Kharazmi, J. | Search bioRxiv

bioRxiv2026

Functional dissection of Drosophila Myc cis-regulatory modules (Myc-CRMs) reveals developmentally active DNA-protein interactions

Abstract Precise regulation of Drosophila Myc is essential for growth and homeostasis, yet regulation of its transcriptional control remains incompletely understood. We investigated the Myc cis-regulatory landscape using in vivo reporter assays, EMSA, and LC-MS/MS-based identification of DNA-associated proteins. By truncating Myc cis-regulatory modules (CRMs), we delineated the activity of conserved non-coding elements across adult female tissues and larval stages. Specific DNA-protein interactions were confirmed by EMSA using nuclear embryonic extracts. We developed a Solid Surface Magnetic Enrichment protocol (SSMEP) to pull down DNA-protein complexes formed on Myc cis-elements. Affinity purification followed by LC-MS/MS enabled the identification of candidate transcriptional regulators associated with Myc-CRMs. This integrative approach provides new insights into promoter structure and trans-regulatory architecture of Myc and their roles in developmental gene expression programs. Our study identifies a distal enhancer required for larval and pupal patterning, with activation dependent on specific spacing relative to the TATA-box core promoter, a strong enhancer cluster within 5'-UTR cis-elements active in ovaries and embryos, and a DPE-core promoter requiring nearby enhancer action. The DPE-linked enhancer can function with both Inr-DPE and TATA-box promoters.The identified Myc-CRMs interact with conserved signaling pathways to tightly control Myc during development.

molecular biology↗

bioRxiv2025

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, MYCs transcriptional regulation remains incompletely understood. Our previous work demonstrated that key regulatory elements controlling Drosophila Myc reside within its 5 UTR and intergenic regions. In this study, we developed a highly sensitive and selective Solid Surface Magnetic Enrichment Protocol (SSMEP) to purify DNA-protein complexes formed on Myc cis-elements. Using analysis of truncated reporters, Electrophoretic-Mobility Shift Assay (EMSA), and mass spectrometric protein identification, we identified a cis-regulatory module (CRM) within the proximal 5 UTR that appears sufficient to activate lacZ reporter expression in the ovary and embryos. Furthermore, combining CRMs from either the upstream region or the intergenic Downstream Promoter Element (DPE) region with the proximal 5-UTR regulatory unit recapitulates expression patterns previously observed in larval tissues, ovaries, and embryos. The DPE itself exhibits promoter activity when fused in-frame to an adjacent binding site cluster, and drives expression mimicking that of wild-type Myc. Further in vivo validation is necessary to confirm the functional relevance of the associated factors and identified signaling cascades as Myc regulators. Additionally, this highly selective and sensitive protocol has potential applications in other model organisms and various fields of biomedical and synthetic biology research.

molecular biology↗