FIG. 1.

MBD1-mediated transcriptional repression is resistant to HDAC inhibitors. (A) Effect of HDAC inhibitors on transcriptional repression by MBD proteins. The reporter constructs contain both the yeast GAL4 DNA binding site (5xGAL) and sequences from the human promoter-associated CpG island upstream of the luciferase cDNA. Effectors express the TRDs of MeCP2 and MBD1 and the MBD2a and MBD3 in a fusion to the GAL4 DNA binding domain. An effector (0.3, 0.6, and 1.2 μg) and the insertless pCMV-GAL4 (0.9, 0.6, and 0 μg) were introduced into HeLa and U2OS cells together with the reporter containing VHL promoter (1.0 μg) and an internal control pRL-CMV (0.02 μg). At 30 h after transfection, the cells were treated for 12 h with the HDAC inhibitors, TSA and sodium butyrate (SB), and with the solvent alone (black). The luciferase activities in combination with pCMV-GAL4 (mock) were normalized to 100 (as indicated by a line). (B) Little influence of HDAC inhibitors on repression by TRD of MBD1 from a distance. GAL4 binding motifs were inserted more than 3 kb upstream of the transcription start site in the reporter constructs. Four gene promoters were used: p16 (black), VHL (hatched), E-cadherin (gray), and SNRPN (white). An effector (0 to 1.0 μg) was transfected into HeLa cells. (C) Induction of acetylated histones H3 and H4 by HDAC inhibitors. Lane C, the solvent alone.