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Figure 3.

Figure 3. From: Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster.

Sequence composition of the H-probes. (A) Sequence composition of H-probes mapped to different heterochromatic regions. (B,C) Bar graphs summarizing the satellite DNA populations (B) and TE populations (C, grouped into clades) within H-probes mapped to different heterochromatic regions. (Xp) X1; (Xd) X2-4.

Bing He, et al. Genome Res. 2012 December;22(12):2507-2519.
Figure 5.

Figure 5. From: Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster.

Generation of small RNA from the heterochromatic sequences. Sequence reads of small RNA libraries produced from 0 to 1-h embryos, 2 to 6-h embryos, 6 to 10-h embryos, imaginal discs, female bodies, female heads, male bodies, or male heads (Chung et al. 2008) were aligned to the probe sequences, and the number of reads that match each probe was measured. (A) Heterochromatic regions differ in their production of small RNAs corresponding to different types of repetitive sequences. All show overall higher levels of small RNA than that of the euchromatic genes (Eu), but between different heterochromatic regions the overall abundance of small RNAs and the type of repetitive sequences they originate from vary substantially. Inset highlights the log2(small RNA reads) of the major satellite DNAs at the region. (B) Temporal profile of small RNA expression. Heat map showing log2 (small RNA reads) at different developmental stages.

Bing He, et al. Genome Res. 2012 December;22(12):2507-2519.
Figure 1.

Figure 1. From: Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster.

Sequence analysis of candidate H-probes. (A) Distribution of candidate H-probes according to their sequence similarity to the reference genome shown here as BLAST e-values. The pie diagrams on the right show the distribution of the best BLAST hit for each category. H-probes in category 1 map to heterochromatic sequences, whereas most H-probes in category 2 have their best matches evenly distributed to euchromatic regions. (B) Comparison of H-probes with chromosome U sequences. 60mer pseudo probes were generated spanning the entire arm U with a 30-nt overlap. Their best alignments on the assembled euchromatin (including “h”) or annotated TEs were assessed. Our H-probes show low similarity to euchromatic sequences in the reference genome (blue) or to TEs (red), whereas a significant fraction of the pseudo arm U probes show such homology. (C) Distribution of arm U contigs according to the percentage of the contig sequence that is covered by H-probes. (D) Two examples showing the typical patterns of H-probe location relative to the euchromatin-like and TE-like sequences in arm U contigs. Some U contigs are predominantly composed of sequences that are highly similar to the euchromatin and therefore only contain a small number of H-probes (top), whereas others show little sequence similarity to the euchromatin and are populated by H-probes (bottom).

Bing He, et al. Genome Res. 2012 December;22(12):2507-2519.
Figure 4.

Figure 4. From: Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster.

Repetitiveness of the H-probes and their underreplication in polytene chromosomes. (A) CGH analysis of polytene DNA purified from larval salivary glands and DNA from blastoderm stage embryos collected prior to tissue differentiation and chromosome polytenization. The data were normalized such that the mean log2-ratios of polytene DNA and embryonic DNA were zero for single-copy euchromatic genes (Eu). The H-probes were strongly underrepresented in the polytene DNA, and the level of underreplication varied for H-probes mapped to different chromosomal regions. (B) Comparing the degree of underreplication in salivary glands and ovaries. The colored lines are the linear fits to the data for the indicated category of H-probes. Note that the slope of linear regression fit for the satellite-like H-probes is less steep compared with the non-satellite-like H-probes. (C) Comparing sequence repetitiveness with level of underreplication in polytene chromosomes. To measure repetitiveness, sequence reads from an independent D. melanogaster genomic library were aligned to the probe sequences and the number of reads per probe was taken as an indication for repetitiveness. (Eu) Annotated euchromatic genes. (Het) H-probes. (D) H-probes from different sequence element families within individual heterochromatic regions show characteristic patterns of repetitiveness and underreplication in salivary gland polytene chromosomes.

Bing He, et al. Genome Res. 2012 December;22(12):2507-2519.
Figure 6.

Figure 6. From: Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster.

Transcription profile of the heterochromatic sequences. cDNA were prepared from manually staged wild-type Oregon R embryos at various developmental stages and analyzed by microarray using cDNA prepared from Oregon R embryos with mixed developmental stages (0–16 h) as reference for hybridization. The hybridization intensities of the test cDNAs were normalized according to the reference cDNA and used as a measure of transcription levels. The cutoff for background (nonspecific) hybridization intensity was determined using control probes that do not hybridize with Drosophila sequences. (A, left) Percentage of H-probes that are transcribed during the examined stages. (Right) The average transcription level of the H-probes that are transcribed. Annotated euchromatic genes (anno-eu), annotated heterochromatic genes (anno-het), and genomic euchromatic sequences (genomic-eu) were included as controls. (B) Temporal pattern of expression shown as fold changes compared to the reference. H-probes from each chromosomal region were grouped by hierarchical clustering according to their expression profiles (left and middle). Only transcribed probes are shown. Prevalent temporal patterns were detected for H-probes mapped to Xd-het and Xp-het and the satellite-like H-probes mapped to 3CEN-het, but not the non-satellite-like H-probes mapped to autosomal heterochromatin. Annotated heterochromatic genes (Het genes) and 2000 randomly selected euchromatic genes were clustered and shown as controls (right). (C) Histone modifications associated with H-probes. ChIP-seq reads for H3K9Me3 and H3K27Me3 (modENCODE) were aligned to H-probes and the number of reads for each H-probe was normalized to the input. Heat map demonstrates enrichment of each modification by showing log2(normalized reads). H-probes within the same category were clustered by hierarchical clustering. Note that H-probes mapped to the same subdivision (X2, X3, or X4) of Xd-het share similar patterns of H3K27Me3 enrichment. Annotated euchromatic and heterochromatic genes (Het genes) were included as controls. (D, left) Bivariate scatter plots comparing H3K9Me3 enrichment with level of transcription during early embryogenesis. The x-axis is the average of log2(normalized reads for H3K9Me3) at 0–4 h, 4–8 h, and 8–12 h of the embryonic development. The y-axis is the log2 scale of the highest transcription level during 2.5–5 h of the embryonic development detected by microarray. (Right) Distribution of the H-probes according to their enrichment for H3K9Me3 during 0–12 h.

Bing He, et al. Genome Res. 2012 December;22(12):2507-2519.
Figure 2.

Figure 2. From: Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster.

Assignment of unmapped H-probes to specific chromosomal regions by CGH analysis of chromosome deletions. CGH analysis of embryos lacking specific chromosomes or chromosome arms. (A) Single-copy euchromatic reference probes localized by their hybridization behavior to DNA deficient for individual chromosomes or chromosome fragments. Probes are clustered according to their chromosomal localizations as indicated on the right of the image. (Green) Fold decrease relative to Oregon R normal diploid reference. (Red) Fold increase. (B) Locations of H-probes that align to arm Het. Probes are clustered according to the position of their best match on arm Het. Each probe was mapped with either high confidence (H), low confidence (L), or unmappable (U). Note that we detected a previously reported misassignment of scaffold CP000217 from 3L to 2R (arrow) (Hoskins et al. 2007). (C) New assignment of H-probes that map to arm U or Uextra. Chromosome regions and number of probes mapped to each region are shown on the right. (D) Identification of novel H-probes from probes that do not have a perfect match in the reference genome but were mapped to a specific chromosome or chromosome arm. Novel H-probes mapping to 2CEN, 3CEN were identified based on their absence in deficiency DNA from compound entire stocks. For three chromosome arms (X, 2L, and 3L), novel H-probes are identified by CGH analysis of translocations breaking at the euchromatin/heterochromatin boundary. Numbers of H-probes mapped to the heterochromatin (het) or euchromatin (eu) are indicated. (E) CGH analysis of translocations with breakpoints in 2R heterochromatin. (Top) Cytological map of 2R heterochromatin and a model showing the translocation breakpoints. The cytological positions of the breakpoints were determined based on H-probes that match to 2R Het. (Bottom) Hierarchical clustering of H-probes that were mapped to 2R and 2CEN. These H-probes were further positioned into six nonoverlapping regions on 2R heterochromatin (R1–R6). (F) CGH analysis of the X chromosome rearrangements, including (1) Y duplicated for a piece of proximal X and (2) X deficiencies encompassing part or all of the X heterochromatin. Using hierarchical clustering, we were able to classify the X-specific H-probes into four nonoverlapping categories (X1–X4). Note that X4 contains almost all H-probes that match to X Het (h26). (G) Distinguishing Y-specific H-probes from those shared by X and Y by CGH analysis of polytene chromosomes from ovaries (Ov) and salivary glands (SG). Both groups were initially mapped to Y because they are depleted in X-Y−, but present in X-Y+. Y-specific H-probes are expected to be absent in female tissues and therefore show greater degree of depletion in ovaries than in salivary glands, whereas XY-shared H-probes are expected to be less depleted in the ovaries than in salivary glands because of the higher degree of polytenization in the latter (Fig. 4B). H-probes mapped to Y are clustered into two populations. One population that overlaps with autosomal H-probes and shows greater log2(Ovary/embryo) and log2(Ovary/Salivary gland) values is categorized as XY-shared, while the other population that overlaps with most control probes from YHet is categorized as Y-specific. (H) The position of chromosome regions on the cytogenetic map of Drosophila heterochromatin with numbered divisions (h1–h58) and centromeres (c). Modified from Gatti et al. (1994). The breakpoints of the compounds 2 and 3 chromosomes were determined according to the cytological location of the arm “h” and arm Het scaffolds (double arrows) and the sets of H-probes that match to them.

Bing He, et al. Genome Res. 2012 December;22(12):2507-2519.

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