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. 2004 Sep;24(18):7914–7930. doi: 10.1128/MCB.24.18.7914-7930.2004

FIG. 8.

FIG. 8.

Chromatin architecture of the GM-CSF enhancer. (A to E) Indirect end-labeling assays of MNase and DNase I sites within the GM-CSF enhancer in nuclei prepared from cells that were either left unstimulated (nil) or stimulated for 4 h with 20 ng of PMA per ml and 2 μM A23187 (PMA/I). Analyses were performed by Southern blot hybridization using either 10 μg of human T-cell DNA per lane (A to C) or 1 μg of Gm-CSF transgenic mouse T-cell DNA per lane (D and E). The positions of nucleosomes N1 and N2 and the DH site (DHS) are indicated by brackets. Band positions are calculated relative to the upstream BglII site at position 1 in the 717-bp BglII enhancer sequence. Asterisks indicate the positions of new inducible MNase sites at positions 260 and 470. Panel A depicts the ethidium bromide staining pattern for the analyses performed in panels B and C and the positions of bands containing two to six nucleosomes (indicated to the left of the gel). The restriction enzyme marker in panels B and C consists of DNA partially digested with BglII, PstI, and ApaI, with the positions of these sites depicted on the right. (F) Map of the major nuclease cleavage sites within the 1,660-bp BglI fragment encompassing the GM-CSF enhancer, showing the predicted positions of nucleosomes, the DH site, and the indirect end-labeling probes used to map cleavage sites. HS, hypersensitive. (G) LM-PCR mapping of nucleosome boundaries in nucleosome length DNA fragments purified from human T cells that were either left nonstimulated (nil) or stimulated for 4 h with 20 ng of PMA per ml and 2 μM A23187 (PMA/I). The leftmost panel depicts agarose gel electrophoresis of 146-bp nucleosome-length DNA (N) and 167-bp chromatosome length DNA (C) purified from MNase digestion products of T-cell nuclei. Twenty-five nanograms of purified nucleosomal DNA was assayed by LM-PCR using the 2R2 and 4F primer sets. DMS-treated DNA samples to align the sequence (G) and a control MNase digest of purified DNA (M) were also included. The map shows the relative positions of the primer sets used to map nucleosome boundaries.