The Ink4/Arf locus is a barrier for iPS reprogramming

By Han Li, M. Collado, Aranzazu Villasante, K. Strati, Sagrario Ortega, M. Cañamero, M. Blasco, Manuel Serrano. Nature, 2009

972 citations recorded by the source corpus.

The mechanisms involved in the reprogramming of differentiated cells into induced pluripotent stem (iPS) cells by the three transcription factors Oct4 (also known as Pou5f1), Klf4 and Sox2 remain poorly understood. The Ink4/Arf locus comprises the Cdkn2a–Cdkn2b genes encoding three potent tumour suppressors, namely p16Ink4a, p19Arf and p15Ink4b, which are basally expressed in differentiated cells and upregulated by aberrant mitogenic signals. Here we show that the locus is completely silenced in iPS cells, as well as in embryonic stem (ES) cells, acquiring the epigenetic marks of a bivalent chromatin domain, and retaining the ability to be reactivated after differentiation. Cell culture conditions during reprogramming enhance the expression of the Ink4/Arf locus, further highlighting the importance of silencing the locus to allow proliferation and reprogramming. Indeed, the three factors together repress the Ink4/Arf locus soon after their expression and concomitant with the appearance of the first molecular markers of ‘stemness’. This downregulation also occurs in cells carrying the oncoprotein large-T, which functionally inactivates the pathways regulated by the Ink4/Arf locus, thus indicating that the silencing of the locus is intrinsic to reprogramming and not the result of a selective process. Genetic inhibition of the Ink4/Arf locus has a profound positive effect on the efficiency of iPS cell generation, increasing both the kinetics of reprogramming and the number of emerging iPS cell colonies. In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming by activation of p53 (encoded by Trp53) and p21 (encoded by Cdkn1a); whereas, in human fibroblasts, INK4a is more important than ARF. Furthermore, organismal ageing upregulates the Ink4/Arf locus and, accordingly, reprogramming is less efficient in cells from old organisms, but this defect can be rescued by inhibiting the locus with a short hairpin RNA. All together, we conclude that the silencing of Ink4/Arf locus is rate-limiting for reprogramming, and its transient inhibition may significantly improve the generation of iPS cells.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/39c12b79c4c363dce72858d5f2cd931ded5f40ab

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Citations in the corpus

Senescence impairs successful reprogramming to pluripotent stem cells. (2009, background): “Fibroblasts from older mice, which express high levels of the Ink4b/Arf/Ink4a locus products, are less efficient in generating iPS cells (Li et al., 2009), further linking ageing with decreased reprogramming efficiency.”
Direct cell reprogramming is a stochastic process amenable to acceleration (2009, background): “, Lin28 modulates cell growth and associates with a subset of cell cycle regulator mRNAs in mouse embryonic stem cells. RNA 15 (3), 357-361 (2009).”
Induced pluripotency: history, mechanisms, and applications. (2010, background): “…have also been derived from a number of different species—including humans (Takahashi et al. 2007; Yu et al. 2007; Park et al. 2008b), rats (W Li et al. 2009a), and rhesus monkeys (Liu et al. 2008)—by expression of the four Yamanaka factors (Table 2), demonstrating that fundamental…”
Cellular senescence and tumor suppressor gene p16 (2011, background): “Oncogene-mediated premature senescence is a tumor suppressor mechanism that has been well described in the RAS activated MAP kinase pathway.(19,91,92) The Ets family of tran-”
The essence of senescence. (2010, background): “…and p53 limit the efficiency of iPS cell formation, it has been suggested that cellular senescence counteracts the induced conversion of primary cells into pluripotent stem cells (Banito et al. 2009; Hong et al. 2009; Kawamura et al. 2009; Li et al. 2009; Marión et al. 2009; Utikal et al. 2009).”
Cellular senescence and its effector programs (2014, background): “Collectively, they suggest that senescence can be a barrier to the reprogramming to generate iPS cells from fibroblasts (Banito et al. 2009; Hong et al. 2009; Kawamura et al. 2009; Li et al. 2009; Marión et al. 2009; Utikal et al. 2009).”
Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. (2010, result): “While our work was in progress, six independent laboratories identified cell senescence as a roadblock for reprogramming (Hong et al., 2009; Kawamura et al., 2009; Li et al., 2009; Marión et al., 2009; Utikal et al., 2009; Zhao et al., 2008), creating significant interest in finding compounds that a”
Epigenetic memory in induced pluripotent stem cells (2010, methodology): “Hematopoietic progenitors and fibroblasts yielded a comparable frequency of reprogrammed colonies (0.02%), which consistent with prior reports 5 , was lower than the yield from fibroblasts of a juvenile mouse (0.1%).”
EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer (2010, background): “…manipulating epithelial plasticity is reinforced by studies showing that depletion of Zeb1, either chemically or by RNA interference, in mesenchymal-like cells results in a partial epithelial metaplasia and either gain of K-Ras dependency or drug sensitivity (Singh et al., 2009; Li et al., 2009b).”
Cellular Senescence: Aging, Cancer, and Injury. (2019, background): “Owing to INK4A/ARF forming a barrier to reprogramming in vitro, it has been suggested that transient silencing of the locus during cellular reprogramming could be an effective approach for regenerative medicine in old individuals without increasing risk of malignancy (204).”