Periodic Reporting for period 1 - qCHROMDEK (Quantitative insight into chromatin nanoscale structure: sub-nuclear organisation of oncoprotein DEK)
Período documentado: 2019-12-01 hasta 2021-11-30
Chromatin organization is the fundamental feature of physiologically functioning eukaryotic cells and, when misbalanced, can cause severe pathological conditions such as cancer. Specific chromatin factors ensure the maintenance of chromatin architecture and the proper functioning of chromatin-related processes. One of such protective chromatin proteins is DEK protein, which plays an important role not only in nuclear processes but also in cancer biology. In recent years, the DEK protein gathered significant attention from the scientific community, thanks to its numerous functions and potential role within cancer progression – features that candidate DEK as a potential cancer biomarker. The cellular functions of the DEK protein are still a subject of ongoing research. So far, multiple studies have shown that DEK has a pleiotropic mode of action by influencing several regulatory pathways.
The qCHROMDEK project aimed to investigate, via 4D advanced fluorescence microscopy, the correlation between the sub-nuclear organization of the DEK protein (connected to overexpression level differences in normal and cancer cells) and the local chromatin organization in a breast cancer model. In particular, the project’s goals include: (a) quantitative analysis of the correlation between DEK protein distribution and the pattern of specific post-translational histone modifications marking eu- or heterochromatin; (b) analysis of the above mention correlation along the process of cancerogenesis (represented by a breast cancer model of three cell lines – MCF10A, MCF7, and MDA-MB-231 cells); (c) the study of the DEK protein nuclear pattern along with the cell cycle progression; (d) the investigation of the role of DEK bodies in the process of the DNA replication; (e) and the connection between DEK bodies formation and the type of chromatin content.
The qCHROMDEK project aimed to investigate, via 4D advanced fluorescence microscopy, the correlation between the sub-nuclear organization of the DEK protein (connected to overexpression level differences in normal and cancer cells) and the local chromatin organization in a breast cancer model. In particular, the project’s goals include: (a) quantitative analysis of the correlation between DEK protein distribution and the pattern of specific post-translational histone modifications marking eu- or heterochromatin; (b) analysis of the above mention correlation along the process of cancerogenesis (represented by a breast cancer model of three cell lines – MCF10A, MCF7, and MDA-MB-231 cells); (c) the study of the DEK protein nuclear pattern along with the cell cycle progression; (d) the investigation of the role of DEK bodies in the process of the DNA replication; (e) and the connection between DEK bodies formation and the type of chromatin content.
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).
The role of DEK protein is thoroughly explored in the subject of cancer biology. However, its nuclear pattern observed with the use of microscopy technique is not investigated enough, given the importance of this oncoprotein. The results of the qCHROMDEK project show the nuclear spatial organization of DEK, which could play a significant function in the DNA replication of hard-to-replicate structures, as well as the correlation of DEK and chromatin marks along the cancerogenesis.