ACF1 improves the effectiveness of nucleosome mobilization by ISWI through PHD-histone contacts - PubMed (original) (raw)

ACF1 improves the effectiveness of nucleosome mobilization by ISWI through PHD-histone contacts

Anton Eberharter et al. EMBO J. 2004.

Abstract

The nucleosome remodelling ATPase ISWI resides in several distinct protein complexes whose subunit composition reflects their functional specialization. Association of ISWI with ACF1, the largest subunit of CHRAC and ACF complexes, improves the efficiency of ISWI-induced nucleosome mobilization by an order of magnitude and also modulates the reaction qualitatively. In order to understand the principle by which ACF1 improves the efficiency of ISWI, we mapped their mutual interaction requirements and generated a series of ACF complexes lacking conserved ACF1 domains. Deletion of the C-terminal PHD finger modules of ACF1 or their disruption by zinc chelation profoundly affected the nucleosome mobilization capability of associated ISWI in trans. Interactions of the PHD fingers with the central domains of core histones contribute significantly to the binding of ACF to the nucleosome substrate, suggesting a novel role for PHD modules as nucleosome interaction determinants. Connecting ACF to histones may be prerequisite for efficient conversion of ATP-dependent conformational changes of ISWI into translocation of DNA relative to the histones during nucleosome mobilization.

PubMed Disclaimer

Figures

Figure 1

Figure 1

ACF1 interacts with the C-terminus of ISWI. (A) Summary of the domain organization of ISWI known to date. HAND (aa 697–795), SANT (aa 796–850) and SLIDE (aa 886–977) domains were recently defined by Grüne et al (2003). ‘+' and ‘–' behind each derivative indicate the ability to bind as determined in (B) and (C). The AID (between aa 962 and 991) was defined here. (B) FLAG-tagged ACF1 was immobilized on M2 anti-FLAG agarose. The resulting affinity resin was extensively washed and then used in pull-down experiments to monitor the interaction of ISWI. Bacterially expressed ISWI derivatives as indicated were incubated with the ACF1 beads. After extensive washes 30% of bound material was separated by SDS–8% PAGE and detected by Western blotting with an ISWI antibody (‘ACF1-bound'). As a control for interaction with full-length ISWI, we used a whole-cell extract of baculoviral-expressed, untagged ISWI. As reference, 10% of the input was loaded. (C) Smaller parts of ISWI (numbers above lanes correspond to first and last amino acids) were expressed in E. coli and tested for interaction with ACF1, as in (B). The upper panel displays the input of ISWI derivatives, and the lower panel reveals the bound protein.

Figure 2

Figure 2

Mapping the ISWI interaction determinants on ACF1. (A) Summary of the known domain organization of ACF1 (Ito et al, 1999). The C-terminal domains extend between the following coordinates: PHD1: aa 1064–1114; PHD2: aa 1240–1300; Brd: aa 1361–1463. ‘+' and ‘−' indicate the ability to bind as determined in (B). (B) Full-length (FL) myc-tagged ACF1 and derivatives bearing various deletions as indicated were coexpressed with FLAG-tagged ISWI in Sf9 cells and resulting complexes were affinity-purified via the FLAG tag. Equal amounts of FLAG-eluted ACF complexes (i.e. ACF1 bound to ISWI) and the corresponding supernatants (i.e. proteins not interacting with ISWI) were separated by SDS–PAGE. ACF1 and ISWI were detected by Western blotting and probing with either monoclonal anti-myc antibody 9E10 for ACF1 detection or with antibody directed against ISWI (provided by J Tamkun).

Figure 3

Figure 3

The PHD fingers of ACF1 are important for nucleosome mobilization. (A) 60 fmol of mononucleosomes positioned at the end of a 248 bp DNA fragment were incubated for 1 h with affinity-purified ACF or variant complexes bearing the indicated deletions of ACF1. In order to visualize nucleosome movement, samples were separated on a native 4.5% polyacrylamide gel. Dried gels were exposed to film overnight at −80°C. The reactions contained the following amounts of ACF complexes: 3 fmol (lanes 2, 7, 12 and 17); 1.5 fmol (lanes 3, 8, 13 and 18); 1 fmol (lanes 4, 9, 14 and 19); 0.75 fmol (lanes 5, 10, 15 and 20) and 0.375 fmol (6, 11, 16 and 21). Lane 1 shows the band corresponding to the mononucleosome in the absence of enzyme. The positions of free DNA, end- and centre-positioned nucleosomes are indicated to the left. (B) Nucleosome sliding reaction as in (A), but starting with 60 fmol of a centrally positioned mononucleosome. Lanes 22–25 show reactions driven by 6, 3, 1.5 and 0.75 fmol of ISWI, respectively. The untreated centre-positioned nucleosome is shown in lane 1. (C) Time course of nucleosome sliding. Reactions as in (A) contained 0.5 fmol of each of the indicated ACF complexes and 60 fmol of nucleosomes. Reactions were stopped at different times (5, 10, 15, 30 and 60 min as indicated) by the addition of 200 ng of unlabelled competitor DNA. (D) Nucleosomal sliding reactions as in (A) in the absence (lane 2) or presence of either 1,10 phenanthroline (0.1, 0.5, 1, 2.5 and 5 mM in lanes 3–7, respectively) or corresponding concentrations of the solvent ethanol (0.5, 1.25 or 2.5% in lanes 8–10, respectively). For controls, mononucleosomes were incubated with either 1, 2.5 or 5 mM of 1,10 phenanthroline or 0.5, 1.25 or 25% ethanol and resolved on the gel in lanes 11–13 and 14–16.

Figure 4

Figure 4

Requirement of the ACF1 PHD fingers for nucleosomal mobilization. (A) Normalization of proteins in ACF wt and ACFΔPHD1-2 complexes to be compared in the following experiments by 6% denaturing PAGE and Coomassie staining. Lane 1: size marker. (B) ATPase assays with 120 ng of naked DNA or 120 ng of nucleosomal DNA using 2 fmol of either wt ACF or the indicated mutant ACF complex. The reactions were performed in the absence (−) or presence of 3 mM of either 1,10-phenanthroline or 1,7-phenanthroline. (C) Nucleosome sliding reactions as in Figure 3A comparing ACF (lanes 2–5) and ACFΔPHD1-2 (lanes 6–9). Protein concentrations were 1 fmol (lanes 2 and 6), 0.5 fmol (lanes 3 and 7), 0.25 fmol (lanes 4 and 8) or 0.125 fmol (lanes 5 and 9). The untreated nucleosome is indicated in lane 1. (D) Nucleosome sliding reactions as in (C) with 1 fmol of either ACF wt (lanes 2–8) or ACFΔPHD1-2 (lanes 9–15). Reactions were carried out in the absence (lanes 2 and 9) or presence of either 1,10-phenanthroline at 2 mM (lanes 3 and 10), 3 mM (lanes 4 and 11) and 4 mM (lanes 5 and 12) or 1,7-phenanthroline at 2 mM (lanes 6 and 13), 3 mM (lanes 7 and 14) and 4 mM (lanes 8 and 15). The untreated nucleosome is indicated in lane 1 (nuc).

Figure 5

Figure 5

The PHD fingers of ACF1 are important for nucleosome recognition. (A) EMSA monitoring the interaction of various remodelling factors with mononucleosome substrates. Affinity-purified ACF complexes bearing the indicated ACF1 deletions, ISWI and ACF1 were incubated with 60 fmol of end-positioned nucleosome for 15 min. The reactions were then separated on a 1.4% agarose gel, which was dried and exposed to film overnight at −80°C. Protein concentrations were 60 fmol (lanes 2, 5, 8, 11, 14 and 17), 30 fmol (lanes 3, 6, 9, 12, 15 and 18) or 15 fmol (4, 7, 10, 13, 16 and 19). Lane 1 shows the migration of the nucleosome alone. (B) EMSA as in (A) using wt ACF complex (lanes 2–7) and ACFΔPHD1-2 (lanes 9–15). Protein concentrations were 120 fmol (lanes 2, 6, 7, 9, 14 and 15), 90 fmol (lanes 3 and 10), 60 fmol (4 and 11), 30 fmol (lanes 5 and 12) and 15 fmol (lane 13). Where indicated, either 3 mM 1,10-phenanthroline (lanes 6 and 14) or 3 mM 1,7-phenanthroline was added to the reaction. Lanes 1 and 8 show the migration of the nucleosome alone. (C) EMSA as in (A) analysing ACF1 full length (lanes 2–6) and ACFΔPHD1-2 (lanes 7–11). Protein concentrations were 90 fmol (lanes 2 and 7), 60 fmol (lanes 3 and 8), 30 fmol (lanes 4 and 9), 15 fmol (lanes 5 and 10) or 7.5 fmol (lanes 6 and 11). The migration of mononucleosomes in the absence of protein is shown in lane 1.

Figure 6

Figure 6

The ACF1 PHD fingers are required for interaction with histones. (A) GST-PHD1-2 and GST-Brd fusion proteins were expressed in bacteria, bound to glutathione-Sepharose 4B and used in ‘pull-down' experiments. About 500 ng of immobilized protein was incubated with 1 μg of Drosophila histone mixtures either purified from embryos (purified histones) or expressed in bacteria (recombinant histones) at the indicated salt concentrations. Histones that remained bound through excessive washes were separated by 15% SDS–PAGE and visualized by Coomassie blue staining. Lane 9 shows the histone input. (B) Indicated domains of ACF1 were expressed as GST fusion proteins and used in ‘pull-down' experiments as in (A). A 1 μg portion of all four recombinant histones (recomb histones) or of recombinant H3/H4 tetramers (tailless H3/H4) or recombinant H2A/H2B dimers (tailless H2A/2B), lacking their N-terminal tail domains, was tested in ‘pull-down' experiments. Bound material (50%) was separated by 15% denaturing PAGE and stained with Coomassie blue. The relative amount of each GST construct is shown in the bottom panel. (C) The indicated domains of ACF1 (upper panel) were expressed and used in pull-downs as described in (A). Before incubation with histones, the indicated GST beads were pretreated for 4 h at RT with buffer alone (−), 3 mM of 1,7-phenanthroline (+1,7) or 3 mM of 1,10-phenanthroline (+1,10). Lane 1 shows the histone input.

Figure 7

Figure 7

Models explaining the increased effectiveness of nucleosome sliding upon interaction of ACF1 with ISWI. (A) ISWI mainly interacts with linker DNA. (B, C) Additional contact of ACF1 with histones provides an anchor on the histone moiety of the nucleosome that allows efficient conversion of ATP-dependent conformational changes of ISWI into translocation of DNA relative to the histones during nucleosome mobilization.

Similar articles

Cited by

References

    1. Aasland R, Gibson TJ, Stewart AF (1995) The PHD finger: implications for chromatin-mediated transcriptional regulation. Trends Biochem Sci 20: 56–59 - PubMed
    1. Adamson AL, Shearn A (1996) Molecular genetic analysis of Drosophila ash2, a member of the trithorax group required for imaginal disc pattern formation. Genetics 144: 621–633 - PMC - PubMed
    1. Akhtar A, Becker PB (2001) The histone H4 acetyltransferase MOF uses a C2HC zinc finger for substrate recognition. EMBO Rep 2: 113–118 - PMC - PubMed
    1. Becker PB, Hörz W (2002) ATP-dependent nucleosome remodeling. Annu Rev Biochem 71: 247–273 - PubMed
    1. Bird AJ, McCall K, Kramer M, Blankman E, Winge DR, Eide DJ (2003) Zinc fingers can act as Zn2+ sensors to regulate transcriptional activation domain function. EMBO J 22: 5137–5146 - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources