Developing a novel Drosophila model to study Menin biological function

Multiple Endocrine Neoplasia type 1 (MEN1) is an autosomal dominant tumour predisposition syndrome caused by loss-of-function mutations in the MEN1 gene, which encodes the scaffold protein Menin. Menin regulates transcription through interactions with histone deacetylases, histone methyltransferases, and transcription factors, but its molecular function in neuroendocrine tissues remains incompletely understood. Drosophila melanogaster offers a tractable in vivo platform in which to study Menin biology, with a conserved orthologue (Mnn1) and a larval neuroendocrine organ, the ring gland, that is functionally analogous to the endocrine tissues affected in MEN1 syndrome. However, the endogenous expression, subcellular localisation, and protein interactions of Drosophila Menin have not previously been characterised at the protein level. This work describes the generation and validation of a Drosophila line carrying an endogenous 3xHA-tagged allele of the principal Mnn1 isoform (C-08), produced by CRISPR/Cas9-mediated insertion via the ExTaSy recombinase-mediated cassette exchange system. Anti-HA immunofluorescence revealed that endogenous Menin is broadly expressed across the larval central nervous system with predominantly nuclear localisation and is detectable throughout embryonic development from the cellular blastoderm stage onwards. Colocalisation with HDAC1 was observed in both contexts, consistent with the conserved mammalian Menin-HDAC1 association. Biochemical characterisation demonstrated that HA-tagged Menin can be immunoprecipitated from dissected larval brain and adult head lysates. Label-free quantitative mass spectrometry of optimised immunoprecipitates identified Menin as the most enriched protein in the larval brain comparison, with Drosophila orthologues of the majority of established mammalian Menin partners detected in the dataset. These findings establish the tagged line as a proof-of-concept tool for studying endogenous Menin in Drosophila, demonstrating the feasibility of localisation and interactome analysis in this system. Future work will extend these findings through NanoDam-based chromatin binding site mapping, introduction of MEN1 patient-derived mutations into the endogenous Mnn1 locus, and refinement of the proteomics approach through cell-type-specific immunoprecipitation.

Grantor dc:publisher

Oxford Brookes University

Author dc:creator

• Ainsworth, Scarlett Amy

Contributors dc:contributor

• Lines, Kate E.
• Jennings, Barbara H.

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/rx0n-e891

OAI identifier oai:identifier

tle:eb2627ea-fd90-432c-a56f-2af12b5de99e:d6bd9758-527a-46cd-bfe2-c433766e8fca:1