Fig. 3: U2AF2 Exon 2 inclusion, induced by IWS1 phosphorylation at Ser720/Thr721, depends on H3K36 trimethylation by SETD2. | Nature Communications

Fig. 3: U2AF2 Exon 2 inclusion, induced by IWS1 phosphorylation at Ser720/Thr721, depends on H3K36 trimethylation by SETD2.

From: RETRACTED ARTICLE: AKT3-mediated IWS1 phosphorylation promotes the proliferation of EGFR-mutant lung adenocarcinomas through cell cycle-regulated U2AF2 RNA splicing

Fig. 3

a ChIP assays showing the abundance of H3K36me3 in the indicated NCI-H522 (left) and NCI-H1299 (right) cells. Bars show the mean fold enrichment in H3K36me3 (anti-H3K36me3 IP, vs IgG control IP) in the indicated regions of the U2AF2 gene, in shControl, shIWS1, shIWS1/WT-R and shIWS1/MT-R cells ±SD. Data were normalized relative to the input (2%). b ChIP assays showing the abundance of H3K36me3 in the indicated NCI-H522 (left) and NCI-H1299 (right) cells, treated with MK2206 (5μM) or DMSO. The bars show the mean fold enrichment of H3K36me3 (anti-H3K36me3 IP, vs IgG control IP) in the indicated regions of the U2AF2 gene, ±SD. Data were normalized relative to the input (2%). c ChIP assays showing the binding of HA-SETD2 in the indicated NCI-H522 (left) and NCI-H1299 (right) cells, transduced with a lentiviral HA-SETD2 construct. The bars show the mean fold enrichment in SETD2 binding (anti-HA IP, vs IgG control IP) ±SD. Data were normalized relative to the input (2%). The expression of HA-SETD2 is shown in Supplementary figure 4a. d (Upper panel) Lysates of NCI-H522 (left) and NCI-H1299 (right) cells transduced with the indicated constructs, were probed with the antibodies, as shown. RT-PCR addressing U2AF2 exon 2 splicing in the same cells. (Middle panel) GAPDH-normalized E2 and E3 bands in the RT-PCR experiment above, were used to calculate the E2/E3 ratio±SD. (Lower panel) Ratio of the U2AF2 exons 2 and 3 levels in the same cells ±SD, as determined by quantitative RT-PCR. E2/E3 ratio is shown relative to shControl (value = 1). All experiments had three biological replicates, all done in triplicate. n.s non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-sided unpaired t-test). e Snapshots of the integrative genomic viewer showing the ChIP-Seq-determined distribution of Flag-IWS1, HA-SETD2, and histone H3K36me3, in U2AF2. Parallel snapshots show the distribution of RNA reads in the RNA-seq experiment. Scale represents reads per million (RPM). Snapshots of peaks detected in the two biological replicates are shown. Black box highlights U2AF2 exons 2, 3 and adjacent regions.

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