Variant Polycomb complexes in Drosophila consistent with ancient functional diversity

Abstract

Polycomb group (PcG) mutants were first identified in Drosophila on the basis of their failure to maintain proper Hox gene repression during development. The proteins encoded by the corresponding fly genes mainly assemble into one of two discrete Polycomb repressive complexes: PRC1 or PRC2. However, biochemical analyses in mammals have revealed alternative forms of PRC2 and multiple distinct types of noncanonical or variant PRC1. Through a series of proteomic analyses, we identify analogous PRC2 and variant PRC1 complexes in Drosophila, as well as a broader repertoire of interactions implicated in early development. Our data provide strong support for the ancient diversity of PcG complexes and a framework for future analysis in a longstanding and versatile genetic system.

Fig. 1.. RYBP and L(3)73Ah copurify orthologs of mammalian vPRC1 subunits.

(A) Mammalian PRC1 complex components. PRC1 complexes contain core subunits RING1A/B and PCGF, and variant PRC1 (vPRC1) complexes commonly contain RYBP or YAF2. Each PRC1 subtype is defined by distinct PCGF proteins and additional accessory proteins, as indicated. (B) Top: Sequence alignment of Drosophila RYBP (NP_001286742.1) with mammalian YAF2 (XP_011536030.1) and RYBP (NP_036366.3). Bottom: Alignment of Psc (NP_001286368.1), Su(z)2 (NP_001260933.1), and L(3)73Ah (NP_001246797.1) with mammalian PCGF orthologs PCGF1 (NP_116062.2), PCGF2 (NP_001356543.1), PCGF3 (NP_001304765.1), PCGF4/BMI1 (NP_005171.4), and PCGF5 (NP_001243478.1). Identical sequences of conserved regions are depicted as black lines, and percent sequence identity and percent similarity (identity + conservative substitutions) between two protein sequences are described in parentheses. Conserved domains are also shown. (C) Enrichment plots (log₁₀ fold enrichment of normalized spectral abundance factors (NSAFs) in pulldown with respect to input) for proteins (individual dots) identified in RYBP and L(3)73Ah BioTAP-XL experiments from Drosophila embryos. Dashed lines denote the 99th percentile threshold of enriched proteins in RYBP pulldown (x axis) and in L(3)73Ah pulldown (y axis). (D) Peptide counts of Drosophila PRC1.1 and PRC1.3 subunit orthologs copurified by RYBP and L(3)73Ah embryonic BioTAP-XL compared to input. Proteins are color-coded according to their mammalian vPRC1 subunit orthologs in (A). Counts of all peptides detected in BioTAP-XL pulldowns and inputs are indicated as total peptides at the bottom of the table. See data file S1 for the full set of results.

Fig. 2.. Drosophila complexes corresponding to mammalian vPRC1.1 and vPRC1.3/5.

(A and B) Scatterplot comparing enriched proteins from Kdm2 and L(3)73Ah BioTAP-XL from 12- to 24-hour embryos (A). Scatterplot comparing enriched proteins from Kdm2 and RYBP pulldowns from 12- to 24-hour embryos (B). Dashed lines on the x axis denote 99th percentile threshold of enriched proteins in Kdm2 pulldown (x axis) and dashed lines on the y axis denote 99th percentile threshold of enriched proteins by L(3)73Ah and RYBP in (A) and (B), respectively. Coordinates represent log₁₀ fold enrichment of NSAF of proteins in BioTAP-XL affinity purification compared to input. Red boxes indicate areas of scatterplots that are enlarged in bottom panels, and protein names are color-coded according to the vPRC1 subunit color scheme in Fig. 1A and (D). (C) Heatmap of enrichment of selected vPRC1 subunits copurified from RYBP, L(3)73Ah, and Kdm2 BioTAP-XL. For relative abundance comparison, the NSAF enrichment value of each protein is divided by the NSAF enrichment value of CG14073 (BCOR), which is the top hit common to all three pulldowns. (D) Illustration of Drosophila vPRC1 complexes from BioTAP-XL mass spectrometry analysis of the three bait proteins RYBP, L(3)73Ah, and Kdm2 organized similarly to their mammalian complexes in Fig. 1A. Usp7 (not copurified by three bait proteins) is depicted by a dashed line. CG8677, indicated by a question mark, is a potential Drosophila-specific subunit of PRC1.1.

Fig. 3.. Sfmbt interacts with orthologs of mammalian PRC1.6 and many additional proteins.

(A) Conserved domains in Drosophila Sfmbt and sequence alignments with human MBT domain orthologs. Sequence identities between Drosophila Sfmbt protein sequence (NP_001137821.1) and two human orthologs, MBTD1 (XP_011523224.1) and L3MBTL2 (NP_001137821.1) are shown as black lines. Percent sequence identity and percent similarity in domains are also described in parentheses. (B and C) Log₁₀ fold enrichment of relative abundance for proteins identified in Sfmbt BioTAP-XL experiments from 12- to 24-hour embryos (B) and from S2 cells (C). (D) Scatterplot of Sfmbt pulldown enrichments from embryos and S2 cells normalized to their inputs. Horizontal and vertical dashed lines represent the 99th percentile of proteins enriched by Sfmbt BioTAP-XL in embryos and in S2 cells, respectively. Red box indicates the area of the scatterplot enlarged in the bottom panel. (E) Total peptide counts of select proteins enriched from Sfmbt BioTAP-XL experiment in embryos and in S2 cells compared to peptide counts from respective inputs. Total peptide counts from pulldowns are indicated at the bottom of table. See data file S1 for the full results. (B to E) Orthologs of mammalian PRC1.6 subunits and PhoRC subunits are color-coded according to color scheme in Figs. 1A and 2D.

Fig. 4.. Embryonic Sfmbt copurifies orthologs of coactivators linked to mammalian YY1 and MBTD1.

(A) Total peptide counts of the top 35 proteins enriched from Sfmbt BioTAP-XL in embryos. See data file S1 for full results. Among non-PcG proteins, those with mammalian orthologs that have known interactions are written in red font and highlighted in color-coded boxes according to the color scheme in (D). (B) STRING protein-protein interaction network for human orthologs of proteins highlighted in (A). Only known interactions (from curated databases and experimentally determined) are used as interaction sources, and the thickness of the gray lines indicates strength of supporting data. L3MBTL2 (human ortholog of Sfmbt) is not connected with the interaction network. (C) Combined interaction network of MBTD1 (human ortholog of Sfmbt) and YY1 (human ortholog of Pho/Phol). (D) Drosophila Sfmbt may have properties of both L3MBTL2 and MBTD1 and may interact with binding partners of each ortholog. Furthermore, fly vPRC1.6 may not be a single entity but instead encompass multiple modules with additional combinatorial capabilities not included here.

Fig. 5.. Drosophila forms mutually exclusive PRC2.1 and PRC2.2 complexes.

(A) Peptide counts of PRC2 subunits recovered from BioTAP-XL pulldowns of bait proteins in embryos compared with peptide counts of input. Total peptides are indicated at the bottom of the table. See data file S1 for the full range of results. (B) Scatterplot showing Jarid2 and Pcl pulldown enrichment (normalized to total embryonic chromatin input). PRC2 core subunits are highlighted in pink, and additional subunits of PRC2.1 and PRC2.2 are highlighted in green and blue, respectively. (C) Schematic of copurification strategies of BioTAP-XL tandem tag and split tag. (D) Peptide counts of PRC2 subunits copurified by BioTAP-XL split tag pulldowns. (E) Genome browser view of a 4.3-Mb region of chromosome 3R showing colocalization of Pcl and Scm with E(z) and H3K27me3 in embryos, while Jarid2 is widely dispersed across the genome. The normalized ChIP/input ratio is presented on a log₂ scale. (F) Cartoon showing conservation of PRC2 complexes between Drosophila and mammals. Scm and PALI are not conserved orthologs but may have functional similarity via their separate interaction with G9A methyltransferase.