Regulation of RNA polymerase III transcription by the oncoproteins ErbB2/Neu, c-Myc and Id2

For cells to proliferate, a certain size needs to be reached, which is mainly determined by the rate of translation. Thus, the constituents of the translational apparatus play an essential role in cell growth and proliferation. Because some of the products transcribed by pol III transcription are constituents of the translational apparatus, the rate of pol III transcription will affect cell growth rate. Mitogenic stimulation induces pol III activity [1], while differentiation represses pol III transcription [2]. The retinoblastoma protein (RB) has been implicated in repression of pol III transcription by inhibiting TFIIIB activity through binding of its hypophosphory-lated form [3,4]. The oncoproteins ErbB2/neu, c-Myc and Id2 have been shown to block RB function by inducing its phosphorylation (ErbB2 and c-Myc) [5,6], or by binding and sequestrating it (Id2) [7,8]. These oncoproteins have been shown to be involved in the upregulation of cell proliferation, growth and the repression of differentiation, processes in which pol III transcription displays similar regulation. For this reason, their involvement in the regulation of pol III output was investigated. The data presented here demonstrate that all three oncoproteins upregulate pol III-transcribed genes. Although these oncoproteins can inhibit RB function, only ErbB2/neu seems to induce pol III activity disrupting TFIIIB-RB complexes. In addition, consitutive activation of ErbB2/neu also produces several other changes that can affect pol III transcription, including protein modifications of the TFIIIB subunits TBP and Brf, as well as induction of TBP, the pol III subunit BN51 and the transcription factor c-Myc. Furthermore, it was demonstrated that by inhibiting expression of c-Myc, the stimulation of pol III activity by ErbB2/neu could be overriden. Together these results suggest that constitutive activation of ErbB2/neu increases pol III transcription through several mechanisms, though induction of c- Myc appears to play an essential role in this upregulation. On the other hand, the activation of pol III transcription by c-Myc and H2 is through an RB-independent mechanism, since their overexpression in the absence of the entire RB family, still produced pol III upregulation. Instead, they activate directly pol III transcription. Chromatin immunoprecipitation revealed that both endogenous c-Myc and Id2 are present at class III genes in cultured mammalian cells. c-Myc and Id2 induction of pol III activity appears to be through direct mechanisms by interaction of these proteins with TFIIIB. In the case of c-Myc, coimmunoprecipitations demonstrated that it interacts with TFIIIB at physiological ratios. Furthermore, both endogenous c-Myc as well as endogenous Id2 were found to cofractionate with samples containing TFIIIB activity prepared on several columns, such as MonoQ gradients. Prom these results it can be concluded that endogenous c-Myc binds stably and specifically to TFIIIB. Further analysis has to be performed for to confirm the interaction between Id2 with TFIIIB. In summary, this work has identified three oncogene products that can increase pol III transcriptional output. These findings have important implications for tumour development in a range of tissue types