High-resolution analysis of Drosophila heterochromatin organization using SuUR Su(var)3-9 double mutants

Abstract

The structural and functional analyses of heterochromatin are essential to understanding how heterochromatic genes are regulated and how centromeric chromatin is formed. Because the repetitive nature of heterochromatin hampers its genome analysis, new approaches need to be developed. Here, we describe how, in double mutants for Su(var)3-9 and SuUR genes encoding two structural proteins of heterochromatin, new banded heterochromatic segments appear in all polytene chromosomes due to the strong suppression of under-replication in pericentric regions. FISH on salivary gland polytene chromosomes from these double mutant larvae allows high resolution of heterochromatin mapping. In addition, immunostaining experiments with a set of antibodies against euchromatic and heterochromatic proteins reveal their unusual combinations in the newly appeared segments: binding patterns for HP1 and HP2 are coincident, but both are distinct from H3diMetK9 and H4triMetK20. In several regions, partial overlapping staining is observed for the proteins characteristic of active chromatin RNA Pol II, H3triMetK4, Z4, and JIL1, the boundary protein BEAF, and the heterochromatin-enriched proteins HP1, HP2, and SU(VAR)3-7. The exact cytological position of the centromere of chromosome 3 was visualized on salivary gland polytene chromosomes by using the centromeric dodeca satellite and the centromeric protein CID. This region is enriched in H3diMetK9 and H4triMetK20 but is devoid of other proteins analyzed.

Fig. 1.

Additional bands are detected in the pericentric region of polytene chromosome 3 from SuUR Su(var)3-9 double mutants. (A) In wild-type strains, pericentric region of chromosome 3 (from 80A to 81F) is represented by a compact and unstructured block. (B and C) In SuUR Su(var)3-9 double mutants, this region shows an extensive and finely banded chromosome segment, which has been referred to as the Plato Atlantis (24).

Fig. 2.

Mapping of BACs, genes, and the centromere region within 3LR heterochromatin. (A–F) FISH of BAC clones CHORI223-40H04 (A, B, D, and E) and RPCI-98-25K12 (C and F) to pericentric region 80A-81F of polytene chromosome 3 from wild-type (A and D) and SuUR, Su(var)3-9⁰⁶ mutant homozygotes (B, C, E, and F). (A–C) FISH. (D–F) Merged images: phase contrast view/FISH. (G–J) FISH of unique DNA sequences from the heterochromatin of chromosome 3. (G) auxillin (red) and Gel (green) (genes auxillin and Gel are located 8 kb apart). (H) α-cat (red) and CG12460 (green). (I) Parp. (J) RpL15. (G–J) Merged image: phase contrast view/FISH. (K–N) Mapping of the centromere of chromosome 3 in SuUR Su(var)3-9⁰⁶ double mutants. (K and L) FISH of BAC CHORI223-11M22 to PA. (K) Merged images: phase contrast view/FISH. (L) FISH and corresponding Hoechst-33258 DNA staining. (M) FISH of dodeca satellite to PA (merged images: phase contrast view/FISH). (N) Immunofluorescence micrograph showing labeling with anti-CID antibodies (merged images: phase contrast view/fluorescent immunostaining). Arrows indicate centromere localization in chromosomes 3 and 2. (O) Correspondence between mitotic and polytene heterochromatin in the third chromosome. At the top, the pericentric heterochromatin (80A to 81F/82A) in polytene chromosome 3 is shown. In the middle, the map of heterochromatin regions (h47 to h58) of chromosome 3 is given. Below and above the mitotic map, the locations of DNA probes are given. These positions are depicted in the polytene chromosome and in the mitotic map with the same color as the one shown in the diagram at the bottom. The isolated BAC contigs, the GenBank scaffolds, and the relative positions of the three different 1.688 satellite variants (353, 356, and 361 bp) are shown in the diagram at the bottom. The wide usage of BACs for heterochromatin mapping is restricted by its composition in repetitive sequences. Thus, the BAC CHORI221-27P10 that derives from h53 almost stains the totality of PA and other euchromatic regions (top).

Fig. 3.

Distribution of heterochromatin proteins within the region 80A-81F in SuUR Su(var)3-9⁰⁶ double mutants. (A, D, and G) Immunostaining of HP1. (B, E, and H) Immunostaining of H3diMetK9. (B, K, and L) H4triMetK20. (C, F, and I) SU(VAR)3-7. (A–C) Phase contrast. (D–F) Fluorescent immunostaining. (G–I and L) Merged images: phase contrast/inverted fluorescent immunostaining. (J) Double fluorescent immunostaining: HP1 (green) and HP2 (red) overlapping binding sites look yellow-colored.

Fig. 4.

Distribution of euchromatin proteins within the region 80A-81F in SuUR Su(var)3-9⁰⁶ double mutants. Immunostaining of proteins RNA Pol II (A, E, and I), H3triMetK4 (B, F, and J), Z4 (C, G, and K), and BEAF (D, H, and L). (A–D) Phase contrast. (E–H) Fluorescent immunostaining. (I–L) Merged images (phase contrast/inverted fluorescent immunostaining).