Reciprocal interactions of human C10orf12 and C17orf96 with PRC2 revealed by BioTAP-XL cross-linking and affinity purification

Abstract

Understanding the composition of epigenetic regulators remains an important challenge in chromatin biology. Traditional biochemical analysis of chromatin-associated complexes requires their release from DNA under conditions that can also disrupt key interactions. Here we develop a complementary approach (BioTAP-XL), in which cross-linking (XL) enhances the preservation of protein interactions and also allows the analysis of DNA targets under the same tandem affinity purification (BioTAP) regimen. We demonstrate the power of BioTAP-XL through analysis of human EZH2, a core subunit of polycomb repressive complex 2 (PRC2). We identify and validate two strong interactors, C10orf12 and C17orf96, which display enrichment with EZH2-BioTAP at levels similar to canonical PRC2 components (SUZ12, EED, MTF2, JARID2, PHF1, and AEBP2). ChIP-seq analysis of BioTAP-tagged C10orf12 or C17orf96 revealed the similarity of each binding pattern with the location of EZH2 and the H3K27me3-silencing mark, validating their physical interaction with PRC2 components. Interestingly, analysis by mass spectrometry of C10orf12 and C17orf96 interactions revealed that these proteins may be mutually exclusive PRC2 subunits that fail to interact with each other or with JARID2 and AEBP2. C10orf12, in addition, shows a strong and unexpected association with components of the EHMT1/2 complex, thus potentially connecting PRC2 to another histone methyltransferase. Similarly, results from CBX4-BioTAP protein pulldowns are consistent with reports of a diversity of PRC1 complexes. Our results highlight the importance of reciprocal analyses of multiple subunits and suggest that iterative use of BioTAP-XL has strong potential to reveal networks of chromatin-based interactions in higher organisms.

Keywords: LC-MS/MS; chromatin IP; formaldehyde cross-linking; protein–protein interactions.

Fig. 1.

Overview of the BioTAP-XL purification strategy. The BioTAP tag includes two epitopes: Protein A and Bio, a 75-amino-acid sequence that is biotinylated in vivo. Lentiviral vectors were used to make stable 293T-REx cells expressing N- and C-terminal BioTAP-tagged human proteins. Expression was induced by adding doxycycline (1 μg/mL) to the medium and incubating for 4 d. Crude nuclear extracts were cross-linked using formaldehyde, sonicated, and subjected to tandem affinity purification, first with rabbit IgG−agarose beads eluted under denaturing conditions and subsequently using streptavidin–agarose beads. The resulting DNA was analyzed by high-throughput sequencing. Peptides from the protein fraction were released by direct on-bead trypsin digestion and then identified by LC-MS/MS.

Fig. 2.

PRC1 and PRC2 show distinct distributions in 293T-REx cells. (A) Representative ChIP-seq profiles in a region of chromosome 2 demonstrate the similar binding patterns of PRC2-associated EZH2-CBioTAP, EZH2-NBioTAP, and H3K27me3. In contrast, PRC1-associated CBX4-CBioTAP and Flag-His–tagged CBX2 display similar binding patterns that are distinct from the PRC2 profiles. H3K27me3 and Flag-His–tagged CBX2 profiles are from ref. . (B) Venn diagram illustrates the substantial overlap (measured in Mbp) of EZH2 and H3K27me3 large enrichment domains.

Fig. 3.

C17orf96 and C10orf12 protein distributions closely match those of EZH2 and H3K27me3 modification. (A) A circos plot showing enrichment (log₂ scale) of C17orf96-CBioTAP and C10orf12-NBioTAP proteins, along with H3K27me3 (4) and EZH2-CBioTAP in the human genome. (B) The enrichment estimates (gray dots) and large enrichment domains (color shading) detected by the Hidden Markov model are shown for the entire human chromosome 2, illustrating similarity of the enrichment patterns, with an enlarged view of a 20-Mb region. (C) Venn diagrams illustrate the substantial overlap (measured in Mbp) of the large enrichment domains.