The Structure and Function of Polycomb-Group (PC-G) Protein Transcriptional Silencing Complexes in Drosophila
We have analyzed the subcellular 3-dimensional distribution of the Polycomb-group (PcG) proteins, Polycomb (Pc), Polyhomeotic (PH), Posterior sex combs (Psc), and Pleiohomeotic (PHO) in fixed whole-mount Drosophila embryos by multicolor confocal fluorescence microscopy. The proteins are localized in complex patterns of 100 or more loci throughout most of the interphase nuclear volume. Genes of the Bithorax complex can be colocalized with single PcG protein loci in repressed regions of the embryo. 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, 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. On the other hand, there is 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. The PcGs are thought to repress by binding to Polycomb Response Elements (PREs), sequences found in the non-coding control regions of responsive genes. Only PHO has been shown to interact directly with a PRE sequence in vitro. Although the PcG proteins maintain tissue specific repression for up to 14 cell generations the PcGs 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 less than 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.
We have recently developed a repressible reporter transgene carrying a PRE which can be manipulated in transiently transfected cells. The chromatin of the transgene is being studied by biochemical and biophysical means to determine the protein composition and structural arrangement under repressive and non-repressive conditions.
D. J. Arndt-Jovin, P. Buchenau*, and L. Trieschmann
Max Planck Institute for Biophysical Chemistry, Dept. of Molecular Biology,