Fig. 4.
Fig. 4. (A) Competition survey of FP probes shows binding sites for CSBP-2, SSP, and YY1. The labeled probe was described in an earlier study and spans a phylogenetic footprint located at −835 upstream from the ε gene.32 This probe binds CSBP-2 and SSP at moderate affinity and YY1 weakly (indicated at left). Each lane contains 4 μg of K562 extract, 0.2 ng of labeled probe (20,000 cpm), 500 ng poly (dIdC), and 100 ng of the specific competitor indicated above the lane. Lane 1 contains no added competitor. (B) Effect of a 4-bp mutation on CSBP-2 binding. The labeled probe spans a characterized CSBP-2 binding site located −698 bp upstream from the ε gene.32 In lane 2 (WT), this probe was used in a binding assay with K562 extract, poly(dIdC) and no specific competitor. In lane 1, the same probe, but containing base changes at the four sites shown in (C) was used in the binding (M). (C) Alignment of five moderate- to high-affinity CSBP-2 sites found at conserved sites in the globin cluster. The −835 ε, −698 ε, and −49 ε sequences were described earlier.32 The location and exact sequence of several mutations that have been shown to disrupt binding of the CSBP-2 complex are shown below the consensus sequence. Mutants 2 and 4 are from Philipsen et al23 and were shown to severely inhibit the binding of factor “X,” here identified as CSBP-2. Effect of the −698 ε mutation is shown in (B). At the bottom is the sequence of the portion of the HS3 core known as “footprint 1” described by Philipsen et al.23 The location of the CSBP-2 consensus on the antisense strand is shown by the long arrow below the sequence. A GATA-1 consensus sequence is also located on the bottom strand (overline). S, G or C; B, not A; H, not G; Y, T or C. (D) Effect of chelators on binding of the zinc finger protein YY1. The probe for (D) is a well-described YY1 binding site (Table 1). Lane 1 is a standard binding reaction with K562 extract (described in Materials and Methods). In lane 2, 100 ng of specific competitor (unlabeled probe) was added to show specificity of binding. In lanes 3 and 4, 3 mmol/L orthophenanthroline and 1 mmol/L EDTA, respectively, was added to the binding reaction. The position of the YY1 complex (large arrowhead) and a minor subband described earlier (small arrowhead)31 is shown. Both complexes are sensitive to the presence of chelators. (E) The CSBP-2 complex is not sensitive to chelation. In lane 1, the −698ε probe was used in a standard binding reaction. Chelators were added to other lanes as follows: lane 2, 3 mmol/L orthophenanthroline; lane 3, 6 mmol/L orthophenanthroline; lane 4, 1 mmol/L EDTA; lane 5, 3 mmol/L EDTA. The position of the CSBP-2 complex is marked with an arrowhead.

(A) Competition survey of FP probes shows binding sites for CSBP-2, SSP, and YY1. The labeled probe was described in an earlier study and spans a phylogenetic footprint located at −835 upstream from the ε gene.32 This probe binds CSBP-2 and SSP at moderate affinity and YY1 weakly (indicated at left). Each lane contains 4 μg of K562 extract, 0.2 ng of labeled probe (20,000 cpm), 500 ng poly (dIdC), and 100 ng of the specific competitor indicated above the lane. Lane 1 contains no added competitor. (B) Effect of a 4-bp mutation on CSBP-2 binding. The labeled probe spans a characterized CSBP-2 binding site located −698 bp upstream from the ε gene.32 In lane 2 (WT), this probe was used in a binding assay with K562 extract, poly(dIdC) and no specific competitor. In lane 1, the same probe, but containing base changes at the four sites shown in (C) was used in the binding (M). (C) Alignment of five moderate- to high-affinity CSBP-2 sites found at conserved sites in the globin cluster. The −835 ε, −698 ε, and −49 ε sequences were described earlier.32 The location and exact sequence of several mutations that have been shown to disrupt binding of the CSBP-2 complex are shown below the consensus sequence. Mutants 2 and 4 are from Philipsen et al23 and were shown to severely inhibit the binding of factor “X,” here identified as CSBP-2. Effect of the −698 ε mutation is shown in (B). At the bottom is the sequence of the portion of the HS3 core known as “footprint 1” described by Philipsen et al.23 The location of the CSBP-2 consensus on the antisense strand is shown by the long arrow below the sequence. A GATA-1 consensus sequence is also located on the bottom strand (overline). S, G or C; B, not A; H, not G; Y, T or C. (D) Effect of chelators on binding of the zinc finger protein YY1. The probe for (D) is a well-described YY1 binding site (Table 1). Lane 1 is a standard binding reaction with K562 extract (described in Materials and Methods). In lane 2, 100 ng of specific competitor (unlabeled probe) was added to show specificity of binding. In lanes 3 and 4, 3 mmol/L orthophenanthroline and 1 mmol/L EDTA, respectively, was added to the binding reaction. The position of the YY1 complex (large arrowhead) and a minor subband described earlier (small arrowhead)31 is shown. Both complexes are sensitive to the presence of chelators. (E) The CSBP-2 complex is not sensitive to chelation. In lane 1, the −698ε probe was used in a standard binding reaction. Chelators were added to other lanes as follows: lane 2, 3 mmol/L orthophenanthroline; lane 3, 6 mmol/L orthophenanthroline; lane 4, 1 mmol/L EDTA; lane 5, 3 mmol/L EDTA. The position of the CSBP-2 complex is marked with an arrowhead.

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