Key points are not available for this paper at this time.
Nucleosomes are highly dynamic macromolecular complexes that are assembled and disassembled in a modular fashion. One important way in which this dynamic process can be modulated is by the replacement of major histones with their variants, thereby affecting nucleosome structure and function. Here we use fluorescence resonance energy transfer between fluorophores attached to various defined locations within the nucleosome to dissect and compare the structural transitions of a H2A.Z containing and a canonical nucleosome in response to increasing ionic strength. We show that the peripheral regions of the DNA dissociate from the surface of the histone octamer at relatively low ionic strength, under conditions where the dimer-tetramer interaction remains unaffected. At around 550 mm NaCl, the (H2A-H2B) dimer dissociates from the (H3-H4)2 tetramer-DNA complex. Significantly, this latter transition is stabilized in nucleosomes that have been reconstituted with the essential histone variant H2A.Z. Our studies firmly establish fluorescence resonance energy transfer as a valid method to study nucleosome stability, and shed new light on the biological function of H2A.Z. Nucleosomes are highly dynamic macromolecular complexes that are assembled and disassembled in a modular fashion. One important way in which this dynamic process can be modulated is by the replacement of major histones with their variants, thereby affecting nucleosome structure and function. Here we use fluorescence resonance energy transfer between fluorophores attached to various defined locations within the nucleosome to dissect and compare the structural transitions of a H2A.Z containing and a canonical nucleosome in response to increasing ionic strength. We show that the peripheral regions of the DNA dissociate from the surface of the histone octamer at relatively low ionic strength, under conditions where the dimer-tetramer interaction remains unaffected. At around 550 mm NaCl, the (H2A-H2B) dimer dissociates from the (H3-H4)2 tetramer-DNA complex. Significantly, this latter transition is stabilized in nucleosomes that have been reconstituted with the essential histone variant H2A.Z. Our studies firmly establish fluorescence resonance energy transfer as a valid method to study nucleosome stability, and shed new light on the biological function of H2A.Z. IntroductionChromatin is built from nucleosomes, the universally repeating protein-DNA complexes in all eukaryotic cells. The crystal structure of the nucleosome core particle (NCP) 1The abbreviations used are: NCP, nucleosome core particle; FRET, fluorescence resonance energy transfer; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; CPM, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin; FM, fluorescein-5-maleimide. (1Luger K. Maeder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-259Google Scholar) reveals an octameric histone core around which 147 base pairs of DNA are wrapped in 1.65 tight superhelical turns. The histone octamer itself is a modular assembly of two copies each of the four histone proteins H2A, H2B, H3, and H4. Two histone pairs, composed either of H2A and H2B, or H3 and H4, form stable heterodimers. In solution, two H3-H4 dimers form a tetramer in the shape of a flat, twisted horseshoe that binds the central 60 base pairs of the nucleosomal DNA around its outside (1Luger K. Maeder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-259Google Scholar, 2Van Holde K.E. Rich A. Chromatin Series in Molecular Biology. Springer-Verlag, New York1988Google Scholar). One (H2A-H2B) dimer is tethered to each face of the (H3-H4)2 tetramer-DNA complex, to a DNA in K. interaction between the two histone two interaction of structure by and is by and (1Luger K. Maeder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-259Google Scholar). The is by and between the of H2A, and the histone of H3 and R.K. K. Scholar). In a is between the of the two H2A which to the two of the DNA at the of the nucleosome R.K. K. Scholar). the (H2A-H2B) dimer with the (H3-H4)2 tetramer in the of DNA Scholar). (H2A-H2B) dimer base pairs either of the The base pairs of nucleosomal DNA on either are by a of H3 that form an of the (H3-H4)2 and that is to DNA in the of the (H2A-H2B) dimer K. Richmond T.J. Scholar). The between and DNA in this are in the K. Richmond T.J. of that the of the (H2A-H2B) dimer with the (H3-H4)2 tetramer-DNA is highly and that this be important the in assembly and biological function of the in and in of (H2A-H2B) dimers at a in Scholar, Scholar, Scholar). been that is in (H2A-H2B) dimers Nature. and this been in Scholar). that at the of the the of histones in nucleosome assembly with the of the (H3-H4)2 tetramer the by the of two (H2A-H2B) dimers to the The assembly of histone complexes DNA is by histone and assembly in K. and Scholar). In the of assembly the in of histone is by (H3-H4)2 (H2A-H2B) and DNA at by a of the ionic by either or The (H3-H4)2 tetramer with the DNA at around (H2A-H2B) dimers with a (H3-H4)2 tetramer-DNA the ionic Scholar). in assembly is is the by which nucleosomes are or disassembled in that in in a of the assembly with the of histone and assembly and all that DNA as a One way to in to histone and is to within the or between nucleosomes, by the of histone Scholar). to the H2A that the H2A in the H2A, to the surface and of the nucleosome by an on the surface of the histone and by a interaction with the (H3-H4)2 tetramer H2A K. Richmond T.J. Scholar). In the of H2A is the of between the two (H2A-H2B) dimers within the NCP, and is the histone variant with a and all histone variants, H2A.Z is the which is and R.K. K. Scholar, Nature. Scholar, K. Scholar, K. Scholar, Scholar). The structure of an in which major H2A been by H2A.Z been R.K. K. Scholar). The structure that in the interaction of H2A.Z with H3 with the interaction between major H2A and H3 (1Luger K. Maeder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-259Google in of the NCP, of of the between the (H2A-H2B) dimer and the (H3-H4)2 the between two H2A.Z within by the to be stabilized with the major of the highly of the between the histone and between histones and is to the of the two nucleosomes from structural as the of a on and and the of in is to Sargent D.F. K. Maeder A.W. Richmond T.J. we that H2A.Z in in Scholar) and that the of H2A.Z by in and in this function of and the in function of histone in is important to compare the of variant and nucleosomes, to which of the are by the of the of the from used as fluorescence Scholar, A. Scholar, 1997; Scholar, K. T.J. Richmond T.J. 1997; Scholar) and Scholar, Scholar, Scholar, Holde K.E. and K. be to of nucleosome and a the dynamic of and with the that histone and of their function by nucleosome structure and stability, the a of nucleosome in have a to dissect the of nucleosomes, and to the that the of histone nucleosome in we a of the of two of nucleosome core either containing H2A or the essential histone variant H2A.Z fluorescence resonance energy transfer Scholar). fluorescence pairs to either of the to and the and to and within the NCP, we can in nucleosome fluorescence resonance energy transfer by fluorophores Scholar, and by the highly of the in the of the structure We have this by a to highly defined K. Scholar). to use histone in which have been to which are with of The of the fluorophores the of the that is a method to dissect the of We show that the peripheral regions of the DNA dissociate from the histone octamer in a at relatively low ionic mm At this ionic strength, the dimer-tetramer interaction remains unaffected. At a of mm NaCl, the (H2A-H2B) dimer dissociates from the (H3-H4)2 tetramer-DNA complex, by the of the DNA from the tetramer at of the of the (H2A-H2B) dimer from the (H3-H4)2 is stabilized in with major the process by the of a and is with the of H2A.Z in of of a have been a T.J. Scholar). The in cells. and from DNA as K. of DNA on a in an mm mm to DNA with the of at The DNA the with mm and with increasing in the of mm in two The of the DNA by of DNA with of of of the DNA of the DNA in mm mm DNA with a this a at the to with the of the The in a in the of and containing and at Scholar, A.W. The in and of Scholar, A. Scholar). The to and by the of a and by on a in to the of of the DNA with by at the of the DNA in mm in The in or in a The used in this study and a of Scholar). The and with and FM, DNA from by a of by and under at The DNA in as by The and are in that with DNA DNA and to the as is are the DNA of of DNA a DNA with a and a on at in mm NaCl, mm mm at to by on at The DNA to to The DNA on a of histone proteins K. T.J. Richmond T.J. 1997; Scholar) in and as we K. Scholar). used to and H3 used to The histones in mm mm and their K. Scholar). the the method used DNA in a of fluorescence to in the of The at in the fluorescence by a as the that within histone proteins as we proteins as by histone and the H2A.Z histone variant R.K. K. Scholar) to histone a K. Scholar). histone octamer from as in and of histone from of by as K. Scholar). nucleosomes 60 at to a of the nucleosomes from DNA and by a studies with fluorescence an with The at that histones and DNA form stable and the of and by of fluorescence by DNA K. T.J. Richmond T.J. 1997; Scholar, A. Scholar). In all of the in mm mm and mm to the with and at fluorescence at an of and of and the fluorescence in mm at at an of and an of The from the and fluorescence Two of the fluorescence at and of the and is the of the to the of the fluorescence the used to the of the to and to and at The used in all and at the a of in mm mm NaCl, mm mm The in the used in all to the and in each In all at each to of to in that an been within with NCP, and to on the and the as by We used at to the the of the of the (H2A-H2B) dimer the of DNA we the to compare the of The the base the and the regions Scholar). The of the is the of and and are by of the of the and regions of the and we the and on the of where is the of the of on and DNA the of a and an within the of transfer or energy transfer is an of the of the fluorescence with the of the fluorescence In this we used as an and as a an of a that is the of the the fluorophores with the in that of the fluorophores have to energy and that fluorescence and are within the to be to of the and we are that in either histones or the within a by the between the of the in the crystal structure and the of the CPM, FM, to in a new a of with the in the crystal (1Luger K. Maeder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-259Google Scholar, R.K. K. we used histones in with H2A.Z. we in the of the interaction between the (H3-H4)2 tetramer and either the (H2A-H2B) or the dimer within the we that to in between the of the in between of the DNA and the (H3-H4)2 and in between the (H3-H4)2 tetramer and the (H2A-H2B) the of fluorophores on the and the are in of the of the NCP, two copies of each are in and to and and are surface and are in in the of we the in of the four H3 to is within the that the and structural function. is an in and the structure of the in this is R.K. K. of each with the histone and by the of to histone K. Scholar). of the of histone from a and of histone by and with the and of the histone a of of and H2B, and the of fluorescence on histones H3 and H2A and in of the to and to the and of a DNA we used an DNA from the in all K. T.J. Richmond T.J. 1997; Scholar, T.J. Scholar). by under highly conditions and DNA with a and the DNA The with the of fluorescence and as in and fluorescence of the by with of and fluorophores in by The of the by which is a the structural of nucleosomes K. T.J. Richmond T.J. Scholar) of the fluorophores and the of the histone octamer to in a on the DNA process to as K. T.J. Richmond T.J. compare and with The new that the is that we with nucleosome and is in the that used the that the of DNA is important DNA as a the (H2A-H2B) dimer and the of with fluorescence at the and can be with on structure or dynamic reconstituted with either histones or or with H3, H4, H2B, and H2A.Z by nucleosome are by a to with and DNA are and on a by with and as major and and and with and at the of the DNA nucleosomes and are as major and and and with on histone H4, and with on the of the DNA is as DNA is major and with and on and H4, is as a and and nucleosome and an that is in nucleosome are on the nucleosome and of at an of and of and the the of are to the of the in way by the of the In a histone fluorescence is by the of the DNA and the of is with nucleosome K. T.J. Richmond T.J. 1997; Scholar, A. Scholar). We fluorescence in response to ionic and in which H2B, or been with The are in within in the are between and the that the nucleosomes are to all from their the of the attached fluorescence with and histone complexes with various The are in In all the of of all in light of the of the In the between at the DNA and on the histone proteins that the fluorophores are in regions of the used the the is to the in the two in a new on we that fluorophores attached to histone proteins and DNA are to be and and be the of fluorophores the of nucleosomes, as by by and fluorescence fluorescence of each by fluorescence a nucleosomes with of at In the fluorescence as a of is by the of an the at is DNA is the by the of a DNA is the this In NaCl, the of the DNA are from the surface of the histone the between the fluorophores attached to of the DNA the transfer that of The in the the nucleosome is an of the with its with the two that and are in two pairs the two of the of the nucleosome core of the are each can each We that by the in the of with DNA and nucleosomes In we of and DNA at and at is an of nucleosomal DNA have been with and FM, The at to is at and in are as the of fluorescence at in the two in the of with of as a nucleosome we in in response to increasing ionic strength. the is the nucleosome dissociates its as is by an in of FRET, are by the in the reconstituted with the in of can be by all by fluorescence at are major are the in by and and and nucleosomes and DNA with fluorescence the with on the of the DNA with on its with on histone H4. and a nucleosome core particle from in response to ionic strength. at and at at that the of the DNA by of between fluorophores attached to the two of nucleosomal a relatively and an is mm and mm NaCl, the peripheral regions of the DNA transition is under the conditions of the to The transition is as the of or (H2A-H2B) dimers from the the conditions of the this transition is is with the that the of between the (H3-H4)2 tetramer and the DNA in two transitions The the transition is with that nucleosomes with and of the DNA from the histone of or (H2A-H2B) dimers from the DNA (H3-H4)2 the DNA to in of between the of the DNA and histone H4. is between the (H2A-H2B) dimer and the DNA is with the the of the (H2A-H2B) dimer from the (H3-H4)2 tetramer-DNA in a with a at mm to that the is an of in as a of ionic strength. we and that fluorescence in the of by ionic strength. we of an by and and that as to the that of between two nucleosomes in solution, we of nucleosomes either with fluorescence or with fluorescence at of the of two nucleosomes with increasing under conditions We can of or of of at In on the are in within and by a with which we can the of within a we the of with of major to the that the in the dimer-tetramer in the of the variant The DNA and histone used assembly of major and a the of the is The is the two of the to be is that is stabilized with major is in a of the The the transition with this is mm major NCP, and mm The two the that the of protein-DNA are in the two of is stabilized of of and by of between the of the DNA The a the central of the as the of between and the DNA as by the of between and increasing ionic the of between the of the DNA and histone is the transition is the two nucleosomes, the transition a in can be by a interaction between the dimer and the (H3-H4)2 tetramer-DNA complex, with the (H2A-H2B) tetramer The in the of the between the two of nucleosomes either a of in dimer or in the of the In we a transition around mm in the with which is in major is of a transition at that is in major that the tetramer interaction be stabilized in is by the of between the (H2A-H2B) and the (H3-H4)2 tetramer within the NCP, In this in a transition that is The the of between the (H2A-H2B) dimer and the (H3-H4)2 tetramer are mm major and mm the of the the (H2A-H2B) dimer and (H3-H4)2 tetramer between and major that the of the major and variant dimer be at is the between the (H2A-H2B) dimer and (H3-H4)2 tetramer is a of and within the of are highly modular that to histone of histone variants, of proteins and and to DNA and Scholar, Nature. Scholar, Scholar, Scholar, Scholar, Scholar, Scholar). The of DNA and the dynamic of histone are to be in the biological function of nucleosomes Scholar, Scholar, K. Scholar). the of the nucleosomal DNA are to dissociate and with the histone core in a to of to nucleosomal DNA Scholar). The (H2A-H2B) dimer can in of assembly Scholar, Scholar). The replacement of histone H2A with histone be a and way to the of the and the and structure of K. Scholar). The of the between the DNA and the histone and between the (H2A-H2B) dimer and the (H3-H4)2 tetramer within the nucleosome the of as as the by which DNA is to the in the of histone and on is important to study the of of have that the of the nucleosome in response to can be by nucleosomes K. Scholar, K. T.J. Richmond T.J. fluorophores can be attached on the and on the of the The structure and of nucleosomes remains as by and fluorescence this we that the of nucleosomal DNA dissociate in a at relatively low by of the (H2A-H2B) dimer at is in the of an in the of between the of the and between the DNA and the (H3-H4)2 tetramer is The latter transition is by between the (H2A-H2B) dimer and (H3-H4)2 which We this to compare nucleosomes containing the histone variant H2A.Z with canonical nucleosomes containing major and that the of the dimer from the nucleosome at and a with the (H2A-H2B) of DNA as a of of the of the DNA from the surface of the histone core been and in an of by and Scholar, and in between the of the we show that of DNA with the (H2A-H2B) dimer and (H3-H4)2 tetramer firmly in of the DNA from the surface of the histone octamer been as an important in nucleosome been In a we used this to that the nucleosomal in of the DNA and K. The of the (H2A-H2B) dimer at relatively low ionic is with in Scholar, Scholar, studies have the structural in nucleosomes in response to ionic strength, a of and at Holde K.E. Scholar, and studies that all four core histones are to the DNA to mm NaCl, and that of the (H2A-H2B) dimer from the DNA that of the (H3-H4)2 tetramer Scholar, Scholar, and A. Scholar). to mm NaCl, the nucleosomal DNA to its Scholar). of nucleosomal DNA are to with are with that the structure of the DNA in the nucleosome remains to mm with and Scholar) by that the remains to mm NaCl, and in the of of the of been of the nucleosome structure been at the studies that the H3 is to structural of the (H3-H4)2 The between the DNA and a attached to of H4, with the and Scholar) a transition around NaCl, of in the of the (H3-H4)2 by between the two this been with and A. Scholar) and of fluorescence by DNA as a the of nucleosome and transitions in the an structural NaCl, of the (H2A-H2B) dimer NaCl, and of the (H3-H4)2 tetramer transitions are at ionic The this in the of histone complexes to with DNA in a are from the nucleosome which in fluorescence The of the in the of nucleosomes, and in the defined of to study the dynamic of histone this to a between canonical nucleosomes and nucleosomes in which H2A been by the essential histone variant H2A.Z R.K. K. Scholar, Nature. Scholar, K. Scholar, K. Scholar). We that nucleosomes reconstituted with H2A.Z are stabilized as a of the in the interaction between the dimer and (H3-H4)2 of the crystal of with major a of the tetramer as a of of two between the H2A.Z and H3 is by an in the of between the of H2A.Z R.K. K. Scholar). Our that the dimer-tetramer interaction is stabilized the of this in nucleosome Our are in with an that the dimer at from the (H2A-H2B) can that of H2A.Z a nucleosomal in nucleosome K. Scholar). The in the of the tetramer interaction that replacement of a dimer with a dimer containing H2A.Z of by and is with that H2A.Z an important in an structure at Scholar). in with studies in which structural transitions in H2A.Z Scholar). that the ionic of the of a and this to the stable of the dimer with the of the We to in the way nucleosomes in be the of nucleosomes containing histone and histone can be or to the of the nucleosome and under studies on the structural transitions highly IntroductionChromatin is built from nucleosomes, the universally repeating protein-DNA complexes in all eukaryotic cells. The crystal structure of the nucleosome core particle (NCP) 1The abbreviations used are: NCP, nucleosome core particle; FRET, fluorescence resonance energy transfer; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; CPM, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin; FM, fluorescein-5-maleimide. (1Luger K. Maeder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-259Google Scholar) reveals an octameric histone core around which 147 base pairs of DNA are wrapped in 1.65 tight superhelical turns. The histone octamer itself is a modular assembly of two copies each of the four histone proteins H2A, H2B, H3, and H4. Two histone pairs, composed either of H2A and H2B, or H3 and H4, form stable heterodimers. In solution, two H3-H4 dimers form a tetramer in the shape of a flat, twisted horseshoe that binds the central 60 base pairs of the nucleosomal DNA around its outside (1Luger K. Maeder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-259Google Scholar, 2Van Holde K.E. Rich A. Chromatin Series in Molecular Biology. Springer-Verlag, New York1988Google Scholar). One (H2A-H2B) dimer is tethered to each face of the (H3-H4)2 tetramer-DNA complex, to a DNA in K. interaction between the two histone two interaction of structure by and is by and (1Luger K. Maeder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-259Google Scholar). The is by and between the of H2A, and the histone of H3 and R.K. K. Scholar). In a is between the of the two H2A which to the two of the DNA at the of the nucleosome R.K. K. Scholar). the (H2A-H2B) dimer with the (H3-H4)2 tetramer in the of DNA Scholar). (H2A-H2B) dimer base pairs either of the The base pairs of nucleosomal DNA on either are by a of H3 that form an of the (H3-H4)2 and that is to DNA in the of the (H2A-H2B) dimer K. Richmond T.J. Scholar). The between and DNA in this are in the K. Richmond T.J. of that the of the (H2A-H2B) dimer with the (H3-H4)2 tetramer-DNA is highly and that this be important the in assembly and biological function of the in and in of (H2A-H2B) dimers at a in Scholar, Scholar, Scholar). been that is in (H2A-H2B) dimers Nature. and this been in Scholar). that at the of the the of histones in nucleosome assembly with the of the (H3-H4)2 tetramer the by the of two (H2A-H2B) dimers to the The assembly of histone complexes DNA is by histone and assembly in K. and Scholar). In the of assembly the in of histone is by (H3-H4)2 (H2A-H2B) and DNA at by a of the ionic by either or The (H3-H4)2 tetramer with the DNA at around (H2A-H2B) dimers with a (H3-H4)2 tetramer-DNA the ionic Scholar). in assembly is is the by which nucleosomes are or disassembled in that in in a of the assembly with the of histone and assembly and all that DNA as a One way to in to histone and is to within the or between nucleosomes, by the of histone Scholar). to the H2A that the H2A in the H2A, to the surface and of the nucleosome by an on the surface of the histone and by a interaction with the (H3-H4)2 tetramer H2A K. Richmond T.J. Scholar). In the of H2A is the of between the two (H2A-H2B) dimers within the NCP, and is the histone variant with a and all histone variants, H2A.Z is the which is and R.K. K. Scholar, Nature. Scholar, K. Scholar, K. Scholar, Scholar). The structure of an in which major H2A been by H2A.Z been R.K. K. Scholar). The structure that in the interaction of H2A.Z with H3 with the interaction between major H2A and H3 (1Luger K. Maeder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-259Google in of the NCP, of of the between the (H2A-H2B) dimer and the (H3-H4)2 the between two H2A.Z within by the to be stabilized with the major of the highly of the between the histone and between histones and is to the of the two nucleosomes from structural as the of a on and and the of in is to Sargent D.F. K. Maeder A.W. Richmond T.J. we that H2A.Z in in Scholar) and that the of H2A.Z by in and in this function of and the in function of histone in is important to compare the of variant and nucleosomes, to which of the are by the of the of the from used as fluorescence Scholar, A. Scholar, 1997; Scholar, K. T.J. Richmond T.J. 1997; Scholar) and Scholar, Scholar, Scholar, Holde K.E. and K. be to of nucleosome and a the dynamic of and with the that histone and of their function by nucleosome structure and stability, the a of nucleosome in have a to dissect the of nucleosomes, and to the that the of histone nucleosome in we a of the of two of nucleosome core either containing H2A or the essential histone variant H2A.Z fluorescence resonance energy transfer Scholar). fluorescence pairs to either of the to and the and to and within the NCP, we can in nucleosome fluorescence resonance energy transfer by fluorophores Scholar, and by the highly of the in the of the structure We have this by a to highly defined K. Scholar). to use histone in which have been to which are with of The of the fluorophores the of the that is a method to dissect the of We show that the peripheral regions of the DNA dissociate from the histone octamer in a at relatively low ionic mm At this ionic strength, the dimer-tetramer interaction remains unaffected. At a of mm NaCl, the (H2A-H2B) dimer dissociates from the (H3-H4)2 tetramer-DNA complex, by the of the DNA from the tetramer at of the of the (H2A-H2B) dimer from the (H3-H4)2 is stabilized in with major the process by the of a and is with the of H2A.Z in
Park et al. (Fri,) studied this question.