Chromatin-directed proteomics identifies cell vulnerabilities in DNA repair and elucidates the impact of signal transduction on the DNA-bound proteome

Fundamental biological processes ranging from DNA replication and repair to cell cycle and gene transcription are tightly controlled in time and space through the coordinate interplay of among different chromatin layers including DNA accessibility, histone post-translational modifications (hPTMs), and chromatin-associated proteins. Yet a comprehensive understanding of chromatin dynamics downstream of signaling cascades is elusive.

We here develop chromatin-directed proteomic approaches to provide an unbiased perspective of the chromatin composition dynamics upon signaling pathway perturbation. In particular we adopt our multi-layered chromatin proteomic strategy to quantify and dynamically profile different chromatin layers at high temporal resolution during DNA damage repair (DDR) signaling. In particular, we a) use ChIP to enrich for known repair proteins and characterize their functional chromatin-mediated interactions via the Selective Isolation of Chromatin-Associated Proteins (or ChIP-SICAP), b) dissect the composition of the DNA-bound proteome by the isolation of Proteins on Chromatin (or iPOC), and c) profile signatures of hPTMs at mononucleosome resolution with Native Chromatin Proteomics (N-ChroP).

Through our multilayered approach we quantify a high number of both expected and novel determinants deregulated during the DDR, and validated them by imaging- and FACS-based orthogonal approaches. These results allow us to attribute novel candidates to the main DDR repair pathways. In particular, we identified a role for the histone reader ATAD2, the microtubule organizer TPX2 and the histone methyltransferase G9A as novel important regulators of the homologous recombination (HR) repair pathway, and showed that their depletion sensitizes to PARP inhibitors, thus prompting for innovative combinatorial treatments. Furthermore, during the DDR we profile 33 hPTMs at mononucleosomes bearing the DNA damage marker gamma H2AX (or γH2AX). Integration of these data with the complementary results from iPOC shed light on, among the others, G9A-mediated monomethylation of H3K56 in HR repair. Moreover, all multi-layered chromatin proteomic data can be easily queried via a shinyApp at chromatin-proteomics.dkfz.de.

In addition, we further optimized and applied iPOC to investigate the DNA-bound proteome dynamics upon perturbation of the cancer-relevant PI3K/AKT/mTOR signaling pathway. Our results show how the selective inhibition of PI3K, AKT, or mTOR has a distinctive effect on the DNA-bound proteome. Moreover, we provide evidence that the PI3K/AKT/mTOR signaling cascade acts on the DNA-bound status of the epigenetic regulator SUZ12, thereby modulating H3K27me3 levels.

Collectively, our results provide novel insight on the chromatin dynamics in highly studied signaling pathways, thereby demonstrating the added value of chromatin-directed proteomics in decoding the complexity of biological processes.