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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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RSC preferentially repositions tetra-acetylated H3 nucleosomes. (a) 20 fmol of RSC were incubated with 1pmol unmodified S28C octamers assembled onto Cy3-labelled 54A18 DNA and 1pmol H3 tetra-acetylated nucleosomes on the same DNA labelled with Cy5 and incubated for the specified length of time at 30 °C in the presence of 1 mM ATP. RSC shows a dramatic preference for H3 tetra-acetylated nucleosomes: compare lanes 1–6 and 7–12; see also (d). On this fragment a proportion of nucleosomes are deposited at an alternative location, indicated by an asterisk (*), that has been characterised previously.61 Inclusion of nucleosomes deposited at this location had little effect on the calculated initial rate of sliding so they were excluded from quantitative analysis. (b) H4 tetra-acetylated nucleosomes, in contrast, are not repositioned faster by the RSC complex. (c) Nucleosomes that are both H3 and H4 tetra-acetylated are not repositioned any faster than H3 tetra-acetylated nucleosomes, confirming that H4 tetra-acetylation does not promote RSC catalysed repositioning. (d) Table indicating the average initial rate of repositioning relative to control and standard deviation from three independent experiments for the acetylated constructs described above.
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fig3: RSC preferentially repositions tetra-acetylated H3 nucleosomes. (a) 20 fmol of RSC were incubated with 1pmol unmodified S28C octamers assembled onto Cy3-labelled 54A18 DNA and 1pmol H3 tetra-acetylated nucleosomes on the same DNA labelled with Cy5 and incubated for the specified length of time at 30 °C in the presence of 1 mM ATP. RSC shows a dramatic preference for H3 tetra-acetylated nucleosomes: compare lanes 1–6 and 7–12; see also (d). On this fragment a proportion of nucleosomes are deposited at an alternative location, indicated by an asterisk (*), that has been characterised previously.61 Inclusion of nucleosomes deposited at this location had little effect on the calculated initial rate of sliding so they were excluded from quantitative analysis. (b) H4 tetra-acetylated nucleosomes, in contrast, are not repositioned faster by the RSC complex. (c) Nucleosomes that are both H3 and H4 tetra-acetylated are not repositioned any faster than H3 tetra-acetylated nucleosomes, confirming that H4 tetra-acetylation does not promote RSC catalysed repositioning. (d) Table indicating the average initial rate of repositioning relative to control and standard deviation from three independent experiments for the acetylated constructs described above.

Mentions: RSC is an abundant and essential yeast chromatin remodelling complex that is closely related to SWI/SNF.28 A notable feature of the RSC complex is that it contains almost half of the known bromodomains in the S. cerevisiae genome. As bromodomains within other proteins have been found to recognise specific patterns of histone acetylation,29,30 a distinct possibility is that histone acetylation will influence the action of the RSC complex. To test this, the ability of RSC to remodel either H3 or H4 acetylated chromatin relative to unmodified control chromatin was assayed. RSC showed a dramatic preference for H3 (K9, 14, 18, 23) tetra-acetylated chromatin compared to an unmodified control (Figure 3(a)). From the initial rates of remodelling derived from the plotted data, this was calculated to be 16(±1.5)-fold faster than control (Figure 3(d)). When the rate of H4 (K5, 8, 12, 16) tetra-acetylated nucleosomes was measured, this was found to be indistinguishable from the rate of an unmodified control (Figure 3(b) and (d)).


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

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

RSC preferentially repositions tetra-acetylated H3 nucleosomes. (a) 20 fmol of RSC were incubated with 1pmol unmodified S28C octamers assembled onto Cy3-labelled 54A18 DNA and 1pmol H3 tetra-acetylated nucleosomes on the same DNA labelled with Cy5 and incubated for the specified length of time at 30 °C in the presence of 1 mM ATP. RSC shows a dramatic preference for H3 tetra-acetylated nucleosomes: compare lanes 1–6 and 7–12; see also (d). On this fragment a proportion of nucleosomes are deposited at an alternative location, indicated by an asterisk (*), that has been characterised previously.61 Inclusion of nucleosomes deposited at this location had little effect on the calculated initial rate of sliding so they were excluded from quantitative analysis. (b) H4 tetra-acetylated nucleosomes, in contrast, are not repositioned faster by the RSC complex. (c) Nucleosomes that are both H3 and H4 tetra-acetylated are not repositioned any faster than H3 tetra-acetylated nucleosomes, confirming that H4 tetra-acetylation does not promote RSC catalysed repositioning. (d) Table indicating the average initial rate of repositioning relative to control and standard deviation from three independent experiments for the acetylated constructs described above.
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fig3: RSC preferentially repositions tetra-acetylated H3 nucleosomes. (a) 20 fmol of RSC were incubated with 1pmol unmodified S28C octamers assembled onto Cy3-labelled 54A18 DNA and 1pmol H3 tetra-acetylated nucleosomes on the same DNA labelled with Cy5 and incubated for the specified length of time at 30 °C in the presence of 1 mM ATP. RSC shows a dramatic preference for H3 tetra-acetylated nucleosomes: compare lanes 1–6 and 7–12; see also (d). On this fragment a proportion of nucleosomes are deposited at an alternative location, indicated by an asterisk (*), that has been characterised previously.61 Inclusion of nucleosomes deposited at this location had little effect on the calculated initial rate of sliding so they were excluded from quantitative analysis. (b) H4 tetra-acetylated nucleosomes, in contrast, are not repositioned faster by the RSC complex. (c) Nucleosomes that are both H3 and H4 tetra-acetylated are not repositioned any faster than H3 tetra-acetylated nucleosomes, confirming that H4 tetra-acetylation does not promote RSC catalysed repositioning. (d) Table indicating the average initial rate of repositioning relative to control and standard deviation from three independent experiments for the acetylated constructs described above.
Mentions: RSC is an abundant and essential yeast chromatin remodelling complex that is closely related to SWI/SNF.28 A notable feature of the RSC complex is that it contains almost half of the known bromodomains in the S. cerevisiae genome. As bromodomains within other proteins have been found to recognise specific patterns of histone acetylation,29,30 a distinct possibility is that histone acetylation will influence the action of the RSC complex. To test this, the ability of RSC to remodel either H3 or H4 acetylated chromatin relative to unmodified control chromatin was assayed. RSC showed a dramatic preference for H3 (K9, 14, 18, 23) tetra-acetylated chromatin compared to an unmodified control (Figure 3(a)). From the initial rates of remodelling derived from the plotted data, this was calculated to be 16(±1.5)-fold faster than control (Figure 3(d)). When the rate of H4 (K5, 8, 12, 16) tetra-acetylated nucleosomes was measured, this was found to be indistinguishable from the rate of an unmodified control (Figure 3(b) and (d)).

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