Molecular Biology and Genetics MS Thesis Defense by Eray Enüstün

KOÇ UNIVERSITY

GRADUATE SCHOOL OF SCIENCES & ENGINEERING

MOLECULAR BIOLOGY AND GENETICS

MS THESIS DEFENSE BY ERAY ENUSTUN

Title: The role of chromatin modifying enzymes in fibroblast-to-hepatocyte direct lineage conversion

Speaker: Eray Enüstün

Time: July 17, 2018, 10:00

Place: ENG 127

Koç University

Rumeli Feneri Yolu

Sariyer, Istanbul

Thesis Committee Members:

Asst. Prof. Dr. Tamer T. Onder (Advisor, Koç University)

Asst. Prof. Dr. Tugba Bagcı Onder (Koç University)

Prof. Dr. Esra S. Erdal (Dokuz Eylül University, Izmir Biomedicine and Genome Center)

Abstract:

Transdifferentiation is a reprogramming method to directly convert a differentiated cell into another differentiated cell type by overexpressing lineage-specific transcription factors. Compared to reprogramming of differentiated cells into induced pluripotent stem cells (iPSC), it is faster and potentially safer for clinical applications. However, the efficiency of lineage conversion remains low and needs to be improved for potential therapeutic applications. Reprogramming requires extensive remodeling cellular epigenetic state and chromatin modifiers have emerged as important regulators of iPSC generation. We hypothesized that such regulators may also play crucial roles in direct lineage conversion. To investigate the role of chromatin modifiers in transdifferentiation, we utilized an established protocol of direct conversion of human fibroblasts to induced hepatocytes via overexpression of FOXA3, HNF1A and HNF4A. A lentiviral GFP-reporter for Albumin expression was generated to monitor conversion efficiency. Using this system, we observed that CRISPR/CAS mediated knockout and chemical inhibition of the H3K79 methyltransferase Dot1L increases direct conversion to hepatocytes at early time points. Dot1L inhibition accelerated the induction of hepatic markers and silencing of the fibroblast markers. In addition, iHeps obtained through Dot1L inhibition displayed basic hepatic functions such as glycogen storage. Encouraged by these results, we performed a compound screen that targets broad range of chromatin writers, erasers and readers. Using the albumin reporter, we identified compounds that increases transdifferentiation efficiency at day 6, day 9 and day 12 after the overexpression of required transcription factors. Among these Rocilinostat, SAHA, GSK8815, MS023, Valproic Acid, GSK8814, LP99 and IOX2 can increase transdifferentiation efficiency at early stages, while 3-DZNEP, Repsox and CHR6494 have a role in later stages. These results provide important insights into how chromatin modifiers and epigenetic modifications affect transdifferentiation and may contribute to new strategies of obtaining iHeps with higher efficiencies for potential therapeutic applications.