GAL4 directs nucleosome sliding induced by NURF - PubMed (original) (raw)

GAL4 directs nucleosome sliding induced by NURF

Ju-Gyeong Kang et al. EMBO J. 2002.

Abstract

The Drosophila nucleosome remodeling factor (NURF) is an imitation switch (ISWI)-containing chromatin remodeling complex that can catalyze nucleosome repositioning at promoter regions to regulate access by the transcription machinery. Mononucleosomes reconstituted in vitro by salt dialysis adopt an ensemble of translational positions on DNA templates. NURF induces bi-directional 'sliding' of these nucleosomes to a subset of preferred positions. Here we show that mononucleosome sliding catalyzed by NURF bears similarity to nucleosome movement induced by elevated temperature. Moreover, we demonstrate that the GAL4 DNA-binding domain can extend NURF-induced nucleosome movement on a GAL4-E4 promoter, expanding the stretch of histone-free DNA at GAL4 recognition sites. The direction of NURF-induced nucleosome movement can be significantly modulated by asymmetric placement of tandem GAL4 sites relative to the nucleosome core particle. As such, sequence-specific, transcription factor-directed nucleosome sliding is likely to have substantial influence on promoter activation.

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Figures

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Fig. 1. Reconstituted nucleosomes occupy multiple positions on the GAL4-E4 promoter. (A) Native PAGE of mononucleosomes reconstituted on a 369 bp fragment carrying the GAL4-E4 promoter. Major nucleosome species are indicated as N1–N7′. Nucleosome positions and GAL4-binding sites determined by Exo III footprinting (precise to ± 2 bp) are illustrated on the right. The TATA box and restriction enzyme sites are indicated. (B) Exo III footprinting. Gel bands corresponding to individual N1–N7 nucleosome species (DNA was radiolabeled at either end of GAL4-E4 promoter) were excised, nucleosomes were eluted and digested with Exo III (400 U/ml) for 2 min at 37°C. DNAs were analyzed by electrophoresis in a 6% polyacrylamide gel containing 8 M urea. M indicates pBR322 _Hpa_II fragments as markers. Dots indicate major Exo III pauses. The Exo III footprint of GAL4 (–153 to –44) extends beyond the recognition sites (–140 to –56) (Carey et al., 1989; Carey and Smale, 2000).

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Fig. 2. NURF mediates nucleosome sliding on the GAL4-E4 promoter. 369 bp GAL4-E4 mononucleosomes (40 nM) were incubated with NURF (0.4 nM) in the absence or presence of ATP for 30 min at 26°C (lanes 1 and 2), followed by native 4.5% PAGE in 0.5× TBE. N1, N2 and N4 nucleosomes were eluted from the gel slice and incubated with NURF and ATP as indicated, before electrophoresis on a second native gel (lanes 3–8).

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Fig. 3. Comparison of heat- and NURF-induced nucleosome sliding. Mononucleosomes reconstituted on GAL4-E4 (A) or hsp70 (B) radiolabeled DNAs were incubated for 30 min at the indicated temperatures in TE buffer and 1 mg/ml BSA. Samples were cooled on ice, and analyzed by native PAGE as in Figure 2 legend. Mononucleo somes were incubated with NURF (0.4 nM), [(A) lane 7; (B) lane 4] or ACF (1.6 nM), [(A) lane 8; (B) lane 5] in nucleosome sliding buffer at 26°C. Samples were electrophoresed as in Figure 2 legend.

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Fig. 4. GAL4-DBD and NURF-induced extension of nucleosome sliding—Exo III footprinting analysis. Mononucleosomes were reconstituted on the end-labeled 369 bp GAL4-E4 promoter. Gel-purified nucleosomes (0.025 nM) and carrier DNA (20 µg/ml), N1 (lanes 1–6), N4 (lanes 7 and 8) were incubated with NURF (0.2 nM) and GAL4-DBD (0.3 nM) in the presence or absence of ATP as indicated. Reaction mixtures were digested by Exo III (400 U/ml) and analyzed on an 8 M urea–6% polyacrylamide gel. Dots indicate major Exo III pauses. Diagrams show the position of nucleosomes (open circles), GAL4 (filled ovals) and Exo III pauses (arrowheads).

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Fig. 5. GAL4-DBD and NURF-induced extension of nucleosome sliding—MNase and restriction enzyme analysis. Nucleosomes were reconstituted on 32P uniformly labeled 369 bp GAL4-E4 fragment and incubated with GAL4 and/or NURF prior to MNase digestion. To map boundaries of MNase protection, DNA was purified and digested with _Ban_I (B) or _Fnu_4H1 (F). (A) Schematic illustration showing mononucleosomes and GAL4-DBD binding on 369 bp GAL4-E4 promoter. Arrowheads mark the boundaries of MNase protection. (B) Mononucleosome fractions (0.7 nM) purified by glycerol gradient centrifugation were incubated with NURF (0.2 nM) and/or GAL4 (30 nM monomer) for 30 min at 26°C, followed by MNase (25 U/ml) digestion (lanes 1–4). DNA was purified and further digested with _Ban_I (1300 U/ml, lanes 5 and 6) or _Fnu_4H1 (333 U/ml, lanes 7 and 8) and analyzed by 8% PAGE in TBE and autoradiography. Dots (filled and unfilled) indicate fragments protected by complexes of GAL4-DBD and N7 or N7′ nucleosomes, respectively. Binding of GAL4-DBD to free DNA or nucleosomes generates 109 and 145–240 bp MNase resistant fragments, respectively. In the absence of GAL4-DBD, MNase digestion of N1–N7 nucleosomes yields 145–155 bp fragments from core particles. (C) Mononucleosomes were incubated with GAL4(1–94) and GAL4(1–147) (20 nM monomer) in the presence or absence of NURF, and analyzed by MNase digestion and DNA gel electrophoresis. MNase cleavage at sites flanking the tandemly bound GAL4-DBDs appears to be more efficient for GAL4(1–94).

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Fig. 6. Placement of GAL4 sites modulates direction of nucleosome sliding. (A) Gel-purified 369 bp GAL4-E4 mononucleosomes N2 (upper panel) and N4 (lower panel) were incubated with NURF in the presence of ATP and digested with MNase; DNA was purified, further digested with _Ban_I and _Fnu_4H1, and analyzed by PAGE and auto radiography as in the legend to Figure 5. The lower part of the gel (indicated by the box) was exposed to film ∼3 times longer than the upper part and contrast enhanced to visually reveal small DNA fragments. Quantitation of radioactive band intensity was performed by phosphoimager analysis. The percentage restriction enzyme cleavage was calculated by subtraction of the residual ∼150 bp nucleosome core particle DNA from an equivalent, uncleaved sample (lanes 1 and 4). Restriction fragments derived from N7 and N7′ nucleosomes are indicated by unfilled and filled dots, respectively. The 81 bp fragment derived from _Ban_I cleavage of N7′ core particle DNA is not visible at this exposure; note that the N7 and N7′ percentage values given in the text are approximate. (B) Gel-purified 369 bp GAL4-E4 mononucleo somes N2 (upper panel) and N4 (lower panel) were incubated with NURF and GAL4-DBD, in the presence or absence of ATP, and analyzed as above. Dots (unfilled and filled) indicate fragments corresponding to, or derived from, N7 and N7′ nucleosomes, respectively. The distribution of N7 and N7′ nucleosomes was calculated by phosphoimager analysis of radioactive fragments, and averaged from three sets of experiments.

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