Figure 1
Figure 1. Cyclic amplification of selected sequences yields consensus C/EBP-ϵ and C/EBP-ϵ–ATF4 binding sites. (A) Definition of the consensus C/EBP-ϵ binding DNA sequences. Analysis of 103 individual clones derived from 7 cycles of site selection by anti–C/EBP-ϵ antibodies. Invariable AA and TT sequences within each C/EBP-binding half-site were used to align the selected sequences. Half-sites are indicated by arrows. (B) Definition of the consensus C/EBP-ϵ/ATF4 heterodimer binding DNA sequences. Analysis of 43 individual clones derived from site selection experiments using C/EBP-ϵ/ATF4 heterodimers produced in COS1 cells. Selected sequences were aligned using an invariable AA sequence within the C/EBP-binding half-site. (C) Activation of promoter containing C/EBP-ϵ–ATF4 heterodimer binding site by coexpressed ATF4 and C/EBP-ϵ in Jurkat cells. Jurkat cells were transfected with C/EBP-ϵ and ATF4 expression constructs and pCATP-ATCEBP reporter in chloramphenicol acetyl transferase (CAT) reporter. The cells were harvested 48 hours after transfection and CAT and β-galactosidase assays were performed. These results are shown as percentage of chloramphenicol conversion to acetylated form, adjusted for transfection efficiency as monitored by cotransfected β-galactosidase reporter assay. Experiments were done in triplicate; and the mean and SD are shown by black bars and brackets, respectively. (D) DNA binding of C/EBP-ϵ to DNA is enhanced in the presence of nuclear lysates. Samples of dsDNA prior to and after several cycles of selection were analyzed by EMSA. The number of CASTing cycles for each radiolabeled probe is indicated. C/EBP-ϵ and C/EBP-ϵ–ATF4 were derived from nuclear lysates of transfected COS1 cells, whereas MBP–C/EBP-ϵ fusion protein was produced and purified from E coli. Specificity of binding was confirmed by addition of 50-fold molar excess of unlabeled (“cold”) C/EBP-ϵ binding oligonucleotide. In the 2 right lanes, nuclear lysates derived from KCL22 and COS1 cells were added to EMSA reaction containing bacterially produced MBP–C/EBP-ϵ.

Cyclic amplification of selected sequences yields consensus C/EBP-ϵ and C/EBP-ϵ–ATF4 binding sites. (A) Definition of the consensus C/EBP-ϵ binding DNA sequences. Analysis of 103 individual clones derived from 7 cycles of site selection by anti–C/EBP-ϵ antibodies. Invariable AA and TT sequences within each C/EBP-binding half-site were used to align the selected sequences. Half-sites are indicated by arrows. (B) Definition of the consensus C/EBP-ϵ/ATF4 heterodimer binding DNA sequences. Analysis of 43 individual clones derived from site selection experiments using C/EBP-ϵ/ATF4 heterodimers produced in COS1 cells. Selected sequences were aligned using an invariable AA sequence within the C/EBP-binding half-site. (C) Activation of promoter containing C/EBP-ϵ–ATF4 heterodimer binding site by coexpressed ATF4 and C/EBP-ϵ in Jurkat cells. Jurkat cells were transfected with C/EBP-ϵ and ATF4 expression constructs and pCATP-ATCEBP reporter in chloramphenicol acetyl transferase (CAT) reporter. The cells were harvested 48 hours after transfection and CAT and β-galactosidase assays were performed. These results are shown as percentage of chloramphenicol conversion to acetylated form, adjusted for transfection efficiency as monitored by cotransfected β-galactosidase reporter assay. Experiments were done in triplicate; and the mean and SD are shown by black bars and brackets, respectively. (D) DNA binding of C/EBP-ϵ to DNA is enhanced in the presence of nuclear lysates. Samples of dsDNA prior to and after several cycles of selection were analyzed by EMSA. The number of CASTing cycles for each radiolabeled probe is indicated. C/EBP-ϵ and C/EBP-ϵ–ATF4 were derived from nuclear lysates of transfected COS1 cells, whereas MBP–C/EBP-ϵ fusion protein was produced and purified from E coli. Specificity of binding was confirmed by addition of 50-fold molar excess of unlabeled (“cold”) C/EBP-ϵ binding oligonucleotide. In the 2 right lanes, nuclear lysates derived from KCL22 and COS1 cells were added to EMSA reaction containing bacterially produced MBP–C/EBP-ϵ.

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