Gao, Yuan

Abstract Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein–protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2 (pA-APEX2) labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a pA-APEX2 fusion protein. Activation of APEX2 labels the nearby proteins with biotin; the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry. We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac, and H4K12ac, as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient method to identify proteins that are proximal to modified histones.

Significance ES cells possess the unique capacity to self-renew as well as differentiate into specialized cell types. It is known that transcription factors and chromatin regulators regulate the cell-fate choices during differentiation. We report unexpectedly that Asf1a, a histone chaperone involved in nucleosome assembly, regulates mouse ES cell differentiation. Mechanistically, we show that Asf1a functions in nucleosome disassembly to resolve the bivalent chromatin domains at lineage-specific genes for gene activation during differentiation. These insights will likely be applicable for understanding human ES cell differentiation and regenerative medicine.