Nuclear envelope transmembrane proteins in differentiation systems

Abstract

Historically, our perception of the nuclear envelope has evolved from a simple barrier isolating the genome from the rest of a cell to a complex system that regulates functions including transcription, splicing, DNA replication and repair and development. Several recent proteomic studies uncovered a great variety of nuclear envelope transmembrane proteins (NETs). Diseases associated with several nuclear envelope proteins, mostly NETs, affect many tissues e.g. muscle, adipose tissue, skin, bones. Many NETs of the inner nuclear membrane have been shown to interact with chromatin, suggesting that their influencing gene expression might explain NET roles in disease. This work is focused on finding novel interactions of NETs with chromatin. First, SUN2 post-translational modifications were analysed and the effect of phosphomimetic and phospho-null mutants on heterochromatin and the cytoskeleton was tested by overexpression. However, no obvious changes were found. Second, several tissue-preferential NETs were tested in an adipocyte differentiation system. NET29 changed chromosome 6 position in pre-adipocytes. This matched changes in chromosome positioning that occur during adipocyte differentiation when NET29 is normally induced. Post-translational modifications of NET29 are likely to play a vital role in this process because a phospho-null mutant dominantly blocked chromosome repositioning. The effect of over-expression and down-regulation of NET29 on transcription was tested and results suggest that NET29 negatively regulates expression of myogenic genes during adipogenesis. This thesis is split into six chapters. Chapter I is an overview of the nuclear envelope, adipogenesis and chromatin remodelling, Chapter II is a detailed description of methods used in this study. Chapter III focuses on post-translational modifications of SUN2, as well as trials to identify novel partners of SUN2. Chapter IV and V deal with a novel nuclear envelope transmembrane protein and its role in adipogenesis. Finally, the last chapter includes a discussion and recommended future directions.