Deacetylation and demethylation are functionally cooperative in vivo. (A) Analysis of dimethyl K4 H3 levels on the synapsin promoter by quantitative chromatin immunoprecipitation (ChIP) assay after transient transfection of HEK 293 cells with plasmids encoding BHC110 or its mutant (K661A). (B) Analysis of acetyl H3 levels on synapsin promoter by quantitative ChIP assay after transient transfection of HEK 293 cells with plasmids encoding BHC110 or its mutant (K661A). (C) Analysis of dimethyl K4/acetyl H3 levels on synapsin promoter by quantitative ChIP assay after trichostatin A treatment (100 ng/ml) of HEK 293 cells.

Recombinant HDAC1 fails to deacetylate nucleosomes. (A) Analysis of recombinant HDAC1 (rHDAC1) isolated from Sf21 insect cells by colloidal staining (left) and analysis of the BHC110 complex isolated from nuclear extracts (NE) by silver staining (right). (B) Comparison of histone deacetylation activities between recombinant HDAC1 and the BHC110 complex. Bulk histones were used as substrates, and reaction mixtures were analyzed by SDS-PAGE, followed by Western blotting. (C) Comparison of nucleosomal deacetylation activities between recombinant HDAC1 and the BHC110 complex. Nucleosomes were used as substrates, and reaction mixtures were analyzed by SDS-PAGE, followed by Western blotting. “1×” corresponds to approximately 5 ng of recombinant HDAC1 or HDAC1/2 in the complex. “Acetyl H3” represents acetyl K9/K14 H3.

Demethylation of nucleosomes enhances deacetylation. (A) Analysis of recombinant BHC110 (rBHC110) and rCoREST isolated from Sf21 insect cells by colloidal staining. (B) Effect of recombinant BHC110 and CoREST on nucleosomal deacetylation. The amounts of recombinant proteins used were approximately as follows: BHC110, 800 ng (+) and 1200 ng (++); HDAC1, 400 ng (+); CoREST, 400 ng (+) and 800 ng (++). Nucleosomes were used as substrates, and reaction mixtures were analyzed by SDS-PAGE, followed by Western blotting. (C) Quantitation of acetyl H3 levels shown in panel B. The acetyl H3 levels in the absence of recombinant proteins (−) were set as 100%. Data are means plus standard deviations from three experiments.

The ELM2 domain of CoREST confers nucleosomal deacetylation. (A) Map of five mammalian expression constructs for CoREST and its mutants containing FLAG tag (ΔELM2, ΔSANT2, ΔSANT1, and ELM2). All CoREST constructs lack 30 amino acids from position 31 to 60. (B) Western blot analysis of BHC110 and HDAC2 coimmunoprecipitated with CoREST and its mutants. HEK-293 cells were transfected with CoREST and its mutants, and whole-cell extracts were immunoprecipitated with anti-FLAG M2 resin. The eluates were analyzed by Western blotting using anti-BHC110, anti-HDAC2, and anti-FLAG antibodies. (C) Effect of ELM2 domain on nucleosomal deacetylation. The amounts of recombinant proteins used were approximately as follows: rBHC110, 800 ng (+) and 1600 ng (++); rHDAC1, 400 ng (+); rCoREST, 400 ng (+); rELM2, 150 ng (+). Nucleosomes were used as substrates, and reaction mixtures were analyzed by SDS-PAGE, followed by Western blotting.

While abrogation of demethylation by K661A mutation decreases deacetylation activity, nucleosomal deacetylation enhances demethylation. (A) Effect of the K661A mutation on histone deacetylation. Bulk histones were used as substrates. (B) Effect of the K661A mutation on nucleosomal deacetylation. Nucleosomes were used as substrates. (C) Quantitation of acetyl H3 levels shown in panel B. The acetyl H3 levels in the absence of the complex (−) were set as 100%. Data are means plus standard deviations from three experiments. “1×” corresponds to approximately 50 ng of HDAC1/2 in the complex. (D) Effect of recombinant HDAC1 and CoREST on nucleosomal demethylation. The amounts of recombinant proteins used were approximately as follows: BHC110, 800 ng (+); HDAC1, 400 ng (+) and 800 ng (++); CoREST, 200 ng (+). (E) Quantitation of dimethyl H3 levels shown in panel D. The dimethyl K4 H3 levels in the absence of recombinant proteins (−) were set as 100%. Data are means plus standard deviations from three experiments. For panels A, B, and D, reaction mixtures were analyzed by SDS-PAGE, followed by Western blotting.

Inhibition of deacetylation reduces nucleosomal demethylation. (A) Effect of sodium (Na⁺) butyrate (100 mM) on histone demethylation. Bulk histones were used as substrates. (B) Effect of sodium butyrate (100 mM) on nucleosomal demethylation. Nucleosomes were used as substrates. “1×” corresponds to approximately 50 ng of HDAC1/2 in the complex. (C) Immunoprecipitation of the BHC110 complexes in the absence or presence of trichostatin A or sodium (Na⁺) butyrate. The BHC110 complexes were incubated without or with TSA (500 ng/ml) and sodium butyrate (Na⁺Bu, 100 mM), immunoprecipitated, and analyzed by Western blotting. (D) Effect of TSA (500 ng/ml) on histone (left) and nucleosomal demethylation (right). Bulk histones and nucleosomes, respectively, were used as substrates. (E) Quantitation of dimethyl K4 H3 levels shown in panel D, right panel. The dimethyl K4 H3 levels in the absence of the complex (−) were set as 100%. Data are means plus standard deviations from three different experiments. (F) Effect of different concentrations of trichostatin A on nucleosomal demethylation/deacetylation. Data are the averages of two different points. “1×” corresponds to approximately 50 ng of HDAC1/2 in the complex. In panels A, B, and D, reaction mixtures were analyzed by SDS-PAGE, followed by Western blotting.