Elucidation of the structural determinants responsible for the specific formation of heterodimeric Mxd1/Max b-HLH-LZ and its binding to E-box sequences

The proteins of the Mxd family (formally known as Mad) are antagonists of the oncoprotein c-Myc. They compete with c-Myc for their obligate partner Max to prevent the c-Myc/Max heterodimer from binding to E-box sequences in the target gene promoters. In cancer cells, where Myc is overexpressed, the expression of Mxd proteins is usually insufficient or abrogated. However, the reintroduction of Mxd1 expression in these cells prevents growth and proliferation. While the antagonism of c-Myc functions by Mxd proteins is of potential relevance for the development of cancer treatment strategies, the structural determinants responsible for the specific heterodimerization between the Mxd and the Max b-helix-loop-helix-leucine zippers are not fully understood. Moreover, whether the heterodimer is assembled on DNA or in the nucleoplasm prior to DNA binding is under debate. In this article, we demonstrate that Mxd1 D112a and Max N78a and H81d, which are located in the leucine zippers of the proteins, can dictate the specificity of heterodimerization and whether or not the Mxd1/Max/DNA complex forms. Our results also indicate that additional specific determinants exist in the helix-loop-helix domains of Max and Mxd1. Finally, we provide evidence that heterodimerization must precede DNA binding in vivo.     

DNA-binding domain of human c-Myc produced in Escherichia coli.

We have identified the domain of the human c-myc protein (c-Myc) produced in Escherichia coli that is responsible for the ability of the protein to bind sequence-nonspecific DNA. Using analysis of binding of DNA by proteins transferred to nitrocellulose, DNA-cellulose chromatography, and a nitrocellulose filter binding assay, we examined the binding properties of c-Myc peptides generated by cyanogen bromide cleavage, of mutant c-Myc, and of proteins that fuse portions of c-Myc to staphylococcal protein A. The results of these analyses indicated that c-Myc amino acids 265 to 318 were responsible for DNA binding and that other regions of the protein (including a highly conserved basic region and a region containing the leucine zipper motif) were not required. Some mutant c-Mycs that did not bind DNA maintained rat embryo cell-cotransforming activity, which indicated that the c-Myc property of in vitro DNA binding was not essential for this activity. These mutants, however, were unable to transform established rat fibroblasts (Rat-1a cells) that were susceptible to transformation by wild-type c-Myc, although this lack of activity may not have been due to their inability to bind DNA.     

Expression and DNA-binding activity of MYCN/Max and Mnt/Max during induced differentiation of human neuroblastoma cells

Amplification of MYCN is one of the most important prognostic markers for neuroblastoma and is correlated with rapid tumor progression and poor prognosis. MYCN belongs to the Myc/Max/Mad/Mnt network of proteins that regulate proliferation, apoptosis, and differentiation. It is well established that MYCN is downregulated during induced differentiation of neuroblastoma cells carrying an amplified MYCN gene, but very little is known about other components of the network, i.e., the Max, Mad, and Mnt proteins, during this process. In this study we show that Mad and Mnt expression was only modestly regulated in differentiating SK-N-BE(2) neuroblastoma cells, while MYCN was rapidly downregulated. This downregulation was reflected in a decreased MYCN/Max DNA-binding activity while the Mnt/Max binding did not change during differentiation. In parallel experiments we also analyzed the Myc/Max/Mad expression and DNA binding capacity during induced differentiation in the MYCN single copy neuroblastoma cell line SH-SY5Y. In this cell line only modest changes in expression of the components of the MYCN/Max/Mad/Mnt network was detected, but since the cell line expresses relatively low levels of MYCN and c-Myc, these changes might be of functional significance. Cell cycle analyses of SK-N-BE(2) demonstrated an increase in the G1-phase fraction after RA-treatment. These data show that the decreased MYCN expression and MYCN DNA-binding is correlated with retarded cell cycle progression. Furthermore, when Mad1 or Mnt was overexpressed in SK-N-BE(2) cells they retained the capacity to differentiate, underscoring the notion that MYCN downregulation, and not changes in Mad/Mnt expression, is essential for neuroblastoma cell differentiation.