A Native Peptide Ligation Strategy for Deciphering Nucleosomal Histone Modifications

Abstract

Post-translational modifications of histones influence both chromatin structure and the binding and function of chromatin-associated proteins. A major limitation to understanding these effects has been the inability to construct nucleosomes in vitro that harbor homogeneous and site-specific histone modifications. Here, we describe a native peptide ligation strategy for generating nucleosomal arrays that can harbor a wide range of desired histone modifications. As a first test of this method, we engineered model nucleosomal arrays in which each histone H3 contains a phosphorylated serine at position 10 and performed kinetic analyses of Gcn5-dependent histone acetyltransferase activities. Recombinant Gcn5 shows increased histone acetyltransferase activity on nucleosomal arrays harboring phosphorylated H3 serine 10 and is consistent peptide in to analyses peptide we that the histone acetyltransferase activity of the is H3 in the of nucleosomal peptide and that the to nucleosomal arrays a for understanding the effects of histone modifications. Post-translational modifications of histones influence both chromatin structure and the binding and function of chromatin-associated proteins. A major limitation to understanding these effects has been the inability to construct nucleosomes in vitro that harbor homogeneous and site-specific histone modifications. Here, we describe a native peptide ligation strategy for generating nucleosomal arrays that can harbor a wide range of desired histone modifications. As a first test of this method, we engineered model nucleosomal arrays in which each histone H3 contains a phosphorylated serine at position 10 and performed kinetic analyses of Gcn5-dependent histone acetyltransferase activities. Recombinant Gcn5 shows increased histone acetyltransferase activity on nucleosomal arrays harboring phosphorylated H3 serine 10 and is consistent peptide in to analyses peptide we that the histone acetyltransferase activity of the is H3 in the of nucleosomal peptide and that the to nucleosomal arrays a for understanding the effects of histone modifications. of the of a in a wide range of and histones these to in wide range of and of these modifications to the for that to effects peptide to that of serine 10 histone H3 the of to the histone histone to for major limitation for of of histone modifications is the inability to nucleosomes in vitro that harbor histone modifications. a histone that and homogeneous modifications can and a in understanding the of histone modifications the of and histone which to in nucleosomes can nucleosomes homogeneous of we describe a native ligation strategy that the of nucleosomal arrays that harbor a wide range of of histone serine and this method, peptide is to a histone that contains at desired ligation is to which is histone and nucleosomal describe the first test of this in which we engineered model nucleosomal arrays harboring a phosphorylated serine at position 10 of each histone H3 histone native ligation that a and a that these a a in which the is to the a peptide of H3 histone H3 modifications the first peptide can to histone that a wide range of modifications in this these we to serine at position 10 of the desired a strategy of and the peptide peptide on of the the the which can to a to the desired H3 peptide in the to phosphorylated H3 peptide of the and and histone H3 native ligation is for histone of histone H3 A ligation histone H3 peptide and histone H3 on contains histone shows the shows of H3 the H3 and phosphorylated H3 proteins. of histone histone the of histone on that histones in the and histones and of histone of the of histone and and to the of each histone is in the to the of the shows a of of and H3 the of histone H3 we of a histone H3 construct to the at position a this is to structure is the of the histone and is the of is a to the at position of histone H3 the to the desired H3 and of this and and phosphorylated histone H3 the peptide and in the of the contains the ligation which of a that histone H3 of the that the of the to the for histone H3 the ligation performed of peptide and the to to the to and histone the the H3 and histones and in and the histone in the of histones for H3 that the of performed on the of the histone and the to the of the phosphorylated histone H3 that this histone is homogeneous for H3 serine 10 histone to model nucleosomal arrays a of to of a can and position a vitro a of nucleosomes contains a in the of the we the nucleosomal arrays each in the is a a that can to native of the that can the to that of and of to in in the of and to these that histones to nucleosomal arrays serine 10 phosphorylated histone H3 of the nucleosomal is of of a the contains a of nucleosomal arrays arrays on a native and nucleosomal arrays of histone to is of nucleosomal arrays histone performed and nucleosomal arrays H3 and the of the and to of of and a and of the the of in the and of performed a of performed to the nucleosomal arrays histones and to 10 nucleosomal arrays and and and a of to which a of the of the the of nucleosomal arrays histones of arrays and the histone to to to we that of the arrays for and and each this to the of these that H3 serine 10 the of to peptide that H3 serine 10 the histone acetyltransferase of and native the of nucleosomal arrays the activity of we first performed and nucleosomal arrays which the nucleosomal activity of of the nucleosomal arrays a function of and in the of the to for nucleosomal H3 and the for the phosphorylated nucleosomal increased to the consistent activity phosphorylated peptide H3 serine 10 of nucleosomal arrays the of activity on phosphorylated and nucleosomal acetyltransferase performed and nucleosomal arrays H3 of histone at the of the in a at and the of a function of for each of activity on phosphorylated and nucleosomal and of performed to and nucleosomal histone of activity on phosphorylated and nucleosomal arrays a range of nucleosomal histone H3 for arrays histone H3 for arrays performed histone for each for performed at each and the for a function of histone H3 for nucleosomal arrays H3 to a a of and a for nucleosomal arrays histone of histone for phosphorylated and nucleosomal performed and nucleosomal arrays H3 for and to to in and this increased activity phosphorylated nucleosomal arrays to a native we performed nucleosomal the to the increased activity of on the phosphorylated the for activity of a the phosphorylated and this to that and phosphorylated a kinetic that the performed in binding a wide range of nucleosomal for of of activity a function of that for nucleosomal of of for phosphorylated and arrays a range of of in of these that the H3 the and that in the of is the of and to nucleosomes of histones for the of nucleosomal arrays histones native the to phosphorylated nucleosomal arrays has to that the nucleosomal activity of is H3 and that the nucleosomal activity of the native is to H3 that peptide of activity H3 the of is that the of activity is to increased for the peptide to the of the of H3 to nucleosomal activity to to a of histone nucleosomes that peptide for histone the of to to to a of nucleosomal that consistent that that histone H3 is to histone in native peptide ligation to phosphorylated nucleosomal this strategy has the for generating and nucleosomal arrays that harbor a wide range of histone the ligation is that harbor nucleosomal arrays at position of histone H3 arrays for of of nucleosomal arrays harboring at position of histone H3 to this the of the the of this native peptide ligation strategy a for the of histone modifications the structure and function of the chromatin the of a in a wide range of and histones these to in wide range of and of these modifications to the for that to effects peptide to that of serine 10 histone H3 the of to the histone histone to for A major limitation for of of histone modifications is the inability to nucleosomes in vitro that harbor histone modifications. a histone that and homogeneous modifications can and a in understanding the of histone modifications the of and histone which to in nucleosomes can nucleosomes homogeneous of modifications. we describe a native ligation strategy that the of nucleosomal arrays that harbor a wide range of of histone serine and this method, peptide is to a histone that contains at desired ligation is to which is histone and nucleosomal describe the first test of this in which we engineered model nucleosomal arrays harboring a phosphorylated serine at position 10 of each histone H3 histone native ligation that a and a that these a a in which the is to the a peptide of H3 histone H3 modifications the first peptide can to histone that a wide range of modifications in this these we to serine at position 10 of the desired a strategy of and the peptide peptide on of the the the which can to a to the desired H3 peptide in the to phosphorylated H3 peptide of the and H3 the of histone H3 we of a histone H3 construct to the at position a this is to structure is the of the histone and is the of is a to the at position of histone H3 the to the desired H3 and of this and and phosphorylated histone H3 the peptide and in the of the contains the ligation which of a that histone H3 of the that the of the to the for histone H3 the ligation performed of peptide and the to to the to and histone the the H3 and histones and in and the histone in the of histones for H3 that the of performed on the of the histone and the to the of the phosphorylated histone H3 that this histone is homogeneous for H3 serine 10 histone to model nucleosomal arrays a of to of a can and position a vitro a of nucleosomes contains a in the of the we the nucleosomal arrays each in the is a a that can to native of the that can the to that of and of to in in the of and to these that histones to nucleosomal arrays serine 10 phosphorylated histone H3 of the nucleosomal is of of a the contains a of nucleosomal arrays arrays on a native and nucleosomal arrays of histone to is of nucleosomal arrays histone performed and nucleosomal arrays H3 and the of the and to of of and a and of the the of in the and of performed a of performed to the nucleosomal arrays histones and to 10 nucleosomal arrays and and and a of to which a of the of the the of nucleosomal arrays histones of arrays and the histone to to to we that of the arrays for and and each this to the of these that H3 serine 10 the of to peptide that H3 serine 10 the histone acetyltransferase of and native the of nucleosomal arrays the activity of we first performed and nucleosomal arrays which the nucleosomal activity of of the nucleosomal arrays a function of and in the of the to for nucleosomal H3 and the for the phosphorylated nucleosomal increased to the consistent activity phosphorylated peptide H3 serine 10 of nucleosomal arrays the of activity on phosphorylated and nucleosomal acetyltransferase performed and nucleosomal arrays H3 of histone at the of the in a at and the of a function of for each of activity on phosphorylated and nucleosomal and of performed to and nucleosomal histone of activity on phosphorylated and nucleosomal arrays a range of nucleosomal histone H3 for arrays histone H3 for arrays performed histone for each for performed at each and the for a function of histone H3 for nucleosomal arrays H3 to a a of and a for nucleosomal arrays histone of histone for phosphorylated and nucleosomal performed and nucleosomal arrays H3 for and to to in and this increased activity phosphorylated nucleosomal arrays to a native we performed nucleosomal the to the increased activity of on the phosphorylated the for activity of a the phosphorylated and this to that and phosphorylated a kinetic that the performed in binding a wide range of nucleosomal for of of activity a function of that for nucleosomal of of for phosphorylated and arrays a range of of in of these that the H3 the and that in the of is the of and to nucleosomes of histones for the of nucleosomal arrays histones native the to phosphorylated nucleosomal arrays has to that the nucleosomal activity of is H3 and that the nucleosomal activity of the native is to H3 that peptide of activity H3 the of is that the of activity is to increased for the peptide to the of the of H3 to nucleosomal activity to to a of histone nucleosomes that peptide for histone the of to to to a of nucleosomal that consistent that that histone H3 is to histone in native peptide ligation to phosphorylated nucleosomal this strategy has the for generating and nucleosomal arrays that harbor a wide range of histone the ligation is that harbor nucleosomal arrays at position of histone H3 arrays for of of nucleosomal arrays harboring at position of histone H3 to this the of the the of this native peptide ligation strategy a for the of histone modifications the structure and function of the chromatin histone native ligation that a and a that these a a in which the is to the a peptide of H3 histone H3 modifications the first peptide can to histone that a wide range of modifications in this these we to serine at position 10 of the desired a strategy of and the peptide peptide on of the the the which can to a to the desired H3 peptide in the to phosphorylated H3 peptide of the and H3 the of histone H3 we of a histone H3 construct to the at position a this is to structure is the of the histone and is the of is a to the at position of histone H3 the to the desired H3 and of this and and phosphorylated histone H3 the peptide and in the of the contains the ligation which of a that histone H3 of the that the of the to the for histone H3 the ligation performed of peptide and the to to the to and histone the the H3 and histones and in and the histone in the of histones for H3 that the of performed on the of the histone and the to the of the phosphorylated histone H3 that this histone is homogeneous for H3 serine 10 histone to model nucleosomal arrays a of to of a can and position a vitro a of nucleosomes contains a in the of the we the nucleosomal arrays each in the is a a that can to native of the that can the to that of and of to in in the of and to these that histones to nucleosomal of performed to the nucleosomal arrays histones and to 10 nucleosomal arrays and and and a of to which a of the of the the of nucleosomal arrays histones of arrays and the histone to to to we that of the arrays for and and each this to the of these that H3 serine 10 the of to peptide that H3 serine 10 the histone acetyltransferase of and native the of nucleosomal arrays the activity of we first performed and nucleosomal arrays which the nucleosomal activity of of the nucleosomal arrays a function of and in 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phosphorylated and this to that and phosphorylated a kinetic that the performed in binding a wide range of nucleosomal for of of activity a function of that for nucleosomal of of for phosphorylated and arrays a range of of in of these that the H3 the and that in the of is the of and to nucleosomes of histones for the of nucleosomal arrays histones native the to phosphorylated nucleosomal arrays has to that the nucleosomal activity of is H3 and that the nucleosomal activity of the native is to H3 that peptide of activity H3 the of is that the of activity is to increased for the peptide to the of the of H3 to nucleosomal activity to to a of histone nucleosomes that peptide for histone the of to to to a of nucleosomal that consistent that that histone H3 is to histone in native peptide ligation to phosphorylated nucleosomal this strategy has the for generating and nucleosomal arrays that harbor a wide range of histone the ligation is that harbor nucleosomal arrays at position of histone H3 arrays for of of nucleosomal arrays harboring at position of histone H3 to this the of the the of this native peptide ligation strategy a for the of histone modifications the structure and function of the chromatin of H3 histone H3 modifications the first peptide can to histone that a wide range of modifications in this these we to serine at position 10 of the desired a strategy of and the peptide peptide on of the the the which can to a to the desired H3 peptide in the to phosphorylated H3 peptide of the and H3 the of histone H3 we of a histone H3 construct to the at position a this is to structure is the of the histone and is the of is a to the at position of histone H3 the to the desired H3 and of this and and phosphorylated histone H3 the peptide and in the of the contains the ligation which of a that histone H3 of the that the of the to the for histone H3 the ligation performed of peptide and the to to the to and histone the the H3 and histones and in and the histone in the of histones for H3 that the of performed on the of the histone and the to the of the phosphorylated histone H3 that this histone is homogeneous for H3 serine 10 histone to model nucleosomal arrays a of to of a can and position a vitro a of nucleosomes contains a in the of the we the nucleosomal arrays each in the is a a that can to native of the that can the to that of and of to in in the of and to these that histones to nucleosomal of performed to the nucleosomal arrays histones and to 10 nucleosomal arrays and and and a of to which a of the of the the of nucleosomal arrays histones of arrays and the histone to to to we that of the arrays for and and each this to the of these that H3 serine 10 the of to peptide that H3 serine 10 the histone acetyltransferase of and native the of nucleosomal arrays the activity of we first performed and nucleosomal arrays which the nucleosomal activity of of the nucleosomal arrays a function of and in the of the to for nucleosomal H3 and the for the phosphorylated nucleosomal increased to the consistent activity phosphorylated peptide this increased activity phosphorylated nucleosomal arrays to a native we performed nucleosomal the to the increased activity of on the phosphorylated the for activity of a the phosphorylated and this to that and phosphorylated a kinetic that the performed in binding a wide range of nucleosomal for of of activity a function of that for nucleosomal of of for phosphorylated and arrays a range of of in of these that the H3 the and that in the of is the of and to nucleosomes of histones for the of nucleosomal arrays histones native the to phosphorylated nucleosomal arrays has to that the nucleosomal activity of is H3 and that the nucleosomal activity of the native is to H3 that peptide of activity H3 the of is that the of activity is to increased for the peptide to the of the of H3 to nucleosomal activity to to a of histone nucleosomes that peptide for histone the of to to to a of nucleosomal that consistent that that histone H3 is to histone in native peptide ligation to phosphorylated nucleosomal this strategy has the for generating and nucleosomal arrays that harbor a wide range of histone the ligation is that harbor nucleosomal arrays at position of histone H3 arrays for of of nucleosomal arrays harboring at position of histone H3 to this the of the the of this native peptide ligation strategy a for the of histone modifications the structure and function of the chromatin of H3 histone H3 modifications the first peptide can to histone that a wide range of modifications in this these we to serine at position 10 of the desired a strategy of and the peptide peptide on of the the the which can to a to the desired H3 peptide in the to phosphorylated H3 peptide of the and histone H3 modifications the first peptide can to histone that a wide range of modifications in this these we to serine at position 10 of the desired a strategy of and the peptide peptide on of the the the which can to a to the desired H3 peptide in the to phosphorylated H3 peptide of the and H3 the of histone H3 we of a histone H3 construct to the at position a this is to structure is the of the histone and is the of is a to the at position of histone H3 the to the desired H3 and of this and the of histone H3 we of a histone H3 construct to the at position a this is to structure is the of the histone and is the of is a to the at position of histone H3 the to the desired H3 and of this and H3 and phosphorylated histone H3 the peptide and in the of the contains the ligation which of a that histone H3 of the that the of the to the for histone H3 the ligation performed of peptide and the to to the to and phosphorylated histone H3 the peptide and in the of the contains the ligation which of a that histone H3 of the that the of the to the for histone H3 the ligation performed of peptide and the to to the to and histone the the H3 and histones and in and the histone in the of histones for H3 that the of performed on the of the histone and the to the of the phosphorylated histone H3 that this histone is homogeneous for H3 serine 10 histone to model nucleosomal arrays a of to of a can and position a vitro a of nucleosomes contains a in the of the we the nucleosomal arrays each in the is a a that can to native of the that can the to that of and of to in in the of and to these that histones to nucleosomal histone the the H3 and histones and in and the histone in the of histones for H3 that the of performed on the of the histone and the to the of the phosphorylated histone H3 that this histone is homogeneous for H3 serine 10 histone to model nucleosomal arrays a of to of a can and position a vitro a of nucleosomes contains a in the of the we the nucleosomal arrays each in the is a a that can to native of the that can the to that of and of to in in the of and to these that histones to nucleosomal of performed to the nucleosomal arrays histones and to 10 nucleosomal arrays and and and 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of a the phosphorylated and this to that and phosphorylated a kinetic that the performed in binding a wide range of nucleosomal for of of activity a function of that for nucleosomal of of for phosphorylated and arrays a range of of in of these that the H3 the and that in the of is the of and to nucleosomes of histones for the of nucleosomal arrays histones native the to phosphorylated nucleosomal arrays has to that the nucleosomal activity of is H3 and that the nucleosomal activity of the native is to H3 that peptide of activity H3 the of is that the of activity is to increased for the peptide to the of the of H3 to nucleosomal activity to to a of histone nucleosomes that peptide for histone the of to to to a of nucleosomal that consistent that that histone H3 is to histone in peptide that H3 serine 10 the histone acetyltransferase of and native the of nucleosomal arrays the activity of we first performed and nucleosomal arrays which the nucleosomal activity of of the nucleosomal arrays a function of and in the of the to for nucleosomal H3 and the for the phosphorylated nucleosomal increased to the consistent activity phosphorylated peptide this increased activity phosphorylated nucleosomal arrays to a native we performed nucleosomal the to the increased activity of on the phosphorylated the for activity of a the phosphorylated and this to that and phosphorylated a kinetic that the performed in binding a wide range of nucleosomal for of of activity a function of that for nucleosomal of of for phosphorylated and arrays a range of of in of these that the H3 the and that in the of is the of and to nucleosomes of histones for the of nucleosomal arrays histones native the to phosphorylated nucleosomal arrays has to that the nucleosomal activity of is H3 and that the nucleosomal activity of the native is to H3 that peptide of activity H3 the of is that the of activity is to increased for the peptide to the of the of H3 to nucleosomal activity to to a of histone nucleosomes that peptide for histone the of to to to a of nucleosomal that consistent that that histone H3 is to histone in native peptide ligation to phosphorylated nucleosomal this strategy has the for generating and nucleosomal arrays that harbor a wide range of histone the ligation is that harbor nucleosomal arrays at position of histone H3 arrays for of of nucleosomal arrays harboring at position of histone H3 to this the of the the of this native peptide ligation strategy a for the of histone modifications the structure and function of the chromatin native peptide ligation to phosphorylated nucleosomal this strategy has the for generating and nucleosomal arrays that harbor a wide range of histone the ligation is that harbor nucleosomal arrays at position of histone H3 arrays for of of nucleosomal arrays harboring at position of histone H3 to this the of the the of this native peptide ligation strategy a for the of histone modifications the structure and function of the chromatin and the of peptide for to the for the of and and for on the