527 siRNA targeting of thymidylate synthase and thymidine kinase for anti-cancer therapy

Abstract

Expression of DNA topoisomerase (topo) IIα is cell-cycle-regulated, with its peak in G2/M and its lowest level in G0/G1. In agreement with this expression pattern, we have shown that the topo IIα gene promoter shows cell-cycle-dependent activity, which is repressed in G0/G1 and activated exclusively in G2/M. However, the promoter sequence reveals no canonical CDE/CHR motifs, repressor elements commonly found in promoters of late S/G2-activated genes. Here, we show that at least two of the three proximal inverted CCAAT boxes (ICBs) are responsible for the G2/M-specific activation of the topo IIα promoter. Using antibody supershift experiments, we identify NF-Y as the ICB-binding transcription factor. However, the expression profile and binding capacity of NF-Y were constant during the cell cycle, suggesting a more global mechanism in topo IIα promoter regulation. Interestingly, we find that trichostatin A (TSA), a specific histone deacetylase inhibitor, greatly enhances topo IIα promoter activity in an ICB-dependent manner. In addition, the effect of TSA is predominant in G0/G1 and less obvious in G2/M. Our data, along with the recent findings that NF-Y associates in vivo with histone acetyltransferases (HATs), strongly suggest a mechanism, in which histone deacetylation plays a crucial role in the G0/G1-specific repression of the topo IIα promoter, and NF-Y recruits HATs to the promoter region, thereby stimulating histone acetylation and activating transcription in G2/M.