FIG. 6.

e(y)2 activates transcription on chromatin template. (A) Transcription was performed on (17M)5β2G (β2G) chromatin or naked templates (200 pM) using a HeLa cell nuclear extract (100 μg) in the presence or absence of GAL4-VP16 (1 nM) and e(y)2 (5 nM [lanes 2, 4, 7, and 9] or 1 nM [lane 5]) in a final reaction volume of 50 μl. The ratio of added recombinant e(y)2 to endogenous e(y)2 was 10 in lanes 2, 4, 7, and 9 or 2 in lane 5. S1 nuclease analysis was performed after deproteinization (see Materials and Methods). (B) Analysis of the structure of chromatin reconstituted in the presence or absence of e(y)2 (5 nM) and GAL4-VP16 (1 nM). The template was digested with various concentrations of micrococcal nuclease in a final volume of 80 μl, separated on a 1.5% gel, and Southern blotted using a ³²P-labeled probe corresponding to the region from positions −40 to +5 of the β2G proximal promoter. Hybridization with the probes corresponding to a region 5′ of the GAL4 binding sites gave a similar result (data not shown).