A role for Z-DNA in transcriptional activation of the human c-myc oncogene by the vaccinia virus E3L protein

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The proto-oncogene c-myc plays a pivotal role in cell growth control, differentiation, and apoptosis. Regulation of the c-myc gene has implications for tumorigenesis and its transcription can be activated by viral oncoproteins. The vaccinia virus (VV) E3L protein, which is essential for virulence, has oncogenic and anti-apoptotic activities. The left-handed Z-DNA-binding activity of the N-terminal domain (Zα) of E3L is required for VV lethality, anti-apoptoic effect, and transactivation activity on several genes, including, as we report here, c-myc.
 Sequences favorable for Z-DNA-formation occur with great frequency near the transcription start site of many genes and Z-DNA formation near the promoter region can regulate promoter activity. In the human CSF-1 promoter, the presence of a Z-DNA forming sequence is critical for the activation of CSF-1 gene by the SWI/SNF-like BAF chromatin remodeling complex. Sequences in the c-myc promoter are in the Z form when the gene is actively transcribed but not when it is silenced.
 Here, we report that E3L, through the Z-DNA binding of its Zα domain, activates transcription of the human c-myc gene, which contains three Z-forming sequences in its promoter region. Using transient transfection analysis, we demonstrated that E3L activates transcription of the human c-myc gene, and increases its mRNA level in HeLa cells. We have also demonstrated the crucial role of Z-DNA binding by E3L in this transactivation activity.
 In the human c-myc gene, three regions with high Z-DNA forming potential are clustered just upstream of the major c-myc P2 promoter. Functional analyses of the human c-myc promoter by serial deletion mutations and the minimal TATA box-containing promoter system revealed that these Z-forming sequences are required for the transactivation of the human c-myc promoter by E3L. Chromatin immunoprecipitation assays show that E3L binds to the human c-myc promoter via Z-DNA binding. In addition, the chromatin remodeling factor INI1/hSNF5 is recruited to the c-myc promoter by E3L in vivo, as shown by co-immunoprecipitation. E3L and INI1/hSNF5 act synergistically to upregulate c-myc in cotransfection experiments.
 The demonstration that E3L acts via Z-DNA binding and chromatin remodeling to regulate the expression of c-myc provides a satisfying explanation for the oncogenic properties of VVE3L. Further, it provides a mechanistic relevance for the formation of Z-DNA in the active c-myc promoter. The role of binding to Z-DNA forming sequences by the conformation specific Zα domain suggests that Z-DNA binding domains may play a broad role in the regulation of the many genes that can form Z-DNA within their promoter regions.