Biology subjects

Brody, T.

Publications and source records attributed to Brody, T..

3 recordsLinked to original sources

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↗

bioRxiv2019

Ultraconserved non-coding DNA within insect phyla

Presence of ultra-conserved sequence elements in vertebrate enhancers suggest that transcription factor regulatory interactions are shared across phylogenetically diverse species. To date evidence for similarly conserved elements among evolutionarily distant insects such as flies, mosquitos, ants and bees, has been elusive. This study has taken advantage of the availability of the assembled genomic sequence of these insects to explore the presence of ultraconserved sequence elements in these phylogenetic groups. To investigate the integrity of fly regulatory sequences across ~100 million years of evolutionary divergence from the fruitfly Drosophila melanogaster, we compared Drosophila non-coding sequences to those of Ceratitis capitata, the Mediterranean fruit fly and Musca domestica, the domestic housefly. Using various alignment techniques, Blastn, Clustal, Blat, EvoPrinter and Needle, we show that many of the conserved sequence blocks (CSBs) that constitute Drosophila cis-regulatory DNA, recognized by EvoPrinter alignment protocols, are also conserved in Ceratitis and Musca. We term the sequence elements shared among these species ultraconserved CSBs (uCSBs). The position of the uCSBs with respect to flanking genes is also conserved. The results suggest that CSBs represent the point of interaction of multiple trans-regulators whose functions and interactions are conserved across divergent genera. Blastn alignments also detect putative cis-regulatory sequences shared among evolutionarily distant mosquitos Anopheles gambiae and Culex pipiens and Aedes aegypti. We have also identified conserved sequences shared among bee species. Side by side comparison of bee and ant EvoPrints identify uCSBs shared between the two taxa, as well as more poorly conserved CSBs in either one or the other taxon but not in both. Analysis of uCSBs in dipterans, mosquitos and bees will lead to a greater understanding of their evolutionary origin and the function of their conserved sequences.

bioinformatics↗