Fig. 2

hNAP1 opposes the repressive function of linker histone H1 to enhance activated transcription in vitro. a hNAP1 reverses H1-induced transcriptional repression. In vitro transcription reactions were performed on the 588 bp HTLV-1/G-less cassette fragment as described (Fig. 1b), in the presence of activators and in the absence or presence of Ac-CoA, hNAP1, or rH1⁰. Reactions were supplemented with increasing amounts of rH1⁰ (0.03, 0.06, and 0.12 µM) at the start of the PIC assembly step (lanes 5–7). Reactions containing a constant amount of rH1⁰ (0.06 µM) were supplemented with increasing amounts of hNAP1 (0.17, 0.34 and 0.67 µM) (lanes 8–10). Asterisk denotes the rH1⁰ concentration used in reactions supplemented with varying amounts of histone chaperone. b Transcription from unassembled (naked) promoter templates is only modestly affected by H1 and hNAP1. The experiment was performed as described in panel a, except only two amounts of hNAP1 (0.34 and 0.67 µM) were added to reactions containing rH1⁰ (lanes 8, 9). c rH1⁰-induced chromatin compaction. Chromatin was assembled on the 900 bp HTLV-1/G-less cassette fragment in the absence or presence of equimolar amounts of rH1.0 (H1:octamer), as indicated. The chromatin templates were incubated with increasing amounts of MgCl₂, precipitated, and the percentage of DNA from chromatin prepared in the absence ([−H1], blue) or presence ([+H1], red) remaining in the supernatant following centrifugation was plotted. The graph shown is representative of three experiments. d hNAP1 relieves potent transcriptional repression induced by H1⁰-chromatin. Chromatin was assembled on the 900 bp HTLV-1/G-less cassette fragment in the absence or presence of equimolar rH1.0 (H1:octamer) (see “Methods”). hNAP1 (0.67 µM) was added to in vitro transcription reactions performed on chromatin (lanes 1–4) or H1-chromatin (lanes 5–8) templates, in the presence of activators, and in the absence or presence of Ac-CoA. Note that the 900 bp fragment produces an RNA transcript 390 nt in length