Designing Transcription Factors for Efficient Hematopoietic Reprogramming

September 21, 2017
MSc. Thesis - University of Coimbra

Rita Alexandra Silvério Alves

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Hematopoietic stem cell transplantation (HSCT) has been used as a treatment for a variety of haematological disorders, due to the ability of hematopoietic stem cells (HSCs) to self-renew and differentiate into all blood cell lineages. Insufficient number of cells and matching incompatibilities between donors and recipients hinder the broad application of this therapy. Expansion of HSCs has met limited success and additional strategies for the in vitro generation of HSCs are required to overcome transplant-associated limitations. Somatic cell reprogramming mediated by transcription factors (TFs) is opening new avenues for regenerative medicine and allowed the design of new approaches to convert one differentiated cell type directly into another. In the hematopoietic system, direct reprogramming of fibroblasts to HSC-like cells has been shown through ectopic expression of Gata2, Gfi1b and cFos, providing an alternative method to generate patient tailored HSCs in vitro. A better understanding of the mode of action of these three critical TFs during reprogramming is needed in order to increase the efficiency of the process.Here, I have defined potential reprogramming domains of hematopoietic TFs by homologous gene (paralog) and deletion construct substitution during hematopoietic reprogramming. First, paralogs of Gata2, Gfi1b and cFos and Gata2 deletion constructs were cloned into a lentiviral gene delivery system to induce fibroblast cell identity towards the hematopoietic lineage. Secondly, hematopoietic reprogramming efficiency was assessed by hCD34/H2BGFP reporter activation. Interestingly, Gata1 did not substitute Gata2 for hematopoietic reprogramming, despite evidences of overlapping function during hematopoiesis. Notwithstanding, Gfi1b and cFos were partially replaced by their respective paralogs, indicating a determinant role for non-homologous domains of Gata2 during reprogramming. Consistently, hematopoietic reprogramming with Gata2 deletion constructs revealed the requirement of the transactivation domains (TADs), the negative regulatory domain (NRD) and the C-terminal zinc finger (C-ZF) for successful reprogramming. Remarkably, I have also unveiled that Gata2 display mitotic bookmarking activity. This epigenetic feature may be important for the acquisition and maintenance of the HSC fate as well as the inheritance of the reprogrammed cell state to daughter cells. Overall, this study identified functional reprogramming features of Gata2, Gfi1b and cFos and sheds new light on how the HSC fate is acquired and preserved. Hereafter, these reprogramming modules will be critical for the design of enhanced synthetic TFs to increase hematopoietic reprogramming efficiency bringing this technology one step closer to clinical translation.