Role of the nuclear envelope transmembrane protein, Samp1, in chromatin organization, differentiation and diseaseKašnik, Urška (Avtor) Vardjan, Nina (Mentor) Hallberg, Einar (Komentor) chromatin organizationdifferentiationSamp1Emery-Dreifuss muscular dystrophynuclear envelope proteinsThe nucleus, a hallmark of eukaryotic cells, contains the genetic material called chromatin. Chromatin is organized and structured as transcriptionally inactive heterochromatin, most of which is found in the nuclear periphery, and transcriptionally active euchromatin, most of which is found in the nuclear interior. The nucleoplasm is separated from the cytoplasm by a nuclear envelope (NE) consisting of two concentric lipid membranes, nuclear pores, and the nuclear lamina. The nuclear lamina is formed by intermediate filament proteins, called lamins, and the nuclear envelope transmembrane proteins (NETs), which organize chromatin in the nuclear periphery. Out of several hundred NETs, only a small minority have been characterized, most of which show a high tissue diversity. Mutations of some of these nuclear envelope proteins cause a wide range of diseases called envelopathies or laminopathies. In this thesis, we focused on chromatin reorganization during differentiation and the roles of an inner nuclear membrane protein, Samp1, during differentiation, and Emery-Dreifuss muscular dystrophy (EDMD). We improved the previously developed method for quantitative monitoring of the epigenetic state of chromatin in live cells, called Fluorescent Ratiometric Imaging of Chromatin (FRIC). FRIC uses pTandemH vector that assures stoichiometrically constant expression of histones 3.3-EGFP and H2B-mCherry, markers for euchromatin and general chromatin, respectively. With the improved presentation of FRIC, it was possible for the first time to demonstrate an increase in heterochromatin in the nuclear periphery during the late-stage of neuronal differentiation and the role of Samp1 in promoting peripheral heterochromatin organization. The results also suggest that Samp1 can interact directly with the chromatin without the help of its binding partners at the nuclear envelope. We were also interested in Samp1 mutations that occur in patients with EDMD. In a recent study, three EDMD-connected mutations were found in the TMEM201 gene, encoding Samp1. While no effect of the mutants was traced in our experimental system, in HeLa and U2OS cells over-expression of YFP Samp1 caused a mislocalization of emerin, but in each of these lines, it had an opposite effect on the centrosome-to-nucleus distance, supporting the idea of the tissue-specific functions of Samp1.20212021-02-16 15:30:03Magistrsko delo/naloga124773sl