs (in intervals of 100 bp [for PH] and 50 bp [for H3K4me3]). Note the strong binding of PHO and TRX-C and the lack of PHOL binding at PH-associated TSS. The shoulder of the H3K4me3 peak in Figure 4C (left panel) likely corresponds to promoter regions of divergently transcribed genes, because we generally do not detect H3K4me3 enrichment 5′ of the TSS of isolated genes. (D) Average fold change of PHO at TSS associated either with PH or H3K4me3.

Figure 5

Figure 5. Overrepresented Sequence Motifs of PcG Recruitment Factors in ChIP on Chip Bound Regions Genome Wide

(A) Overrepresented DNA motifs of GAF, DSP1, PHO, and PHOL (No motif-length parameter) (B) Overrepresented DNA motifs of DSP1, DSP1, PHO, and PHOL (motif-length parameter 5–10 bp). Sequence logo representation of the consensus is shown for top motif of each profile. MEME E-value for the motif is given below the name of the factor. Note that even though PHO and PHOL regions have the same overrepresented motif, the motif in PHOL is weakly enriched and may be a consequence of the basal PHOL-PHO overlap.

Figure 6

Figure 6. Overrepresented Sequence Motifs in the Different Spatial Clusters

Shown are data for motifs that distinguish clusters or groups of clusters. The motifs were identified with no prior assumptions, but include the known GAF site [32]; PHO site [33]; Sp1/KLF site [38]; E-box [37] Max, Mad/Mnt site; and DRE site [36]. For each inferred position weight matrix (PWM), we computed the predicted binding energy for bins of 100 bp [35] and plotted a color-coded representation of it in the 8 kb around the center of each cluster (yellow indicates stronger binding). We polarized the clusters according to the strand of the nearest TSS. For each motif and cluster, we also plotted the percentage of probes with predicted binding strength in the top 5% (_y_-axis) in the 6 kb around the clusters' centers (_x_-axis).

Figure 7

Figure 7. Differential PHO and PHOL Binding Ratios at PcG Target Genes in ON and OFF States

(A) Profiles of H3K27me3, PH, the PHO/PHOL ratio, PHO, and PHOL are shown along part of chromosome 2R. Significantly enriched fragments (_p_-value <1E−04) are shown in red. Note that at PcG binding sites, the PHO/PHOL ratio is significantly increased. Apt, apontic; bs, blistered; Dll, Distal-less; fd59A, forkhead domain 59A; gsb, gooseberry; Kr, Kruppel; retn, retained; Tkr, Tyrosine kinase-related protein; Twi, twist. (B–F) ChIP-qPCR performed with PH, PHO, and PHOL antibodies of haltere/third leg imaginal discs (HD) and eye imaginal discs (ED). Ubx is expressed in haltere/third leg imaginal discs and is repressed in eye imaginal discs. so (sine oculis) and toy (twin of eyeless) both show low expression levels in haltere/third leg imaginal discs and are highly expressed in eye imaginal discs. The ChIP yield (qPCR) of the examined regions was normalized to input DNA and an internal control (robo3). Data are expressed as the ratio of ChIP enrichments in haltere/third leg discs versus eye discs. The standard deviation, as indicated by the error bars, was calculated from three independent experiments. At the Ubx gene (B–D), a small decrease in the levels of PH was detected in haltere/third leg discs compared to eye discs. Lower levels of PH in haltere/third leg discs correlated with a lower PHO/PHOL ratio. In contrast, slightly higher levels of PH binding were detected in haltere/third leg discs at so and toy (E and F), which are repressed in these discs. Higher levels of PH in haltere/third leg discs correlate with a higher PHO/PHOL ratio.

Figure 8

Figure 8. Changes in Transcription Levels of PHO Target Genes in pho1 Mutants

Fold changes of Ubx, Antp, Rp49, and Chc expression levels in eye, haltere/third leg and wing imaginal discs in pho1 homozygous mutant larvae (green histograms) compared to wild type (wt; blue histograms). (A) Expression of homeotic genes in eye discs, where both Ubx and Antp genes are OFF. (B) Expression of homeotic genes in haltere/third leg discs (Ubx) and wing discs (Antp) where genes are ON. (C) Fold changes in expression levels of Rp49 and Chc in eye (i) and haltere/third leg discs (ii). The standard deviation, as indicated by the error bars, was calculated from at least two independent experiments.

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