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. 2010 Aug 9;30(20):4922–4939. doi: 10.1128/MCB.00354-10

FIG. 5.

FIG. 5.

T-cell silencer activity mapped to a conserved core region. (A) Diagram of reporters with CE4A truncations flanked by the CE9-CE8 enhancer and the promoter. (B) The L98 plus 4A5 reporter contains the core silencer sufficient for silencing (red bar). Data shown are from a representative transient experiment performed in duplicate with Adh.2C2 T cells. (C) Multigenome alignment of the CE4A core silencer. Nucleotides conserved in all 11 organisms are marked by an asterisk. Six nucleotide blocks mutated in the scanning mutagenesis analysis are labeled (M1 to M15). Sequences with >0.925 similarity to TRANSFAC predicted binding sites are shown. The region affected by M5 is designated site X. (D) Scanning mutagenesis revealed multiple sites contributing to core silencer activity. Data shown are averages from four or more independent Adh.2C2 transfections ± standard deviations, with relative light units (RLU) normalized against L98 activity. Red bars mark reporters with mutations blocking silencer activity. *, P < 0.02 (Bonferroni corrected) compared to wild-type reporter data.