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. 2001 Dec 15;15(24):3342–3354. doi: 10.1101/gad.946501

Figure 4.

Figure 4

p300 and RMF enhance transcription from fully assembled chromatin templates by LEF-1 and β-catenin in vitro. (A) Chromatin transcription reactions either lacked enhancer factors (lane 1), or contained ΔAD-LEF (120 nM, lanes 2,3,5,6), β-cat (120 nM, lanes 2,5), or β-cat ΔC (120 nM, lanes 3,6). Where indicated, purified recombinant p300 (60 nM, lanes 4–6) was incubated together with LEF-1 and β-cat after completion of pBRE chromatin assembly. (B) Transcription reactions either lacked enhancer factors (lane 1), or contained 120 nM ΔAD-LEF and 120 nM β-cat (lanes 5–14) added after pBRE chromatin assembly. Where indicated, reactions also contained p300 (60 nM, lanes 3,4,9–14), RMF (1 μg, lanes 2,4,6–8,12–14), GST–CT (4.2 μg, lanes 7,10,13), or GST–CT–ARM (4.8 μg, lanes 8,11,14), added simultaneously with LEF-1 and β-cat. (C) Binding of LEF-1–β-cat to assembled pBRE chromatin templates in the presence or absence of p300, RMF, and the CT–ARM inhibitor. Binding reactions either lacked enhancer factors (lanes 1,9) or contained ΔAD-LEF (120 nM, lanes 3–8,10–11,13–14), β-cat (120 nM, lanes 4–8,10–11,13–14), p300 (60 nM, lanes 12–14), RMF (1 μg, lanes 2,5–8,11,14), or a 10-fold excess of GST–CT (lane 7), GST–CT–ARM (lane 8), and apyrase (0.5 unit; lane 6), added with the LEF-1–β-cat complex after chromatin assembly.