SIRT1 Regulates HIV Transcription via Tat Deacetylation
Figure 5
Transcriptional Activity of AcTat Depends on Deacetylation by SIRT1
(A) AcTat functions through TAR and cyclinT1 binding. Nuclear microinjection of increasing amounts of synthetic Tat or AcTat together with wild-type (wt TAR), TAR Δbulge, or TAR Δloop mutant HIV LTR luciferase constructs into HeLa cells. Cells were coinjected with CMV-GFP, and luciferase activity was calculated per GFP-positive cell. An average of three experiments is shown (± SEM).
(B) AcTat transactivation requires CDK9. HeLa cells microinjected with Tat or AcTat (each 30 ng/μl) and the HIV LTR luciferase reporter were treated with increasing amounts of DRB, a known CDK9 inhibitor, for 4 h.
(C) AcTat transcriptional activity is inhibited by nicotinamide, but not TSA. HeLa cells injected with HIV LTR luciferase and increasing amounts of AcTat were treated with TSA (400 nM) or nicotinamide (5 mM) for 4 h. The average of two experiments is shown.
(D) SIRT1 is necessary for AcTat, but not Tat function. HeLa cells were transfected with siRNAs specific for SIRT1 or GL3 control siRNAs 48 h before microinjection of HIV LTR luciferase and Tat or AcTat (each 30 ng/μl). The average of three experiments is shown (± SEM).