RelB proximity proteomics and CRISPR screening define chromatin regulators of noncanonical NF-κB control of HIV latency and reactivation

Activation of the non-canonical NF-{kappa}B pathway via RelB/p52 signaling by SMAC mimetics such as AZD5582 is a promising strategy to induce HIV expression from latency, but the chromatin mechanisms linking RelB/p52 to proviral regulation remain poorly defined. Here, we combine RelB BioID proteomics, targeted HIV-CRISPR screening, and pharmacologic validation to identify ncNF-{kappa}B-associated regulators of HIV expression. RelB BioID revealed an extensive interaction network of chromatin and transcriptional regulators in basal and AZD5582-activated states. Basally associated RelB proteins included NSD2, SWI/SNF components, UHRF1, and DNMT1, while AZD5582-enriched proteins included p300, USP7, LSD1/KDM1A, NuRD components, SIN3A, and HBO1/KAT7. Functional screening using a custom guide RNA library targeting all BioID-identified factors identified regulators that promote HIV reactivation, including HBO1/KAT7, NSD2, and SIN3A, and regulators that restrict HIV expression, including p300, CHD4, and USP7. Because KAT7/HBO1 and p300 encode acetyltransferases with opposing screen phenotypes, we tested whether their catalytic activities contribute to HIV transcriptional regulation and found that, consistent with the CRISPR screen, KAT7/HBO1 inhibition reduced AZD5582-induced reactivation, whereas p300 inhibition enhanced it. Together, these data define a resource linking the RelB-associated chromatin landscape to HIV latency and reactivation.