Journal:

Article Title: An Ikaros-Containing Chromatin-Remodeling Complex in Adult-Type Erythroid Cells

doi:

Figure Lengend Snippet: PYR complex DNA-binding activity cofractionates from MEL nuclear extract with Ikaros and a subpopulation of SWI/SNF and NuRD protein. (A) Purification scheme. The PYR complex was purified from MEL nuclear extract in four chromatographic steps using High Q Macroprep (Bio-Rad; 0.1 to 0.4 M NaCl step elution), reactive yellow 3-agarose (Sigma; 0.1 to 1.0 M NaCl gradient), double-stranded (ds) DNA-cellulose (calf thymus DNA-cellulose [Sigma], 0.1 to 0.7 M NaCl step gradient), and Superose 6 (Amersham Pharmacia) columns. (B) Chromatography on reactive yellow 3-agarose shows peak PYR complex DNA-binding activity by gel shift assay (PYR) eluting in fractions 64 through 70 (upper panel), corresponding precisely with the elution pattern of Ikaros by Western blotting (lower panel). The majority of NuRD (Mi-2) and SWI/SNF (BRG1, SRG3, and BAF57) proteins elute prior to fraction 64, with some overlap into fractions 64 through 68. Fractions 65 through 70 were pooled, concentrated, adjusted to 0.1 M NaCl, and loaded onto the DNA-cellulose column (C). Peak PYR complex DNA-binding activity elutes off the DNA cellulose column in fractions 0.3 through 0.45 (upper panel), again corresponding precisely with the elution pattern of Ikaros (lower panel). Much of the NuRD (Mi-2 and RbAp46/48) and SWI/SNF (BRG1, SRG3, and BAF57) proteins elutes prior to fraction 0.3, but there is greater overlap into the PYR DNA-binding fractions than seen with reactive yellow 3-agarose. Fractions 0.3 through 0.45 were pooled, concentrated, and loaded onto the Superose 6 gel filtration column (D). Again, PYR complex DNA-binding activity, which elutes in high-molecular-mass fractions (1 to 2 MDa, upper panel), corresponds exactly with the elution pattern of Ikaros by Western blotting (lower panel). Here, however, both NuRD (Mi-2 and RbAp46/48) and SWI/SNF (BRG1 and BAF57) subunits cofractionate very closely with PYR DNA-binding activity and Ikaros. FT, failure to bind.

Article Snippet: The PYR complex was purified from MEL nuclear extract in four chromatographic steps using High Q Macroprep (Bio-Rad; 0.1 to 0.4 M NaCl step elution), reactive yellow 3-agarose (Sigma; 0.1 to 1.0 M NaCl gradient), double-stranded (ds) DNA-cellulose (calf thymus DNA-cellulose [Sigma], 0.1 to 0.7 M NaCl step gradient), and Superose 6 (Amersham Pharmacia) columns. (B) Chromatography on reactive yellow 3-agarose shows peak PYR complex DNA-binding activity by gel shift assay (PYR) eluting in fractions 64 through 70 (upper panel), corresponding precisely with the elution pattern of Ikaros by Western blotting (lower panel).

Techniques: Binding Assay, Activity Assay, Purification, Chromatography, Electrophoretic Mobility Shift Assay, Western Blot, Filtration

Journal:

Article Title: An Ikaros-Containing Chromatin-Remodeling Complex in Adult-Type Erythroid Cells

doi:

Figure Lengend Snippet: The PYR complex is associated with nucleosome remodeling and histone deacetylase activities. (A) Calf thymus DNA cellulose column fractions with peak PYR complex DNA-binding activity remodel mononucleosomes in an ATP-dependent manner. DNA cellulose column fractions (Fig. ​(Fig.4C)4C) were tested for the ability to remodel a radiolabeled probe (IFNβ-110) packaged into a mononucleosome template in the presence (+) or absence (−) of ATP. Remodeling activity, indicated by the combination of increased (filled arrows) and decreased (open arrows) DNase I sensitivity in the presence of ATP, is found in the 0.3 and 0.4 M NaCl fractions. A different pattern of activity seen in the 0.2 M fraction, suggestive of DNase I protection, is also indicated. (B) Superose 6-purified PYR complex is associated with DNA-dependent ATPase activity. DNA-dependent ATPase activity, as measured by percent ATP hydrolysis, is detected in all of the fractions off the Superose 6 column (Fig. ​(Fig.4D)4D) except fraction 12. Two different activities elute off the column, a high-molecular-weight activity that prefers chromatin as its substrate (fractions 13 through 17) and a lower-molecular-weight activity that prefers naked DNA (fractions 18 through 22). No appreciable ATPase activity is seen in control reactions that use purified histones as the substrate. (C) Superose 6-purified PYR complex is associated with histone deacetylase activity. Histone deacetylase activity, as measured by counts per minute of released [3H]acetate, is detected in the high-molecular-weight fractions (peak activity in fractions 13 through 18).

Article Snippet: The PYR complex was purified from MEL nuclear extract in four chromatographic steps using High Q Macroprep (Bio-Rad; 0.1 to 0.4 M NaCl step elution), reactive yellow 3-agarose (Sigma; 0.1 to 1.0 M NaCl gradient), double-stranded (ds) DNA-cellulose (calf thymus DNA-cellulose [Sigma], 0.1 to 0.7 M NaCl step gradient), and Superose 6 (Amersham Pharmacia) columns. (B) Chromatography on reactive yellow 3-agarose shows peak PYR complex DNA-binding activity by gel shift assay (PYR) eluting in fractions 64 through 70 (upper panel), corresponding precisely with the elution pattern of Ikaros by Western blotting (lower panel).

Techniques: Histone Deacetylase Assay, Binding Assay, Activity Assay, Purification, Molecular Weight