Fig. 1

NEAT ChIP-MS reveals the HDAC3 liver nuclear interactome. a Schematic illustration of the NEAT ChIP-MS protocol. HA-tagged HDAC3 or EGFP was expressed with or without Cre in Hdac3 ^fl/fl mice specifically in hepatocytes via adeno-associated virus (AAV8 TBG). After isolation of nuclei and cross-linking with formaldehyde, HDAC3 or EGFP control were captured by anti-HA immunoprecipitation. Protein complexes and associated DNA sequences were analyzed by mass spectrometry or high-throughput sequencing, respectively. b Volcano plot of mass spectrometry analysis of HDAC3 interacting proteins (HDAC3 n = 11, EGFP n = 13). The x axis indicates log₂ ratio of normalized intensity (iBAQ) of proteins discovered in HDAC3 to EGFP control. Red box indicates fold-change (10-fold) and P-value (0.01) cutoffs for interactors. Core NCoR complex components (blue), selected high scoring interactors (red), and Rev-erbα (green) are indicated. c Heatmap of normalized intensity (iBAQ) of HDAC3 interactors from b in the presence or absence of micrococcal nuclease (MNase) or Benzonase nuclease. Each lane represents an independent experiment. d Co-IP experiments confirming interaction of HDAC3 with PROX1 from liver expressing tagged HDAC3 (top) or endogenous IPs (bottom). e Protein–protein interaction network analysis. Circle color represents enrichment over control and the size of the circle represents −log₁₀(P-value). Nodes displayed met eightfold enrichment cutoff after Benzonase treatment and lines indicate validated interactions (STRING, active interaction sources include experiments and databases, minimum interaction score 0.6). Interactors are grouped by known type and circles indicate known functional complexes