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A Motherly Gift to Reprogrammers : Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1

From Nature
By Stuart P. Atkinson

Of the most widely used transcription factors in reprogramming experiments, MYC is often considered the “problem child”. Although MYC boosts the efficiency of induced pluripotent stem cell (iPSC) generation, it also increases the tumorigenic nature of the resultant cells thereby presenting clear cautionary implications for clinical use (Nakagawa et al). It is for this reason that studies have attempted to describe protocols for iPSC generation which remove MYC from the reprogramming cocktail or attempt to replace it with alternative reprogramming factors or small-molecules in order to generate “safer” iPSCs. Maekawa et al from the lab of Shinya Yamanaka now show that GLIS1, a GLI-related Kruppel-like zinc finger protein that functions both as an activator and repressor of transcription (Kim et al) and which is enriched in oocytes and 1-cell embryos, can effectively replace MYC in the reprogramming of mouse and human fibroblasts when used alongside OCT4, SOX2 and KLF4 (OSK), resulting in enhanced generation of fully pluripotent iPSCs. This study is published in Nature.

GLIS1 was discovered as a potent replacement for MYC in iPSC generation during a screening of human transcription factors using mouse fibroblasts with a GFP-reporter under the control of the Nanog promoter and enhancer. Alongside OSK, GLIS1 (OSKG) showed comparable abilities in the generation of GFP-positive colonies to MYC (OSKM), but GLIS1 preferentially promoted the generation of GFP-positive colonies and not GFP-negative colonies which represent partially reprogrammed colonies. iPSCs generated with OSKG were more similar to mouse embryonic stem cells (mESC) with regards to morphology, pluripotency marker expression, teratoma formation and they also produced germline-competent chimaeras. The effects of GLIS1 were mirrored when reprogramming human adult fibroblasts and additional DNA microarray analysis of resultant iPSCs showed that OSKG iPSCs where very similar to OSKM iPSCs. Also, in OSKG iPSCs the OCT4 promoter was hypo-methylated and cells were able to differentiate into cells indicative of each of the three germ layers. Further support for a role for GLIS1 in reprogramming was shown by the reduction of GFP-positive colonies generated by OSK when Glis1-levels were reduced by siRNA in mouse fibroblasts. Overall, these data show that GLIS1 can replace MYC in reprogramming with OSK and leads to the generation of fully pluripotent iPSCs.

But how does GLIS1 function? Microarray analysis demonstrated that Glis1 expression in OSK-mediated reprogramming of p53-knockout MEFs leads to an increase in genes which have previously been shown to enhance iPSC generation (Esrrb, Wnt3, 6, 8a and 10a, Lin28a, Nanog, Mycn and Mycl1), while Myc itself was shown to be down-regulated. The group have previously shown that Mycn and Mycl1 expression leads to an increase ESC-like colonies during reprogramming (Nakagawa et al), so the change in balance of Myc-family members expression could lead to the increase efficiency of GLIS1 over MYC observed in this study. Further, Foxa2 expression was increased, a factor known to antagonise the epithelial-to-mesenchymal transition which is required for iPSC generation (Samavarchi-Tehrani et al and Li et al). ChIP analysis showed that only Mycn, Mycl1 and Myc were direct targets of Glis1, suggesting both direct and indirect effects for Glis1. Finally, protein-protein interaction analyses showed that Glis1 physically associates with Oct4, Sox2 and Klf4 and may have its effects through the promotion of gene activation as part of a larger complex.

The use GLIS1, a factor highly expressed in cells with a very high developmental potential, links what we know about early development to the reprogramming process, a process which we are still attempting to delineate. Perhaps further studies of the earliest stages of reprogramming will uncover new transcription factors or other proteins which may make the generation of iPSCs a safer and easier undertaking.



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Li R, Liang J, Ni S, Zhou T, Qing X, Li H, He W, Chen J, Li F, Zhuang Q, Qin B, Xu J, Li W, Yang J, Gan Y, Qin D, Feng S, Song H, Yang D, Zhang B, Zeng L, Lai L, Esteban MA, Pei D.
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