The Ink4/Arf locus is a barrier for iPS cell reprogramming

Abstract

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 p16(Ink4a), p19(Arf) and p15(Ink4b), 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.

Figure 1. Functional reprogramming of the Ink4/Arf locus

a. Expression of the indicated genes in iPS compared with their parental MEFs and with ES cells measured by quantitative RT-PCR. b. Epigenetic marks present at the indicated promoters. Sequential ChIP, first of H3K27me3 and then of H3K4me3, is in the leftmost panel. Data correspond to the average ± s.d. of a representative assay from at least 2 independent assays. c. Expression of the indicated genes in iPS and in ES cells undergoing differentiation by addition of retinoic acid in the absence of LIF. Data correspond to the average ± s.d. of at least 2 independent assays. d. Re-expression of Arf in a teratoma developed in a chimeric-iPS mouse detected by immunohistochemistry.

Figure 2. Silencing of the Ink4/Arf locus during reprogramming

a. Experimental layout and day numbering. b. Kinetics of expression of the Ink4/Arf locus in mock-infected (mock) and 3F-infected (3F) MEFs, measured by quantitative RT-PCR. c. Repression of Ink4a and Arf during 3F-reprogramming of MEFs expressing large-T (MEF-LT+3F), measured by qRT-PCR. Data correspond to the average ± s.d. of at least 2 independent assays.

Figure 3. Impact of Ink4a/Arf on reprogramming efficiency

a. Reprogramming efficiencies of MEFs of the indicated genotypes relative to wt MEFs. Data correspond to the average ± s.e.m.; n, independent assays with different MEF isolates. b. Fold change of reprogramming efficiency of primary wt MEFs retrovirally infected with 3F plus empty vector (e.v.) or the indicated shRNAs. Data correspond to the average ± s.d. Protein levels were analyzed 48 h after infection. c. Fold change of reprogramming efficiency measured in newborn keratinocytes of the indicated genotypes. d. Kinetics of expression of pluripotency markers AP and SSEA1 during 3F-reprogramming, measured by FACS. e. Representative images of colonies at days 7 and 14. f. Schematic representation of the kinetics of Ink4/Arf locus suppression and marker expression during reprogramming. g. Reprogramming efficiencies of human diploid fibroblasts IMR90/hTERT using 3F or 4F plus the indicated shRNAs. Data correspond to the average ± s.d. The right panels show representative iPS colonies.

Figure 4. Association between age of the parental cells, expression of the Ink4/Arf locus and reprogramming efficiency

a. Expression of the Ink4/Arf locus in mouse skin fibroblasts (MSF) from 2-month old (young) or 2-year old (old) mice, compared to MEF, iPS and ES cells, measured by qRT-PCR. b. Reprogramming efficiencies of old MSFs by 3F plus or minus shInk4a/Arf. Data correspond to the average ± s.d. Statistical significance: **p < 0.01.