Fig. 4: The long-term hypoxia adaptation induces novel truncated Wt1 mRNA transcripts from an intronic HRE-driven promoter. | Cancer Gene Therapy

Fig. 4: The long-term hypoxia adaptation induces novel truncated Wt1 mRNA transcripts from an intronic HRE-driven promoter.

From: Long-term severe hypoxia adaptation induces non-canonical EMT and a novel Wilms Tumor 1 (WT1) isoform

Fig. 4

A LTHY read coverage of the Wt1 locus, at 0.1% O2 of the LTHY time course, generated in IGV. Number ranges are coverage depths at the nucleotide level. Histogram is representative of replicates (n = 2, n2 shown). Introns 1–4 are condensed for visual clarity. B Transcription Factor Binding Site analysis of murine Wt1 intron 5 from beginning of intron 5 to beginning of RNAseq read coverage for Wt1. Only considered TFBSs with a score ≥ 0.95. Intron 5 sequence is broken into 40 bins, ~500 bp/bin. Analysis done using TFBStools in R. C FACS samples of the empty promoter-reporter construct, and the wild-type (WT) across the LTHY time course. Gate represents mCherry+ gate used for promoter activity calculations. FACS plots are representative of their triplicates. D Functional investigation into tWT1 promoter subregions. “Dist”: Distal region. “P1”: Proximal subregion 1. “P2”: Proximal subregion 2. “pT”: Poly-Thymine stretch. +: DNA region is present. −: DNA region is not present. M: DNA region has specific TFBSs scrambled. Promoter activity calculated using a ratio ZsGreen expression in transduced cells relative to untransduced cells, normalized to their normoxic counterparts. Significance calculated using 2-way ANOVA with Tukey’s multiple comparisons. Black stars represent intra-construct statistical comparisons; only reporting statistics relative to 5% O2. Blue stars represent significance relative to WT at 0.1% O2. Other comparisons are not shown for visual clarity. E Functional investigation into the P1 subregion of the tWt1 promoter at 0.1% O2. Promoter activity was calculated as in D. Statistics are a 2-way ANOVA with Tukey’s multiple comparisons test. Black stars represent statistical comparisons. Blue stars represent significance relative to WT at 0.1% O2. Other comparisons not shown for visual clarity. D, E “-“: an absence of subregion. “+“: presence of wild-type sequence. “M”: Transcription factor binding sites listed in Fig. S4F are scrambled. *p < 0.05. **p < 0.01. ***p < 0.001 ****p < 0.0001.

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