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. 1998 Feb 1;12(3):331–342. doi: 10.1101/gad.12.3.331

Figure 2.

Figure 2

Figure 2

Figure 2

Figure 2

 Ligand-dependent activation by ER with chromatin but not with nonchromatin templates. (A) Ligand-dependent transcriptional activation by ER with chromatin templates in vitro. The plasmid pERE was assembled into chromatin and then subjected to in vitro transcription–primer extension analysis. Where indicated, ER and E2 were added to naked DNA before chromatin assembly (added during assembly) or to preassembled chromatin (added after assembly). The final concentrations of ER and E2 in the transcription reactions were 4.5 and 30 nm, respectively. (B) ER is not degraded during chromatin assembly. Aliquots of assembly reaction mixtures containing the indicated components were taken either before (0 hr) or after (4.5 hr) chromatin assembly was complete. The samples were subjected to Western blot analysis with anti-ER antibodies. (C) The efficiency of chromatin assembly is not affected by ER and E2. Chromatin assembled in the presence or the absence of ER (10 nm) and E2 (100 nm) was subjected to micrococcal nuclease digestion and DNA supercoiling analyses. Where noted, the DNA supercoiling gel contained 2 μm chloroquine. The positions of nicked circular (N) and supercoiled (S) DNAs are indicated. (D) Ligand-stimulated transcription by ER requires packaging of the DNA template into chromatin. Chromatin assembly and in vitro transcription reactions were performed as in A. To inhibit assembly of the pERE tem-plate DNA into chromatin, a nontemplate competitor DNA (pUC118; at a pUC118:pERE mass ratio of 3) was added to the assembly reactions 30 min prior to the addition of pERE template DNA (before assembly). As a control, pUC118 was added to the assembly reactions subsequent to assembly of pERE into chromatin (after assembly). The amounts of transcription in each lane are directly comparable in terms of autoradiography exposure time and the amounts of reaction products that were applied to each gel lane. The relative transcription levels for each set of reaction conditions (i.e., the presence or absence of competitor DNA added before or after chromatin assembly) are normalized to the transcription reactions in which ER and E2 were not included, as designated by (1). The final concentrations of ER and E2 in the transcription reactions were 4.5 and 30 nm, respectively.