FIG. 3.

Synergy between histone deacetylation and Mbd2 in silencing the Xist gene. (A) TSA collaborates with Mbd2 deficiency to cause enhanced activation of Xist transcription as measured by both RT-PCR analysis of Xist RNA using primers XIST-7F/XIST-11R (upper panel) and counting of Xist-positive (+ve) cells in the microscope (lower panel). Xist RNA was visualized in treated and untreated cells by in situ hybridization as described in Materials and Methods. (B) Xist-positive foci (red) within TSA-treated Mbd2-null cell nuclei stained with DAPI (4′,6′-diamidino-2-phenylindole). (C) Enhanced histone acetylation in Mbd2-null cells treated with TSA as measured by ChIP with an antibody against pan-acetyl H4 and quantitative RT-PCR of the Xist gene (primers XIST-7F/XIST-11R; see Fig. 1C). Values are expressed as the percentage of input precipitated. As a control, samples were precipitated with an irrelevant antibody and processed the same way (right-hand bar in each pair).