Structural basis for targeting the chromatin repressor Sfmbt to Polycomb response elements

Abstract

Polycomb group (PcG) protein complexes repress developmental regulator genes by modifying their chromatin. How different PcG proteins assemble into complexes and are recruited to their target genes is poorly understood. Here, we report the crystal structure of the core of the Drosophila PcG protein complex Pleiohomeotic (Pho)-repressive complex (PhoRC), which contains the Polycomb response element (PRE)-binding protein Pho and Sfmbt. The spacer region of Pho, separated from the DNA-binding domain by a long flexible linker, forms a tight complex with the four malignant brain tumor (4MBT) domain of Sfmbt. The highly conserved spacer region of the human Pho ortholog YY1 binds three of the four human 4MBT domain proteins in an analogous manner but with lower affinity. Comparison of the Drosophila Pho:Sfmbt and human YY1:MBTD1 complex structures provides a molecular explanation for the lower affinity of YY1 for human 4MBT domain proteins. Structure-guided mutations that disrupt the interaction between Pho and Sfmbt abolish formation of a ternary Sfmbt:Pho:DNA complex in vitro and repression of developmental regulator genes in Drosophila. PRE tethering of Sfmbt by Pho is therefore essential for Polycomb repression in Drosophila. Our results support a model where DNA tethering of Sfmbt by Pho and multivalent interactions of Sfmbt with histone modifications and other PcG proteins create a hub for PcG protein complex assembly at PREs.

Keywords: Pho; PhoRC; Polycomb; Polycomb response element; Sfmbt; YY1.

Figure 1.

Biophysical and structural characterization of the Pho spacer:Sfmbt 4MBT interaction. (A, top) Sequence alignment of the Drosophila melanogaster Pho spacer region (dm, Q8ST83, orange) with the YY1 orthologs from Danio rerio (dr, Q7T1S3), Xenopus laevis (xl, Q6DDI1), mice (mm, Q00899), and humans (hs, P25490). Residues involved in the interaction with the Sfmbt 4MBT domain are indicated with asterisks. (Bottom) Pho and Sfmbt domain architecture. Pho spacer:Sfmbt 4MBT-interacting regions are enclosed by a dashed rectangle, and the first and last residue of the interacting regions are given. Sfmbt MBT repeats 1–4 are colored. (B) ITC data of the Pho spacer:Sfmbt 4MBT interaction. (C) Overview of the miniPhoRC complex crystal structure as a ribbon diagram presentation. (D) Close-up view of the Pho spacer:Sfmbt 4MBT interaction. Interacting residues of the Pho spacer and the Sfmbt 4MBT domain are depicted. Gly635 and Ala638 in the Sfmbt clamping helix are highlighted (purple). (E) Schematic representation of the Pho spacer:4MBT domain interaction.

Figure 2.

In vitro mutagenesis analysis of the Pho:Sfmbt interaction. (A) GST pull-down of recombinant GST-Pho spacer and untagged Sfmbt 4MBT wild-type and structure-based mutant proteins. (B) SPR measurements of biotin-labeled Pho and Sfmbt 4MBT wild-type or mutant proteins. Results are shown as affinities relative to the Pho spacer:Sfmbt 4MBT wild-type affinity. (C) Anti-Flag affinity purifications of full-length Pho-Flag:Sfmbt wild-type or mutant complexes detected by Western blot. Antibodies used for the detection are indicated at right. (D) EMSA experiments of full-length Pho or full-length Pho:Sfmbt 4MBT wild-type and mutant complexes using a ³²P end-labeled double-stranded Pho DNA-binding site. Arrows indicate full-length Pho:DNA and supershifted Pho:Sfmbt 4MBT:DNA complexes. Lanes containing only DNA and the Sfmbt 4MBT domain were used as control. The asterisk indicates the Pho DNA-binding domain/DNA complex resulting from the degradation of full-length Pho protein as confirmed by mass spectrometry (MS) (data not shown). Binding reactions were performed with 5 ng of DNA probe and 50 ng of Pho full-length protein; 50-fold, 100-fold, and 500-fold molar excess of Sfmbt 4MBT wild-type or mutant protein was added to fixed amounts of Pho protein.

Figure 3.

Pho:Sfmbt interaction is critical for Sfmbt function during Drosophila development. (A) Western blot analyses with the indicated antibodies of input (“I”; 2.5% of total) and eluted (“E”; 100% of total) material of IgG sepharose pull-downs of Sfmbt-CTAP proteins from nuclear extracts of 0- to 12-h-old embryos expressing the indicated Sfmbt-CTAP protein or from nontransgenic animals (“not TG”). The tagged proteins were expressed from UAS_Gal4:Sfmbt-CTAP transgenes under the control of the daughterless:Gal4 driver. Blots were first probed with anti-Sfmbt, and then stripped and reprobed with anti-peroxidase antibody to specifically detect the Sfmbt-CTAP fusion proteins. Wild-type Sfmbt-CTAP protein coimmunoprecipitates Pho but not the PRC2-subunit E(z). Significantly lower levels of Pho are coimmunoprecipitated with the Sfmbt^G635K/A638E-CTAP and Sfmbt^ΔMBT1-CTAP proteins (cf. lanes 6,8 and 4). (B) ChIP analysis monitoring binding of the indicated Sfmbt-CTAP proteins at PcG target genes in chromatin of wild-type larvae that express Sfmbt-TAP proteins under the control of the ubiquitous daughterless:Gal4 driver. Graphs show results from three independent immunoprecipitation reactions with anti-peroxidase antibody that binds to the protein A moiety of the TAP tag. ChIP signals, quantified by quantitative PCR, are presented as the mean percentage of input chromatin precipitated at each region; error bars indicate ±SD. Locations of PREs (purple boxes) and other regions relative to the transcription start sites are indicated in kilobases; control regions C3 in euchromatin and C4 in heterochromatin are located remotely from PcG target genes. Sfmbt-CTAP is specifically enriched at PREs. Levels of Sfmbt^G635K/A638E-CTAP protein binding are reduced at several PREs, whereas binding of Sfmbt^ΔMBT1-CTAP is reduced twofold to fivefold at all analyzed PREs. (C) Wing imaginal discs from third instar larvae stained with antibody against the HOX protein Ubx (red, top row) and anti-peroxidase antibody to detect the Sfmbt-CTAP proteins (purple, bottom row). Clones of Sfmbt¹ homozygous cells are marked by the absence of GFP (green) and were induced in animals lacking a transgene (“no TG”) or expressing the indicated Sfmbt-CTAP proteins under the control of the 69B:Gal4 driver. For unknown reasons, the Sfmbt^ΔMBT1-CTAP is expressed at higher levels (see Supplemental Fig. S8) than the Sfmbt-CTAP and Sfmbt^G635K/A638E-CTAP proteins. Note that only Sfmbt¹ homozygous cell clones (“no TG”) in the wing pouch but not in the notum or hinge show strong misexpression of Ubx, as described previously (Klymenko et al. 2006). To evaluate the capacity of the transgene-encoded Sfmbt proteins to repress Ubx in Sfmbt¹ mutant cell clones, we therefore only analyzed mutant clones in the wing pouch area for the presence of Ubx protein. For each genotype, multiple wing imaginal discs were analyzed, and in animals expressing a CTAP fusion protein, only clones in which the fusion protein was detected by immunofluorescence labeling (shown in the bottom panel) were scored. In the “no TG” animals, 94% of Sfmbt¹ homozygous clones (n = 98 clones) show misexpression of Ubx. In Sfmbt-CTAP animals, repression of Ubx is rescued in most Sfmbt¹ homozygous clones, and only 4% of the clones (n = 78 clones) show misexpression of Ubx. In Sfmbt^G635K/A638E-CTAP animals, 81% of Sfmbt¹ homozygous clones (n = 97 clones) show misexpression of Ubx. In Sfmbt^ΔMBT1-CTAP animals, 87% of the clones (n = 31 clones) show misexpression of Ubx. The large proportion of Ubx-expressing Sfmbt¹ mutant clones in Sfmbt^G635K/A638E-CTAP and Sfmbt^ΔMBT1-CTAP animals suggests that these two proteins are largely nonfunctional in Polycomb repression.

Figure 4.

TAP of Sfmbt protein complexes from Drosophila embryonic nuclear extracts identifies a larger PhoRC assembly that resembles mammalian PRC1.6/E2F.6 complexes. (A) Sfmbt complexes isolated by TAP from wild-type (wt) or α-tubulin-Sfmbt-CTAP transgenic embryos. (Left) Input material for purification was normalized by protein concentration, and equivalent amounts of eluate from calmodulin affinity resin were separated on a 4%–12% polyacrylamide gel and visualized by silver staining; the molecular weight marker is indicated on the left. Sfmbt bait protein containing the calmodulin-binding tag (Sfmbt-CBP), its degradation products, and bands representing Mga (CG3363), Hdac1/Rpd3, Pho, Nap1, and HP1b were identified by MS (Supplemental Table S2). (Right) The Nap1 and HP1b proteins were undetectable as bands on silver-stained gels, and their presence was verified by Western blot analysis: total embryonic nuclear extract input material from wild-type (wt) and Sfmbt-CTAP transgenic embryos (lanes 1,2) and material eluted from the calmodulin affinity resin after purification (lanes 3,4), probed with the indicated antibodies. The Nap1 and HP1b panels come from the same batches of input and eluate material, and the same ratio of input versus eluate was loaded in both cases. (B) The Drosophila Sfmbt assembly resembles human PRC1.6/E2F.6 assemblies. Graphic representation of the larger Drosophila Sfmbt–Pho assembly with the additional proteins identified in A and therefore called PhoRC-L and the PRC1.6 assembly described in Gao et al. (2012). Note that the PRC1.6 assembly is identical to the E2F.6 assembly described in Ogawa et al. (2002) but was reported to also contain HDAC1/2 and WDR5. Drosophila Sfmbt and human L3MBTL2 proteins are labeled in green, the orthologous subunits identified in both Drosophila and human assemblies are labeled in blue, and Pho is labeled in orange. The Drosophila genome does not encode orthologs of E2F.6 and MBLR (asterisks), and this might explain why Drosophila PhoRC-L assemblies do not contain the RING1A/B ortholog Sce, the RYBP/YAF2 ortholog Rybp, and the DP-1 ortholog Dp.

Figure 5.

Pho:Sfmbt interaction is structurally and biochemically conserved in humans. (A) Sequence alignment of the Pho spacer-binding pocket from Sfmbt (dm, Q9VK33) and mouse and human L3MBTL2 (mm, P59178; hs, Q969R5), MBTD1 (mm, Q6P5G3; hs, Q05BQ5), SFMBT2 (mm, Q5DTW2; hs, Q5VUG0), and SFMBT1 (mm, Q9JMD1; hs, Q9UHJ3). Sfmbt MBT repeats are colored as above. The same color code is used for MBTD1, where we solved the crystal structure bound to YY1. The remaining human 4MBT proteins are depicted in gray. Pho spacer:Sfmbt 4MBT-interacting residues are marked with asterisks. Glycine and alanine residues in the Sfmbt clamping helix are highlighted (purple). (B) Structural superposition of the Drosophila Pho spacer-binding pocket with the corresponding regions in the human L3MBTL2 and MBTD1 4MBT domains (PDB ID: 3f70 and 3feo). (C) Dissociation constants of YY1 or Pho spacers for D. melanogaster or human 4MBT wild-type and mutant proteins. Note that K_D values measured by SPR were consistently lower than those measured by ITC, presumably due to the immobilization of the spacer peptides required for SPR. (D) Domain architecture of human 4MBT proteins. The first and last residues of the 4MBT domain constructs used in the experiments in C are indicated. The N-terminal FCS Zn finger and the C-terminal SAM domains are represented as white boxes. (E) Crystal structure of the YY1 spacer:MBTD1 4MBT complex. Color scheme of the MBT repeats according to D with the YY1 spacer depicted in orange. (F) Stereo view of the YY1 spacer σ_A-weighted simulated annealing omit electron density map contoured at 0.7 σ. YY1 spacer residues are depicted and colored according to temperature factors (increasing from blue to red).

Figure 6.

The PhoRC complex is a hub for multiple interactions. The Pho:Sfmbt interaction is required for the PcG-repressive function on HOX genes. The Pho/YY1 protein (orange) is recognizing the Pho-binding sites (gray and white) in a PRE through its DNA-binding domain (PDB ID: 1ubd) (Houbaviy et al. 1996) and recruits the Sfmbt 4MBT domain through its spacer region (miniPhoRC crystal structure) (this study). Pho regions flanking the spacer and connecting it to the DNA-binding domain are predicted to be disordered (dotted line). The Sfmbt MBT repeats are colored as above. Sfmbt also interacts with Scm and thereby tethers PRC1. Interaction of the fourth MBT repeat of Sfmbt with mono- or dimethylated lysines in histone tails (red) (PDB ID: 3h6z) (Grimm et al. 2009) links the PhoRC complex with nucleosomes.