p53-directed translational control can shape and expand the universe of p53 target genes

Zaccara, Sara; Tebaldi, Toma; Pederiva, Chiara; Ciribilli, Yari; Bisio, Alessandra; Inga, Alberto

doi:10.1038/cdd.2014.79

Cited by 52 publications

(65 citation statements)

References 60 publications

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“…5A). However, we also noted concomitant changes in gene pathways associated with T cell survival, in particular the upregulation of cells that promote cell death, including Bax, Fas, and Casp6 (p53 pathway) and Cabin1 and Mef2 (Nur77 signaling pathway) (71) (Supplementary Table 1). In addition, disruption of cell survival mechanisms was further shown by the down-regulation of BCL-2 (Fig.…”

Section: Resultsmentioning

confidence: 81%

Effect of Anti–IL-15 Administration on T Cell and NK Cell Homeostasis in Rhesus Macaques

DeGottardi

Okoye

Vaidya

et al. 2016

The Journal of Immunology

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IL-15 has been implicated as a key regulator of T and NK cell homeostasis in multiple systems; however, its specific role in maintaining peripheral T and NK cell populations relative to other gamma-chain (γc) cytokines has not been fully defined in primates. Here, we address this question by determining the effect of IL-15 inhibition with a rhesusized, anti-IL-15 mAb on T and NK cell dynamics in rhesus macaques. Strikingly, anti-IL-15 treatment resulted in rapid depletion of NK cells, and both CD4+ and CD8+ effector memory T cells (TEM) in blood and tissues, with little to no effect on naïve or central memory T cells. Importantly, whereas depletion of NK cells was nearly complete and maintained as long as anti-IL-15 treatment was given, TEM depletion was countered by the onset of massive TEM proliferation, which almost completely restored circulating TEM numbers. Tissue TEM, however, remained significantly reduced, and most TEM maintained very high turnover throughout anti-IL-15 treatment. In the presence of IL-15 inhibition, TEM became increasingly more sensitive to IL-7 stimulation in vivo, and transcriptional analysis of TEM in IL-15-inhibited monkeys revealed engagement of the JAK/STAT signaling pathway, suggesting alternative γc cytokine signaling may support TEM homeostasis in the absence of IL-15. Thus, IL-15 plays a major role in peripheral maintenance of NK cells and TEM. However, whereas most NK cell populations collapse in the absence of IL-15, TEM can be maintained in the face of IL-15 inhibition by the activity of other homeostatic regulators, most likely IL-7.

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Section: Resultsmentioning

confidence: 81%

Effect of Anti–IL-15 Administration on T Cell and NK Cell Homeostasis in Rhesus Macaques

DeGottardi

Okoye

Vaidya

et al. 2016

The Journal of Immunology

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“…Polysomal fractions were prepared as described previously (Zaccara et al 2014). Total RNA was extracted using TRI Reagent, according to the manufacturer's instructions.…”

Section: Rna-seqmentioning

confidence: 99%

Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity

et al. 2017

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The tumor suppressor TP53 is the most frequently mutated gene product in human cancer. Close to half of all solid tumors carry inactivating mutations in the TP53 gene, while in the remaining cases, TP53 activity is abrogated by other oncogenic events, such as hyperactivation of its endogenous repressors MDM2 or MDM4. Despite identification of hundreds of genes regulated by this transcription factor, it remains unclear which direct target genes and downstream pathways are essential for the tumor suppressive function of TP53. We set out to address this problem by generating multiple genomic data sets for three different cancer cell lines, allowing the identification of distinct sets of TP53-regulated genes, from early transcriptional targets through to late targets controlled at the translational level. We found that although TP53 elicits vastly divergent signaling cascades across cell lines, it directly activates a core transcriptional program of ∼100 genes with diverse biological functions, regardless of cell type or cellular response to TP53 activation. This core program is associated with high-occupancy TP53 enhancers, high levels of paused RNA polymerases, and accessible chromatin. Interestingly, two different shRNA screens failed to identify a single TP53 target gene required for the anti-proliferative effects of TP53 during pharmacological activation in vitro. Furthermore, bioinformatics analysis of thousands of cancer genomes revealed that none of these core target genes are frequently inactivated in tumors expressing wild-type TP53. These results support the hypothesis that TP53 activates a genetically robust transcriptional program with highly distributed tumor suppressive functions acting in diverse cellular contexts.

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“…34,77 RNA associated with 2 or more ribosomes, labeled as POL fraction, were collected and analyzed separately from RNA co-sedimenting with individual ribosomal subunits or with the 80S, labeled as SUB fraction. Fractions were monitored for RNA content recovered after ultracentrifugation using UVC scanning with a Teledyne Isco system (Isco, Inc.., Lincoln, Nebraska, USA).…”

Section: Sucrose Gradient Fractionation Of Cytoplasmic Cell Lysates Amentioning

confidence: 99%

“…SDS-PAGE was performed as previously described using the following antibodies: PTC1-L (patched, G-19 sc-6149 from Santa Cruz), PCNA (F-2, Santa Cruz), b-tubulin (3F3-G2, Santa Cruz) and the ECL select reagent (Amersham, GE-Health Care, Milan, Italy) with a ChemiDoc XRSC documentation system with ImageLab software (BioRad, Milan, Italy). 77 Statistical analysis Differences in luciferase activity and mRNA expression between various plasmids were analyzed by one-way analysis of variance (ANOVA), followed by Tukey's post-hoc test for multiple comparisons. Expression of differently sized PTCH1b 5'UTRs under basal level and endogenously activated Hh-Gli pathway, and the effect of hypoxia on the activity of pRuF-type plasmids were analyzed by regular 2-way ANOVA, followed by Tukey's post-hoc test.…”

Section: Western Blot Analysismentioning

confidence: 99%

“…RNA was recovered by organic solvent extraction and salt/ethanol precipitation and processed for qPCR analysis, following the protocol previously described. 34,77 The expression of the PTCH1b transcripts containing a 5'UTR of at least 188nt length or of at least 300nt were quantified using the primers described in the previous section.…”

Section: Sucrose Gradient Fractionation Of Cytoplasmic Cell Lysates Amentioning

confidence: 99%

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Regulation of humanPTCH1bexpression by different 5' untranslated regioncis-regulatory elements

et al. 2015

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Inga (2015) Regulation of human PTCH1b expression by different 5' untranslated region cis-regulatory elements, RNA Biology, 12:3, 290-304, DOI: 10.1080/15476286.2015 PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG) n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed variability in the length of 5'UTR and changes in abundance of the associated transcripts after pathway activation. We show that upstream AUG codons (uAUGs) present only in longer 5'UTRs could negatively regulate the amount of PTC1 isoform L (PTC1-L). The existence of an internal ribosome entry site (IRES) observed using different approaches and mapped in the region comprising the CGG repeats, would counteract the effect of the uAUGs and enable synthesis of PTC1-L under stressful conditions, such as during hypoxia. Higher relative translation efficiency of PTCH1b mRNA in HEK 293T cultured hypoxia was observed by polysomal profiling and Western blot analyses. All our results point to an exceptionally complex and so far unexplored role of 5'UTR PTCH1b cis-element features in the regulation of the Hedgehog-Gli signaling pathway.

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p53-directed translational control can shape and expand the universe of p53 target genes

Cited by 52 publications

References 60 publications

Effect of Anti–IL-15 Administration on T Cell and NK Cell Homeostasis in Rhesus Macaques

Effect of Anti–IL-15 Administration on T Cell and NK Cell Homeostasis in Rhesus Macaques

Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity

Regulation of humanPTCH1bexpression by different 5' untranslated regioncis-regulatory elements

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