Transcriptional regulation of Cyclin A2 by RASSF1A through the enhanced binding of p120E4F to the Cyclin A2 promotor

Recent advances in the study of RASSFIA, the candidate tumor suppressor gene, indicate a possible role of RASSFIA in cell cycle regulation; however, very little is known regarding molecular mechanisms underlying this control. Using small interfering RNA to knockdown endogenous RASSFIA in the breast tumor cell line 11132 and in the cervical cancer cell line HeLa, we identify that a key player in cell cycle progression, cyclin A2, is concomitantly increased at both protein and mRNA levels. In A549 clones stably expressing RASSFIA, cyclin A2 levels were diminished compared with vector control. A known transcriptional regulator of cyclin A2, p120(E4F) (a repressor of cyclin A2), has been shown previously by our group to interact with RASSFIA. We show that levels of p120(E4F) are not affected by RASSFIA small interfering RNA in HB2 and HeLa cells. However, electrophoretic mobility shift assays indicate that knockdown of endogenous RASSFIA in HB2 and HeLa cells leads to a reduction in the binding capacity of p120(E4F) to the cyclin A2 promoter, whereas in the A549 clone stably expressing RASSFIA the binding capacity is increased. These data are further corroborated in vitro by the luciferase assay and in vivo by chromatin immunoprecipitation experiments. Together, these data identify the cyclin A2 gene as a cellular target for RASSFIA through p120(E4F) and for the first time suggest a transcriptional mechanism for RASSFIA-dependent cell cycle regulation.