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

Han Li ¹, Manuel Collado ¹, Aranzazu Villasante ¹, Katerina Strati ², Sagrario Ortega ³, Marta Cañamero ⁴, Maria A. Blasco ² and Manuel Serrano ¹

¹ Tumor Suppression Group,
² Telomeres and Telomerase Group,
³ Transgenic Mice Unit,
⁴ Comparative Pathology Unit, Spanish National Cancer Research Centre (CNIO), 3 Melchor Fernandez Almagro Street, Madrid E-28029, Spain

Correspondence to: Manuel Serrano ¹ Correspondence and requests for materials should be addressed to M.S. (Email: mserrano@cnio.es).

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 understood1. 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 signals2, 3, 4. 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 locus2, 5 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 cells compared with their parental MEFs and with ES cells measured by quantitative reverse transcription PCR (qRT–PCR).

b, Epigenetic marks present at the indicated promoters. Sequential ChIP, first of H3K27me3 and then of H3K4me3, is in the right panel. Data correspond to the average and s.d. of a representative assay from at least two independent assays.

c, Expression of the indicated genes in iPS and ES cells undergoing differentiation by the addition of retinoic acid and in the absence of LIF. Data correspond to the average and s.d. of at least two independent assays.

d, Re-expression of p19Arf in a teratoma developed by a chimaeric-iPS-cell mouse and 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 three-factor-infected (3F) MEFs, measured by qRT–PCR.

c, Repression of Ink4a and Arf during three-factor-reprogramming of MEFs expressing large-T (MEF-LT + 3F), measured by qRT–PCR. Data correspond to the average and s.d. of at least two independent assays.

FIGURE 3. Effect of Ink4a/Arf on reprogramming efficiency.

a, Reprogramming efficiencies of MEFs of the indicated genotypes relative to wild-type (WT) MEFs. Data correspond to the average and s.e.m.; n = independent assays with different MEF isolates. 3F, three factors; 4F, four factors.

b, Fold change of reprogramming efficiency of primary wild-type MEFs retrovirally infected with three factors plus empty vector (EV) or the indicated shRNAs. Data correspond to the average and s.d. Protein levels were analysed 48 h after infection.

c, Fold change of reprogramming efficiency measured in newborn keratinocytes (kerat.) of the indicated genotypes. Tissue culture plate diameters are indicated.

d, Kinetics of expression of pluripotency markers alkaline phosphatase (AP) and SSEA1 during three-factor-reprogramming, measured by FACS.

e, Representative images of colonies at days (D) 7 and 14. Original magnification, 20.

f, Schematic representation of the kinetics of Ink4/Arf locus suppression and marker expression during reprogramming.

g, Reprogramming efficiencies of human diploid fibroblasts IMR90-TERT using three or four factors plus the indicated shRNAs. Data correspond to the average and s.d. The right panels show representative iPS cell colonies. Original magnification, 20.

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 MSFs from 2-month-old (young) or 2-year-old (old) mice, compared to MEFs, iPS and ES cells, measured by qRT–PCR.

b, Reprogramming efficiencies of old MSFs by three factors plus or minus Ink4a/Arf shRNA. Data correspond to the average and s.d. **P < 0.01.

References:

1. Krizhanovsky V, and Lowe SW,
"The promises and perils of p53".

2. Marión RM, Strati K, Li H, Murga M, Blanco R, Ortega S, Fernandez-Capetillo O, Serrano M, Blasco MA.
"A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity".

3. Utikal J, Polo JM, Stadtfeld M, Maherali N, Kulalert W, Walsh RM, Khalil A, Rheinwald JG, and Hochedlinger K.
"Immortalization eliminates a roadblock during cellular reprogramming into iPS cells".

Additional References:

1. Frenster JH, and Hovsepian JA,  (October, 2007c)
 “Models of Embryonic Gene-Induced Initiation and Reversion of Adult Neoplasms”.

2. Berx G, Raspe E, Christfori G, Thiery JP, and Sleeman JP, (November, 2007)
"Pre-EMTing metastasis? Recapitulation of morphogenetic processes in cancer".
Clin Exp Metastasis, 2007;24(8):587-97, Epub 2007 Nov3.

3. Sarrió D, Rodriguez-Pinilla SM, Hardisson D, Cano A, Moreno-Bueno G, and Palacios J,  (Feb. 2008)
"Epithelial-Mesenchymal Transition in Breast Cancer Relates to the Basal-like Phenotype",
Cancer Research 68, 989-997, February 15, 2008.

4. Kumar MS, Erkeland SJ, Pester RE, Chen CY, Ebert MS, Sharp PA, and  Jacks T., (March, 2008)
"Suppression of non-small cell lung tumor development by the let-7 microRNA family".

5. Haigis KM, Kendall KR, Wang Y, Cheung A, Haigis MC, Glickman JN, Niwa-Kawakita M,  Sweet-Cordero A, Sebolt-Leopold J, Shannon KM, Settleman J, Giovannini M,  and  Jacks T.  (March 30, 2008)
"Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon".
Nature Genetics 40, 600 - 608 (2008). Published online: 30 March 2008 | doi:10.1038/ng.115)

6. Boyerinas B, Park S-M, Shomron N, Hedegaard MM, Vinther J, Andersen JS, Feig C, Xu J,  Burge CB, and Peter ME,  (April, 15, 2008)
"Identification of Let-7–Regulated Oncofetal Genes",
Cancer Research vol. 68, no. 8, pp. 2587-2591  (April 15, 2008).

7. Marcucci G, Radmacher MD, Maharry K, Mrózek K, Ruppert AS, Paschka P, Vukosavljevic T, Whitman SP, Baldus CD, Langer C, Liu C-G, Carroll AJ, Powell BL, Garzon R, Croce CM, Kolitz JE, Caligiuri MA, Larson RA, and Bloomfield CD,  (May 1, 2008)
"MicroRNA Expression in Cytogenetically Normal Acute Myeloid Leukemia",
New England Journal of Medicine vol. 358: no. 18, pp. 1919-1928 May 1, 2008.)

8. Mani SA, Guo W, Liao M-J, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F,  Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, and Weinberg RA,  (May 16, 2008)
"The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells".
Cell, vol: 133, pp. 704-715 (May 16,  2008).

Further Topics in:  Euchromatin,  active DNA, and  RNA  ribo-regulators:

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A Brief History of Activator RNA:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".
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