Functional roles of two chromatin factors (USP22 ve MENIN) in reprogramming and pluripotency

Overexpression of OCT4, SOX2, KLF4 and MYC (OSKM) factors can reprogram somatic cells to induced pluripotent cells (iPSC). Process of somatic cell reprogramming is inherently inefficient, pointing to the cell's intrinsic barriers that safeguards somatic cell identity. Previously conducted CRISPR-Cas9-based knockout screens during reprogramming revealed USP22 and MLL1 as barriers to reprogramming. In the first part of this thesis, overexpression of wild-type and catalytic mutant USP22 in USP22 KO cell lines were performed which showed that increased reprogramming efficiency is indeed related to USP22 loss, and this effect is not related to its deubiquitination activity. Reprogramming of USP22 knock-out primary fibroblasts under different primed pluripotency culture conditions proved that increased reprogramming efficiency is independent of cell line or culture condition of choice. Transcriptome analysis at specific time-points during reprogramming revealed that loss of USP22 represses fibroblast-specific genes such as COL1A2, POSTN, FOXC2 that occurs throughout reprogramming. In addition, USP22 loss results in upregulation of general pluripotency markers such as SOX2, LIN28A and naïve pluripotency markers such as DNMT3L, GDF3, ALPPL2, ARGFX as early as 3 days after OSKM expression. To investigate a potential role of USP22 in attaining naïve pluripotency, I reprogrammed somatic cells under naïve culture conditions which resulted in an increased number of naïve colonies as quantified by TRA 1-60/KLF17 double staining. In addition, USP22 loss enhanced conversion from primed to naive pluripotency indicated by higher expression levels of naive pluripotency markers DNMT3L and ALPPL2. In the second part of the thesis, I showed that MEN1 loss enhances reprogramming efficiency in primary fibroblasts. Rescue experiments utilizing overexpression of H433A mutant MENIN which cannot recognize H3K79me2, indicated that MENIN acts as a barrier partially through its ability to read this chromatin mark. To test whether knocking-out MEN1 affects pluripotency, single clone iPSCs were generated from MEN1 KO fibroblasts. Characterization assays showed that these clones express endogenous OCT4, SOX2 and KLF4 while silencing C-MYC and other exogenous transgenes. Also, MEN1 iPSCs stably express pluripotency related proteins and contribute to all three germ layers when subjected to teratoma formation assay. Finally, MENIN-MLL1 Complex inhibition via VTP50469 enhanced reprogramming efficiency in primary fibroblast lines. In addition, VTP50469 supplementation increased reprogramming efficiency of 3-factor (OSK) reprogramming, and enabled iPSC generation with only 2 factos (OS). Taken together, this work demonstrates important roles for two distinct chromatin factors in reprogramming and pluripotency.