Limits...
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.

Show MeSH
Chd1 requires the H4 tail for efficient nucleosome remodelling. (a) 40 fmol of Chd1 were incubated with 1pmol of wild- type octamers deposited on Cy3-labelled 54A0 DNA and 1pmol of octamers containing H4 with the first 19 amino acid residues deleted assembled on the same DNA labelled with Cy5 and incubated for the specified lengths of time at 30 °C in the presence of 1 mM ATP. Deletion of the H4 tail results in poor nucleosome remodelling by Chd.1 (b) Mutation of amino acid residues 16–19 to alanine significantly reduces the rate at which Chd1 repositions nucleosomes. (c) Tetra-acetylation of H4 causes nucleosomes to be repositioned at a slower rate by. (d) Quantification of the effect of alterations to histones on the initial rate of nucleosome repositioning by Chd1 shown in (a)–(c). (e) Effect of truncating the H4 tail on the ATPase activity of Chd1. The reaction conditions are as for Figure 4 except using 1 nM Chd1 and 54W0 nucleosomes initial rates of ATP hydrolysis were measured. A non-linear fit of these data to the Michaelis–Menton equation (with R2 confidence values above 0.98) allows Km and Kcat to be determined for nucleosomes containing intact H4 and truncated H4. There is little effect on Km, but Kcat is significantly reduced, suggesting that the H4 tail is an allosteric effector for Chd1.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2279226&req=5

fig6: Chd1 requires the H4 tail for efficient nucleosome remodelling. (a) 40 fmol of Chd1 were incubated with 1pmol of wild- type octamers deposited on Cy3-labelled 54A0 DNA and 1pmol of octamers containing H4 with the first 19 amino acid residues deleted assembled on the same DNA labelled with Cy5 and incubated for the specified lengths of time at 30 °C in the presence of 1 mM ATP. Deletion of the H4 tail results in poor nucleosome remodelling by Chd.1 (b) Mutation of amino acid residues 16–19 to alanine significantly reduces the rate at which Chd1 repositions nucleosomes. (c) Tetra-acetylation of H4 causes nucleosomes to be repositioned at a slower rate by. (d) Quantification of the effect of alterations to histones on the initial rate of nucleosome repositioning by Chd1 shown in (a)–(c). (e) Effect of truncating the H4 tail on the ATPase activity of Chd1. The reaction conditions are as for Figure 4 except using 1 nM Chd1 and 54W0 nucleosomes initial rates of ATP hydrolysis were measured. A non-linear fit of these data to the Michaelis–Menton equation (with R2 confidence values above 0.98) allows Km and Kcat to be determined for nucleosomes containing intact H4 and truncated H4. There is little effect on Km, but Kcat is significantly reduced, suggesting that the H4 tail is an allosteric effector for Chd1.

Mentions: Chd1 belongs to a phylogenetic subfamily that is distinct from either of the previously studied enzymes1 and contains a chromodomain that has the potential to recognise histone modifications. To investigate the histone dependence of this enzyme, we first tested the effect of deleting individual histone tails. We found that Chd1 is unable to efficiently slide nucleosomes lacking the H4 amino terminal tail (approximately sixfold slower than control: Figure 6(a), compare lanes 1–6 with 7–12). This requirement for the H4 tail is similar to that seen with Iswi remodellers such as Isw2, although the magnitude of the effect is lower. Mutation of residues 16–19 to alanine caused a 4.4-fold reduction in Chd1 activity (Figure 6(b)). Although this effect is significant it does not fully match the effect of a H4 truncation raising the possibility that the amino acids recognised by Chd1 include residues in addition to those within the 16–19 region.


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

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

Chd1 requires the H4 tail for efficient nucleosome remodelling. (a) 40 fmol of Chd1 were incubated with 1pmol of wild- type octamers deposited on Cy3-labelled 54A0 DNA and 1pmol of octamers containing H4 with the first 19 amino acid residues deleted assembled on the same DNA labelled with Cy5 and incubated for the specified lengths of time at 30 °C in the presence of 1 mM ATP. Deletion of the H4 tail results in poor nucleosome remodelling by Chd.1 (b) Mutation of amino acid residues 16–19 to alanine significantly reduces the rate at which Chd1 repositions nucleosomes. (c) Tetra-acetylation of H4 causes nucleosomes to be repositioned at a slower rate by. (d) Quantification of the effect of alterations to histones on the initial rate of nucleosome repositioning by Chd1 shown in (a)–(c). (e) Effect of truncating the H4 tail on the ATPase activity of Chd1. The reaction conditions are as for Figure 4 except using 1 nM Chd1 and 54W0 nucleosomes initial rates of ATP hydrolysis were measured. A non-linear fit of these data to the Michaelis–Menton equation (with R2 confidence values above 0.98) allows Km and Kcat to be determined for nucleosomes containing intact H4 and truncated H4. There is little effect on Km, but Kcat is significantly reduced, suggesting that the H4 tail is an allosteric effector for Chd1.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Chd1 requires the H4 tail for efficient nucleosome remodelling. (a) 40 fmol of Chd1 were incubated with 1pmol of wild- type octamers deposited on Cy3-labelled 54A0 DNA and 1pmol of octamers containing H4 with the first 19 amino acid residues deleted assembled on the same DNA labelled with Cy5 and incubated for the specified lengths of time at 30 °C in the presence of 1 mM ATP. Deletion of the H4 tail results in poor nucleosome remodelling by Chd.1 (b) Mutation of amino acid residues 16–19 to alanine significantly reduces the rate at which Chd1 repositions nucleosomes. (c) Tetra-acetylation of H4 causes nucleosomes to be repositioned at a slower rate by. (d) Quantification of the effect of alterations to histones on the initial rate of nucleosome repositioning by Chd1 shown in (a)–(c). (e) Effect of truncating the H4 tail on the ATPase activity of Chd1. The reaction conditions are as for Figure 4 except using 1 nM Chd1 and 54W0 nucleosomes initial rates of ATP hydrolysis were measured. A non-linear fit of these data to the Michaelis–Menton equation (with R2 confidence values above 0.98) allows Km and Kcat to be determined for nucleosomes containing intact H4 and truncated H4. There is little effect on Km, but Kcat is significantly reduced, suggesting that the H4 tail is an allosteric effector for Chd1.
Mentions: Chd1 belongs to a phylogenetic subfamily that is distinct from either of the previously studied enzymes1 and contains a chromodomain that has the potential to recognise histone modifications. To investigate the histone dependence of this enzyme, we first tested the effect of deleting individual histone tails. We found that Chd1 is unable to efficiently slide nucleosomes lacking the H4 amino terminal tail (approximately sixfold slower than control: Figure 6(a), compare lanes 1–6 with 7–12). This requirement for the H4 tail is similar to that seen with Iswi remodellers such as Isw2, although the magnitude of the effect is lower. Mutation of residues 16–19 to alanine caused a 4.4-fold reduction in Chd1 activity (Figure 6(b)). Although this effect is significant it does not fully match the effect of a H4 truncation raising the possibility that the amino acids recognised by Chd1 include residues in addition to those within the 16–19 region.

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