Biology subjects

Moshfegh, C.

Publications and source records attributed to Moshfegh, C..

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↗

bioRxiv2021

Differentiation of mouse embryonic stem cells into cells with spermatogonia-like morphology with chemical intervention-dependent increased gene expression of LIM homeobox 1 (Lhx1).

During the development of the male germline, spermatogonial stem cells (SSCs) originate from gonocytes that differentiate from primordial germ cells (PGCs). In the developing and regenerating mouse testis, expression of the gene LIM homeobox 1 (Lhx1) marks the most undifferentiated SSCs. However, an enrichment of Lhx1 expression in spermatogonia-like cells generated in vitro has not been reported so far. Previously, it was shown that a chemical intervention in male mouse embryonic stem (ES) cells in serum culture, including a timed combination of the SIRT1 inhibitor Ex-527, the DNA methyltransferase inhibitor RG-108 and the electrophilic redox cycling compound tert-butylhydroquinone (tBHQ), was associated with molecular markers of the PGC to gonocyte differentiation process. Here, we report the in vitro differentiation of male mouse ES cells, cultured under dual chemical inhibition of GSK3{beta} and MEK (termed 2i) with leukemia inhibitory factor (LIF) (termed 2iL) and serum, into cells with spermatogonia-like morphology (CSMs) and population-averaged expression of spermatogonia-specific genes. This was achieved by the removal of 2iL and a specific schedule of 2 partial medium replacements per day with alternating 8-hour and 16-hour intervals over a period of 32 days. Combination of this new cell culture protocol with the previously reported chemical intervention in ES cells changed the population-averaged expression of spermatogonia- and gonocyte-specific genes during the differentiation process and increased the population-averaged gene expression of Lhx1 in the resulting CSMs compared to CSMs without chemical intervention. Furthermore, we detected single CSMs with a strong nuclear LHX1/5 protein signal only in the chemical intervention group. Our results provide the first experimental evidence for the generation of CSMs with an enrichment of Lhx1 expression in vitro. We propose that further investigation of the CSMs generated with this in vitro system may provide new insights into male germline and stem cell development.

cell biology↗