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Identifier

000351373

Title

Γενετική και επιγενετική ανάλυση της γονιδιακής έκφρασης των εμβρυ'ικών βλαστοκυττάρων του ποντικού κατά τη διαφοροποίηση / Καράντζαλη Ευθυμία.

Alternative Title

Genetic and epigenetic analysis of gene expression during mouse embryonic stem cell differentiation

Author

Καράντζαλη, Ευθυμία

Thesis advisor

Παπαματθαιάκης, Ιωσήφ

Abstract

In the present study we developed a new way of mESC differentiation by treating cells with the deacetylase inhibitor, Trichostatin A (TSA). This treatment resulted in rearrangements of chromatin structure and modifications as well as changes in the expression profile. Pluripotency factors were downregulated and differentiation factors were induced. These changes are similar to those observed during differentiation via the formation of embryoid bodies (EB), but they occur within 12 hours instead of 8 days that the EB need to form. Nevertheless, the effect of TSA was not permanent since after removal of the reagent from the culture, cells were partly reversed in the undifferentiated state, restoring the initial levels of certain factors and certain histone modifications on their promoters. Combination of the above results demonstrated that genes that were transcriptionally down-regulated with simultaneous appearance of 3meK27H3 levels on their promoters, were the ones that could not restore their initial exepression levels upon TSA removal. However, even with the partial reversal, cells remained pluripotent as they were capable of inducing differentiation factors of all 3 germ layers (endoderm, mesoderm, ectoderm) during subsequent differentiation (EB). Among the genes that were severely downregulated during differentiation was sall1. Silencing of this gene resulted in downregulation of Nanog, one of the master pluripotency regulators. After in vitro and in vivo protein interaction experiments it was found that Sall1 protein could physically interact with Nanog and another master pluripotency regulator Sox2. ChIP-on-chip experiments for Nanog and Sall1 revealed that these two factors had a large number of common target genes. Finally, maintenance of Sall1 expression during differentiation prevented certain differentiation factors from being induced, showing that Sall1 plays a role in their suppression in the undifferentiated state and placing it among the factors that a have a role in pluripotency maintenance.

Language

Greek

Subject

Chromatin

Differentiation

Nanog

Salli

Trichostatin A CTSA

Διαφοροποίηση

Επιγενετικές τροποποιήσεις

Τριχοστατίνη Α

Χρωματίνη