Rapid changes in genome organisation during exit from pluripotency and the role of the nuclear envelope in maintaining the pluripotent state
The majority of Nuclear Envelope Transmembrane Proteins (NETs) are tissue specific and many of these facilitate tissue-specific genome organization. Genome organization changes dramatically during differentiation and these NETs impact this process: muscle-specific genome-organizing NETs NET39, WFS1 and TMEM38A are important for myogenesis (Robson et al, 2016) while fat-specific genome-organizing NETs TMEM120A and B are important for adipogenesis (Batrakou et al, 2015). Although during lineage specification of mouse embryonic stem cells (Peric-Hupkes et al, 2010), we do not yet understand the temporal dynamics of these changes nor the components of the nuclear envelope that orchestrate these changes during early stages of exit from pluripotency. In this thesis, I investigate the temporal dynamics of genome organization changes during pluripotency exit stimulated by LIF withdrawal. Using Fluorescence in-situ Hybridization (FISH) to label DNA, I demonstrate that some of the earliest changes in genome organization occur within the first hour of exit from pluripotency with the relocation of a locus containing three genes Triml1, Triml2 and Zfp42 (that encodes REX1, a well-known marker of pluripotency) from the nuclear interior to the nuclear periphery. The RNA and protein levels of these genes persist for several hours post exit, suggesting that reorganisation of the genome is among the very first of events occurring during lineage specification and is perhaps a higher order mechanism controlling differentiation as a change in genome organisation could affect the transcriptional profile of these cells. To try and identify the proteins involved in tethering the locus and the mechanism of release I also investigated the changes in the nuclear envelope composition as cells undergo an exit from pluripotency. I show that while certain proteins undergo post translational modifications such as phosphorylation, other new proteins are synthesised during the first two hours of exit. Using phospho-null mutants for LBR and LAP2α, I show that these play a role in the relocation of this genomic locus. Finally, I introduced tissue-specific genome-organizing NETs such as NET39 (muscle), TAPBPL (blood) and TMEM120A(fat) into embryonic stem cells and found that their introduction causes a forced exit from pluripotency. Interestingly, these NETs show specificity in their ability to affect the position of genomic loci encoding pluripotency factors like Rex1 and Nanog, strengthening the idea that these tissue specific NETs act as tethers to very specific genomic regions in order to maintain a tissue specific genome organization. The results discussed here present for the first time, a temporal view of the changes in genome organisation during such early stages of in vitro differentiation. While Rex1 repositioning has been studied in greater detail in this thesis, a more comprehensive study over the early stages of exit might reveal additional genomic loci that reposition during this phase. The rapid reorganisation of the genome following LIF withdrawal highlights the importance of tightly controlling and maintaining appropriate culture conditions for the study of pluripotency using embryonic stem cells as a model system. The study leads to conceptual advancement in stem cell biology by describing early events following exit from pluripotency and in the field of nuclear biology by identifying the NE composition in ES cells. Collectively the results demonstrate the role of the nuclear envelope in the maintenance of pluripotency and in orchestrating genome organisation changes during exit.