Uncovering the Domain-Specific Interactome of the TAF1 Tandem Reader Using Site-Specific Azide-Acetyllysine Photochemistry.

Combinatorial readout of histone post-translational modifications by tandem reader modules mediates crosstalk among different histone modifications. To identify the domain-specific interactome of the tandem reader, we engineered the dual bromodomain of TATA-binding protein-associated factor-1 (TAF1) to carry a photoactivatable unnatural amino acid, 4-azido-l-phenylalanine (AzF), via amber suppressor mutagenesis. Using computational approaches, we modeled the targeted residues of TAF1 with AzF to predict the cross-linking distance between the reactive arylazide and its interacting partner. We developed three photoactivatable TAF1 tandem-bromodomain analogues, viz., Y1403AzF in bromodomain 1 (), W1526AzF in bromodomain 2 (), and Y1403AzF/W1526AzF in both and . Circular dichroism and a thermal shift assay were used to confirm the structural integrity of the engineered readers. Using the TAF1 tandem-bromodomain analogues, we characterized their histone ligand binding properties by isothermal titration calorimetry and photo-cross-linking experiments. We found that the dual bromodomain of TAF1 independently binds and cross-links to different acetylated histone ligands. We further used the engineered and analogues of the TAF1 tandem readers to identify their domain-specific interacting partners at the cellular level. Both and independently cross-link to a unique interactome, and importantly, the dual cross-linker carrying TAF1 analogue could capture both - and -specific interactomes. Our work concludes that and of the TAF1 tandem reader independently recognize their interacting partners to regulate downstream cellular functions.