Oncogenic splicing factor SRSF1 is a critical transcriptional target of MYC

Abstract

The SR protein splicing factor SRSF1 is a potent proto-oncogene that is frequently upregulated in cancer. Here, we show that SRSF1 is a direct target of the transcription factor oncoprotein MYC. These two oncogenes are significantly coexpressed in lung carcinomas, and MYC knockdown downregulates SRSF1 expression in lung-cancer cell lines. MYC directly activates transcription of SRSF1 through two noncanonical E-boxes in its promoter. The resulting increase in SRSF1 protein is sufficient to modulate alternative splicing of a subset of transcripts. In particular, MYC induction leads to SRSF1-mediated alternative splicing of the signaling kinase MKNK2 and the transcription factor TEAD1. SRSF1 knockdown reduces MYC's oncogenic activity, decreasing proliferation and anchorage-independent growth. These results suggest a mechanism for SRSF1 upregulation in tumors with elevated MYC and identify SRSF1 as a critical MYC target that contributes to its oncogenic potential by enabling MYC to regulate the expression of specific protein isoforms through alternative splicing.

Figure 1

SRSF1 expression correlates with MYC levels in human lung tumors and cell lines. (A) SRSF1 expression profile from microarray analysis of 132 lung tumors (expO). The data were normalized to Z-score and divided into two categories: tumors expressing high or low MYC levels. The dot plot shows the distribution and the median (horizontal line). Mann-Whitney test ***P<0.0001. (B) Immunoblotting of MYC and SRSF1 in lung-cancer cell lines and lung primary fibroblasts, showing significant correlation between the expression of the two oncoproteins (r=0.75, one-tailed t-test *P=0.05). (C) RT-PCR and (D) Immunoblotting of MYC and SRSF1 in NCI.H460 and NCI.H1666 cells transfected with control siRNA (luciferase) or one of two siRNAs against MYC. See also Figure S1.

Figure 2

MYC regulates SRSF1 expression and alternative splicing SRSF1 Target Genes. (A) RT-PCR and (B) Immunoblotting of SRSF1 from IMR90-ER.MYC or IMR90-ER.MYCΔMBII cells induced with 4-OHT. Error bars, s.d.; n=3; t-test **P<0.01. (C) RT-PCR of MKNK2 and TEAD1 mRNA isoforms in IMR90-ER.MYC cells induced with 4-OHT, with or without SRSF1 knockdown. IMR90 cells overexpressing SRSF1 or hnRNPA1 are shown as controls. Error bars, s.d.; n=3, *P<0.05. See also Figure S2.

Figure 3

MYC binds to and activates the human SRSF1 promoter. (A) RT-PCR of IMR90-ER.MYC cells treated with 4-OHT, with or without cycloheximide. Error bars, s.d.; n= 3; **P<0.01. (B) MYC chromatin immunoprecipitation analysis at the SRSF1 promoter locus in the lung-carcinoma NCI-H460 cell line. Diagram of the SRSF1 gene indicating the E-boxes and amplicons (A-E) used for ChIP assays. The results are expressed as DNA enrichment in fragmented chromatin immunoprecipitated with anti-MYC antibody (relative to anti-rabbit IgG immunoprecipitation) and normalized to the amplicon E signal, as measured by quantitative PCR. The horizontal gray line represents no change in MYC-specific enrichment. Error bars, s.d.; n=3; t-test *P<0.05; n.s., not significant. (C) Diagram of the wild-type SRSF1 promoter, comprising three non-canonical E-boxes, and the E-Box mutants generated for reporter assays. Mutant E-boxes and residues are indicated in red. (D) Luciferase assay of reporter constructs in (C) co-transfected with MYC cDNA or vector control into NIH3T3 cells. Luciferase activity was normalized to co-transfected GFP, and the relative activity is plotted. Error bars, s.d.; n=3; t-test **P<0.01; n.s., not significant.

Figure 4

SRSF1 knockdown impairs anchorage-independent growth of MYC-transformed cells. (A) Immunoblotting of MYC and SRSF1 in the Rat1a-pBabe-Luc control cell line, Rat1a-MYC, and Rat1a-MYC cells transduced with one of two shRNAs against SRSF1. (B) Growth curves of the four cell lines from (A). Error bars, s.d.; n=3. (C) Anchorage-independent growth of cell lines from (A) in soft-agar colony-formation assays. Error bars, s.d.; n=3; t-test *P<0.05.