Fig. 1.
Deacetylation of promoter-associated histones H3 and/or H4 in BCL11A-transfected cells and BCL11A-mediated transcriptional repression are partially reversed by nicotinamide. (A) HEK293 cells were transfected with 3 μg (17-mer)4-tk-CAT reporter and 20 μg of an expression vector encoding Gal4-BCL11A. Twenty-four hours after transfection, cells were treated with histone deacetylase inhibitors, TSA (100 ng/ml; open bars), and nicotinamide (NAM; 15 mM; hatched bars) as indicated, for 24 h before collection. Transfection efficiency was normalized by total amount of the transfected (17-mer)4-tk-CAT reporter present as determined by PCR amplification using template-specific primers (input lanes 1 and 2; 5% of total). Lanes 3–6 represent template amplification reactions from samples immunoprecipitated with or without antibodies specific for acetylated histones H3 and H4 as indicated. Amplification reactions were separated on a 1% agarose gel that was stained with ethidium bromide to visualize DNA products. The indicated band is the expected, 327 bp amplification product from the reporter gene template. Results are representative of three independent experiments. (B) 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 as indicated. The treatments with vehicle (water; solid bars), TSA (open bars), and NAM (hatched bars) were carried out as described above. Transfection efficiency was normalized by use of a co-transfected β-galactosidase expression vector. CAT activity determined in the presence of Gal4 DBD and TSA (lane 2) 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.