Supervisors info:
Δημήτριος Στραβοπόδης, Αναπληρωτής Καθηγητής Βιολογίας Κυττάρου & Ανάπτυξης, Τμήμα Βιολογίας, ΕΚΠΑ
Δημήτρης Θάνος, Ακαδημαικός, Ερευνητής Α', ΙΙΒΕΑΑ
Ισιδώρα Παπασιδέρη, Καθηγήτρια Κυτταρικής & Αναπτυξιακής Βιολογίας, Τμήμα Βιολογίας, ΕΚΠΑ
Summary:
Cellular reprogramming of somatic cells to induced Pluripotent Stem Cells (iPSCs) is a stochastic process accomplished by the exogenous expression of the transcription factors Oct3/4, Sox2, Klf4 and c-Myc. Prior experimental work in our lab has discovered the assembly of a 9-transcription factor network (9 TR network), which if assembled until the 6th day of reprogramming, helps increase the probability of successful reprogramming. The goal of this study is to investigate the role of one of the transcription factors of the 9-TR network, namely Ovol1, and the ways in which its expression is regulated during the process. At first, we examined the activation of putative regulatory regions of the Ovol1 gene during reprogramming through reporter assays, and we found that the activation profile of the 1kb region upstream of the transcription start site follows the expression profile of the endogenous gene. This 1kb region was being activated in cell subpopulations, which had increased the probability for pluripotency retrieval. In order to confirm this observation, we divided the cell culture at Day 6 of reprogramming into two subpopulations, according to the state of 1kb region- active or inactive. We found that cells with active 1kb region were being more efficiently reprogrammed. Our results show that 1kb region upstream of the transcription start site acts as an enhancer of the Ovol1 gene during reprogramming, and that its activation upregulates the efficiency of the process. Secondly, in order to study the role of Ovol1 at reprogramming, we carried out ChIP-seq and RNA-seq experiments, targeting Ovol1, at days 0 and 6 of the process in cell subpopulations, again for active and inactive 1kb region. Our results from RNA-seq experiments confirm the existence of an enhancer in this 1kb region. They also support that upregulation of Ovol1 has a positive effect on reprogramming efficiency by regulating more efficiently the MET procedure. Finally, ChIP-seq results reveal that Ovol1 binds synergistically with Tead4 on its target genes and that most of the Ovol1 target genes are already occupied by Tead4 at Day 0.
