The Yin and the Yang of YY1: Deciphering the Role of YY1 in iPS Cell Reprogramming

Induced pluripotent stem cell (iPSC) reprogramming involves the generation of pluripotent stem cells from patient-derived somatic cells, via the ectopic expression of four transcription factors: Oct4, KLf4, Sox2 and c-Myc (OKSM). iPSCs represent a potential treatment for genetic and degenerative disorders; if realized, patient-derived cells could be cultured and repaired in vitro then re-administered to the patient.  Unfortunately, the translation of iPSCs to a clinical setting is in part impeded by the low efficiency and slow kinetics associated with the cellular reprogramming. Furthermore, the mechanisms engendering the rare conversion of somatic cells to pluripotency remain largely unidentified.  This elusive phenomenon has led to the search for additional factors that influence iPSC generation. To identify prospective mediators of reprogramming, we conducted a preliminary short hairpin RNA (shRNA) screen consisting of 23 chromatin remodelers; we observed that the knockdown (KD) of the Yin Yang 1 (YY1) gene enhanced reprogramming efficiency. This result led us to generate the hypothesis that YY1’s biological activities negatively regulate iPSC reprogramming. YY1 is a ubiquitously expressed transcription factor involved in a gamut of biological processes such as cell proliferation and differentiation, histone modification, and DNA-damage repair—thus to test our hypothesis, we selected downstream targets TGF-β, IFN-β, p21, and PARP-1: each representative of the signaling pathways specific to these YY1  biological functions. In the experiments reported here, we sought to characterize the YY1 effect on reprogramming by interrogating mRNA levels of TGF-β, IFN-β, p21, and PARP-1 in the context of a reprogramming time-point study. Our time-point dependent analyses were performed on shYY1 KD human fibroblasts (dH1f) as they were reprogrammed to iPS cells. Specifically, dH1f cells were first infected by shControl or by one of two experimental shYY1 lentiviral constructs. Infected dH1f cells were then divided into three time-point specific populations set to undergo RNA extraction at Day 0 (pre-reprogramming), Day 6 (mid-reprogramming) or Day 16 (end of reprogramming). Wells predetermined for Day 6 and Day 16 extraction were next infected with the OKSM cocktail, marking the commencement of the reprogramming process. Finally, RNA extracts were collected for each time-point and reverse transcripted to cDNA for q-PCR analysis. We found that, while YY1 knockdown resulted in the upregulation of p21 and IFN-β levels as well as the diminished expression of PARP-1, YY1 silencing showed a striking correlative with TGF- β in promoting its downregulation.  These results suggest that YY1 KD may be mediating its pro-pluripotency effects via the repression of the TGF- β signaling pathway. Our collective data provide preliminary insights into the mechanism by which YY1 regulates iPSC fate decisions.