Dissertation committee:
Δρ. Διαμάντης Σίδερης, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ,
Δρ. Δημήτρης Θάνος, Ακαδημαϊκός, Ερευνητής Α’, Ίδρυμα Ιατροβιολογικών Ερευνών της Ακαδημίας Αθηνών (ΙΙΒΕΑΑ)
Δρ. Δημήτριος Στραβοπόδης, Αναπληρωτής Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ,
Δρ. Διδώ Βασιλακοπούλου, Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ,
Δρ. Παναγιώτα Παπαζαφείρη, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ,
Δρ. Δήμητρα Θωμαΐδου, Ερευνήτρια Α’, Ελληνικό Ινστιτούτο Παστέρ,
Δρ. Παναγιώτης Πολίτης, Ερευνητής Β’, Ίδρυμα Ιατροβιολογικών Ερευνών της Ακαδημίας Αθηνών (ΙΙΒΕΑΑ)
Summary:
Cellular reprogramming of somatic cells towards induced Pluripotent Stem Cells (iPSCs) is achieved through the over-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM). Due to the high complexity of the process, the vast number of the involved molecules and its stochastic and inefficient nature, the molecular background of reprogramming is only partially revealed. The aim of this Doctoral Thesis was the identification of genomic regulatory elements which are induced during cellular reprogramming, the examination of their action and their association with the expression of important genes. To achieve this, we used a combination of different molecular, imaging, high-throughput, and bioinformatics techniques.
First, we constructed a combined ChIP-seq OSKM dataset using experiments on MEFs (Mouse Embryonic Fibroblasts) from multiple published studies, in order to examine the universal OSKM binding upon the genome, irrespective of minor discrepancies originating from the different reprogramming systems and protocols used. Initially, we focused on the ESC-related sites (Embryonic Stem Cells, natural equivalent of iPSCs) and found out that almost 1/3 of them are bound by OSKM as early as at day 1, or since MEFs («Early-bound ESC sites»). Our analysis highlighted the significant role of Myc in reprogramming, which is often overlooked. We saw that, along with Klf4, they “mark” positions where Oct4 and/or Sox2 will bind after reprogramming initiation. Furthermore, we discovered that various genes are regulated by multiple genomic elements, where OSKM bind at different timepoints in reprogramming. For example, many genes involved in the MET pathway (Mesenchymal to Epithelial Transition) are located near both ESC-, MEF- and Transient-OSKM sites, resulting in stably high expression levels which temporarily increase during the intermediate timepoints. Finally, we observed that OSKM often bind on the chromatin in a dynamic fashion. This “on-and-off” association of OSKM with the genome is related to the need of chromatin reorganization to achieve pluripotency.
We wished to take advantage of the results of our genomics analysis in order to identify DNA regulatory elements which are induced in cellular reprogramming (Reprogramming Inducible Enhancers, RIEs) and use their activation as a marker of successful pluripotency acquisition. In other words, we wanted to use the RIEs as “traps” (enhancer traps) for the identification of cells which reprogram with higher efficiency. For this, we focused on the ESC sites which are bound by OSKM early, but only after the MEF stage (day 1, «de novo Early-bound ESC sites»). Among these inducible elements we chose the regions with high number of OSKM peaks, which reside near genes (0-2.5kb upstream/downstream) which are over-expressed in reprogramming, in order to end up with genomic loci with enhancer characteristics. We randomly chose three of these regions for downstream analyses. In particular, we cloned three elements residing upstream of Lefty1, Pou5f1 (Oct4) and Upp1 genes next to a GFP gene, in order to perform reporter assays. Our results showed that all three loci (Lefty1-700, Pou5f1-1800, Upp1-800) act as enhancers and are induced in MEFs which acquire an epithelial phenotype (MET) and often form early iPSC colonies. Importantly, the Upp1-800 enhancer, contrary to the other two, possesses all the necessary sequences for the regulation of the respective gene throughout reprogramming. Cells sorted at day 4 based on Upp1-800 activation (Upp1-800-GFP(+) cells) get reprogrammed with double efficiency rate compared to the rest of the population (Upp1-800-GFP(-) cells). In conclusion, using our unbiased method for the detection of RIE enhancers, we identified a new early reprogramming marker, the Upp1-800 enhancer.
Next, we wished to use this new reprogramming marker to study the cells within which it is induced and thus reveal new, unknown mechanisms of cellular reprogramming. In this context, we performed RNA-seq analysis of the Upp1-800-GFP(+) and GFP(-) cells during the early stages of reprogramming (day 2-4). We found out that the Upp1-800 enhancer marks cells which have induced gene networks involved in MET and which present an increased expression of the 9TR factors Gli2 and Irf6 and of the metallopeptidase Mmp9. Previous experiments in our laboratory have shown that the 9TR network plays a key role in the activation of Nanog and the achievement of pluripotency, and also, that Mmp9 is important for the successful completion of reprogramming. Based on the RNA-seq results and on other analyses, we produced a putative model describing the molecular events involved in the effective emergence of iPSC colonies from the Upp1-800-GFP(+) cells. In more detail, the efficient over-expression of the OSKM transgenes during the first 2 days of reprogramming leads to the temporary induction of Gli2 and Irf6, via the direct OSKM binding at the respective regulatory regions (day 2-4). In parallel, OSKM activate the Upp1-800 enhancer-reporter, leading to the expression of GFP (Upp1-800-GFP(+) cells). Irf6, then, binds at the Mmp9 and Nanog genes’ regulatory regions and induces them. This contributes to the efficient phenotypical transition of cells and the establishment of pluripotency. On the contrary, OSKM are expressed at relatively lower levels during the first 2 days in the GFP(-) cells, resulting in the delayed Irf6 and Gli2 over-expression and in the inability of Mmp9 and Nanog to reach sufficiently high expression levels.
Finally, we got interested in investigating how Mmp9 contributes to the achievement of pluripotency. Transcriptomic analysis of Mmp9 knocked-down cells, or cells treated with an MMP9 inhibitor confirmed previous results of our laboratory, highlighting the role of MMP9 in MET. In more detail, we saw that in the absence of active MMP9 in the cells classic mesenchymal markers are over-expressed, while epithelial genes and reprogramming markers are suppressed. Furthermore, we found out that Mmp9 forms a positive regulatory feedback loop with Irf6 and Nanog. These two factors bind to the Mmp9 regulatory region and induce its expression, while Mmp9 preserves and/or ensures the expression of Irf6 and Nanog genes. Apart from MET, Mmp9 affects multiple other cellular functions, like cell death, metabolism and proliferation. This is achieved by the direct or indirect effect of MMP9 activity on the expression of multiple central genes like Cdh1 (E-cad), Acta2 and Mapk3.
To conclude, in the present Doctoral Thesis we studied the OSKM activity in cellular reprogramming and developed an unbiased methodology for the identification and functional study of DNA elements acting as RIE enhancers. Using this methodology we identified a novel early reprogramming marker and discovered important molecular mechanisms which take place in cells undergoing reprogramming. We proposed a model connecting the activity of OSKM, the 9TR factors and Mmp9 and showcased for the first time in detail the central and pleiotropic mode of action of the metallopeptidase 9 in reprogramming.
