Fig 1: SMYD5 methylates histones and Tat in vitro and binds TAR RNA(A and B) In vitro methylA) recombinant histones and (B) synthetic Tat peptide (aa 1–72), recombinant NF-?B RelA, Sp1, Cyclin T1, or CDK9 incubated with recombinant full-length SMYD5 enzyme and radiolabeled H3-S-adenosyl-L-methionine (SAM). All in vitro methylation assays on isolated histones, Tat peptides, NF-?B, SP1, and pTEFb were repeated at least three times, and representative Coomassie staining (top) and corresponding autoradiographs (bottom) are shown.(C) Gel shift assays of recombinant SMYD5 (0, 50, 150 and 600 ng) and radiolabeled wild-type (WT), bulge-mutant (?Bulge), or loop-mutant (?Loop) TAR RNA probes.(D) Gel shift assay with radiolabeled wild-type (WT) TAR RNA and increasing amounts of SMYD5 in the absence (left) and presence (right) of a 6× excess of non-radiolabeled TAR RNA. All EMSAs were repeated at least three times, and representative autoradiographs are shown.
Fig 2: SMYD5 is expressed and upregulated upon activation in primary CD4+ T cells, and its knockdown inhibits HIV-1 expression(A) SMYD5 protein and mRNA expression in CD4+ T cells from the blood of two independent human donors. The cells were treated with 20 ng/mL TNF-a or aCD3/CD28 Dynabeads (1 bead per 2 cells) for 24 h, then lysed in FLAG immunoprecipitation (IP) buffer for western blot analysis or in RLT+ lysis buffer to isolate mRNA (QIAGEN). RNA levels were analyzed by qRT-PCR and normalized to RPL13A RNA. The average (mean ± SEM) from triplicate experiments performed with two different donors is shown.(B) shRNA-mediated knockdown of SMYD5 in CD4+ T cells. Shown is a schematic of the workflow. Primary CD4+ T cells isolated from uninfected blood donors were activated with aCD3/CD28 Dynabeads for 3 days, infected first with lentiviral particles encoding shRNA against SMYD5 or a non-targeting control, and second with a GFP-tagged reporter virus in the background of HIVNL4–3.(C–E) Impact of non-targeting or SMYD5-targeting shRNAs on the percentage of HIV-1 expressing (GFP+) cells (C), the percentage of live cells as measured by forward scatter analysis and viability stain (D), and the level of SMYD5 mRNA as determined by RT-PCR normalized to RPL13A mRNA (E) among activated CD4+ T cells from three independent donors. The average (mean ± SEM) from triplicate experiments performed with three different donors is shown.
Fig 3: Tat physically associates with SMYD5 and stabilizes SMYD5(A) SMYD5 co-immunoprecipitates with Tat. Cellular extracts from HEK293T cells expressing V5-SMYD5 or empty vector, alone or together with FLAG-Tat, were prepared 48 h after transfection and subjected to purification with anti-M2 FLAG affinity gel. The eluates were resolved by SDS-PAGE and subjected to western blotting.(B) Tat co-immunoprecipitates with SMYD5. Cellular extracts from HEK293T cells expressing V5-SMYD5 or empty vector, alone or together with FLAG-Tat, were prepared 48 h after transfection and subjected to purification with anti-V5 affinity gel. The eluates were resolved by SDS-PAGE and subjected to western blotting.(C) Tat increases SMYD5 protein expression. HEK293T cells were transfected with 500 ng V5-SMYD5 and 0–200 ng of FLAG-Tat or 0–200 ng FLAG-GFP plasmids. After 48 h, cells were lysed and subjected to western blot analysis.(D and E) TNF-a increases SMYD5-Tat co-immunoprecipitation but not SMYD5 RNA production. J-Lat 5A8 cells were treated with increasing amounts of TNF-a. After 48 h, cells were lysed in FLAG-IP buffer and subjected to western blot analysis (C) or lysed in RLT+ buffer to isolate mRNA (QIAGEN) (D). RNA levels were analyzed by qRT-PCR and normalized to RPL13A RNA. All IPs, western blots, and qPCRs were repeated at least three times. Representative results of IPs and western blots are shown. qPCR results of three biological replicates (±SEM) are shown.
Fig 4: SMYD5 is recruited to the HIV-1 LTR and activates HIV-1 transcription(A and B) Luciferase assays in HeLa cells transfected with an HIV-1 LTR luciferase construct and increasing amounts of an expression vector for SMYD5 (0, 5, 50, and 500 ng) in (A) the absence of Tat or (B) the presence of Tat. Expression of SMYD5 strongly activates the HIV-1 LTR and increases in the presence of Tat. Results of three biological replicates (±SEM) are shown. Insets show western blots, with arrows pointing to the SMYD5 band.(C and D) ChIP assays with antibodies against SMYD5, RNA Pol II, and the IgG control at the HIV-1 LTR, followed by qPCR using primers specific for (C) the HIV-1 5' LTR or (D) the RPL30 control. Chromatin was prepared from J-Lat 5A8 cells treated with TNF-a to stimulate the LTR or left untreated. The average of three independent experiments analyzed in triplicate ± SEM is shown and compared with no-treatment control samples by ANOVA: **p < 0.005, ***p < 0.001.(E–H) ChIP assays with antibodies against RNA Pol II, CDK9, H3, H3K4me3, H3K36me3, and the IgG control at the HIV-1 LTR, followed by qPCR using primers specific for the HIV-1 LTR Nuc0, Nuc1, or Nuc2 regions. Chromatin was prepared from J-Lat A72 SMYD5 knockdown or non-targeting shRNA-expressing cells treated with TNF-a to stimulate the LTR. All ChIPs and qPCRs were repeated at least three times, and representative results of three technical replicates are shown.
Fig 5: Model of SMYD5’s activator function at the HIV-1 LTRSMYD5 can activate the HIV-1 LTR alone through its intrinsic TAR RNA-binding capacity and in conjunction with Tat.
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