Cocaine-induced DNA-PK relieves RNAP II pausing by promoting TRIM28 phosphorylation

Drug abuse continues to pose a significant challenge in HIV control efforts. In our investigation, we discovered that cocaine not only upregulates the expression of DNA-dependent protein kinase (DNA-PK) but also augments DNA-PK activation by enhancing its phosphorylation at S2056. Moreover, DNA-PK phosphorylation triggers the translocation of DNA-PK into the nucleus. The finding that cocaine promotes nuclear translocation of DNA-PK further validates our observation of enhanced DNA-PK recruitment at the HIV long terminal repeat (LTR) following cocaine exposure. By activating and facilitating the nuclear translocation of DNA-PK, cocaine effectively orchestrates multiple stages of HIV transcription, thereby promoting HIV replication. Additionally, our study indicates that cocaine-induced DNA-PK promotes hyper-phosphorylation of RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) at Ser5 and Ser2 sites, enhancing both initiation and elongation phases, respectively, of HIV transcription. Cocaine's enhancement of transcription initiation and elongation is further supported by its activation of cyclin-dependent kinase 7 (CDK7) and subsequent phosphorylation of CDK9, thereby promoting positive transcriptional elongation factor b (P-TEFb) activity. We demonstrate for the first time that cocaine, through DNA-PK activation, promotes the specific phosphorylation of TRIM28 at Serine 824 (p-TRIM28, S824). This modification converts TRIM28 from a transcriptional inhibitor to a transactivator for HIV transcription. Additionally, we observe that phosphorylation of TRIM28 (p-TRIM28, S824) promotes the transition from the pausing phase to the elongation phase of HIV transcription, thereby facilitating the production of full-length HIV genomic transcripts. This finding corroborates the observed enhanced RNAP II CTD phosphorylation at Ser2, a marker of transcriptional elongation, following cocaine exposure. Accordingly, upon cocaine treatment, we observed elevated recruitment of p-TRIM28-(S824) at the HIV LTR. Overall, our results have unraveled the intricate molecular mechanisms underlying cocaine-induced HIV transcription and gene expression. These findings hold promise for the development of highly targeted therapeutics aimed at mitigating the detrimental effects of cocaine in individuals living with HIV.