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. 2015 Feb 20;6(5):2992-3002.
doi: 10.18632/oncotarget.2996.

Transcriptional regulation of Sox2 by the retinoblastoma family of pocket proteins

Jéssica M Vilas  1 , Alba Ferreirós  1 , Carmen Carneiro  2 , Lluis Morey  3 , Sabela Da Silva-Álvarez  1 , Tânia Fernandes  2 , María Abad  4 , Luciano Di Croce  3   5 , Tomás García-Caballero  6 , Manuel Serrano  4 , Carmen Rivas  7   8 , Anxo Vidal  2 , Manuel Collado  1
Affiliations

Transcriptional regulation of Sox2 by the retinoblastoma family of pocket proteins

Jéssica M Vilas et al. Oncotarget. .

Abstract

Cellular reprogramming to iPSCs has uncovered unsuspected links between tumor suppressors and pluripotency factors. Using this system, it was possible to identify tumor suppressor p27 as a repressor of Sox2 during differentiation. This led to the demonstration that defects in the repression of Sox2 can contribute to tumor development. The members of the retinoblastoma family of pocket proteins, pRb, p107 and p130, are negative regulators of the cell cycle with tumor suppressor activity and with roles in differentiation. In this work we studied the relative contribution of the retinoblastoma family members to the regulation of Sox2 expression. We found that deletion of Rb or p130 leads to impaired repression of Sox2, a deffect amplified by inactivation of p53. We also identified binding of pRb and p130 to an enhancer with crucial regulatory activity on Sox2 expression. Using cellular reprogramming we tested the impact of the defective repression of Sox2 and confirmed that Rb deficiency allows the generation of iPSCs in the absence of exogenous Sox2. Finally, partial depletion of Sox2 positive cells reduced the pituitary tumor development initiated by Rb loss in vivo. In summary, our results show that Sox2 repression by pRb is a relevant mechanism of tumor suppression.

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Figures

Figure 1
Figure 1. Cells lacking Rb or p130 express higher levels of Sox2
a, Sox2 (left) or Nanog (right) mRNA levels in wt, Rb-null, p107-null, and p130-null primary MEFs as assessed by TaqMan expression analysis. Absolute values are referenced to the levels obtained with primary wt cells. b, Sox2 mRNA levels in wt, RB-null, p107-null, and p130-null immortalized fibroblasts assessed as in (a). Absolute values are referenced to the levels obtained with wt cells. c, Sox2 mRNA (upper panel) and protein expression by Western blot (lower panel) in cell extracts from wt and SV40 large-T antigen (LT) immortalized fibroblasts. Absolute values are referenced to the levels obtained with wt cells. d, Graph showing the analysis of GFP expression by flow cytometry from EOS pluripotency reporter plasmid introduced in reprogrammable primary (i4F-MEFs, black line) or immortalized (i4F-MEFs-shp53, green line) cells (upper panel). The settings were previously adjusted to consider i4F-MEFs without EOS plasmid as GFP negative. Sox2 mRNA levels expressed by wt or immortalized wt-shp53 MEFs, i4F or immortalized i4F-shp53 MEFs, and ESCs, measured by qRT-PCR (lower panel). Values are referenced to the levels obtained using wt primary MEFs and in log10 scale. All data correspond to the average ± s.d. of qRT-PCR data. Statistical significance was assessed by the two-tailed Student's t-test: *** p < 0.001; ** p < 0.01; * p < 0.05; n.s. non significant.
Figure 2
Figure 2. Binding of the Rb family of pocket proteins to the Sox2-SRR2 enhancer and effect of their absence on histone marks
a, Chromatin immunoprecipitation (ChIP) assay using antibodies against p107, p130, and pRb followed by semi-quantitative PCR using primers amplifying the Sox2-SRR2. Primers amplifying β-actin promoter and IgG were used as negative controls. b, ChIP of repressive H3K27me3 and active H3K4me3 histone marks in the Sox2-SRR2 enhancer of wt, Rb-null, and p130-null MEFs, using two different sets of primers amplifying the Sox2-SRR2 enhancer (upper and middle panels). Control ChIP assay using primers amplifying Nanog promoter is shown at the bottom panel. All data correspond to the average ± s.d. of qPCR data. Statistical significance was assessed by the two-tailed Student's t-test: *** p < 0.001; ** p < 0.01; * p < 0.05; n.s. non significant.
Figure 3
Figure 3. Absence of Rb allows two-factor (Oct4 and Klf4) reprogramming
a, Representative pictures of iPSC colonies expressing GFP from the EOS pluripotency reporter plasmid (top panels), and stained for alkaline phosphatase (AP, bottom panels). Shown are iPSC colonies obtained in wt MEFs after three-factor expression (Oct4, Klf4, and Sox2; 3F-OKS; left panel), and Rb-null MEFs after three-factor (3F-OKS; middle panel) or two-factor (Oct4, Klf4; 2F-OK; right panel) expression. b, Pluripotency factor (Oct4, Sox2, Klf4, and Nanog) mRNA expression by qRT-PCR in iPSCs obtained from wt primary MEFs reprogrammed by 3F-OKS, or Rb-null with 3F-OKS or 2F-OK. Null expression from MEFs is shown as negative control, and expression in ESCs as positive control. c, Western blot analysis of the expression of pluripotency factors (Oct4, Sox2, and Nanog) in the same set of cells as in (b). d, Representative pictures of embryoid bodies (EBs) obtained after in vitro spontaneous differentiation of iPSCs generated from wt primary MEFs reprogrammed by 3F-OKS, or Rb-null with 3F-OKS or 2F-OK. e, Differentiation factor (Nkx2.5, Dlx3, and Gata4) mRNA expression by qRT-PCR in EBs obtained from iPSCs generated from Rb-null primary MEFs reprogrammed by 2F-OK. Values are referred to the expression obtained for the corresponding iPSCs. All data correspond to the average ± s.d. of qRT-PCR data. Statistical significance was assessed by the two-tailed Student's t-test: *** p < 0.001; ** p < 0.01; * p < 0.05. f, Representative pictures of H&E stained sections of EBs obtained from iPSCs generated from Rb-null primary MEFs reprogrammed by 2F-OK, and embedded in paraffin. Pictures show examples of ectodermal, endodermal and mesodermal differentiation.
Figure 4
Figure 4. In vivo effect of partial depletion of Sox2 positive cells in Rb+/− mice
a, Sox2 mRNA levels in the pituitary of wt (n=4) and Rb-het (n=4) mice. b, Schematic representation of the experimental protocol of gancyclovir (GCV) treatment of compound Rb-het/Sox2-TK mice (upper panel). Intraperitoneal injections were administered at the times indicated (weeks) and mice were sacrificed when they were 42 weeks old. Pituitary mass (n=4 for HBSS; n=3 for GCV) of Rb-het/Sox2-TK mice (lower panel). Means ± s.e.m. are shown. c, Representative pictures of paraffin sections from control HBSS-treated (upper panels) or GCV-treated (lower panels) tissues from Rb-het/Sox2-TK mice. The first three rows of pictures correspond to pituitary sections stained for H&E, proliferative marker Ki67 and Sox2, respectively. The two rows of pictures at the most right part correspond to control tissues, esophagus and trachea, stained for Sox2. Below is shown the quantification of Ki67 and Sox2 positive cells.
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