Fig. 4.

SIRT1 enhances BCL11A-mediated transcriptional repression. (A) Wild-type SIRT1, but not SIRT1 H363Y, stimulates BCL11A-mediated transcriptional repression. HEK293 cells were transiently transfected with 5 μg of the (17-mer)₄-tk-CAT reporter along with 5 μg of expression vectors encoding either Gal4-BCL11A or Gal4 DBD, and increasing amounts (0.125, 0.25, and 0.5 μg) of expression vectors encoding either SIRT1 WT or SIRT1 H363Y, as indicated. Transfection efficiency was normalized as described in the legend of Fig. 1B. The activity of the CAT reporter in the presence of Gal4 DBD alone (lane 1) was taken to be maximal and that against which all other determined CAT activities were compared. The results presented represent means (±SEM) of three independent experimental determinations. (Inset) The catalytically inactive Myc-SIRT1 H363Y, coimmunoprecipitates with Flag-BCL11A in a manner indistinguishable from wild-type SIRT1 (see Fig. 2A). Transfections, immunoprecipitations, and immunoblotting were conducted as described in Fig. 2A. (B) SIRT1 stimulates deacetylation of template-associated histones H3 and/or H4 in BCL11A-transfected cells. HEK293 cells were transfected with 3 μg of the (17-mer)₄-tk-CAT reporter along with expression vectors encoding Gal4- BCL11A (10 μg) and SIRT1 WT or SIRT1 H363Y (0.5 μg) as indicated. The level of acetylated histones H3 and H4 that were associated with the promoter region of the reporter gene template was determined by a ChIP assay as described in Materials and methods. Transfection efficiency was normalized as described in the legend of Fig. 1A. Input lanes (1–4) correspond to amplification reactions conducted using 3.75% (upper panel) and 5% (lower panel) of the lysates used for IP reactions. Lanes 5–12 represent template amplification reactions from samples immunoprecipitated with or without anti-acetylated histone H3/H4 antibodies as indicated. Results are representative of three independent experiments.