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. 2011 Apr 5;108(14):5572-7.
doi: 10.1073/pnas.1007916108. Epub 2011 Mar 17.

Polycomb Purification by in Vivo Biotinylation Tagging Reveals Cohesin and Trithorax Group Proteins as Interaction Partners

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Free PMC article

Polycomb Purification by in Vivo Biotinylation Tagging Reveals Cohesin and Trithorax Group Proteins as Interaction Partners

Gero Strübbe et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The maintenance of specific gene expression patterns during cellular proliferation is crucial for the identity of every cell type and the development of tissues in multicellular organisms. Such a cellular memory function is conveyed by the complex interplay of the Polycomb and Trithorax groups of proteins (PcG/TrxG). These proteins exert their function at the level of chromatin by establishing and maintaining repressed (PcG) and active (TrxG) chromatin domains. Past studies indicated that a core PcG protein complex is potentially associated with cell type or even cell stage-specific sets of accessory proteins. In order to better understand the dynamic aspects underlying PcG composition and function we have established an inducible version of the biotinylation tagging approach to purify Polycomb and associated factors from Drosophila embryos. This system enabled fast and efficient isolation of Polycomb containing complexes under near physiological conditions, thereby preserving substoichiometric interactions. Novel interacting proteins were identified by highly sensitive mass spectrometric analysis. We found many TrxG related proteins, suggesting a previously unrecognized extent of molecular interaction of the two counteracting chromatin regulatory protein groups. Furthermore, our analysis revealed an association of PcG protein complexes with the cohesin complex and showed that Polycomb-dependent silencing of a transgenic reporter depends on cohesin function.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Establishment of in vivo biotinylation tagging in Drosophila. (A) Pc is fused at its C terminus to a TEV-cleavable BioTag allowing proteolytic elution from streptavidin-coated beads. Gal4 induction of BirA biotin ligase expression leads to biotinylation of the BioTag. Biotinylated Pc-Bio is incorporated into the PRC1 complex and binds to chromatin. Streptavidin pull-down (PD) allows purification of Pc-Bio and detection of interaction partners by mass spectrometry. Experiments B, C, and D were performed with 7.5 mg of input nuclear extract. (B) Analysis of Pc-Bio PD efficiency comparing 30 μg of IN (input) and UB (unbound) with 1/5 of a PD eluate. The lower band corresponds to endogenous Pc, the upper band to the Pc-Bio fusion protein, and the middle band to Pc-Bio after TEV cleavage. (C and D) Quantification of background biotinylation after streptavidin PDs. Numbers indicate the fold-dilution of the eluate obtained from an IP of 7.5 mg nuclear extract that were loaded to the gel. (C) Comparing da-Gal4 induced versus uninduced strains reveals leaky biotinylation in the absence of the driver. (D) Comparing uninduced strains with or without UAS-BirA transgenes identifies the latter as the source of leaky biotinylation.
Fig. 4.
Fig. 4.
Verification of Pc-interaction partners by Co-IP. Equal amounts of IN and UB (30 μg) were loaded to gels together with 1/6 of the PD eluate. (A) Western blot analysis after streptavidin PD of 7.5 mg of NE using antibodies against Rad21, Pho, dSfmbt, and Fs(1)h. In the following Co-IPs, 1 mg of NE was used as IN and 50% of the PD eluates were loaded to the gels. (B) Wild-type nuclear extracts were immunoprecipitated with Pho antibody. Immunoblotting revealed copurification of dSfmbt and Pc. (C) Wild-type nuclear extracts were immunoprecipitated with Pc antibody. Immunoblotting revealed copurification of Pho as well as the three core cohesin complex members Rad21, Smc3, and Smc1. (D) Coimmunoprecipitation experiments using nuclear extract from flies expressing myc-tagged Rad21, which was captured on myc-sepharose. Immunoblotting revealed copurification of Smc1, Pc, dRING, and Psc. (E) Gel filtration chromatography on a Superose 6 column for size estimation of the fly cohesin complex and comparison to the elution profile of PcG proteins. The Pc profile shows multiple peaks of which the middle peak coelutes with cohesin at about 1–1.5 MD.
Fig. 2.
Fig. 2.
Identification of Pc-interaction partners. (A) After streptavidin PD of 7.5 mg NE, TEV eluates of a Pc-Bio purification were separated by SDS/PAGE and Western blotting of PRC1 core proteins shows their specific enrichment over uninduced and untagged controls. Again, equivalent amounts of IN and UB were loaded (30 μg) together with 1/5 of the PD eluates. (B) Silver staining of a representative Pc-Bio purification. Controls missing Pc-Bio or Gal4 induction are shown in the right two lanes as controls. Asterisks indicate specific bands in the Pc-Bio test sample. A gel run in parallel was used to excise whole lanes for tryptic in-gel digestion and subsequent LC-MS/MS analysis. (C) Venn diagram depicting the overlap of proteins identified in the BioTag test sample (red), in the control without BioTag (gray), and in the control without the Gal4 driver (black). The final group of 20 specific interaction partners of Pc-Bio identified with ≥4 peptides total is highlighted.
Fig. 3.
Fig. 3.
MS results for the identification of Pc-interaction partners. List of identified Pc-Bio interacting proteins ranked by the total number of peptides found by mass spectrometry. The six top scoring proteins are known members of the PRC1 core complex. Note that all core and many auxiliary proteins show peptides in the uninduced control sample due to leaky biotinylation by low-level UAS-BirA expression (Fig. 1C). Other proteins specifically enriched in the Pc-Bio pull-down are members of the cohesin complex (blue), PhoRC complex (green) and a Moz/Morf-like complex (yellow). Homologous proteins from vertebrates were retrieved using HomoloGene (http://www.ncbi.nlm.nih.gov/pubmed/). Known biochemical interactions among the recognized proteins identified by literature mining are indicated. Genetic interactions with PcG/TrxG mutations are listed including references. A protein/gene is classified as a PcG member if it enhances PcG and suppresses TrxG phenotypes. Conversely, a protein behaves as a TrxG protein if it genetically interacts in the opposite manner. The last column lists known molecular functions of the respective proteins.
Fig. 5.
Fig. 5.
Genetic interaction and suppression of pairing-sensitive silencing (PSS). (A) Genetic interactions of several roadkill and ebi mutants with the Pc1 and Pc3 alleles revealed by suppression of the extra sex combs Pc phenotype identifies these genes as TrxG members. (B) Illustration of the transgenic reporter used to analyze PSS. (C and D) Heterozygous Rad21 and Pc mutations suppress PSS of the transgenic miniwhite reporter containing a Fab7-PRE. In the absence of the PRE, the homozygous transgene is fully expressed (dark red eye). PSS is observed for the homozygous reporter in a PRE-dependent manner (white eye). (E) Quantitative spectrometric measurements of eye pigment levels confirm derepression of the reporter.

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