Taylor W et al. (2022), The Splicing Factor PTBP1 Represses TP63 γ Isof...

XB-ART-59704

Cancer Res Commun 2022 Dec 01;212:1669-1683. doi: 10.1158/2767-9764.CRC-22-0350.

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The Splicing Factor PTBP1 Represses TP63 γ Isoform Production in Squamous Cell Carcinoma.

Taylor W , Deschamps S , Reboutier D , Paillard L , Méreau A , Audic Y .

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UNLABELLED: The TP63 gene encodes the p63 transcription factor. It is frequently amplified or overexpressed in squamous cell carcinomas. Owing to alternative splicing, p63 has multiple isoforms called α, β, γ, and δ. The regulatory functions of p63 are isoform specific. The α isoform inhibits the epithelial-to-mesenchymal transition (EMT) and controls apoptosis, while the γ isoform promotes EMT. Using The Cancer Genome Atlas data, we observed that a higher proportion of the TP63γ isoform is a detrimental factor for the survival of patients with head and neck squamous cell carcinoma (HNSCC) and is accompanied by the downregulation of desmosomal genes. By a correlation-based approach, we investigated the regulation of the production of the TP63γ isoform. According to our analysis of GTEx data, the expression of the RNA-binding protein PTBP1 (polypyrimidine tract binding protein 1) is negatively correlated with the abundance of TP63γ in several tissues. Accordingly, we demonstrated that PTBP1 depletion in HNSCC cell lines, keratinocyte or Xenopus embryos leads to an increase in TP63γ isoform abundance. By RNA immunoprecipitation and in vitro interaction assays, we showed that PTBP1 directly binds to TP63 pre-mRNA in close proximity to the TP63γ-specific exon. Intronic regions around the TP63γ-specific exon were sufficient to elicit a PTBP1-dependent regulation of alternative splicing in a splice reporter minigene assay. Together, these results identify TP63γ as an unfavorable prognostic marker in HNSCC, and identify PTBP1 as the first direct splicing regulator of TP63γ production and a potential route toward TP63 isoform control. SIGNIFICANCE: Quantifying TP63γ isoforms in patients' tumors could allow for the early detection of patients with HNSCC with an early loss in desmosomal gene expression and poor prognostic. The identification of PTBP1 as a transacting factor controlling TP63γ production may allow to control TP63γ expression.

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Species referenced: Xenopus laevis
Genes referenced: cpa1 eno2 itk pcna ptbp1 ptbp2 tp63
GO keywords: epithelial cell development [+]
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Phenotypes: Xla Wt + ptbp1 MO (Fig. S6 B C)

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	FIGURE 1 TP63 splicing from patients with HNSCC. A,TP63 exon/intron structure (top diagram) and corresponding encoded protein isoforms with color-encoded peptidic domains. TA, ΔN denote the promoters. Exons are numbered according to GTEX. Alternatively spliced junctions and CPA events are indicated with solid lines joining exons and pA, respectively. Terminal exon 16 is present in isoforms α, β, or δ, and terminal exon 12 in isoform γ. The cassette exons 14 and 15 are present in isoform α while the isoform β lacks exon 15 and the isoform δ lacks both. The exons are colored according to the domain structure. TA, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain; SAM, sterile alpha motif; TID, transactivation inhibitory domain; PD, phosphodegron. B, Genewise expression of TP63 in normal and tumor samples from patients with HNSCC. C, Quantification of TP63 splicing by measuring the inclusion of specific exons (PSI) in normal and tumor samples. The median PSI is indicated. Differential expression between normal and tumor samples is assessed using Wilcoxon rank-sum test. D, Patients were segregated on the basis of the high or low inclusion of exon 12 (threshold = 4.07%) or exon 14+15 (threshold = 98.49%) in tumor samples. Patient survival was assessed on a Kaplan–Meier graph in the two classes and statistical differences appraised by a log-rank test.
	FIGURE 2 DEG and GO enrichment analysis of patients tumors with high TP63 expression. A, Volcano plot of gene expression in the groups of patients with a high or a low percentage of TP63 isoform. The red dashed lines represent the threshold at BenjaminiHoschberg adjusted P value <0.05. The favorable and unfavorable prognosis marker genes are highlighted blue and red, respectively. B, GO term enrichment analysis performed on DEGs (Padjusted < 0.05) shown in A, the enrichment (observed/expected) is shown on top of the bar. C, Same as A with desmosomal gene highlighted in black. D, Same as A with SWI/SNF complex gene highlighted in black.
	FIGURE 3 Identification of PTBP1 as an RBP correlated to TP63γ terminal exon splicing. A,TP63 gene expression in samples from TP63-expressing tissues (normalized reads count). se, nse, (non) sun exposed. B, Quantification of the usage of junctions 11–12 and 11–13 pertinent to γ-terminal exon inclusion. C, RBP expression heatmap and hierarchical clustering of tissues based on RBP expression from GTEX data. D, Top 10 RBPs most correlated to the junctions involved in γ exon inclusion (junction 11–12) or γ exon exclusion (junction 11–13). E, Expression of SFRS9, PTBP1, and GRSF1 in two classes of patients with high or low γ exon 12 inclusion in tumors (threshold = 4.07%).
	FIGURE 4 PTBP1 controls TP63 splicing in HNSCC cell lines. A, The abundance of p63 protein was evaluated in HaCaT, HeLa, and HNSCC cells by Western blot analysis and immunodetection using a pan-p63 antibody (4A4) or a p63α-specific antibody (D2K8X; representative results of three independent replicates). Even loading was controlled using proliferating cell nuclear antigen (PCNA) and β-tubulin antibodies as indicated. B, Total TP63 RNA was measured by qRT-PCR with one pair of primers detecting all isoforms. Quantification was normalized to a calibration curve obtained from TP63 plasmid DNA dilutions (n = 3). C, Quantification of TP63α, β, or γ proportions using isoform-specific primer pairs (n = 3). The amount of each isoform was normalized by the total abundance of TP63 mRNA as in B. D, Top, Evaluation of PTBP1 depletion by Western blot analysis, PCNA serves as a loading control. Remaining PTBP1 levels in the three independent experiments are presented below each panel. Bottom, Quantification of TP63γ isoform and PTBP1 RNA in control and PTBP1-depleted HNSCC cell lines (n = 3). , P < 0.05; , P < 0.01; **, P < 0.001; Dunnett test.
	FIGURE 5 PTBP1 binds specifically to TP63 premRNA sequences. A,TP63 exon 12 genomic locus and positions of the RNAs tested in RNA/protein shift assays. Top lane, RBPmap predicted PTBP1 binding sites. Middle, Positions and names of the RNA fragments tested along the TP63 exon 12 genomic sequence. Bottom lane, nucleotidic conservation among vertebrates. B, Immunopurification of PTBP1/RNA complexes or control experiments (Ig and beads only) from HaCaT cell extracts. Immunopurification was controlled by PTBP1 and PCNA Western blot analysis as indicated in input (10%) or immunoprecipited (IP) samples (top). Protein MW are indicated on the side of the membranes. The asterisk denotes the light chain of the IgG. Bottom, RT-PCR detection of TP63 pre-mRNA with primers located in intron 11 and exon 12. C, Same as B from SCC9 cell extracts. D, qRT-PCR detection in HaCaT cells of different mRNA or pre-mRNA portions of TP63 from IP or control samples relative to input signal. Statistical assessment between PTBP1 and control IP was measured by a Student t test (n = 3). E, PTBP1/RNA-binding curves [bound/(bound + free)] obtained for the indicated RNAs (n ≥ 4). RNA mutated to remove PTBP1 binding sites are shown in gray in A. The calculated Kd and their SD is shown for each plot.
	FIGURE 6 PTBP1 represses endogenous TP63 γ exon inclusion in HaCaT cells. A, Detection of PTBP1 in HaCaT cells after PTBP1 depletion mediated by two different siRNA. We show here a representative experiment of a triplicate. B, qRT-PCR quantification of endogenous PTBP1, PTBP2, TP63all and TP63α, β, and γ isoforms levels after siRNA-mediated PTBP1 depletion. n = 3; , P < 0.05; , P < 0.01; **, P < 0.001; Dunnett test (n = 3, Dunnet test).
	FIGURE 7 A TP63γ minigene recapitulates the PTBP1-dependent regulation of exon γ inclusion. A, Structure of the TP63 minigene stably transfected in HaCaT cells. β-globin gene sequences are shown in orange, TP63 gene sequences are shown in green. B, qRT-PCR quantification of PTBP1, and the RNA isoforms produced from the TP63 minigene after siRNA-mediated PTBP1 depletion. C, Delta PSI are computed as the fold difference between test and siCTRL as the reference condition. In all panels, n = 6; , P < 0.05; , P < 0.01; **, P < 0.001; Dunnett test.
	FIGURE 8 PTBP1-dependent regulation of TP63γ in HNSCC. Graphical abstract of the main conclusion and hypothesis. Left side, PTBP1 by binding on 3′SS and CPA of the alternative terminal exon γ repress its inclusion in the mature RNA. Right side, upon decreased PTBP1 expression, accumulation of TP63γ promotes formation of TP63 α/γ heterotetramers that repress desmosomal genes expression in primary tumors, alter epithelial characteristics, and promote an unfavorable prognostic.

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