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Histone modifications influence the action of Snf2 family remodelling enzymes by different mechanisms.

Ferreira H, Flaus A, Owen-Hughes T - J. Mol. Biol. (2007)

Bottom Line: Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways.In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment.These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes.

View Article: PubMed Central - PubMed

Affiliation: Division of Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.

ABSTRACT
Alteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes.

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Effects of histone acetylation on intrinsic nucleosome mobility. (a) Outline of competitive remodelling assay used to accurately measure differences between nucleosomes. This setup has the advantage that the reaction times and conditions are exactly the same for two nucleosomes and avoids the possibility of observing effects due to different nucleosome reconstitution efficiencies. (b) Histone H4 tetra-acetylation does not alter the rate at which nucleosomes reposition thermally. Two pmol of H4 acetylated and H4 V21C unmodified nucleosomes assembled on 54A54 DNA were mixed and incubated at 47 °C for the specified amount of time. The images represent the Cy3 (H4 V21C) and Cy5 (H4 acetylated) scans of the same gel. The amount of remodelling is plotted to the right of the gels and a hyperbolic curve fitted to the data points. From this the initial rate of repositioning is calculated and the average of three independent repeats displayed in (e). (c) Tetra-acetylation of H3 results in a twofold increase in the rate of intrinsic nucleosome mobility. Reaction conditions are as for (b). (d) The increase in nucleosome mobility by H3 acetylation is not solely due to charge neutralisation. Two pmol of H3 wild-type and H3 in which the lysine residues at position 9, 14, 18 and 23 had been substituted to alanine were assembled on 54A18 DNA were mixed and incubated at 47 °C for the specified amounts of time. Substituting the lysine residues with alanine, which will reduce the basic charge of the tail, had no effect on nucleosome mobility. (e) Table indicating the initial rate of repositioning relative to control for the three constructs described above.
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fig2: Effects of histone acetylation on intrinsic nucleosome mobility. (a) Outline of competitive remodelling assay used to accurately measure differences between nucleosomes. This setup has the advantage that the reaction times and conditions are exactly the same for two nucleosomes and avoids the possibility of observing effects due to different nucleosome reconstitution efficiencies. (b) Histone H4 tetra-acetylation does not alter the rate at which nucleosomes reposition thermally. Two pmol of H4 acetylated and H4 V21C unmodified nucleosomes assembled on 54A54 DNA were mixed and incubated at 47 °C for the specified amount of time. The images represent the Cy3 (H4 V21C) and Cy5 (H4 acetylated) scans of the same gel. The amount of remodelling is plotted to the right of the gels and a hyperbolic curve fitted to the data points. From this the initial rate of repositioning is calculated and the average of three independent repeats displayed in (e). (c) Tetra-acetylation of H3 results in a twofold increase in the rate of intrinsic nucleosome mobility. Reaction conditions are as for (b). (d) The increase in nucleosome mobility by H3 acetylation is not solely due to charge neutralisation. Two pmol of H3 wild-type and H3 in which the lysine residues at position 9, 14, 18 and 23 had been substituted to alanine were assembled on 54A18 DNA were mixed and incubated at 47 °C for the specified amounts of time. Substituting the lysine residues with alanine, which will reduce the basic charge of the tail, had no effect on nucleosome mobility. (e) Table indicating the initial rate of repositioning relative to control for the three constructs described above.

Mentions: Although nucleosomes are stable with respect to dissociation, they can undergo a range of dynamic fluctuations in their structure. For example, following thermal incubation nucleosomes are frequently observed to redistribute to thermodynamically favoured locations.25 This movement of nucleosomes from one translational position on DNA to another can be followed using native PAGE.26 Acetylated and control nucleosomes were reconstituted onto differentially labelled fluorescent DNA, mixed, incubated thermally and run on a native gel (Figure 2(a)). The different nucleosomes can then be visualised separately using selective excitation and emission filters. From this the proportion of nucleosomes at the initial and final position can be measured at each time point. The rate at which nucleosomes accumulate at their destination can then be plotted and a hyperbolic curve fitted to the data enabling the initial rate of redistribution to be estimated (Figure 2(b)–(e)). This approach has proved more reliable at directly comparing the behaviour of different nucleosomes.27


Histone modifications influence the action of Snf2 family remodelling enzymes by different mechanisms.

Ferreira H, Flaus A, Owen-Hughes T - J. Mol. Biol. (2007)

Effects of histone acetylation on intrinsic nucleosome mobility. (a) Outline of competitive remodelling assay used to accurately measure differences between nucleosomes. This setup has the advantage that the reaction times and conditions are exactly the same for two nucleosomes and avoids the possibility of observing effects due to different nucleosome reconstitution efficiencies. (b) Histone H4 tetra-acetylation does not alter the rate at which nucleosomes reposition thermally. Two pmol of H4 acetylated and H4 V21C unmodified nucleosomes assembled on 54A54 DNA were mixed and incubated at 47 °C for the specified amount of time. The images represent the Cy3 (H4 V21C) and Cy5 (H4 acetylated) scans of the same gel. The amount of remodelling is plotted to the right of the gels and a hyperbolic curve fitted to the data points. From this the initial rate of repositioning is calculated and the average of three independent repeats displayed in (e). (c) Tetra-acetylation of H3 results in a twofold increase in the rate of intrinsic nucleosome mobility. Reaction conditions are as for (b). (d) The increase in nucleosome mobility by H3 acetylation is not solely due to charge neutralisation. Two pmol of H3 wild-type and H3 in which the lysine residues at position 9, 14, 18 and 23 had been substituted to alanine were assembled on 54A18 DNA were mixed and incubated at 47 °C for the specified amounts of time. Substituting the lysine residues with alanine, which will reduce the basic charge of the tail, had no effect on nucleosome mobility. (e) Table indicating the initial rate of repositioning relative to control for the three constructs described above.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2279226&req=5

fig2: Effects of histone acetylation on intrinsic nucleosome mobility. (a) Outline of competitive remodelling assay used to accurately measure differences between nucleosomes. This setup has the advantage that the reaction times and conditions are exactly the same for two nucleosomes and avoids the possibility of observing effects due to different nucleosome reconstitution efficiencies. (b) Histone H4 tetra-acetylation does not alter the rate at which nucleosomes reposition thermally. Two pmol of H4 acetylated and H4 V21C unmodified nucleosomes assembled on 54A54 DNA were mixed and incubated at 47 °C for the specified amount of time. The images represent the Cy3 (H4 V21C) and Cy5 (H4 acetylated) scans of the same gel. The amount of remodelling is plotted to the right of the gels and a hyperbolic curve fitted to the data points. From this the initial rate of repositioning is calculated and the average of three independent repeats displayed in (e). (c) Tetra-acetylation of H3 results in a twofold increase in the rate of intrinsic nucleosome mobility. Reaction conditions are as for (b). (d) The increase in nucleosome mobility by H3 acetylation is not solely due to charge neutralisation. Two pmol of H3 wild-type and H3 in which the lysine residues at position 9, 14, 18 and 23 had been substituted to alanine were assembled on 54A18 DNA were mixed and incubated at 47 °C for the specified amounts of time. Substituting the lysine residues with alanine, which will reduce the basic charge of the tail, had no effect on nucleosome mobility. (e) Table indicating the initial rate of repositioning relative to control for the three constructs described above.
Mentions: Although nucleosomes are stable with respect to dissociation, they can undergo a range of dynamic fluctuations in their structure. For example, following thermal incubation nucleosomes are frequently observed to redistribute to thermodynamically favoured locations.25 This movement of nucleosomes from one translational position on DNA to another can be followed using native PAGE.26 Acetylated and control nucleosomes were reconstituted onto differentially labelled fluorescent DNA, mixed, incubated thermally and run on a native gel (Figure 2(a)). The different nucleosomes can then be visualised separately using selective excitation and emission filters. From this the proportion of nucleosomes at the initial and final position can be measured at each time point. The rate at which nucleosomes accumulate at their destination can then be plotted and a hyperbolic curve fitted to the data enabling the initial rate of redistribution to be estimated (Figure 2(b)–(e)). This approach has proved more reliable at directly comparing the behaviour of different nucleosomes.27

Bottom Line: Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways.In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment.These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes.

View Article: PubMed Central - PubMed

Affiliation: Division of Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.

ABSTRACT
Alteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes.

Show MeSH