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A Drosophila Polycomb group complex includes Zeste and dTAFII proteins

Abstract

A goal of modern biology is to identify the physical interactions that define ‘functional modules’1 of proteins that govern biological processes. One essential regulatory process is the maintenance of master regulatory genes, such as homeotic genes, in an appropriate ‘on’ or ‘off’ state for the lifetime of an organism. The Polycomb group (PcG) of genes maintain a repressed transcriptional state, and PcG proteins form large multiprotein complexes2,3, but these complexes have not been described owing to inherent difficulties in purification. We previously fractionated a major PcG complex, PRC1, to 20–50% homogeneity from Drosophila embryos. Here, we identify 30 proteins in these preparations, then further fractionate the preparation and use western analyses to validate unanticipated connections. We show that the known PcG proteins Polycomb, Posterior sex combs, Polyhomeotic and dRING1 exist in robust association with the sequence-specific DNA-binding factor Zeste and with numerous TBP (TATA-binding-protein)-associated factors that are components of general transcription factor TFIID (dTAFIIs). Thus, in fly embryos, there is a direct physical connection between proteins that bind to specific regulatory sequences, PcG proteins, and proteins of the general transcription machinery.

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Figure 1: Identification and analysis of proteins identified in PRC1 preparations.
Figure 2: TAFII proteins co-precipitate PRC1 PcG proteins from partially purified Drosophila embryo extracts.

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Acknowledgements

We thank A. Nazarian and A. Grewal for help with mass spectrometric analysis, and the numerous people mentioned in ‘Methods’ for their gifts of antibodies. We also thank N. Francis, K.-M. Lee, S. Levine and A. Weiss for critical reading of the manuscript and E. Duprez and members of the Kingston lab for discussions and comments. A.J.S. is a Human Frontier Science Program Fellow. This work was supported by a NCI Cancer Center grant to P.T. and a NIH grant to R.E.K.

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Correspondence to Robert E. Kingston.

Supplementary information

Methods

Gel filtration chromatography. 200 ml PRC1 (~8 nM) was loaded onto a Sephacryl S-400 HR column (18.5 ml; 0.7 x 50 cm) in HEGN buffer (25 mM HEPES, K+ [pH 7.9], 0.1 mM EDTA, 10% glycerol, 0.1% NP-40, 1 mM DTT, 0.1 mM PMSF) containing 0.3 M KCl (0.3-HEGN) and 50 mg/ml insulin (Sigma, Inc., St. Louis, MO. USA) and fractionated with a linear flow rate of 4 cm/hour. Subsequent fractions were precipitated with 10% trichloroacetic acid with 0.015% deoxycholic acid included as co-precipitant. Following centrifugation at 40,000 rpm, pellets were washed with ice-cold acetone and resolved by 8% SDS-PAGE and visualised by silver staining. Note, a number of protein species appeared during the precipitation process (for example, see asterisk-marked protein in Fig. S1), which are attributed to contaminated insulin (included as carrier protein), solutions or centrifuge rotors. Calibration of the column was achieved using a selection of protein markers from high and low molecular weight gel filtration calibration kits (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Due to the lack of suitable size markers, the void volume was estimated from the manufacturer’s specifications.

Figure S1

(JPG 28 KB)

Fractionation of PRC1 by gel filtration. Silver stain analysis of every third fraction following TCA precipitation of gel filtration-fractionated PRC1 shows proteins that fractionate away from the large PRC1 complex-containing peak fractions. The identities of the protein bands determined by mass spectrometry analysis are indicated. The presence of proteins (an example shown with an asterisk) in fractions eluting ahead of the void volume are contaminants of the column and/or precipitation procedure (see Methods). In; ~80 fmol M2-purified PRC1. Peak elutions of molecular weight standards during gel filtration are indicated.

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Saurin, A., Shao, Z., Erdjument-Bromage, H. et al. A Drosophila Polycomb group complex includes Zeste and dTAFII proteins. Nature 412, 655–660 (2001). https://doi.org/10.1038/35088096

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