Identification and characterization of posttranslational modification-specific binding proteins in vivo by mammalian tethered catalysis Tanya M. Spektor and Judd C. Rice1 Department of Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine, Los Angeles, CA 90033 Communicated by C. David Allis, The Rockefeller University, New York, NY, July 14, 2009 (received for review February 26, 2009) Increasing evidence indicates that an important consequence of To overcome some of the limitations of in vitro approaches, a protein posttranslational modification (PTM) is the creation of a high previously undescribed in vivo method called yeast tethered catal- affinity binding site for the selective interaction with a PTM-specific ysis was developed (1). Briefly, an expressed fusion protein con- binding protein (BP). This PTM-mediated interaction is typically re- taining a target peptide sequence was tethered to an enzyme quired for downstream signaling propagation and corresponding resulting in the constitutive PTM of the peptide and, thereby, biological responses. Because the vast majority of mammalian pro- served as the bait in yeast two-hybrid screens for putative PTMBPs. teins contain PTMs, there is an immediate need to discover and Although this technique was used successfully to identify yeast characterize previously undescribed PTMBPs. To this end, we devel- PTMBPs, the ability to detect PTMBPs in higher eukaryotes is oped and validated an innovative in vivo approach called mammalian constrained by the limitations of the yeast two-hybrid system. tethered catalysis (MTeC). By using methylated histones and methyl- By expanding on the principles of tethered catalysis, we have specific histone binding proteins as the proof-of-principle, we deter- developed and validated a previously undescribed in vivo approach mined that the new MTeC approach can compliment existing in vitro designed specifically for the discovery and characterization of binding methods, and can also provide unique in vivo insights into endogenous PTMBPs in mammalian cells, which we termed mam- PTM-dependent interactions. For example, we confirmed previous in malian tethered catalysis (MTeC). Methylated histones were cho- SCIENCES vitro findings that endogenous HP1 preferentially binds H3K9me3. sen as the proof-of-principle for MTeC, because increasing evi- However, in contrast to recent in vitro observations, MTeC revealed dence indicates that the major role of histone methylation is to bind APPLIED BIOLOGICAL that the tandem tudor domain-containing proteins, JMJD2A and distinct PTMBPs that, in turn, function to regulate a specific 53BP1, display no preferential H4K20 methyl-selectivity in vivo. Last, DNA-templated process such as transcription, replication, or repair by using MTeC in an unbiased manner to identify H3K9 methyl- (2). By testing various MTeC bait fusion proteins, we demonstrate specific PTMBPs, we determined that endogenous G9a binds meth- that this approach can be consistently used to predictably modify ylated H3K9 in vivo. Further use of MTeC to characterize this inter- MTeC bait fusion proteins in vivo. We also demonstrate that this action revealed that G9a selectively binds H3K9me1 in vivo, but not new technique can compliment existing in vitro binding assays, and H3K9me2, contrary to recent in vitro findings. Although this study importantly, can provide previously undescribed in vivo insights focused solely on methylated histones, we demonstrate how the into PTM-dependent interactions. Last, we show that MTeC was innovative MTeC approach could be used to identify and characterize used in an unbiased manner to identify an H3K9me1-specific any PTMBP that binds any PTM on any protein in vivo. binding protein in vivo. Collectively, our findings indicate that MTeC could be used as a new tool to identify and characterize ͉ ͉ ͉ ͉ 53BP1 G9a histone JMJD2A methylation PTMBPs that bind any PTM on any protein in vivo. ukaryotic cells have developed intricate and distinct cellular Results Esignaling cascades to translate particular intra or extracellular Principles and Implementation of MTeC. The overall goals of MTeC are stimuli into an appropriate biological response. These signaling to discover previously undescribed proteins, or validate those, that pathways rely heavily on enzymes that create specific posttransla- selectively bind to a target peptide sequence in vivo only when the tional modifications (PTMs) on certain proteins of the pathway. sequence possesses a specific PTM. As shown in Fig. 1A, MTeC These PTMs, themselves, are typically required for signal propa- begins with the creation of a bait expression plasmid that includes, gation and the desired biological response, indicating that the PTM in tandem, an affinity epitope tag followed by a peptide sequence of proteins is a central component of most normal cellular pro- containing the target amino acid to be modified and, finally, the grams. Recent advances in proteomics demonstrate that the vast catalytic domain of an enzyme known to modify the target residue majority of eukaryotic proteins are posttranslationally modified in of interest within the peptide sequence. The MTeC bait plasmid is vivo, presenting investigators with the formidable challenge of then introduced into the cells of choice where, once in the cell, the identifying the enzymes responsible for each PTM and, impor- catalytic domain of the expressed fusion protein is able to selectively tantly, determining the biological significance of each PTM on each posttranslationally modify the target residue within the peptide protein. sequence. The expressed MTeC fusion protein is then available to Increasing evidence indicates that one common outcome of interact in vivo with proteins that bind this particular modified protein PTM is the creation of a high affinity binding site for the peptide sequence. The cellular proteins bound to the modified selective interaction with a specific PTM-specific binding protein MTeC fusion protein can then be purified from cells by using (BP). The interaction between the PTMBP and the modified standard biochemical techniques, including fractionation of specific protein is often a critical step for downstream signaling and the biological response. Based on these observations, many have at- tempted to discover and characterize PTMBPs by using various Author contributions: J.C.R. designed research; T.M.S. performed research; T.M.S. and J.C.R. classic in vitro approaches. Although these methods are typically analyzed data; and T.M.S. and J.C.R. wrote the paper. amenable to high throughput screens, they are also subject to The authors declare no conflict of interest. numerous inherent limitations and because of these restrictions, 1To whom correspondence should be addressed. E-mail: [email protected]. have resulted in the identification of only a relatively small number This article contains supporting information online at www.pnas.org/cgi/content/full/ of bona fide PTMBPs. 0907799106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907799106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 27, 2021 Fig. 1. Principles of MTeC. (A) An MTeC bait plasmid is composed of an affinity tag fused in tandem with a peptide sequence possessing an amino acid to be post- translationally modified in vivo followed by the catalytic domain of an enzyme known to modify the residue. (B) The MTeC bait plasmids are expressed in the cells of choice and, after the MTeC bait fusion protein is con- firmed to be properly modified in vivo, the PTMBPs are biochemically purified. After SDS/PAGE of the purified material, visible protein bands can be isolated and iden- tified by MS, or alternatively, complete sample mixtures can be submitted for protein identification. MTeC can also be used to verify and characterize PTM-dependent binding in vivo. subcellular compartments, affinity immunoprecipitation, and/or MTeC Bait Fusion Proteins Achieve Specific Degrees of H3K9 Methylation various other available chromatographic steps (Fig. 1B). Once this in Vivo. To test the ability of MTeC bait fusion proteins to acquire material is isolated, the purified proteins are analyzed by MS for the appropriate PTM in vivo, we capitalized on recent discoveries identification of the bound proteins. Importantly, the experiments and characterizations of enzymes that modify specific histone are performed in parallel with two critical controls. The first is an residues. In particular, we first focused on the well-described MTeC bait fusion protein lacking the catalytic domain of the methylation of histone H3 lysine 9 (H3K9) by the G9a methyl- enzyme, allowing subtraction of proteins that may bind both the transferase (3, 4). An MTeC bait plasmid containing a FLAG modified and unmodified peptide sequence. The second control is epitope and the first 44 aa of histone H3 were cloned upstream of an MTeC bait fusion protein where the modified target residue is the catalytic SET domain of the human G9a. Because we previously eliminated and, therefore, unavailable to be posttranslationally demonstrated that G9a is responsible for global dimethylation modified, allowing subtraction of interacting proteins that may bind (H3K9me2) in mammalian cells, and, in vitro, has also been shown another portion of the peptide sequence and/or the catalytic to cause trimethylation (H3K9me3), it was unclear which H3K9 domain of the tethered enzyme. Once the putative PTMBPs are methylated form would be observed in an H3-G9a WT MTeC bait identified,