Locus-specific chromatin proteomics using dCas-guided proximity labelling in Aspergillus nidulans

Area of Science:

• Molecular Biology
• Proteomics
• Genetics

Background:

• Proximity labelling using promiscuous biotin ligases (BirA) fused to bait proteins identifies in vivo protein interactions.
• Enzymatically-disabled Cas9 (dCas9) fused to BirA, guided by sgRNAs, can target specific chromatin locations for protein identification.

Purpose of the Study:

• To adapt and validate the CasID method for locus-specific protein identification in fungal cells.
• To utilize an estrogen-inducible dCas9-BirA fusion for condition-specific labelling in Aspergillus nidulans.
• To identify chromatin-associated proteins and transcription factor interactors at the nitrate assimilation gene cluster.

Main Methods:

• Adaptation of the CasID method in Aspergillus nidulans using an estrogen-inducible dCas9-BirA fusion.
• Chromatin immunoprecipitation (ChIP) to verify dCas9-BirA and transcription factor presence at the target locus.
• Optimization of CasID for efficient labelling and background reduction using control strains (lacking sgRNA or CasID system).
• Affinity purification of biotinylated proteins using streptavidin resin followed by LC-MS/MS analysis.

Main Results:

• Successful verification of dCas9-BirA and AreA transcription factor presence at the target locus via ChIP.
• Proof-of-concept demonstrating enrichment of biotinylated proteins in CasID strains compared to controls.
• Identification of 32 proteins exclusively enriched in the CasID-sgRNA strain after background reduction, including NmrA and chromatin-associated proteins.
• High background of biotinylated proteins necessitates rigorous controls for accurate analysis.

Conclusions:

• The CasID method is suitable for locus-specific labelling and identification of chromatin-associated proteins and transcription factors in Aspergillus nidulans.
• The study provides a detailed protocol for applying CasID in fungi.
• Further optimization is needed to address high background levels in biotinylation and purification steps.