1. The necdin promoter is a direct target of NSCL‐2.
The necdin promoter is a direct target of NSCL‐2. (A) EMSA with in vitro‐translated NSCL‐2 using E‐box1 (lanes 1–7) and E‐box2 oligonucleotides (lanes 8–14) derived from the necdin promoter. Specificity of binding was demonstrated by addition of 100‐ to 1000‐fold molar excess of wild‐type E‐box1 (lanes 2–5) and 200‐ to 500‐fold molar excess of a mutated E‐box1 (lanes 6 and 7), and by addition of 100‐ to 1000‐fold molar excess of wild‐type E‐box2 (lanes 9–12) and 200‐ to 500‐fold molar excess of a mutated E‐box2 (lanes 13 and 14). An NS complex that formed with E‐box1 and E‐box2 was specifically competed with mutated E‐boxes (lanes 6, 7 and 13, 14). Unbound oligonucleotides, which were always in excess, are not shown. (B) ChIP of the necdin promoter by myc‐tagged NSCL‐2. Samples of sonicated and purified chromatin isolated from GT1‐trk and HEK293 cells transfected with the empty vector pCS2‐MT or a combination of NSCL‐2, E12, CLIM2, and LMO‐4 were immunoprecipitated with the myc‐tag antibody. DNAs isolated from immunoprecipitated material were amplified by PCR with primers specific for the human and mouse necdin promoter and resolved on an agarose gel. (C) Schematic outline of mouse and human necdin promoters, localization of E‐boxes, and position of primers used to identify immunoprecipitated chromatin.
Marcus Krüger et al. EMBO J. 2004;23:
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