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

UNLABELLED: 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.

HIGHLIGHTS: Myc Oocyte Element: an eRNA-producing enhancer in ovaries and early embryos DPE promoter synergizes with nearby enhancer to drive Drosophila Myc transcription Late enhancer licenses TATA promoter for larval tissue-specific Myc transcription Promoter-enhancer dynamics differentially drive Myc during development SSMEP protocol helps purify and enrich low-abundance DNA-protein complexes.