Molecular Biology and Genetics PhD Thesis Defense by Deniz Uğurlu Çimen

KOÇ UNIVERSITY

GRADUATE SCHOOL OF SCIENCES & ENGINEERING

MOLECULAR BIOLOGY and GENETICS

PhD THESIS DEFENSE BY DENİZ UĞURLU ÇİMEN

Title: Determining the effects of DOT1L interacting proteins on cellular reprogramming

Speaker: Deniz Uğurlu Çimen

Time: 31 August 2018, 10:00

Place: SNA A52

Koç University

Rumeli Feneri Yolu

Sariyer, Istanbul

Thesis Committee Members:

Assist. Prof. Dr. Tamer Önder (Advisor, Koç University)

Assoc. Prof. Dr. Nurhan Özlü (Koç University)

Assist. Prof. Dr. Bilal Kerman (Medipol University)

Assist. Prof. Dr. Nathan Lack (Koç University)

Assoc. Prof. Dr. Tolga Emre (Boğaziçi University)

Abstract: Fully differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by ectopic expression of transcription factors. However, the mechanism behind this cell fate change is not fully elucidated. Epigenetic regulators have important roles during embryonic development as well as somatic cell reprogramming. Previously, it has been shown that inhibition of DOT1L, the histone H3 lysine 79 methyltransferase, increases the efficiency of reprogramming via regulation of lineage specific genes. DOT1L is recruited to chromatin and act in concert with a number of additional chromatin regulators. However, the role of such DOT1L-interacting proteins in reprogramming remains unknown.

In the first part of this thesis, novel DOT1L interactors were identified using the BioID method in which a promiscuous BirA ligase (BirA*) was employed to biotinylate DOT1L-proximal proteins, in vivo. Biotinylated proteins were pulled-down by Streptavidin and identity of the proteins was determined by LC-MS/MS. The resulting novel interaction candidates were investigated for their effects on reprogramming. Candidate genes were knocked-down in human fibroblasts via shRNAs followed by reprogramming. Our results indicated that knock-down of AF10 (MLLT10), significantly increased the iPSC generation efficiency, suggesting that it acts as a barrier to reprogramming similar to DOT1L. This finding was verified by CRISPR/Cas9 mediated knockout of AF10. Combining DOT1L inhibition or knockout with AF10 suppression did not result in an additive enhancement of reprogramming, suggesting that these two chromatin factors act in the same pathway.

In the second part of this thesis, known direct and functional interactors of DOT1L were curated from the literature and their effects on reprogramming was investigated through loss of function experiments. Suppression of Mixed Lineage Leukemia 1 (MLL1) expression via RNA interference or CRISPR/Cas9 significantly increased reprogramming efficiency. To determine how MLL1 prevents reprogramming, RNA-sequencing was performed. MLL1 suppression resulted in downregulation of fibroblast-specific genes and accelerated the activation of pluripotency-related genes.

Taken together, this study uncovered two important chromatin factors that act as barriers to reprogramming and contributed to our understanding of epigenetic mechanisms that maintain cell identity.