Alteration of cyclin D1 transcript elongation by a mutated transcription factor up-regulates the oncogenic D1b splice isoform in cancer

Abstract

Pre-mRNA splicing and polyadenylation are tightly connected to transcription, and transcriptional stimuli and elongation dynamics can affect mRNA maturation. However, whether this regulatory mechanism has a physio/pathological impact is not known. In cancer, where splice variant expression is often deregulated, many mutated oncogenes are transcriptional regulators. In particular, the Ewing sarcoma (EwSa) oncogene, resulting from a fusion of the EWS and FLI1 genes, encodes a well characterized transcription factor. EWS-FLI1 directly stimulates transcription of the CCND1 protooncogene encoding cyclin D1a and a less abundant but more oncogenic splice isoform, D1b. We show that, although both EWS and EWS-FLI1 enhance cyclin D1 gene expression, they regulate the D1b/D1a transcript ratio in an opposite manner. Detailed analyses of RNA polymerase dynamics along the gene and of the effects of an inhibitor of elongation show that EWS-FLI1 favors D1b isoform expression by decreasing the elongation rate, whereas EWS has opposite effects. As a result, the D1b/D1a ratio is elevated in EwSa cell lines and tumors. The endogenous D1b protein is enriched in nuclei, where the oncogenic activity of cyclin D1 is known to occur, and depleting D1b in addition to D1a results in a stronger reduction of EwSa cell growth than depleting D1a only. These data show that elevated expression of a splice isoform in cancer can be due to an alteration of the transcription process by a mutated transcriptional regulator and provide evidence for a physio/pathological impact of the coupling between transcription and mRNA maturation.

Fig. 1.

EWS and EWS-FLI1 affect the expression of cyclin D1 isoforms. (A–C) MCF-7 and A673 cells transfected with siGL2 (negative control) and siEWS (A), as well as A673-shEF1 and A673-Ctrl cells grown for 2 days with or without Dox (B), were analyzed for cyclin D1a and D1b mRNA levels by RT-qPCR. The effects of siEWS and shEF1 on the D1b/D1a transcript ratio are plotted in C. (D and E) A673-shEF1 and A673-Ctrl cells grown for 2 days with or without Dox were analyzed by Western blot for cyclin D1a and D1b proteins using the sc-718 and α-D1b antibodies, respectively (D), and by 3′RACE on nuclear RNA using sense primers in intron 4 and exon 5 (E). Nucleotidic positions of polyA sites (pA) in intron 4 and exon 5 are indicated. In addition to the previously reported polyA site at position 571 in intron 4 (15), we identified a novel polyA site at position 1097 (Fig. S4). The detection of transcripts using intron 4 polyA sites required more PCR cycles than that of transcripts using the exon 5 polyA site, in agreement with the low D1b/D1a mRNA ratio.

Fig. 2.

EWS-FLI1 associates with the cyclin D1 gene and affects Pol II phosphorylation at the gene 5′ end. (A) Analysis of EWS-FLI1 association with the cyclin D1 gene 5′ end by ChIP using an antibody against EWS-FLI1 (α-EF1) and qPCR with the E1-I1 primer pair (Fig. S1). A673-shEF1 cells were grown with or without Dox, as indicated. Signals were normalized to input, and background levels in IP with control immunoglobulins (IgG) were assigned 1. (B and C) Effects of shEF1 (B) and siEWS (C) on the abundance of Pol II (CTD4H8) and Pol II phospho-Ser-5 (H14) on the cyclin D1 gene at position E1-I1 in A673 cells as determined by ChIP assay. In B, the effect of Dox in A673-shEF1 cells was normalized to its effect in A673-Ctrl cells. In C, the effect of siEWS was determined relative to siGL2 negative control.

Fig. 3.

The effect of EWS-FLI1 on cyclin D1 processing is due to an effect on transcription elongation. (A) Pattern of Pol II on the cyclin D1 gene in untreated A673 cells, as determined by ChIP using the CTD4H8 antibody and qPCR at various positions from 5′ to 3′ along the cyclin D1 gene. (B and C) Effects of shEF1 (B) and siEWS (C) on the abundance of Pol II (CTD4H8) at various positions along the cyclin D1 gene relative to their effects at the gene 5′ end, which were assigned 1. The effect of siEWS was determined relative to the siGL2 negative control. (D) Effects of shEF1 on the abundance of 5′ and 3′ parts of cyclin D1 pre-mRNA (E1-I1 and I4-E5, respectively) in nuclear extracts of A673 cells. (E) Effects of CPT on cyclin D1 mRNA levels in MCF-7 cells. (F) Effects of CPT in MCF-7 cells and of shEF1 in A673 cells on the ratio of 3′ to 5′ parts of indicated introns in cyclin D1 pre-mRNA. To calculate the effects of shEF1 (B, D, and F), the effects of Dox in A673-shEF1 cells were normalized to its effects in A673-Ctrl cells. All primer pairs are described in Fig. S1.

Fig. 4.

Expression and biological significance of the cyclin D1b isoform in EwSa. (A) RT-qPCR analysis of cyclin D1 transcripts in total RNA from the indicated samples. Each dot represents a sample. The average ± SEM is also indicated. CLs, cell lines. (B) Total proteins from various EwSa cell lines, tumors, and A673 cells transfected with the indicated siRNAs were analyzed by Western blot using antibodies against D1a, D1b, and actin. (C and D) Cytosolic (C) and nuclear (N) proteins from EW7 and A673 cells were analyzed by Western blot using the indicated antibodies. A lower exposure of the EW7 cytosol hybridized with the DCS6 antibody is shown. (E) A673 cells transfected for 3 days with the indicated siRNAs were analyzed for cell growth.