Figure 5

Sp1 binds to the Dlk2 promoter. A) Schematic representation of the six GC-boxes in the Dlk2 promoter, the putative Sp1 binding sites (named 1, 2, 3, 4, 5, and 6). The oligonucleotides used for the ChIP analyses (arrows), and the approximate positions of three oligonucleotides used in EMSA assays are also shown. Oligonucleotide Sp1-A contains the GC boxes located at positions -130/-140 (GCBox-1); -108/-115 (GCBox-2); oligonucleotide Sp1-B contains the GC box located at position -61/-70 (GCBox-3); and oligonucleotide Sp1-C contains the GC boxes located at positions +62/+68 (GCBox-4); +76/+82 (GCBox-5); and +81/+90 (GCBox-6). B) Chromatin IP analysis was performed using native chromatin from 3T3-L1 cells, incubated with normal rabbit IgG (IgG), with antibodies against RNA-polymerase II (pol-II), or with antibodies against Sp1. The PCR analysis and the corresponding agarose gel electrophoresis of a representative experiment is shown. C) and D) EMSA analyses were performed using 8 μg of nuclear protein extracts from NIH3T3 cells, which were incubated with the ³²P-labeled oligonucleotides Sp1-A, Sp1-B or Sp1-C. For competition and super-shift assays, the reaction was preincubated with a 100-fold excess of the indicated cold oligonucleotides, or with 2 μg of Sp1 antibody before the addition of the labeled oligonucleotide. The locations of the Sp1 and Sp1 supershifted (SS) bands are indicated by arrows.