The Drosophila Corto protein interacts with Polycomb-group proteins and the GAGA factor

Abstract

In Drosophila, PcG complexes provide heritable transcriptional silencing of target genes. Among them, the ESC/E(Z) complex is thought to play a role in the initiation of silencing whereas other complexes such as the PRC1 complex are thought to maintain it. PcG complexes are thought to be recruited to DNA through interaction with DNA binding proteins such as the GAGA factor, but no direct interactions between the constituents of PcG complexes and the GAGA factor have been reported so far. The Drosophila corto gene interacts with E(z) as well as with genes encoding members of maintenance complexes, suggesting that it could play a role in the transition between the initiation and maintenance of PcG silencing. Moreover, corto also interacts genetically with Trl, which encodes the GAGA factor, suggesting that it may serve as a mediator in recruiting PcG complexes. Here, we show that Corto bears a chromo domain and we provide evidence for in vivo association of Corto with ESC and with PC in embryos. Moreover, we show by GST pull-down and two-hybrid experiments that Corto binds to E(Z), ESC, PH, SCM and GAGA and co-localizes with these proteins on a few sites on polytene chromosomes. These results reinforce the idea that Corto plays a role in PcG silencing, perhaps by confering target specificity.

Figure 1

Structure of the Corto protein. (A) Hydrophobic cluster analysis plot (reviewed in 34) of Corto (Swiss-Prot identity code P41046). The clusters of hydrophobic residues are shown. The symbols used in the plot are: open square, threonine; dotted square, serine; diamond, glycine; star, proline. The three main globular domains, located at positions 127–203, 418–455 and 480–550, are framed. (B) Putative Corto chromo domain three-dimensional structure as obtained with MODELLER using the MoMOD1 chromo domain (PDB identity code 1ap0) as a template. (C) Alignment of the N-terminal globular domain of Corto and the chromo domains of human HP1α (P45973), mouse modifier protein 1 (MoMOD1) (PDB identity code 1ap0), D.melanogaster HP1 (P05205), S.pombe Clr4 protein (O60016), D.melanogaster SU(VAR)3-9 (P45975), mouse M33 (P30658) and D.melanogaster PC (P26017). Identical amino acids between Corto and the other sequences are on a blue blackground, similar ones on a gray background, hydrophobic residues in dark gray and charged residues in light gray. Positions of the secondary structures (b for β-sheets and a for α-helix), as experimentally determined for MoMOD1 (36), are indicated below the alignment.

Figure 2

Self-association of Corto. (A) GST pull-down experiments: binding of ³⁵S-labeled Corto to GST, GST-C127/203 and GST-Corto as revealed by autoradiography. (B) Two-hybrid experiments. Fusion proteins indicated horizontally (the negative control B42, B42AD-T, which binds to LexA-53, and B42-Corto) were expressed in the RFY206 strain (MATa). Fusion proteins indicated vertically (the negative control LexA, LexA-53, LexA-C127/203, LexA-C440/550 and LexA-Corto) were expressed in the RFY231 (MATα) strain that contains the LacZ reporter plasmid pSH18-34 (top). LexA-C1/324 was expressed in the EGY48SHΔSpe (MATα) strain which contains an endogenous LacZ gene (bottom). Strains were mated on appropriate medium and the activity of LacZ in the diploid yeasts was checked as described in Materials and Methods.

Figure 3

Co-immunoprecipitation of LexA-ESC or LexA-PC and Corto. Nuclear extracts of heat-shocked 0–4 h embryos from the hs-LexA-ESC strain (A) or overnight embryos of the α1T-LexA-PC strain (B) were immunoprecipitated with anti-LexA beads (LexA IP) or with beads containing no antibody (Mock IP). Western blots were revealed with anti-LexA or with anti-Corto antibodies as indicated.

Figure 4

Corto binds to ESC and E(Z). (A) Schematic representations of the ESC and E(Z) proteins and of the areas subcloned in-frame with B42 in plasmid pJG4-5. The ESC protein consists of seven WD repeats spread all along the protein. ESC* exhibits two point mutations in the second WD repeat (indicated by an asterisk). The E(Z) protein contains a SET domain at the C-terminal end preceded by a cystein-rich region, CXC. (B) GST pull-down experiments: binding of ³⁵S-labeled ESC and E(Z) to GST, GST-C127/203 and GST-Corto as revealed by autoradiography. (C) Two-hybrid experiments. Mating was performed as described in Figure 2.

Figure 5

Corto directly binds to PH and SCM but does not bind to PC. (A) Schematic representations of PC, PH and SCM and of the areas subcloned in-frame with B42 in pJG4-5. The PC chromo domain (CH) is located at the N-terminal end of the protein and the SAM motifs of PH and SCM are located at the C-terminal ends of these proteins. (B) GST pull-down experiments: binding of PC, PH and SCM to GST, GST-C127/203 and GST-Corto. PC was produced in bacteria and the GST pull-down assays were revealed with anti-PC antibodies; ³⁵S-labeled PH or SCM were produced by in vitro transcription/translation and the GST pull-down assays were revealed by autoradiography. (C) Two-hybrid experiments. Mating was performed as described in Figure 2.

Figure 6

Corto directly binds to the BTB/POZ domain of the GAGA factor. (A) Schematic representation of the GAGA 519 amino acid isoform. The BTB/POZ domain is located in the N-terminal end of the protein. (B) GST pull-down experiments showing the binding of ³⁵S-labeled GAGA to GST, GST-C127/203 and GST-Corto. (C) Two-hybrid experiments. Mating was performed as described in Figure 2.

Figure 7

Simultaneous immunofluorescence localizations of Corto and ESC (A), Corto and E(Z) (B), Corto and PC (C), Corto and PH (D), Corto and SCM (E) and Corto and GAGA (F) on salivary gland polytene chromosomes. The anti-Corto antibodies were detected with Alexa Fluor® 488 anti-rat IgG antibodies (green) and the anti-LexA, E(Z), PC, PH, SCM and GAGA antibodies were detected with Alexa Fluor® 594 anti-rabbit IgG antibodies (red). Yellow arrows indicate some co-localizations.