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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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H4 tetra-acetylation increases octamer transfer by RSC. (a) Octamer transfer assay: RSC is able to disrupt nucleosomes and transfer the histone octamer from unlabelled donor nucleosomes onto a separate DNA molecule, in this case a radiolabelled 147 bp 0W0 fragment derived from the 601 positioning sequence. This is measured by the shift in mobility of a radiolabelled DNA fragment to that of a nucleosome. (b) Efficiency of octamer transfer from different donor nucleosomes. H3 tetra-acetylated nucleosomes are transferred faster than unmodified nucleosomes consisting of wild-type H3 and H4: compare lanes 4–6 with lanes 1–3. Surprisingly, H4 tetra-acetylated nucleosomes are also transferred faster than control: compare lanes 7–9 with lanes 1–3. When both H3 and H4 are acetylated the effect is additive (compare lanes 10–12 with 1–3). Lane 13 shows an equivalent amount of free DNA in the absence of RSC or nucleosomes and lane 14 is a nucleosome reconstituted separately on the same DNA fragment as a mobility reference. (c) Table plotting the amount of octamer transfer from different donor nucleosomes as the average of three independent repeats. Error bars represent the standard deviation. Although histones of wild-type sequence are used as a control in the data shown, octamers bearing cysteine mutations behaved similarly.
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fig7: H4 tetra-acetylation increases octamer transfer by RSC. (a) Octamer transfer assay: RSC is able to disrupt nucleosomes and transfer the histone octamer from unlabelled donor nucleosomes onto a separate DNA molecule, in this case a radiolabelled 147 bp 0W0 fragment derived from the 601 positioning sequence. This is measured by the shift in mobility of a radiolabelled DNA fragment to that of a nucleosome. (b) Efficiency of octamer transfer from different donor nucleosomes. H3 tetra-acetylated nucleosomes are transferred faster than unmodified nucleosomes consisting of wild-type H3 and H4: compare lanes 4–6 with lanes 1–3. Surprisingly, H4 tetra-acetylated nucleosomes are also transferred faster than control: compare lanes 7–9 with lanes 1–3. When both H3 and H4 are acetylated the effect is additive (compare lanes 10–12 with 1–3). Lane 13 shows an equivalent amount of free DNA in the absence of RSC or nucleosomes and lane 14 is a nucleosome reconstituted separately on the same DNA fragment as a mobility reference. (c) Table plotting the amount of octamer transfer from different donor nucleosomes as the average of three independent repeats. Error bars represent the standard deviation. Although histones of wild-type sequence are used as a control in the data shown, octamers bearing cysteine mutations behaved similarly.

Mentions: The octamer transfer reaction involves incubating a “cold” donor nucleosome with a “hot” 32P-labelled accepter DNA fragment in the presence of RSC and ATP. The reaction products were run on a native polyacrylamide gel and transfer is indicated by a shift in mobility of the “hot” accepter DNA. If the accepter DNA has a histone octamer transferred onto it, it should now have the same mobility as a control nucleosome reconstituted onto the same fragment (Figure 7(a)).


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

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

H4 tetra-acetylation increases octamer transfer by RSC. (a) Octamer transfer assay: RSC is able to disrupt nucleosomes and transfer the histone octamer from unlabelled donor nucleosomes onto a separate DNA molecule, in this case a radiolabelled 147 bp 0W0 fragment derived from the 601 positioning sequence. This is measured by the shift in mobility of a radiolabelled DNA fragment to that of a nucleosome. (b) Efficiency of octamer transfer from different donor nucleosomes. H3 tetra-acetylated nucleosomes are transferred faster than unmodified nucleosomes consisting of wild-type H3 and H4: compare lanes 4–6 with lanes 1–3. Surprisingly, H4 tetra-acetylated nucleosomes are also transferred faster than control: compare lanes 7–9 with lanes 1–3. When both H3 and H4 are acetylated the effect is additive (compare lanes 10–12 with 1–3). Lane 13 shows an equivalent amount of free DNA in the absence of RSC or nucleosomes and lane 14 is a nucleosome reconstituted separately on the same DNA fragment as a mobility reference. (c) Table plotting the amount of octamer transfer from different donor nucleosomes as the average of three independent repeats. Error bars represent the standard deviation. Although histones of wild-type sequence are used as a control in the data shown, octamers bearing cysteine mutations behaved similarly.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: H4 tetra-acetylation increases octamer transfer by RSC. (a) Octamer transfer assay: RSC is able to disrupt nucleosomes and transfer the histone octamer from unlabelled donor nucleosomes onto a separate DNA molecule, in this case a radiolabelled 147 bp 0W0 fragment derived from the 601 positioning sequence. This is measured by the shift in mobility of a radiolabelled DNA fragment to that of a nucleosome. (b) Efficiency of octamer transfer from different donor nucleosomes. H3 tetra-acetylated nucleosomes are transferred faster than unmodified nucleosomes consisting of wild-type H3 and H4: compare lanes 4–6 with lanes 1–3. Surprisingly, H4 tetra-acetylated nucleosomes are also transferred faster than control: compare lanes 7–9 with lanes 1–3. When both H3 and H4 are acetylated the effect is additive (compare lanes 10–12 with 1–3). Lane 13 shows an equivalent amount of free DNA in the absence of RSC or nucleosomes and lane 14 is a nucleosome reconstituted separately on the same DNA fragment as a mobility reference. (c) Table plotting the amount of octamer transfer from different donor nucleosomes as the average of three independent repeats. Error bars represent the standard deviation. Although histones of wild-type sequence are used as a control in the data shown, octamers bearing cysteine mutations behaved similarly.
Mentions: The octamer transfer reaction involves incubating a “cold” donor nucleosome with a “hot” 32P-labelled accepter DNA fragment in the presence of RSC and ATP. The reaction products were run on a native polyacrylamide gel and transfer is indicated by a shift in mobility of the “hot” accepter DNA. If the accepter DNA has a histone octamer transferred onto it, it should now have the same mobility as a control nucleosome reconstituted onto the same fragment (Figure 7(a)).

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
Related in: MedlinePlus