Supporting documents for 'BRD9 functions as an HIV-1 latency regulatory factor'

Despite decades of searching for a solution, AIDS remains largely incurable. A major challenge in achieving a complete cure for AIDS is the presence of HIV-1 reservoirs within the immune cell population of the host. The "Shock & Kill" strategy has been recently explored, which involves combining antiretroviral treatment (ART) with latency reversal agent (LRA) to reactivate HIV-1 latent reservoirs followed by eliminating infected cells through cell cytopathic effects and immune responses. However, current LRA treatments are not able to fully reactivate the HIV-1 latent reservoirs. To this end, investigation on novel HIV-1 latency regulator is crucial in addressing the persistent population of latently-infected cells. In this study, we identified BRD9, which belongs to the bromodomain and extra-terminal domain (BET) family, as a novel regulator of HIV-1 latency. Treatment with an BRD9 inhibitor, I-BRD9, induced HIV-1 latency reactivation in T cell line models, HIV-1-infected human resting memory CD4+ T cells, and peripheral blood mononuclear cells from people living with HIV-1 (PLWH) under ART treatment. Gene knockdown and protein degradation of BRD9 resulted in HIV-1 latency reactivation in T cell line. Additionally, BRD9 inhibition synergized with BRD4 inhibition in inducing HIV-1 viral production. Furthermore, we elucidated the underlying mechanisms of HIV-1 latency regulation by BRD9. We found that BRD9 binds to the HIV-1 genome at the LTR promoter and Gag gene region, suppressing HIV-1 gene transcription. Moreover, BRD9 competes with the HIV-1 Tat protein for binding to the HIV-1 genome. Through Cut&Run followed by DNA sequencing and RNA sequencing methods, we identified cellular pathways and downstream targets of BRD9 that can also modulate HIV-1 latency. ATAD2, MTHFD2, LDHB and BUB1B are novel BRD9 downstream genes and HIV-1 suppressing genes. Their inhibition can also reactivate HIV-1 latency and synergize with BRD9 inhibition in stimulating HIV-1 viral production.