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. 2002 Sep 1;30(17):3831–3838. doi: 10.1093/nar/gkf509

Figure 4.

Figure 4

Figure 4

. Dnmt3L requires the deacetylase HDAC1 for transcriptional silencing and represses transcription in a TSA-sensitive manner. (A) Schematic representation of the reporter and the effector constructs used. The reporter construct 4XGAL4-TK-Luc contains four copies of GAL4 sites upstream of the thymidine kinase promoter driving expression of the luciferase gene. Also indicated are effector constructs expressing only the GAL4 DBD [GAL4(DBD)], the GAL4 DBD fused to the PHD of Dnmt3L (residues 53–145) and Flag-tagged full-length HDAC1 (HDAC1-F). (B) U2OS cells were transiently transfected with 250 ng of the 4XGAL4-TK-luc reporter and, as indicated, with 50 ng of GAL4 Dnmt3L 53–145 and/or 500 ng of HDAC1-F. Cells were then harvested and assayed for luciferase. The basal activity of the reporter is normalised to a value of 100%. Transfection efficiencies were normalized using β-galactosidase activity. The results shown are the average of at least two independent experiments with error bars displaying standard deviations. (C) Dnmt3L-mediated repression is sensitive to TSA. U2OS cells were transfected with 250 ng of the 5XGAL4-SV40-CAT reporter and 50 ng of GAL4 Dnmt3L 53–145. Sixteen hours after transfection, cells were treated (lanes 3 and 4) or not (lanes 1 and 2) with the HDAC inhibitor TSA (200 mM). The basal activity of the reporter is normalised to a value of 100%. Transfection efficiencies were normalized using β-galactosidase activity. The results shown are the average of at least two independent experiments with error bars displaying standard deviations.