Polycomb group protein complexes exchange rapidly in livingDrosophila

Abstract

Fluorescence recovery after photobleaching (FRAP) microscopy was used to determine the kinetic properties of Polycomb group (PcG) proteins in whole living Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary glands). PcG genes are essential genes in higher eukaryotes responsible for the maintenance of the spatially distinct repression of developmentally important regulators such as the homeotic genes. Their absence, as well as overexpression, causes transformations in the axial organization of the body. Although protein complexes have been isolated in vitro, little is known about their stability or exact mechanism of repression in vivo. We determined the translational diffusion constants of PcG proteins,dissociation constants and residence times for complexes in vivo at different developmental stages. In polytene nuclei, the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria, and the results compared with our experimental data. We were able to determine forward and reverse rate constants for complex formation. Complexes exchanged within a period of 1-10 minutes, more than an order of magnitude faster than the cell cycle time,ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that long-term repression primarily reflects mass-action chemical equilibria.

Different polycomb group CBX family proteins associate with distinct regions of chromatin using nonhomologous protein sequences

“…This interpretation is consistent with the observation that a large fraction of CBX proteins is highly mobile in undifferentiated ES cells and in Drosophila embryos (26,28). The CBX proteins that are not chromatin associated could be recruited to new target genes during differentiation.…”

Functional Characterization of the dRYBP Gene in Drosophila

“…Although dRYBP mutations alone do not result in homeotic-like phenotypes, they do when in combination with mutations in some PcG and trxG genes. We have found that the dRYBP protein is localized in a nuclear pattern and colocalizes with some of the Polycomb nuclear bodies (Buchenau et al 1998;Saurin et al 1998;Netter et al 2001;Ficz et al 2005;Grimaud et al 2006a). Moreover, the protein is dynamically distributed during the mitotic cycle in the syncytial embryo.…”

Monitoring dynamic protein interactions with photoquenching FRET

“…For example, the transcription factor C/EBPα localized to regions of constitutive heterochromatin in mouse cells where it interacted with HP1α as part of a metastable protein complex. Although typically associated with transcriptional silencing 7,8 , a number of different transcription factors are known to preferentially localize to regions of centromeric heterochromatin 4,[9][10][11][12][13][14][15][16][17] . These associations may function to establish cell-specific patterns of gene expression 4,5,10 .…”

“…These regions of chromatin are marked by several different proteins, including HP1α, and are typically associated with transcriptional silencing 7,8 factors are also found associated with these intranuclear sites 9-11 . For example, the transcription factor C/EBPα, which has a role in the differentiation of many cell types 12 , localizes to regions of centromeric heterochromatin [13][14][15] .…”

“…The important regulatory role of the association of transcription factors with chromocenters is suggested by changes in this pattern of localization with the cell cycle, cell signaling or stage of cell differentiation 4,[10][11][12][13][14][15][16][17] . Kinetic microscopy techniques have shown that most proteins within the nuclear compartment are mobile, and that dynamic processes drive the assembly of metastable protein complexes at certain intranuclear sites 3,8 . What is needed are live-cell imaging methods that allow these dynamic protein-protein interaction networks to be visualized in their natural context within the intact cell nucleus.…”