A Structured Workflow for Mapping Human Sin3 Histone Deacetylase Complex Interactions Using Halo-MudPIT...

Area of Science:

• Molecular Biology
• Proteomics
• Chromatin Biology

Background:

• Affinity purification mass spectrometry (AP-MS) is widely used but prone to artifacts from protein overexpression.
• Investigating protein complexes like the Sin3 histone deacetylase complex requires validated reagents and accurate interaction mapping.

Purpose of the Study:

• To develop and validate a systematic workflow using the Halo tag for assessing protein localization and behavior before AP-MS analysis.
• To map protein interactions within the Sin3 complex using stably expressed subunits and an advanced complex NSAF (cNSAF) approach.

Main Methods:

• Utilized the Halo tag for validating tagged subunits of the Sin3 complex, including SUDS3, SAP30, and SAP30L.
• Applied a modified complex NSAF (cNSAF) approach with normalization to SIN3A for quantitative comparison of bait-specific interactions.
• Performed affinity purification mass spectrometry (AP-MS) to identify interacting proteins.

Main Results:

• The workflow successfully validated reagents, modifying tagging/expression strategies for 21.7% of constructs.
• SAP30 and SAP30L associate with the core Sin3 complex, with SAP30L uniquely interacting with the proteasome and myelin sheath.
• SUDS3 is found in both SIN3A and SIN3B complexes, while SAP30 is absent from SIN3B complexes. SAP18 and SAP25 were not detected in high-confidence interactions.

Conclusions:

• The Halo tag workflow provides a robust framework for generating validated reagents for quantitative interaction network assembly.
• Novel insights into the Sin3 complex reveal differential subunit usage in SIN3A and SIN3B complexes and highlight unique SAP30L interactions.
• The findings advance the understanding of chromatin remodeling complexes and their subunit composition in human cells.