Repression of γ-gene expression by p14 NF-E4 is dependent on the interaction with CP2. (A) Reverse 2-hybrid assay of wild-type and mutant p22 NF-E4. A p22 NF-E4 mutant that fails to interact with CP2 was identified using the reverse 2-hybrid assay (see “Materials and methods”). A GAL4-AD–based yeast expression vector containing this mutant (p22 NF-E4^m) or the pACT vector containing the wild-type p22 NF-E4 (p22 NF-E4) was cotransformed into the yeast strain MAV203 with the GAL4-DBD–based plasmid pGBT9-CP2. Cotransformation of pGBp53 and pACT SV40 large T antigen served as the positive control (P53+T). The resultant transformants were initially plated on selective media plates lacking leucine/tryptophan but containing FOA. Colonies were then replica plated on selective media plates lacking leucine/tryptophan/histidine but containing 3AT (LTH^-+3AT) or plates lacking leucine/tryptophan but containing FOA (LT^-+FOA) or plates lacking leucine/tryptophan for X-gal assays (β-Gal). (B) Amino acid substitutions in p22 NF-E4^m The predicted amino acid sequence of p22 NF-E4 is shown with the position and identity of the 2 substitutions in p22 NF-E4^m indicated by an arrow. The initiating methionine of p14 NF-E4 is underlined. (C) Enforced expression of p22 NF-E4^m in K562 cells fails to induce γ-gene expression. Total RNA from K562 cells transduced with either the MSCV (lane 1), MSCV-HA-p22 NF-E4 (lane 2), or MSCV-HA-p22 NF-E4^m (lane 3) was analyzed by Northern blot (top panels) using a γ-globin gene probe or a GAPDH control probe as indicated. The expression of the wild-type and mutant NF-E4 proteins in the respective cell line extracts is demonstrated by Western analysis using anti-HA antisera (bottom panel). (D) Coimmunoprecipitation of p14 NF-E4 and p14 NF-E4^m with CP2. Cell extract from 293T cells transduced with an expression vector containing CP2 tagged at the 3′ end with an HA-epitope alone (lane 1) or in combination with either an MSCV p14 NF-E4-FLAG retrovirus (p14 NF-E4-FLAG) (lane 2) or an MSCV p14 NF-E4 mutant (p14 NF-E4^m-FLAG) (lane 3) was immunoprecipitated with antisera to the FLAG epitope. The precipitates were then electrophoresed and transferred to a membrane and blotted with either anti-FLAG antisera to detect wild-type and mutant p14 NF-E4 or anti-HA to detect CP2 (top panels). The input extracts for these experiments were also immunoblotted with antisera to HA and FLAG as loading controls (bottom panels). (E) Enforced expression of p14 NF-E4^m in K562 cells fails to repress γ-globin gene expression. Total RNA from K562 cells transduced with either the MSCV (lane 1), MSCV-HA-p14 NF-E4 (lane 2), or MSCV-HA-p14 NF-E4^m (lane 3) was analyzed by Northern blot (top panels) using a γ-globin gene probe or a GAPDH control probe as indicated. The expression of the wild-type and mutant NF-E4 proteins in the respective cell line extracts is demonstrated by Western analysis using anti-HA antisera (bottom panel). (F) ChIP analysis of CP2 at the γ-promoter in K562 cell lines. Chromatin from K562 cells transduced with either the MSCV (lane 1), MSCV p14 HA-NF-E4 (lane 2), or MSCV p14 HA-NF-E4^m retrovirus was immunoprecipitated using antisera to CP2. Quantitative PCR was performed with primer pairs to amplify the SSE in the γ-promoter (top panels) or the MYOD gene (bottom panels). The PCR of the input for each experiment is shown.