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Reciprocal Interactions of Human C10orf12 and C17orf96 with PRC2 Revealed by Biotap-XL Cross-Linking and Affinity Purification

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Reciprocal Interactions of Human C10orf12 and C17orf96 with PRC2 Revealed by Biotap-XL Cross-Linking and Affinity Purification Reciprocal interactions of human C10orf12 and C17orf96 with PRC2 revealed by BioTAP-XL cross-linking and affinity purification Artyom A. Alekseyenkoa,b, Andrey A. Gorchakova,b,c, Peter V. Kharchenkod,e,1, and Mitzi I. Kurodaa,b,1 aDivision of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; bDepartment of Genetics, Harvard Medical School, Boston, MA 02115; cInstitute of Molecular and Cellular Biology, Novosibirsk 630090, Russia; dCenter for Biomedical Informatics, Harvard Medical School, Boston, MA 02115; and eHematology/Oncology Program, Children’s Hospital, Boston, MA 02115 Contributed by Mitzi I. Kuroda, January 13, 2014 (sent for review December 20, 2013) Understanding the composition of epigenetic regulators remains an it would be ideal to recover information about both strong and weak important challenge in chromatin biology. Traditional biochemical interactions of a particular subunit within its chromatin context. analysis of chromatin-associated complexes requires their release However, a biochemical approach typically requires the release of from DNA under conditions that can also disrupt key interactions. the complex from the DNA to solubilize it, and such parameters Here we develop a complementary approach (BioTAP-XL), in which (salt and/or detergent) might simultaneously compromise complex cross-linking (XL) enhances the preservation of protein interactions integrity. Furthermore, each chromatin complex requires specific and also allows the analysis of DNA targets under the same tandem conditions for release from the DNA, and it is hard to predict this affinity purification (BioTAP) regimen. We demonstrate the power property in advance. Given these considerations, we reasoned that establishing of BioTAP-XL through analysis of human EZH2, a core subunit of cross-links before the first step of affinity purification might polycomb repressive complex 2 (PRC2). We identify and validate two enhance the preservation of protein composition of chromatin- strong interactors, C10orf12 and C17orf96, which display enrichment associated complexes and provide the additional advantage of with EZH2-BioTAP at levels similar to canonical PRC2 components characterizing their DNA targets under the same purification (SUZ12, EED, MTF2, JARID2, PHF1, and AEBP2). ChIP-seq analysis of regimen. We present our progress toward this goal by analyzing BIOCHEMISTRY BioTAP-tagged C10orf12 or C17orf96 revealed the similarity of each human EZH2, CBX4, and two candidate EZH2 interactors in binding pattern with the location of EZH2 and the H3K27me3- cultured cells. Our results provide strong validation for inter- silencing mark, validating their physical interaction with PRC2 actions of canonical PRC2 with two uncharacterized proteins components. Interestingly, analysis by mass spectrometry of encoded by the C10orf12 and C17orf96 genes. We propose that, C10orf12 and C17orf96 interactions revealed that these proteins if extended, our approach will reveal chromatin-associated net- may be mutually exclusive PRC2 subunits that fail to interact with works composed of distinct subcomplexes of epigenetic factors each other or with JARID2 and AEBP2. C10orf12, in addition, shows and their genomic targets. a strong and unexpected association with components of the EHMT1/2 complex, thus potentially connecting PRC2 to another Results histone methyltransferase. Similarly, results from CBX4-BioTAP BioTAP-XL Strategy and Its Application for Human EZH2: Identification protein pulldowns are consistent with reports of a diversity of of Canonical Subunits and C10orf12 and C17orf96 in PRC2 Complexes. PRC1 complexes. Our results highlight the importance of re- To preserve DNA–protein and protein–protein interactions ciprocal analyses of multiple subunits and suggest that iterative within their chromatin context, we developed a cross-linking/tandem use of BioTAP-XL has strong potential to reveal networks of chromatin-based interactions in higher organisms. Significance chromatin IP | formaldehyde cross-linking | LC-MS/MS | The fidelity of gene expression is regulated by chromosome- protein–protein interactions associated protein complexes. A traditional approach to char- acterizing complexes bound to chromosomes requires their he organization of the genome into active and silent domains release from the DNA to solubilize them. Here we develop an Tis integral to the fidelity of gene regulation in higher organisms. alternative approach, BioTAP-XL, that allows identification of Since their discovery in Drosophila, the genetic factors known protein–protein interactions while complexes remain linked to collectively as the Polycomb Group (PcG) (1) have emerged as the DNA. We focus on protein interactions and genome local- the prototypical epigenetic factors, required for the critical ization of human EZH2 and two of its relatively uncharac- maintenance of gene silencing during development in higher terized interactors, C10orf12 and C17orf96. Our results provide organisms. Polycomb group proteins are known to form large strong evidence for diversity in human Polycomb repressive multicomponent complexes that vary in their composition, complexes, which are composed of factors essential for gene with a broadly conserved distinction between PRC1 and PRC2 silencing during development in higher organisms. We complexes (reviewed in ref. 2). How these classes of key epi- propose that BioTAP-XL is an effective general approach for investigating the composition and subunit diversity of genetic factors are targeted to their sites of action, and interact chromosome-associated complexes. with appropriate partners within their chromatin context, remains an important question. Author contributions: A.A.A. and M.I.K. designed research; A.A.A. and A.A.G. performed Biochemical analyses of PRC1 and PRC2 have been invalu- research; A.A.A. and P.V.K. analyzed data; and A.A.A., P.V.K., and M.I.K. wrote the paper. able for the discoveries of enzymatic activities, specific binding The authors declare no conflict of interest. properties, and strong subunit interactions (reviewed in refs. 2, Data deposition: The data reported in this paper have been deposited in the Gene Ex- 3). Recent seminal work on PRC1 has led to an emerging rec- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE53495). ognition of the diversity of PRC1 subcomplexes in mammals and 1To whom correspondence may be addressed. E-mail: [email protected]. their possible individual roles in transcriptional repression (4). edu or [email protected]. These discoveries have highlighted the importance of dissecting This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. chromatin-based complexes using multiple strategies. For example, 1073/pnas.1400648111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1400648111 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 affinity purification approach (BioTAP-XL). First, we fixed of these clones from the CMV/2xtetO promoter, we incubated crude nuclear extracts with formaldehyde to introduce covalent cells in the presence of doxycycline for 4 d. We confirmed the bonds between DNA and proteins. Second, we used a dual tag expected size and presence of the biotin mark on the tagged for high-affinity two-step purification (Fig. 1). The BioTAP tag proteins (Fig. S1 A–C), and proceeded with the BioTAP pulldown includes two epitopes: Protein A (5) and a 75-amino-acid bio- using 1.5 × 109 cells. tinylation targeting sequence that is recognized by endogenous Sequencing of the DNA fraction from the EZH2 BioTAP biotin ligases in both prokaryotic and eukaryotic cells (6). pulldown closely recapitulated the genome-wide ChIP-seq en- To assess the BioTAP-XL approach in mammalian cells, we richment profile of H3K27me3, previously published for the 293T performed pulldowns of both N- and C-terminal–tagged EZH2 cell line (4) (Fig. 2A). Specifically, the log-fold enrichment pro- and C-terminal–tagged CBX4 in human HEK 293T cells. EZH2 files of both N- and C-terminal EZH2 pulldowns showed high correlation with H3K27me3 (Pearson r = 0.70, 0.72, respectively; is a core component of the mammalian PRC2 complex, has been − P value < 10 16) with a substantial overlap of the genomic regions shown to associate with SUZ12 and EED, and is responsible for B the deposition of the H3K27me3 histone modification (reviewed covered by the respective enrichment domains (Fig. 2 ). In in ref. 3). CBX4 belongs to a family of Polycomb-related CBX contrast, the CBX4-BioTAP pattern was distinct and correlated proteins known to be involved in a subset of PRC1, but not well with previous FLAG-CBX2 ChIP-seq results from the PRC2, complexes, providing an important contrast with which to Reinberg laboratory (51.2% of CBX4 sites are cobound by CBX2, and 42.3% of CBX2 sites are bound by CBX4) (4). compare EZH2-BioTAP results. We used lentiviral transduction Affinity purification after cross-linking may recover abundant to generate stable HEK-293 T-REx cell lines carrying EZH2 proteins based solely on their general proximity on chromatin. constructs with the BioTAP tag at either the N or the C terminus Therefore, enrichment over input can be a valuable parameter to and CBX4 with a C-terminal BioTAP tag. To induce transcription identify candidate interactions with the highest specificity. Table 1 reveals the liquid chromatography–mass spectrometry/MS (LC- MS/MS) analysis of the results from
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