The Myc Negative Autoregulation Mechanism Requires Myc-Max Association and Involves the c-myc P2 Minimal Promoter

Abstract

Increasing evidence supports an important biological role for Myc in the downregulation of specific gene transcription. Recent studies suggest that c-Myc may suppress promoter activity through proteins of the basal transcription machinery. We have previously reported that Myc protein, in combination with additional cellular factors, suppresses transcription initiation from the c-myc promoter. To characterize the cis components of this Myc negative autoregulation pathway, fragments of the human c-myc promoter were inserted upstream of luciferase reporter genes and assayed for responsiveness to inducible MycER activation in Rat-1 fibroblasts. We found four-to fivefold suppression of a c-myc P2 minimal promoter fragment upon induction of wild-type MycER protein activity, while induction of a mutant MycER protein lacking amino acids 106 to 143 required for Myc autosuppression failed to elicit this response. This assay is physiologically significant, as it reflects Myc autosuppression of the endogenous c-myc gene with regard to kinetics, dose dependency, cell type specificity, and c-Myc functional domains. Analysis of mutations within the P2 minimal promoter indicated that the cis components of Myc autosuppression could not be ascribed to any known protein-binding motifs. In addition, to address the trans factors required for Myc negative autoregulation, we expressed MycEG and MaxEG leucine zipper dimerization mutants in Rat-1 cells and found that Myc-Max heterodimerization is obligatory for Myc autosuppression. Two models for the Myc autosuppression mechanism are discussed.

An N-Myc truncation analogous to c-Myc-S induces cell proliferation independently of transactivation but dependent on Myc homology box II

“…The mechanism of transcriptional repression is distinct from transactivation and does not require the presence of the Myc consensus site in the vicinity of the transcription initiation site (Patel et al, 2004). However, it is important to emphasize that Myc-dependent repression requires Myc-Max heterodimers and DNAbinding activity (Facchini et al, 1997). Our previous microarray analysis of Myc-binding mutants revealed that Myc-dependent repression is completely abolished by a subtle mutation of the basic region, which inhibits Myc-consensus site binding (Cowling and Cole, 2007b).…”

Additive effect between NF-κB subunits and p53 protein for transcriptional activation of human p53 promoter

“…Several oncoproteins, functioning as transcriptional factors, have been shown to autoregulate, negatively (C-Myc and C-Fos) or positively (C-Myb), the transcription of their encoding gene (Schonthal et al, 1989;Nicolaides et al, 1991;Facchini et al, 1997). p53 gene transcriptional autoregulation has been studied by three di erent groups, who reported con¯icting results.…”

Myc represses the growth arrest gene gadd45

“…Recent structure-function analysis of the aminoterminus of the c-Myc protein shows Myc repression and Myc transactivation are separable, with Myc repression being more closely associated with neoplastic transformation (Brough et al, 1995;Lee et al, 1996;Li et al, 1994;Penn et al, 1990b). Myc can repress C/EBPa (Antonson et al, 1995;Li et al, 1994), cyclin D1 (Philipp et al, 1994), the adenovirus 5 major-late promoter (AdMLP) (Li et al, 1994), and cmyc (Facchini et al, 1997) promoter activities. Myc suppression appears to be mediated through core regulatory elements and does not require the con-sensus Myc/Max binding sites within the target promoter sequence (Antonson et al, 1995;Facchini et al, 1997;Li et al, 1994;Lucas et al, 1993;Philipp et al, 1994).…”

“…Myc can repress C/EBPa (Antonson et al, 1995;Li et al, 1994), cyclin D1 (Philipp et al, 1994), the adenovirus 5 major-late promoter (AdMLP) (Li et al, 1994), and cmyc (Facchini et al, 1997) promoter activities. Myc suppression appears to be mediated through core regulatory elements and does not require the con-sensus Myc/Max binding sites within the target promoter sequence (Antonson et al, 1995;Facchini et al, 1997;Li et al, 1994;Lucas et al, 1993;Philipp et al, 1994). Interestingly, Max heterodimerization is not required for cyclin D1 suppression (Philipp et al, 1994), yet it is obligatory for Myc autosuppression (Facchini et al, 1997) and remains unexplored for C/ EBPa and AdMLP suppression.…”

“…Myc suppression appears to be mediated through core regulatory elements and does not require the con-sensus Myc/Max binding sites within the target promoter sequence (Antonson et al, 1995;Facchini et al, 1997;Li et al, 1994;Lucas et al, 1993;Philipp et al, 1994). Interestingly, Max heterodimerization is not required for cyclin D1 suppression (Philipp et al, 1994), yet it is obligatory for Myc autosuppression (Facchini et al, 1997) and remains unexplored for C/ EBPa and AdMLP suppression. It remains unclear whether Myc repression of these target genes occurs by direct or indirect pathways; however, Myc can clearly repress gene expression by more than one mechanism (Marhin et al, 1996).…”