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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00964

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Nuclear location of a chromatin insulator in Drosophila melanogaster

Department of Cellular Biology, The University of Georgia, Athens, GA 30602, USA

View larger version (83K): [in a new window]   Fig. 1. The effect of the gypsy sequence on the nuclear localization of neighboring chromosomal DNA. (A-C) Confocal images of Drosophila imaginal disc nuclei. Disc tissue was visualized with FISH using Cy3-labeled probes (red) and an antibody against the nuclear lamin (green). The main view panel and the side view bars are separated by white lines. The positions of focal planes are indicated by thin yellow lines. Nuclei from non-suHw-LacZ (NS) transgenic (A) or non-transgenic (B) larvae were hybridized with Cy3-labeled LacZ probe. FISH foci were visible within nuclei from transgenic larva. In the majority of the nuclei from the non-transgenic larvae, no stained foci were observed above background. One or sometimes two fluorescence foci per nucleus were detected at various focal planes in virtually all nuclei, depending on transgene copy number and the status of chromosomal pairing between the homolog transgenes. A small number of Cy3-labeled foci appear outside of the lamin ring due to weak or incomplete lamin staining of the nearby nuclei. Arrowheads in A and C indicate Cy3-labeled foci that appear in the center of the nucleus in the top view but are more peripheral in the side view. (C) Nuclei from a gypsy line were hybridized with a Cy3-labeled probe for an 8 kb genomic region in the cut locus near the retrotransposon (gypsy-Cy3). The gypsy foci (red) are frequently present near the nuclear periphery (green). (D) Histograms of 2D foci-distribution of individual NS transgenes (colored lines) and of 3D foci distribution of pooled NS transgenes (black line). Relative frequency of the sample is plotted against the FISH-lamin distance (% radius) of the sample. (E) Histograms of 2D foci distribution of individual gypsy lines (colored lines) and of 3D foci distribution of all gypsy lines (black line). (F) Summary of foci distribution in a quartile diagram. The relative FISH-lamin distance of each sample group (colored bars) is compared with a model of randomly distributed samples (gray bar) (see Materials and Methods for details). (G) Foci histograms along the nuclear radius of selected sample groups (colored lines) and the probability density function along the nuclear radius for the random distribution model (gray dashed line) (see Materials and Methods).

View larger version (52K): [in a new window]   Fig. 2. The nuclear localization of transgenes containing the suHw insulator in functional or non-functional arrangements. (A) Confocal image of imaginal disc nuclei from a single-suHw (SS) transgenic larva, hybridized with Cy3-labeled lacZ DNA probes (red) and an anti-lamin antibody (green) (SS-Cy3 group). (B) Confocal image of a transgenic line with paired-suHw (DS) (DS-Cy3 group). (C-D) Histogram of foci distribution along the radius of the SS-Cy3 (C) and the DS-Cy3 group (D) in independent transgene lines (colored lines). 3D foci histograms of pooled samples are represented by the black lines. (E) Summary of foci distribution in a quartile diagram. The distance between FISH foci and lamin staining as a fraction of the nuclear radius for each sample group (colored bars) is compared with a model of randomly distributed samples (gray bar). (F) Foci histograms along the nuclear radius of each sample groups (colored lines) and the probability density function along the nuclear radius for the random distribution model (gray dashed line). (G) Diagram of cytological locations of insertion sites in selected transgenes. Small vertical lines represent transgene insertions at various locations in the three major chromosomes (black, gypsy; blue, NS; red, SS; green, DS). Insertion sites for individual lines are listed below.

View larger version (76K): [in a new window]   Fig. 3. The suHw insulator can function in the nuclear interior. (A-D) Serial confocal sections (0.3 µm thick) of larval imaginal disc cells stained with anti-SUHW (red) and anti-lamin (blue) antibodies. The SUHW protein foci are detected at both the nuclear periphery and the interior region. (E) SUHW protein foci histograms along the nuclear radius. (F-H) A confocal section of an imaginal disc containing the 3S2 transgene, triple stained for the transgene (FISH, red; SUHW, green; lamin, blue). (I-K) Heat shock does not disrupt enhancer-blocking activity of the suHw insulator. Whole-mount-RNA in situ hybridizations were performed with embryos containing 3T2 (I), and 3S2 transgenes (J,K). Transgene constructs are shown below the embryos. Colored bars represent the even skipped stripe enhancers (E2 and E3) and the neutral spacer (T), the black oval represents the 340 bp suHw element (S). Enhancers that directed the lacZ stripe expression are indicated above the embryos. Embryos were incubated at 25°C (D,E) or 37°C (F) for 30 minutes before fixation and staining.

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