Post-transcriptional gene expression control by NANOS is up-regulated and functionally important in pRb-deficient cells

Abstract

Inactivation of the retinoblastoma tumor suppressor (pRb) is a common oncogenic event that alters the expression of genes important for cell cycle progression, senescence, and apoptosis. However, in many contexts, the properties of pRb-deficient cells are similar to wild-type cells suggesting there may be processes that counterbalance the transcriptional changes associated with pRb inactivation. Therefore, we have looked for sets of evolutionary conserved, functionally related genes that are direct targets of pRb/E2F proteins. We show that the expression of NANOS, a key facilitator of the Pumilio (PUM) post-transcriptional repressor complex, is directly repressed by pRb/E2F in flies and humans. In both species, NANOS expression increases following inactivation of pRb/RBF1 and becomes important for tissue homeostasis. By analyzing datasets from normal retinal tissue and pRb-null retinoblastomas, we find a strong enrichment for putative PUM substrates among genes de-regulated in tumors. These include pro-apoptotic genes that are transcriptionally down-regulated upon pRb loss, and we characterize two such candidates, MAP2K3 and MAP3K1, as direct PUM substrates. Our data suggest that NANOS increases in importance in pRb-deficient cells and helps to maintain homeostasis by repressing the translation of transcripts containing PUM Regulatory Elements (PRE).

Keywords: Nanos; Pumilio; pRb; post‐transcriptional gene regulation; stress response.

Figure 1. E2F/pRB regulate the expression of the Pumilio complex

1. ChIP-Chip from Drosophila larvae of RBF1, E2F2, E2F1, and IgG controls on the nanos, pumilio, and brat promoters.

2. RT-PCR from ChIP of IgG, E2F1, Rbf1, and E2F2 from wild-type (w¹¹¹⁸) Drosophila larvae surrounding the transcription start site of actin, brat, pumilio, and nanos (mean ± SD, n = 3).

3. RT-PCR of pumilio (pum), brat, and nanos expression from adult females expressing UAS-RNAi constructs targeting Gal4, e2f1, dp, e2f2, and rbf1 (mean ± SD, n = 3).

4. RT-PCR results of PUM components (PUM1, PUM2, NANOS1, NANOS2, and NANOS3), E2F target (Cyc A) and non-E2F target (E2F3) from BJ fibroblast cells transfected with siRNA pools targeting the pocket proteins (Rb1, p107, and p130) (mean ± SD, n = 3).

5. Correlation of the expression of the PUM components with Rb1 in cancer cell lines from the Sanger cancer cell line encyclopedia database.

6. RT-PCR results of NANOS1 expression in normal human retina, primary retinoblastoma tumors, and retinoblastoma tumor cells grown as orthotopic xenografts (mean ± SD, n = 3).

Figure 2. The Pumilio and dREAM complexes genetically interact

1. Phenotypes produced from genetic interaction experiments using RNAi driven by Nub-Gal4 to reduce the levels of dREAM components (e2f2, mip120, mip130, and rbf1) and the pumilio complex members (pum, nos, and brat) in the Drosophila wing pouch. RNAi constructs used in this experiment were PUM RNAi (36676), NANOS RNAi (28300), BRAT RNAi, and luciferase RNAi as a control. Genetic interaction analysis of wings was scored as follows: no phenotype (−), variable minor phenotype (−/+), minor extra wing vein (+), minor blistering (++), and severely blistered and deformed wings (+++).

2. Table outlining the genetic interaction between PUM complex members and dREAM components.

Figure 3. NANOS1 is required for the maintenance of pRb-deficient cells

1. Crystal violet staining of wild-type (wt), Rb1 null (Rb1^−/−), p107 null (p107^−/−), and p130 null (p130^−/−) 3T3 cells infected with shRNAs targeting Nanos1 (Nanos1-3, Nanos1-4), Pum1 (mP1-2, mP1-3), Pum2 (mP2-1, mP2-5), or scrambled sequence (Scr).

2. Quantification of Alamar blue staining of human Y79 retinoblastoma cells after puromycin selection of uninfected (mock) and cells infected with shRNAs targeting NANOS1 or scrambled sequences (Scr) (mean ± SD, n = 3).

3. Crystal violet stain of BJ cells infect with shRNAs targeting scrambled (Scr), pRb (Rb1 E3, Rb1 D4, Rb1 D5), and NANOS1 (hNOS1-1 and hNOS1-3).

4. Quantification of crystal violet staining of BJ cells infected with shRNAs targeting Rb1, NANOS1, and scrambled sequences, including analysis of cell transfected with pCMV-Rb1 post-infection (mean ± SD, n = 3).

Figure 4. p53 activity is necessary for the reduction in cell number upon silencing of Rb1 and NANOS1

1. Crystal violet staining of cancer cell lines from diverse tissue types (fibroblast, retina, head and neck, kidney, bone, bladder, breast, non-small cell lung carcinoma (NSCLC), and colorectal) infected with shRNAs targeting NANOS1 (hNOS1-1 and hNOS1-3) or scrambled sequences.

2. Summary table of the pRb and p53 status of the cells tested in the panel above and a description of the consequence of NANOS1 and scrambled sequence depletion.

3. Crystal violet staining of isogenic HCT116 cells with (p53 wt) and without (p53 mut) p53 infected with shRNAs targeting NANOS1.

Figure 5. Analysis of gene expression changes in retinoblastoma tumors and normal retina tissue

1. Schematic representing the number of the E2F-regulated genes in the PRE transcripts from retinoblastoma tumors.

2. Gene ontology of PUM and non-PUM substrates which are up- and down-regulated in retinoblastoma tumors (Ganguly & Shields, 2010).

Figure 6. NANOS1 post-transcriptionally inhibits Map3K1 and Map2K3 levels preventing p38 activation

1. Schematic of MAP2K3 and MAP3K1 3′UTRs detailing PRE and NRE positions.

2. Relative luciferase levels testing how mutations within the PRE and NREs affect the control of the Map2K3 3′UTR on a downstream luciferase report construct transfected into HCT116 p53 mut cells (mean ± SD, n = 3).

3. Relative luciferase levels testing the affect of depleting PUM1 (PUM1-15, PUM1-19), PUM2 (PUM2-1, PUM2-4), and NANOS1 (NOS1-1, NOS1-3) on luciferase constructs containing the MAP2K3 3′UTR, PRE mutant 3′UTR (PRE mut), or triple NRE mutant 3′UTR (NRE all) (mean ± SD, n = 3).

4. Western blots from HCT116 p53 mut cells of MAP3K1, MAP2K3, NANOS1 (NOS1), and tubulin (TUB) after treatment with shRNAs targeting NANOS1 (N1-1, N1-3) and scrambled sequences (Scr).

5. Western blots from HCT116 p53 mut cells of p38 and phospho-p38 (Pho-p38) after treatment with shRNAs targeting NANOS1 (N1-1, N1-3) and scrambled sequences (Scr).

6. Schematic depicting the model of how NANOS1 contributes to the survival of Rb1-deficient cells by preventing the activation of p53-mediated apoptosis.

Source data are available online for this figure.