Figure 5
Figure 5. Erg regulates HDAC6 expression. (A) HUVECs were transfected with either control-GB or Erg-specific GB for 48 hours. Message RNA levels for HDAC6, determined using real-time PCR, are normalized to GAPDH and expressed relative to control-GB. ***P < .001. Values are mean ± SEM, n = 3. (B) HUVECs were treated with control or Erg-specific GB, as above. Whole-cell protein extracts were analyzed by SDS-PAGE immunoblotting with Abs to Erg, HDAC6, and acetylated tubulin. In Erg-deficient cells, HDAC6 protein expression was reduced while acetylated tubulin was increased compared with controls. Total tubulin levels appear unchanged between treatments. *P < .05, ***P < .001. Values are mean ± SEM, n = 3. (C) ChIP from a confluent HUVEC monolayer was performed as described in “Methods.” Genomic DNA obtained after immunoprecipitation, using a rabbit anti-Erg polyclonal Ab or a negative control rabbit IgG, was used as template in real-time PCR with primers spanning a region of the HDAC6 promoter containing 6 ETS-binding sites (see supplemental Figure). Enhanced enrichment of Erg-immunoprecipitated DNA compared with IgG-immunoprecipitated DNA, relative to total chromatin, indicates that Erg binds to the HDAC6 promoter. Primers annealing in the HDAC6 locus, outside the promoter region, were used as negative control. *P < .05. Values are mean ± SEM, n = 6. (D) An Erg cDNA expression plasmid (pSG5Erg) or an empty expression plasmid (pSG5) were cotransfected with HDAC6 promoter-luciferase constructs (HDAC6-P1 or HDAC6-P2) in HUVECs, and luciferase activity was measured. Values are represented as the fold change in relative luciferase activity over the empty pGL4 vector alone. Erg transactivates both HDAC6 promoter constructs, although to a greater extent using the longer sequence (HDAC6-P1), *P < .05. Values are mean ± SEM, n = 3. (E) HUVECs were transfected with either wtHDAC6-EGFP or an enzymatically dead HDAC6 mutant (mutHDAC6-EGFP) and allowed to adhere to gelatin-coated glass chamber slides for 24 hours before transfection with control GB or Erg-specific GB for an additional 24 hours. Immunofluorescence confocal microscopy was used to visualize cells expressing GFP (green); cells were labeled for acetylated tubulin (red), Erg (purple), and nuclear marker DAPI (blue). Scale bar, 20 μm. For all images, the same intensity settings were used for acetylated tubulin detection which results in some saturation of the signal in the 2 bottom rows. (F) HUVECs were transfected as above. Immunofluorescence confocal microscopy was used to visualize cells expressing GFP (green); cells were labeled for actin (red), Erg (purple), and nuclear marker DAPI (blue). Scale bar, 20 μm. In the Erg-deficient cells, note the cell adjacent to the wtHDAC6-EGFP–expressing one; this untransfected cell still exhibits the peripheral actin redistribution after Erg inhibition as observed in the mutHDAC6-expressing cells and in the Erg GB-treated cells in Figure 3A.

Erg regulates HDAC6 expression. (A) HUVECs were transfected with either control-GB or Erg-specific GB for 48 hours. Message RNA levels for HDAC6, determined using real-time PCR, are normalized to GAPDH and expressed relative to control-GB. ***P < .001. Values are mean ± SEM, n = 3. (B) HUVECs were treated with control or Erg-specific GB, as above. Whole-cell protein extracts were analyzed by SDS-PAGE immunoblotting with Abs to Erg, HDAC6, and acetylated tubulin. In Erg-deficient cells, HDAC6 protein expression was reduced while acetylated tubulin was increased compared with controls. Total tubulin levels appear unchanged between treatments. *P < .05, ***P < .001. Values are mean ± SEM, n = 3. (C) ChIP from a confluent HUVEC monolayer was performed as described in “Methods.” Genomic DNA obtained after immunoprecipitation, using a rabbit anti-Erg polyclonal Ab or a negative control rabbit IgG, was used as template in real-time PCR with primers spanning a region of the HDAC6 promoter containing 6 ETS-binding sites (see supplemental Figure). Enhanced enrichment of Erg-immunoprecipitated DNA compared with IgG-immunoprecipitated DNA, relative to total chromatin, indicates that Erg binds to the HDAC6 promoter. Primers annealing in the HDAC6 locus, outside the promoter region, were used as negative control. *P < .05. Values are mean ± SEM, n = 6. (D) An Erg cDNA expression plasmid (pSG5Erg) or an empty expression plasmid (pSG5) were cotransfected with HDAC6 promoter-luciferase constructs (HDAC6-P1 or HDAC6-P2) in HUVECs, and luciferase activity was measured. Values are represented as the fold change in relative luciferase activity over the empty pGL4 vector alone. Erg transactivates both HDAC6 promoter constructs, although to a greater extent using the longer sequence (HDAC6-P1), *P < .05. Values are mean ± SEM, n = 3. (E) HUVECs were transfected with either wtHDAC6-EGFP or an enzymatically dead HDAC6 mutant (mutHDAC6-EGFP) and allowed to adhere to gelatin-coated glass chamber slides for 24 hours before transfection with control GB or Erg-specific GB for an additional 24 hours. Immunofluorescence confocal microscopy was used to visualize cells expressing GFP (green); cells were labeled for acetylated tubulin (red), Erg (purple), and nuclear marker DAPI (blue). Scale bar, 20 μm. For all images, the same intensity settings were used for acetylated tubulin detection which results in some saturation of the signal in the 2 bottom rows. (F) HUVECs were transfected as above. Immunofluorescence confocal microscopy was used to visualize cells expressing GFP (green); cells were labeled for actin (red), Erg (purple), and nuclear marker DAPI (blue). Scale bar, 20 μm. In the Erg-deficient cells, note the cell adjacent to the wtHDAC6-EGFP–expressing one; this untransfected cell still exhibits the peripheral actin redistribution after Erg inhibition as observed in the mutHDAC6-expressing cells and in the Erg GB-treated cells in Figure 3A.

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