A complete knockout of a single key pluripotency gene may drastically affect embryonic stem cell function and epigenetic reprogramming. In contrast, elimination of only one allele of a single pluripotency gene is mostly considered harmless to the cell. To understand whether complex haploinsufficiency exists in pluripotent cells, we simultaneously eliminated a single allele in different combinations of two pluripotency genes (i.e., Nanog+/−;Sall4+/−, Nanog+/−;Utf1+/−, Nanog+/−;Esrrb+/− and Sox2+/−;Sall4+/−). Although these double heterozygous mutant lines similarly contribute to chimeras, fibroblasts derived from these systems show a significant decrease in their ability to induce pluripotency. Tracing the stochastic expression of Sall4 and Nanog at early phases of reprogramming could not explain the seen delay or blockage. Further exploration identifies abnormal methylation around pluripotent and developmental genes in the double heterozygous mutant fibroblasts, which could be rescued by hypomethylating agent or high OSKM levels. This study emphasizes the importance of maintaining two intact alleles for pluripotency induction.
Bibliographical noteFunding Information:
Y.B. is supported by research grants from EMBO Young Investigator Programme (YIP), Howard Hughes Medical Institute International Research Scholar (HHMI, # 55008727 ), Israel Science Foundation (ISF, 161/23 ), and by a generous gift from Ms. Nadia Guth Biasini. We thank Yuval Nevo and huji core bioinformatics unit for analyzing part of the RNA-seq data.
© 2023 The Author(s)
- knockin/knockout targeting approach
- nuclear transfer
- pluripotent stem cells
- reporter genes
- stochastic expression
- tracing system