Regulation of the MAD1 promoter by G-CSF.

MAD family proteins are transcriptional repressors that antagonize the functions of MYC oncoproteins. In particular, MAD1 has been demonstrated to interfere with MYC-induced proliferation, transformation and apoptosis. The MAD1 gene is expressed in distinct patterns, mainly associated with differentiation and quiescence. We observed that MAD1 is directly activated by G-CSF in promyelocytic cell lines. To investigate the transcriptional regulation of the human MAD1 gene, we have cloned and characterized its promoter. A region of high homology between the MAD1 orthologs of human, mouse and rat contains the core promoter, marked by open chromatin, high GC content and the lack of a TATA box. Using deletion constructs we identified two CCAAT-boxes occupied by C/EBPalpha and beta in the homology region that mediate responsiveness to G-CSF receptor signaling. The necessary signals include the activation of STAT3 and the RAS/RAF/ERK pathway. STAT3 does not bind directly to promoter DNA, but is recruited by C/EBPbeta. In summary, our studies provide a first analysis of the MAD1 promoter and suggest STAT3 functions as a C/EBPbeta cofactor in the regulation of the MAD1 gene. Our findings provide the base for the characterization of additional signal transduction pathways that control the expression of MAD1.