Identification and characterization of posttranslational modification-specific binding proteins in vivo by mammalian tethered catalysis

Abstract

Increasing evidence indicates that an important consequence of protein posttranslational modification (PTM) is the creation of a high affinity binding site for the selective interaction with a PTM-specific binding protein (BP). This PTM-mediated interaction is typically required for downstream signaling propagation and corresponding biological responses. Because the vast majority of mammalian proteins contain PTMs, there is an immediate need to discover and characterize previously undescribed PTMBPs. To this end, we developed and validated an innovative in vivo approach called mammalian tethered catalysis (MTeC). By using methylated histones and methylspecific histone binding proteins as the proof-of-principle, we determined that the new MTeC approach can compliment existing in vitro binding methods, and can also provide unique in vivo insights into PTM-dependent interactions. For example, we confirmed previous in vitro findings that endogenous HP1 preferentially binds H3K9me3. However, in contrast to recent in vitro observations, MTeC revealed that the tandem tudor domain-containing proteins, JMJD2A and 53BP1, display no preferential H4K20 methyl-selectivity in vivo. Last, by using MTeC in an unbiased manner to identify H3K9 methylspecific PTMBPs, we determined that endogenous G9a binds methylated H3K9 in vivo. Further use of MTeC to characterize this interaction revealed that G9a selectively binds H3K9me1 in vivo, but not H3K9me2, contrary to recent in vitro findings. Although this study focused solely on methylated histones, we demonstrate how the innovative MTeC approach could be used to identify and characterize any PTMBP that binds any PTM on any protein in vivo.53BP1 ͉ G9a ͉ histone ͉ JMJD2A ͉ methylation E ukaryotic cells have developed intricate and distinct cellular signaling cascades to translate particular intra or extracellular stimuli into an appropriate biological response. These signaling pathways rely heavily on enzymes that create specific posttranslational modifications (PTMs) on certain proteins of the pathway. These PTMs, themselves, are typically required for signal propagation and the desired biological response, indicating that the PTM of proteins is a central component of most normal cellular programs. Recent advances in proteomics demonstrate that the vast majority of eukaryotic proteins are posttranslationally modified in vivo, presenting investigators with the formidable challenge of identifying the enzymes responsible for each PTM and, importantly, determining the biological significance of each PTM on each protein.Increasing evidence indicates that one common outcome of protein PTM is the creation of a high affinity binding site for the selective interaction with a specific PTM-specific binding protein (BP). The interaction between the PTMBP and the modified protein is often a critical step for downstream signaling and the biological response. Based on these observations, many have attempted to discover and characterize PTMBPs by using various classic in vitro approaches. Although t...

Concerted Activities of Distinct H4K20 Methyltransferases at DNA Double-Strand Breaks Regulate 53BP1 Nucleation and NHEJ-Directed Repair

“…To identify potential PR-Set7-interacting proteins necessary for PR-Set7 recruitment to DSBs, two independent tandem affinity purifications of ectopically expressed FLAG-HA-null and -PR-Set7 in HeLa cells were performed followed by comparative mass spectrometry analyses (Spektor and Rice, 2009). Identification of proteins unique to PR-Set7 included the NHEJ-associated factors Ku70, Ku80 and PARP1 (Table S1).…”

“…An iQ5 iCycler (Bio Rad) was used for qPCR with data presented as fold enrichment (%IP/%Input)/(%IP H3/%Input H3) and statistical significance determined by the Student’s t-test. Western analysis, immunoprecipitations, comparative mass spectrometry analysis and in vitro binding assays were performed as previously described (Spektor and Rice, 2009). Laser irradiation and immunofluorescence studies were performed as previously reported (Kong et al, 2009; Wu et al, 2010).…”

DNA methylation dysregulates and silences the HLA-DQ locus by altering chromatin architecture

“…One such factor is heterochromatin protein-1 (HP-1), which recognizes chromatin that contains histone H3K9me3 posttranslational modifications 32. To determine if the regions bearing DNA methylation carried heterochromatin modifications, ChIP assays were carried out for HP-1 and H3K9me3 (Figure 4).…”

PR-Set7 and H4K20me1: at the crossroads of genome integrity, cell cycle, chromosome condensation, and transcription

“…There are three extensively investigated H4K20me1 binders that each carry a unique binding domain and contribute to distinct cellular functions: L3MBTL1 Trojer et al 2007), 53BP1 (Sanders et al 2004;Oda et al 2009;Spektor and Rice 2009), and Condensin II (Liu et al 2010). L3MBTL1 binds to both mono-and dimethylated H4K20 residues through its MBT domains.…”