Abstract 18212: Unique Exonic RNA Polymerase II Pausing in the Hypoxia Inducible Factor-1 Alpha Gene is Associated With Alternative Splicing
Hypoxia-inducible factor 1α (Hif-1α) is the primary transcription factor that promptly increases upon exposure of the cells to hypoxia. It is mainly regulated by posttranscriptional and posttranslational mechanisms involving miR-199a and prolyl hydroxylase, respectively. In addition, the discovery of multiple Hif-1α isoforms suggests that alternative splicing may play a role in regulating its activity, although little is known about this in the heart. We, thus, hypothesized, that in addition to the known mechanisms, Hif-1α is activated by an alternative splicing step, a process that is tightly coupled to transcription. To test this, we treated myocytes with antimiR-199a, which robustly induces Hif-1α expression, and analyzed its effects on gene transcription using RNA polymerase II Chromatin immunoprecipitation-deep sequencing. The analysis revealed that antimiR-199a induced 1) the upregulation of genes that are predominantly Hif-1α targets including Pdk1, Glut3, Vegfa, Bnip3, Ndrg1, and Gys1, in addition to Pfkm, G0s2 and Sdpr, which are a subject of an independent study, and 2) a unique, very precise RNA pol II pausing (accumulation of pol II at a very precise sites with a density >2x of its flanking sequence) within the exons of only the Hif-1α gene, while RNA pol II at the transcription start site, and within other regions of the gene body, was similar to the control myocytes. In particular, all exons except for exons 1, 10, and 11, exhibited some degree of pausing, where exon 15 had a pol II pausing peak that was 20 fold higher than the flanking pol II density. Deceleration of transcriptional elongation has been associated with RNA splicing, however, these very precise exonic pausing peaks are quite unique. Using quantitative PCR, were able to confirm that during normoxia, Hif-1α was abundant as determined by primers specific for exons 10, 11, and 12, which increased by ~2 folds with miR-199a treatment. On the other hand, exon 15, which encodes part of the transactivation domain, was relatively lower during normoxia but increased 300x with antimiR-199a treatment. The result suggests that alternative splicing of exon 15 is a critical regulatory step required for the transcriptional activity of Hif-1α, which is associated with the increase in its protein.
- © 2013 by American Heart Association, Inc.