Chromatin Biology & Proteomics

Quantitative mass spectrometry-based proteomics is a powerful tool for analyzing cellular protein expression patterns, posttranslational modifications of proteins and protein-protein interactions. We develop and employ mass spectrometry-based methods to support our research focused on the cellular response to DNA damage and nuclear ubiquitin signaling.

Mass spectrometry-based proteomics

In the recent years mass spectrometry-based proteomics has become an indispensable tool for the investigation of cellular signaling. In shotgun proteomics approaches proteins are extracted from cells or tissues and digested using specific proteases. The resulting peptides are separated by reversed-phase liquid chromatography and identified by their characteristic mass and fragmentation pattern. Quantitation methods including stable isotope labeling with amino acids in cell culture (SILAC) allow precise relative quantitation of peptide abundance in different samples.

Identification and quantitation of posttranslational modifications requires efficient enrichment of modified peptides from complex peptide mixtures prior mass spectrometric analysis. We have optimized titanium dioxide-based methods for enrichment of phosphorylated peptides and employed these methods to systematically investigate protein phosphorylation after genotoxic stress (Beli et al. 2012). In addition, we have developed enrichment methods for ubiquitin remnant peptides. Using these methods we were among the first to demonstrate proteome-wide identification of ubiquitylation sites and quantitation of ubiquitylation sites in response to cellular perturbations (Wagner and Beli et al. 2011 and 2012).

Figure 1: Analysis of a complex peptide mixture using ultra high pressure liquid chromatography (UHPLC) coupled to a quadrupole-Orbitrap hybrid mass spectrometer (left panel). Stable isotope labeling with amino acids in cell culture (SILAC) allows accurate relative quantitation of peptides in different samples (right panel).

Cellular DNA damage response mechanisms

Cells have evolved complex DNA repair mechanisms to counteract DNA damage continuously imposed by metabolic activity and environmental factors. Failure to repair DNA damage can contribute to carcinogenesis and is the underlying cause for human syndromes with predisposition to cancer development and premature aging. An in-depth understanding of the cellular response to DNA damage is essential to understand cancer development and to design novel targeted cancer therapies.

Recent studies have shown that the complexity of the DNA damage response extends far beyond the DNA damage repair: In response to DNA damage diverse cellular process including chromatin remodeling, transcription and RNA metabolism, are coordinated to ensure cellular homeostasis. Posttranslational modifications of proteins, including protein phosphorylation and ubiquitylation, play key roles in the coordination of these processes after DNA damage. We have employed quantitative mass spectrometry-based proteomics to investigate the cellular phosphorylation signatures in response to genotoxic stress induced by ionizing radiation and topoisomerase II inhibition. This study has revealed novel regulators of the DNA damage response and highlighted the link between transcription and DNA damage signaling (Beli et al. 2011). Ongoing efforts in the lab are focused on unraveling the relation between DNA damage and RNA metabolism. In addition, we are studying the role of stress kinases in DNA damage signaling.

Figure 2: Flow cytometric analysis of DNA content (7-AAD) and H2A.X phosphorylation in human osteosarcoma cells treated with the topoisomerase I inhibitor camptothecin. Camptothecin induces phosphorylation of histone variant H2A.X and arrests cells in S phase. Phosphorylation of H2A.X is critical for the assembly of DNA repair factors at DNA damage sites (left panel). Sequence motif analysis of phosphorylation sites induced by treatment of cells with camptothecin. Protein kinases of the PIKK family ATM, ATR and DNA-PK phosphorylate substrates on serines or threonines that are followed by a glutamine residue (S/TQ). The S/TQ sequence motif is overrepresented in phosphorylation sites that are induced by camptothecin highlighting the importance of ATM/ATR/DNA-PK (right panel).

Specificity in nuclear ubiquitin signaling

Ubiquitin is a 76-amino acid protein that is covalently conjugated to the ε-amino group of lysines in a highly orchestrated enzymatic cascade involving ubiquitin activating (E1), ubiquitin conjugating (E2) and ubiquitin ligase (E3) enzymes. Ubiquitylation is involved in diverse cellular processes including protein degradation, DNA damage repair and chromatin remodeling. We have recently developed mass spectrometric methods to investigate cellular ubiquitylation patterns and employed these methods to study the response of the ubiquitin system to cellular perturbations including the proteasome inhibition and genotoxic stress (Wagner and Beli et al. 2011 and 2012; Povlsen and Beli et al. 2012). We have shown that DNA damage induces site-specific ubiquitylation and deubiquitylation of DNA repair factors highlighting the regulatory role of protein ubiquitylation in DNA repair.

Ubiquitin ligases play an essential role in ubiquitin system by defining its substrate specificity. However, the protein targets and functions of most ubiquitin ligases remain elusive. At present we are employing mass spectrometric methods for analysis of protein ubiquitylation to reveal the substrate spectrum of ubiquitin ligases implicated in the DNA damage response and chromatin remodeling. 

Figure 3: Functional interaction network of proteins with UV-regulated ubiquitylation sites. Irradiation of cells with UV induces site-specific ubiquitylation and deubiquitylation of DNA repair factors highlighting the regulatory role of protein ubiquitylation in the response to DNA damage.

The group’s research is funded by the Emmy Noether Program of the German Research Foundation (DFG) and the Marie Curie Actions of the European Commission. We are also part of the Collaborative Research Center SFB 1177 Molecular and Functional Characterization of Selective Autophagy (www.sfb1177.de)