New functions of DNA topoisomerase I in the regulation of transcript elongation and maturation in tumor cells.

Unexpected and recent findings suggest a role for DNA topoisomerase I-B (Top1) in the coupling of transcription to pre-mRNA maturation and transport. Short cell treatments with CPT, a specific antitumor inhibitor of Top1, apparently enhance the escape of RNAPII from promoterproximal pausing sites. Moreover, altered patterns of alternative splicing have been reported following CPT treatments of cancer cells. These findings provide circumstantial evidence for a critical and direct involvement of Top1 in the regulation of the transcriptional pausing of RNAPII and transcript maturation. Our working hypothesis is that alterations of alternative splicing patterns by CPT can be explained by a role of Top1 in the regulation of RNAPII pausing, an event that likely allows the coupling of RNA synthesis and maturation. Thus, the aims of the project are: 1. to demonstrate a role of Top1 in the regulation RNAPII pausing at promoter-proximal sites of human genes, including HIF-1a 2. to identify new RNAPII pausing sites regulated by Top1 by the ChIP-chip approach using NimbleGene arrays 3. to establish Top1 protein partners contributing to the regulation of RNAPII pausing 4. to determine internal RNAPII pause sites regulated by Top1, and genomic regions of CPT-activated transcription by genome mapping. Initially, we will focus on specific genes as model systems. Over the last years, Topotecan (a CPT analog) has been shown to inhibit the accumulation of HIF-1 (hypoxia-inducible factor 1) a subunit in a Top 1- and transcription-dependent manner. However, the molecular mechanism has not yet established. Since HIF-1 has emerged as an attractive target for cancer therapy, and we have evidence that RNAPII has a transcriptional pausing site close to the promoter of the human HIF-1a gene, we intend to show that CPT alterations of the RNAPII transcriptional pausing may affect HIF-1a pre-mRNA maturation leading to reduced protein levels in the cell. Then, we will collect a number of genomic Top1 binding sites, and RNAPII pausing sites close to promoters and regulated by Top1 by using the ChIP-chip technology and NimbleGen arrays and service. The findings will provide a global view of Top1 involvement in the new function, and will provide unique data that will help to define the functions and regulatory networks of the enzyme. The demonstration of the role of Top1 in the regulation of promoter-proximal pausing of RNAPII will raise several questions concerning the functions of the enzyme in gene expression and the mechanism of action of the antitumor inhibitors. Thus, we will proceed by defining the protein partners of Top1 at RNAPII pausing sites, and will attempt to identify genomic regions of RNA synthesis activated by CPT and/or Top1 inhibition. The results might have a great impact in the discovery of novel molecular targets for cancer therapy among transcription and maturation factors.