Okita, K. | Search bioRxiv

bioRxiv2022

B1 SINE-binding ZFP266 impedes reprogramming through suppression of chromatin opening by pioneering factors

Induced pluripotent stem cell reprogramming is inherently inefficient and understanding the molecular mechanisms underlying this inefficiency holds the key to successfully control cellular identity. Here, we report 16 novel reprogramming roadblock genes identified by CRISPR/Cas9-mediated genome-wide knockout (KO) screening. Of these, depletion of the predicted KRAB zinc finger protein (KRAB-ZFP) Zfp266 strongly and consistently enhanced iPSC generation in several iPSC reprogramming settings, emerging as the most robust roadblock. Further analyses revealed that ZFP266 binds Short Interspersed Nuclear Elements (SINEs) adjacent to binding sites of pioneering factors, OCT4 (POU5F1), SOX2 and KLF4, and impedes chromatin opening. Replacing the KRAB co-suppressor with a co-activator domain converted ZFP266 from a reprogramming inhibitor to a potent reprogramming facilitator. This work proposes SINE-KRAB-ZFP interaction to be a critical regulator of chromatin accessibility at enhancers for efficient cellular identity changes and also serves as a resource to further illuminate molecular mechanisms hindering reprogramming.

cell biology↗

bioRxiv2021

Generation of iPSCs from endangered Grevy's zebra and comparative transcriptomic analysis of mammalian PSCs

Induced pluripotent stem cells (iPSCs) can provide a biological resource for functional and conservation research in various species. This expectation has led to generation of iPSCs from various species, including those identified as endangered species. However, the understanding of species variation in mammalian iPSCs is largely unknown. Here, to gain insight into the species variation in iPSCs, we the first generated iPSCs from the endangered species Grevys zebra (Equus grevyi; gz-iPSCs) for the first time in the world. We isolated primary fibroblasts cell from an individual that had died of natural causes at a zoo and reprogrammed the fibroblasts into iPSCs. We confirmed their pluripotency and differentiation potential and performed RNA sequencing analysis. The gz-iPSC transcriptome showed that the generated gz-iPSCs robustly expressed genes associated with pluripotency and reprogramming processes, including epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions. Comparative transcriptomics with other species revealed patterns of gene expression among mammalian PSCs and detected evolutionary conservation of pluripotency-associated genes and the plausible importance of the translation process. This study provides new insights into the evolution of mammalian PSCs, and the species conservation and variation of PSCs will advance our understanding of the early development of mammals.

cell biology↗