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

doi:10.1073/pnas.0710748105

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

*Institut National de la Santé et de la Recherche Médicale, Unité 685, Institut Universitaire d'Hématologie, 1, Avenue Claude Vellefaux, 75010 Paris, France;
^†Institut National de la Santé et de la Recherche Médicale, Unité 830, Institut Curie, 26, Rue d'Ulm, 75248 Paris Cedex 05, France; and
^‡Institut Curie, 75248 Paris Cedex 05, France

Edited by Bert W. O'Malley, Baylor College of Medicine, Houston, TX, and approved February 12, 2008 (received for review November 13, 2007)

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.

Footnotes

^§To whom correspondence should be addressed. E-mail: martin.dutertre{at}inserm.fr
Author contributions: O.D., D.A., and M.D. designed research; G.S., D.B., J.B., and M.D. performed research; K.L. and O.D. contributed new reagents/analytic tools; G.S., K.L., O.D., D.A., and M.D. analyzed data; and G.S., D.A., and M.D. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0710748105/DCSupplemental.