The distribution of polycomb-group proteins during cell division and development in Drosophila embryos: impact on models for silencing - PubMed (original) (raw)

The distribution of polycomb-group proteins during cell division and development in Drosophila embryos: impact on models for silencing

P Buchenau et al. J Cell Biol. 1998.

Abstract

The subcellular three-dimensional distribution of three polycomb-group (PcG) proteins-polycomb, polyhomeotic and posterior sex combs-in fixed whole-mount Drosophila embryos was analyzed by multicolor confocal fluorescence microscopy. All three proteins are localized in complex patterns of 100 or more loci throughout most of the interphase nuclear volume. The rather narrow distribution of the protein intensities in the vast majority of loci argues against a PcG-mediated sequestration of repressed target genes by aggregation into subnuclear domains. In contrast to the case for PEV repression (Csink, A.K., and S. Henikoff. 1996. Nature. 381:529-531), there is a lack of correlation between the occurrence of PcG proteins and high concentrations of DNA, demonstrating that the silenced genes are not targeted to heterochromatic regions within the nucleus. There is a clear distinction between sites of transcription in the nucleus and sites of PcG binding, supporting the assumption that most PcG binding loci are sites of repressive complexes. Although the PcG proteins maintain tissue-specific repression for up to 14 cell generations, the proteins studied here visibly dissociate from the chromatin during mitosis, and disperse into the cytoplasm in a differential manner. Quantitation of the fluorescence intensities in the whole mount embryos demonstrate that the dissociated proteins are present in the cytoplasm. We determined that <2% of PH remains attached to late metaphase and anaphase chromosomes. Each of the three proteins that were studied has a different rate and extent of dissociation at prophase and reassociation at telophase. These observations have important implications for models of the mechanism and maintenance of PcG- mediated gene repression.

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Figures

Figure 1

Figure 1

Ubiquitous expression of PcG proteins in developing Drosophila embryos. (A–C) Antibody staining to (A) PC, (B) PH, and (C) PSC (top) and DNA (bottom) in whole mount stage 11 (extended germ band) embryos with anterior right and posterior left. The images are single confocal sections. Objective, Neofluar 25×, NA 0.8 immersion with refractive index correction collar. Field widths are 590 μm. (D) Higher magnification image of a field of 2n nuclei from a whole mount embryo stained for PH. Bar, 5 μm.

Figure 2

Figure 2

Three-dimensional distribution of PcG proteins in embryonic interphase nuclei. (A–C) The stereo images show the distribution of PH (A), PC (B), and PSC (C) in representative interphase nuclei. The images were reconstructed from (A) 30 confocal sections separated by 0.2 μm, (B) 21 sections, 0.2 μm, (C) 15 sections, 0.3 μm, by projection of the maximal pixel values along two viewing lines of the image stack. (D) This overlay of two adjacent confocal sections through a PH-stained interphase nucleus demonstrates one example in which a faint fluorescence between the PH spots suggests the location of the loci on a chromatin fiber. Bars, 2 μm (A–C), 0.5 μm (D).

Figure 3

Figure 3

Interphase localization of PcG proteins in relation to DNA distribution and transcription sites. (A–C) The distribution of PH (A), PC (B), and PSC (C) compared with the DNA concentration as seen in single confocal sections through embryonic interphase nuclei from stage 9 whole-mount embryos. The protein concentration visualized by Cy-3 labeled antibody staining (left, red channel), and the DNA concentration visualized by YOYO-1 staining (middle, green channel) are seen to be relatively uncorrected as demonstrated by the lack of many strongly overlapping intensities (white arrowheads) that appear yellow in the merged images (right). Black arrowheads indicate examples for loci lying within regions of decondensed DNA. (D) The distribution of PH (left, red channel) compared with the distribution of the hnRNPK protein, Hrb57A (middle, green channel) and their merged images (right). Bars, 2 μm (A–C), 5 μm (D).

Figure 4

Figure 4

Distribution of PH in an embryo with various cell cycle phases. (A and B) The distribution of DNA and PH in the anterior region of an embryo of stage 8. The DNA staining (A) allows the identification of the mitotic regions 1–3, 5, 6, and 9 in nuclear cycles 15 and 14, respectively (Foe, 1989). The PH antibody staining (B) reveals that the chromosomal protein concentration is drastically reduced during mitosis. Both images are maximum projection overlays of four confocal sections. Field width, 138 μm. (C and D) Stereo image pairs of a field of cells from an embryo at a later developmental stage. (C) DNA staining; (D) PH staining. Field width, 50 μm.

Figure 5

Figure 5

Distribution of PcG proteins across the cell cycle. A gallery of single confocal sections showing the distribution of PC, PH, and PSC during all distinguishable phases of the cell cycle for 2n nuclei. The images were extracted from whole mount embryos older than stage 8. From left to right: interphase, prophase, metaphase, anaphase, and telophase. The corresponding DNA image is shown above each protein image. Rows from top to bottom: PC, PH, PSC (see text).

Figure 6

Figure 6

Quantitation of the cell cycle–dependent PH fluorescence. The total PH fluorescence intensity within the volume of 53 nuclei (or chromosome sets) and 35 cells was measured as described in Materials and Methods. By inspecting DNA staining, the cells were grouped into a phase of the cell cycle. The bars show the percentage of PH intensity associated with the nuclear mask.

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