Daxx deletion mutant transcriptional repression activity. (A) The indicated hDaxx deletion mutants (10 fmol), with domains indicated as described for Fig. 1, or the GAL4 DBD alone were co-transfected into NIH3T3 cells with a chloramphenicol acetyl transferase (CAT) reporter construct containing one GAL4-DNA-binding site. Forty-eight hours after transfection, CAT activity was determined as described in the Materials and Methods. All values were normalized for co-transfected secreted alkaline phosphatase activity and are presented as the percentage of CAT activity in the absence of hDaxx. Errors represent the standard deviation from four independent determinations. (B) The protein expression levels for the GAL4-hDaxx deletion mutants. Equivalent amounts of total cellular lysate from NIH3T3 cells overexpressing each of the individual GAL4-hDaxx deletion mutants (30 μg)were separated by either 10% SDS-PAGE (left panel) or by a 4-15% SDS-PAGE gradient gel (right panel). The level of protein expression was determined by western analysis using an anti-GAL4-DBD monoclonal antibody.

Co-fractionation of hDaxx, HDAC II, Dek and core histones. (A) Sephacryl S-300 size exclusion chromatography. Total cellular extracts of U937 cells ( Boer et al., 1998) stably expressing FLAG-epitope tagged Dek were sonicated and separated by Sephacryl S-300 size exclusion chromatography. Each fraction was analyzed for the presence of hDaxx, Dek-FLAG, HDACII and acetylated histone H4 by western blot analysis. The elution volumes for molecular weight standards are noted below the gel. The amount of total protein (absorbance 280 nm, dotted line), hDaxx (circle, solid line), HDAC II (triangle, solid line) and acetylated histone H4 (square, solid line) in each fraction was determined by densitometry and is presented graphically as arbitrary densitometric units for each fraction. (B) Resource Q® anion exchange chromatography. Fractions 9-13 of the Sephacryl S-300 column were fractionated over a Resource Q® anion exchange column, and proteins were eluted with a 0.15-1 M KCl linear gradient. Equivalent volumes of protein from each fraction were DOC/TCA/acetone precipitated, separated on a 4-20% SDS-PAGE gel and analyzed for the presence of all four proteins. In addition to HDAC II and acetylated histone H4, both the 70 kDa form of hDaxx (closed circle, solid line) and the 120 kDa form of hDaxx (open circle, solid line) are presented. (C) Superdex HR 200 gel filtration chromatography. Fractions 16-19 of the Resource Q® column were concentrated and fractionated over a Superdex HR 200 gel filtration column. Each fraction was analyzed for the presence of all four proteins as described above. In panels A-C the faster-migrating band observed for Dek-FLAG most probably consists of a degradation product as described by others ( Alexiadis et al., 2000; McGarvey et al., 2000). (D) A Coomassie-blue-stained SDS-PAGE of the purified hDaxx complex. Lane 1 contains 1 μg of each of the purified histones H1, H2A, H2B, H3 and H4. Lane 2 contains 20 μg of DOC/TCA-precipitated protein from fraction 13 of the Superdex HR200 column.

Dek-FLAG co-immunoprecipitates the 70 kDa isoform of hDaxx, HDAC II, acetylated histone H4 and core histones. Fractions from the Sephacryl S-300 column that contained hDaxx, HDAC II, acetylated histone H4 and Dek-FLAG were pooled (fractions 9-13, 25ml total) and immunoprecipitated using an anti-FLAG affinity resin to isolate Dek-FLAG and associated proteins. After extensive washing, the proteins were eluted with a FLAG-specific peptide (5 ml final volume). (A) Silver stain analysis of bound proteins. Bound proteins from lysates not expressing Dek-FLAG (left lane) and lysates expressing Dek-FLAG (right lane) were separated by 4-20% SDS-PAGE and visualized by silver staining. (B) Western blot analysis of bound proteins. Equal volumes of the bound (left lane) and unbound (right lane) fractions were separated by 4-20% SDS-PAGE, and the proteins were detected with a western blot analysis using anti-hDaxx, anti-HDAC II or anti-acetylated histone H4 antibodies.

Co-immunoprecipitation of acetylated histone H4 and HDAC II with GAL4-hDaxx. 293T cells overexpressing equivalent amounts of GAL4-hDaxx, GAL4-hDaxxΔ1-132, GAL4-hDaxxΔ1-352 and GAL4-SPT (A) were lysed in the presence of protease and phosphatase inhibitors. The resulting cell lysates were immunoprecipitated with an anti-GAL4 DBD antibody followed by detection with anti-GAL4 (B), anti-acetylated histone H4 (C) or anti-HDAC II (D) antibodies. The asterisks in panel B indicate the location of the IgG heavy and light chains.

Co-immunoprecipitation of histone deactylase activity with GAL4-hDaxx. Equal amounts (100 μg) of total cell lysates from 293T cells overexpressing equivalent levels of either GAL4-hDaxx or GAL4 alone (data not shown) were immunoprecipitated with an anti-GAL4 DBD antibody. The resulting immune complexes were analyzed for histone deacetylase activity either in the absence (gray bars) or presence (striped bars) of the specific HDAC inhibitor, sodium butyrate (see Materials and Methods). Total HeLa nuclear extracts (10 μg) were used as a positive control for the deacetylase assay, and the results are presented as the total CPM of [³H]-acetate released for each sample.