Targeting Regulatory Factors Associated with the Drosophila Myc cis-Elements by Reporter Expression, Gel Shift Assay, and Mass Spectrometric Protein Identification

Transcription factor MYC is highly responsive to mitogenic stimuli to modulate the expression of several targets involved in cell growth, protein biogenesis, proliferation, and differentiation. However, the regulation of MYC at the level of transcription is still incompletely understood. In previous work, we showed that most of the regulatory elements associated with Drosophila Myc are located within the 5′ UTR and the intergenic regions of Myc. In this study, we developed a highly sensitive and selective Solid Surface Magnetic Enrichment Protocol (SSMEP) and used it to enrich DNA-Protein complexes compounded with the Myc cis-elements. Using smaller truncations, Electrophoretic-Mobility Shift Assay (EMSA) and protein identification by Mass Spec, we identified a cis-regulatory module (CRM) within the proximal 5′ UTR that might be sufficient to activate lacZ reporter in the ovary and embryos. Furthermore, each of the CRMs from either the upstream region or the DPE within the intergenic region in conjunction with the proximal 5′-UTR regulatory unit recapitulates the missing expression in the larval tissues, the ovary, and in embryos. Finally, the Downstream Promoter Element (DPE) shows promoter activity and mimics the Myc wild type expression when fused in frame to the adjacent cluster of binding sites. Further In vivo functionality tests will be required to confirm the relevance of associated factors and identified signaling cascades as Myc regulators. In addition, the highly selective and sensitive protocol can be used for other model organisms and biomedical research projects and/or synthetic biological studies.