The study performed within the project included four Work Packages (WP). Within the WP1, the quantitative analysis of the expression level of DEK protein and H3K9ac, H3K4me3, and H3K27ac was performed using the Western Blot technique together with Pierce BCA Protein Assay. This study was made on a breast cancer model involving MCF10A, MCF7, and MDA-MB-231 cell lines. Confocal and STimulated Emission Depletion (STED) imaging enabled the DEK protein nuclear distribution investigation. The implementation of the Proximity Ligation Assay (PLA) and Image Cross-Correlation Spectroscopy (ICCS) analysis revealed possible protein-protein interactions between DEK protein and particular histone modifications. WP2 included investigating the formation of the DEK protein nuclear bodies, which are observed within DNA replication regions. The experiments based on immunostaining, RNA Fluorescence In Situ Hybridization (RNA-FISH), PLA, and others, show how DEK bodies correspond to different heterochromatin structures during the DNA replication process in MCF10A cells. In WP3, the investigation focus on the live-cell study of the dynamics of DEK bodies formation and DEK-eGFP fusion protein mobility within the cell nucleus, thanks to Fluorescence Recovery After Photobleaching (FRAP) experiments. The last work package (WP4) was based on applying the Single Molecule Localization Microscopy technique in order to perform the super-resolution qualitative analysis of the clusters formed by DEK protein on the single-molecule level. We expect that this study will provide novel insights into how the DEK protein modulates the organization of chromatin, especially for DNA structures that are difficult to replicate. The results of the project were presented at five international conferences: (a) 3rd Nanoscopy & 2nd Molecular Microscopy and Spectroscopy Retreat, Genoa, Italy,(b) EMBO Workshop „Chromatin dynamics and nuclear organization in genome maintenance”, virtual, (c) 65th Biophysical Society Annual Meeting, Virtual, (d) Polish Perspective conference, Polonium Foundation, Zurich & online, (e) EBSA2021, Vienna, Austria and published in the conference proceedings. The Fellow was awarded two Travel Awards (EBSA bursary for attending 2021 Conference and 2022 Travel Award at the BPS meeting).
