PLAMseq enables the simultaneous proteo-genomic characterization of chromatin-associated proteins and protein interactions in a single workflow.

Chromatin Immunoprecipitation (ChIP) and Co-Immunoprecipitation (CoIP) assays are the most common approaches to characterize the genomic localization and protein interactors, respectively for a protein of interest. However, these approaches require the use of specific antibodies, which are costly reagents that often face sensitivity and specificity issues. Based on TurboID, we developed PLAMseq (Proximity Labelled Affinity-purified Mass spectrometry plus sequencing), after a short biotin pulse, DNA-protein crosslinks are induced by formaldehyde and the interactors of a protein of interest together with their associated DNA sequences are purified simultaneously and identified by mass spectrometry-based proteomics and Next Generation Sequencing, respectively. To validate PLAMseq, we performed the proteo-genomic characterization of two proteins which genomic loci are very well characterized, namely, RNA polymerase II and CTCF with excellent robustness and reproducibility. Next, we applied PLAMseq to characterize Histone H1 SUMOylation, a histone post-translational modification which study has remained elusive due to the lack of specific reagents. SETDB1 binds to SUMOylated histone H1 and, accordingly, SUMOylated histone H1 colocalize with H3K9me3 at repetitive regions of the genome. Overall design: PLAMseq (Proximity Labelled Affinity-purified Mass spectrometry plus sequencing) was performed in CTCF-TurboID, POL2RI-TurboID, TurboID-H1.2, TurboID-H1.4, TurboID-H1.0, NTurboID-H1.2 + CTurboID-SUMO1, NTurboID-H1.2 + CTurboID-SUMO2, NTurboID-H1.4 + CTurboID-SUMO1, NTurboID-H1.4 + CTurboID-SUMO2, NTurboID-H1.0 + CTurboID-SUMO1, NTurboID-H1.0 + CTurboID-SUMO2, TurboID-only and Wild Type HeLa cell lines after 10 minutes biotin 1x exposure. All experiments were done in triplicate.