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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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Determining the kinetic parameters of nucleosome remodelling by RSC with a fluorescent ATPase assay. Overview of ATPase assay. (a) Nucleosome remodelling generates the release of inorganic phosphate (Pi) as a result of ATP hydrolysis. (b) This level of Pi is detected by a fluorescently labelled phosphate binding protein (PBP–MDCC), whose fluorescence increases dramatically upon phosphate binding. (c) Chromatin remodelling is initiated by the addition of 0.3 nM RSC to different concentrations of 36W36 nucleosomes and the fluorescence intensity measured in real-time at 1 s intervals over approximately 10 min. (d) Kinetic parameters were calculated by non-linear fitting of the Michaelis–Menton equation to the plotted data. (e) Km and Kcat of remodelling of different nucleosome substrates by RSC. H3 tetra-acetylated nucleosomes show lower Km values without affecting Kcat. H4 tetra-acetylation does not affect either parameter, consistent with data from Figure 3. Mono-acetylation at K14 of H3 significantly affects the Km of remodelling, largely mimicking H3 tetra-acetylation. Although the Km and Kcat shown above were calculated for wild-type histones no difference was detected for H3S28C nucleosomes (Supplementary Data, Figure 4).
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fig4: Determining the kinetic parameters of nucleosome remodelling by RSC with a fluorescent ATPase assay. Overview of ATPase assay. (a) Nucleosome remodelling generates the release of inorganic phosphate (Pi) as a result of ATP hydrolysis. (b) This level of Pi is detected by a fluorescently labelled phosphate binding protein (PBP–MDCC), whose fluorescence increases dramatically upon phosphate binding. (c) Chromatin remodelling is initiated by the addition of 0.3 nM RSC to different concentrations of 36W36 nucleosomes and the fluorescence intensity measured in real-time at 1 s intervals over approximately 10 min. (d) Kinetic parameters were calculated by non-linear fitting of the Michaelis–Menton equation to the plotted data. (e) Km and Kcat of remodelling of different nucleosome substrates by RSC. H3 tetra-acetylated nucleosomes show lower Km values without affecting Kcat. H4 tetra-acetylation does not affect either parameter, consistent with data from Figure 3. Mono-acetylation at K14 of H3 significantly affects the Km of remodelling, largely mimicking H3 tetra-acetylation. Although the Km and Kcat shown above were calculated for wild-type histones no difference was detected for H3S28C nucleosomes (Supplementary Data, Figure 4).

Mentions: The increased rate with which RSC repositions H3 acetylated nucleosome could be due to the modified lysine residues acting to recruit RSC or by allosterically affecting the remodelling reaction. To differentiate between these two options, the kinetic parameters of the ATP hydrolysis reaction were measured using a real-time fluorescent ATPase assay.31 The assay hinges on using a phosphate binding protein (PBP) labelled with a coumarin-based fluorescent dye, 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC), as a sensor for the amount of inorganic phosphate (Pi). On binding Pi, the labelled protein (MDCC-PBP) undergoes a shift in its emission wavelength coupled with a fivefold increase in fluorescence. When performed in a fluorimeter, this assay has the advantage of measuring ATP hydrolysis in real-time, from which kinetic parameters such as Km and Vmax are determined by non-linear fitting to the Michaelis–Menton equation (Figure 4(a)–(d)). We find that RSC has approximately threefold lower Km (tighter binding) for H3 acetylated nucleosome compared to the unmodified control, without affecting the Kcat of ATP hydrolysis (Figure 4(e)). This is consistent with preferential recruitment of RSC to H3 acetylated chromatin and the preferential binding of RSC to H3 acetylated nucleosomes (Supplementary Data, Figure 5). In contrast, RSC does not preferentially bind H4 acetylated nucleosomes, as the Km for these is the same as for the unmodified control (Figure 4(e)). Consistent with this, the Km value of the doubly H3, H4 acetylated nucleosome is indistinguishable from that of the H3 acetylated nucleosome (Figure 4(e)).


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

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

Determining the kinetic parameters of nucleosome remodelling by RSC with a fluorescent ATPase assay. Overview of ATPase assay. (a) Nucleosome remodelling generates the release of inorganic phosphate (Pi) as a result of ATP hydrolysis. (b) This level of Pi is detected by a fluorescently labelled phosphate binding protein (PBP–MDCC), whose fluorescence increases dramatically upon phosphate binding. (c) Chromatin remodelling is initiated by the addition of 0.3 nM RSC to different concentrations of 36W36 nucleosomes and the fluorescence intensity measured in real-time at 1 s intervals over approximately 10 min. (d) Kinetic parameters were calculated by non-linear fitting of the Michaelis–Menton equation to the plotted data. (e) Km and Kcat of remodelling of different nucleosome substrates by RSC. H3 tetra-acetylated nucleosomes show lower Km values without affecting Kcat. H4 tetra-acetylation does not affect either parameter, consistent with data from Figure 3. Mono-acetylation at K14 of H3 significantly affects the Km of remodelling, largely mimicking H3 tetra-acetylation. Although the Km and Kcat shown above were calculated for wild-type histones no difference was detected for H3S28C nucleosomes (Supplementary Data, Figure 4).
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fig4: Determining the kinetic parameters of nucleosome remodelling by RSC with a fluorescent ATPase assay. Overview of ATPase assay. (a) Nucleosome remodelling generates the release of inorganic phosphate (Pi) as a result of ATP hydrolysis. (b) This level of Pi is detected by a fluorescently labelled phosphate binding protein (PBP–MDCC), whose fluorescence increases dramatically upon phosphate binding. (c) Chromatin remodelling is initiated by the addition of 0.3 nM RSC to different concentrations of 36W36 nucleosomes and the fluorescence intensity measured in real-time at 1 s intervals over approximately 10 min. (d) Kinetic parameters were calculated by non-linear fitting of the Michaelis–Menton equation to the plotted data. (e) Km and Kcat of remodelling of different nucleosome substrates by RSC. H3 tetra-acetylated nucleosomes show lower Km values without affecting Kcat. H4 tetra-acetylation does not affect either parameter, consistent with data from Figure 3. Mono-acetylation at K14 of H3 significantly affects the Km of remodelling, largely mimicking H3 tetra-acetylation. Although the Km and Kcat shown above were calculated for wild-type histones no difference was detected for H3S28C nucleosomes (Supplementary Data, Figure 4).
Mentions: The increased rate with which RSC repositions H3 acetylated nucleosome could be due to the modified lysine residues acting to recruit RSC or by allosterically affecting the remodelling reaction. To differentiate between these two options, the kinetic parameters of the ATP hydrolysis reaction were measured using a real-time fluorescent ATPase assay.31 The assay hinges on using a phosphate binding protein (PBP) labelled with a coumarin-based fluorescent dye, 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC), as a sensor for the amount of inorganic phosphate (Pi). On binding Pi, the labelled protein (MDCC-PBP) undergoes a shift in its emission wavelength coupled with a fivefold increase in fluorescence. When performed in a fluorimeter, this assay has the advantage of measuring ATP hydrolysis in real-time, from which kinetic parameters such as Km and Vmax are determined by non-linear fitting to the Michaelis–Menton equation (Figure 4(a)–(d)). We find that RSC has approximately threefold lower Km (tighter binding) for H3 acetylated nucleosome compared to the unmodified control, without affecting the Kcat of ATP hydrolysis (Figure 4(e)). This is consistent with preferential recruitment of RSC to H3 acetylated chromatin and the preferential binding of RSC to H3 acetylated nucleosomes (Supplementary Data, Figure 5). In contrast, RSC does not preferentially bind H4 acetylated nucleosomes, as the Km for these is the same as for the unmodified control (Figure 4(e)). Consistent with this, the Km value of the doubly H3, H4 acetylated nucleosome is indistinguishable from that of the H3 acetylated nucleosome (Figure 4(e)).

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