Changes in promoter structure and occupation have been shown to modify the splicing pattern of several genes, evidencing a coupling between transcription and alternative splicing. It has been proposed that the promoter effect involves modulation of RNA pol II elongation rates. The C4 point mutation of the Drosophila pol II largest subunit confers on the enzyme a lower elongation rate. Here we show that expression of a human equivalent to Drosophila's C4 pol II in human cultured cells affects alternative splicing of the fibronectin EDI exon and adenovirus E1a pre-mRNA. Most importantly, resplicing of the Hox gene Ultrabithorax is stimulated in Drosophila embryos mutant for C4, which demonstrates the transcriptional control of alternative splicing on an endogenous gene. These results provide a direct proof for the elongation control of alternative splicing in vivo.

In search for physiological pathways affecting alternative splicing through its kinetic coupling with transcription, we found that membrane depolarization of neuronal cells triggers the skipping of exon 18 from the neural cell adhesion molecule (NCAM) mRNA, independently of the calcium/calmodulin protein kinase IV pathway. We show that this exon responds to RNA polymerase II elongation, because its inclusion is increased by a slow polymerase II mutant. Depolarization affects the chromatin template in a specific way, by causing H3K9 hyper-acetylation restricted to an internal region of the NCAM gene surrounding the alternative exon. This intragenic histone hyper-acetylation is not paralleled by acetylation at the promoter, is associated with chromatin relaxation, and is linked to H3K36 trimethylation. The effects on acetylation and splicing fully revert when the depolarizing conditions are withdrawn and can be both duplicated and potentiated by the histone deacetylase inhibitor trichostatin A. Our results are consistent with a mechanism involving the kinetic coupling of splicing and transcription in response to depolarization through intragenic epigenetic changes on a gene that is relevant for the differentiation and function of neuronal cells.histone acetylation ͉ neuronal excitation ͉ transcription ͉ mRNA processing O ver the past decade, it has become increasingly clear that all eukaryotic mRNA processing steps (capping, splicing, and 3Ј end formation) are coupled to transcription (1-3). We and others have studied the mechanisms by which transcription can affect alternative splicing, leading to the proposal of 2 different but not exclusive models (4): the recruitment model, by which different factors associated with the transcription complex regulate splicing choices (5-7); and the kinetic model, whereby the rate of RNA polymerase II (pol II) elongation influences splice site selection (8-12). Most of this mechanistic work was performed by experiments involving either promoter swapping or mutant RNA polymerases (5,11,13). Both approaches allow a fine control of transcription properties but are unlikely to reflect physiological conditions in which endogenous alternative splicing is regulated in response to environmental cues.The chromatin structure is likely to play a relevant role in the effects of transcription on alternative splicing, a subject that has received recent attention (14). It has been shown that histone modifications can influence the recruitment of splicing factors to transcription foci (15). Furthermore, the chromatin remodeling complex SWI/SNF has been reported to modulate alternative splicing by taking part in a complex that causes RNA pol II to stall near alternative exons (16). Epigenetic marks such as histone post-translational modifications are possible ways of regulating template properties and transcription quality that, in turn, could influence alternative splicing. Suggestively, induction of a more restrictive intragenic chromatin conformation decreases pol II elongation without affec...