Initial OCT4 engagement with the somatic proteome during reprogramming to iPSC

Cellular reprogramming to pluripotency can be achieved by using a defined cocktail of transcription factors, converting differentiated somatic cells into induced pluripotent stem cells (iPSCs). These cells behave like embryonic stem cells (ES) and can be used to generate all cell types in the body. OCT4, SOX2, KLF4 and cMYC (OSKM) were the first set of reprogramming factors defined based on both their importance on maintaining pluripotency, and their ability to reprogram mouse and human somatic cells to iPSCs. Despite recent progress in refining the reprogramming technique, this process is still highly inefficient and often leads to partially reprogrammed cells. This is even more dramatic in human cells, where efficiency is low (∼0.001–1%) hindering the therapeutic prospects and promise of iPSCs and their clinical applications. Extensive studies have been focused in elucidating the molecular mechanisms by which the transcription factors contribute to reprogramming, but there is still a notable lack of understanding of the reprogramming process at the protein level. Particularly little is known about the protein network by which the reprogramming factors maintain pluripotency in human ES and drive pluripotency during the reprogramming process. Due to the relevance of OCT4 as a core transcription factor for both, the pluripotency network and the reprogramming process, the work present herein focused on studying OCT4 at the proteomic level. By exploiting proteomic approaches focused in on and off-chromatin bound proteins this work described for the first time the OCT4 protein interactors involved in early stages of reprogramming and during pluripotency. For the hES interactome, OCT4 binding partners involved in pluripotency maintenance were identified, in addition to a new set of chromatin associated proteins that have non-previously been described in the pluripotency context. On the other hand, during early reprogramming, OCT4 interactors included somatic transcription factors and other interacting proteins non-previously reported in the reprogramming context, such as proteins involved in cell death, development and differentiation. Further comparison of both interactomes revealed that the initial engagement of OCT4 with the somatic proteome is markedly different from that in ES, illustrating how OCT4 is able to change its chromatin-binding dynamics in order to stablish different phenotypes. Additionally, the analysis was expanded by applying the same on and off chromatin proteomic approaches to three OCT4 mutants bearing deletions of essential or non-essential reprogramming regions located in the transactivation domains (TAD). This allowed the identification of a unique set of seven proteins present in all the OCT4 with reprogramming capacity (WT or mutant). Interestingly, these were not associated with OCT4 in ES nor the deficient OCT4 mutant bearing a deletion of an essential reprogramming domain. This set of proteins included: UFD1L, RAI1, TNIP2, ETV4, XPO6, FBRSL1 and MCMBP and further functional analysis proved that they are necessary for the reprogramming process as their depletion had negative effects in its efficiency. Remarkably, the biological processes these proteins can be involved are quite varied, including nuclear export, signalling response, post-translational modifications recognition, transcriptional regulation, chromatin remodelling and cell cycle; evidencing the versatility of OCT4 involvement in different processes necessary to achieve the pluripotent state. Furthermore, the mutants’ analysis not only allowed the identification of important proteins for reprogramming, but also revealed that removing non-essential reprogramming domains caused more expansive engagement of OCT4 with both the somatic genome and the proteome. These results suggest that different OCT4 regions of the TAD domains contribute to its binding properties, being the essential domains more important for more specific interactions with the genome and more functional interactions with the proteome. Overall, the findings of this thesis helped to get a better understanding of the protein interactions of OCT4 by describing for the first time the OCT4 networks during reprogramming and pluripotency in human, while revealing that focusing in the properties of the transactivation domains can help understand new biochemical properties of OCT4. Finally, applying the same proteomic approach analysis to the remaining reprogramming factors, can further help to understand their molecular mechanisms, expand the reprogramming and pluripotency networks, characterize crucial interactors and, thus provide a better understanding of the proteomic molecular mechanisms. This with the ultimate goal of improving the efficiency and fidelity of human iPSCs reprogramming.