Modulation of Chromatin Folding by Histone Acetylation

A homogeneous oligonucleosome complex was prepared by reconstitution of highly hyperacetylated histone octamers onto a linear DNA template consisting of 12 tandemly arranged 208-base pair fragments of the 5 S rRNA gene from the sea urchin Lytechinus variegatus. The ionic strength-dependent folding of this oligonucleosome assembly was monitored by sedimentation velocity and electron microscopy. Both types of analysis indicate that under ionic conditions resembling those found in the physiological range and in the absence of histone H1, the acetylated oligonucleosome complexes remain in an extended conformation in contrast to their nonacetylated counterparts. The implications of this finding in the context of a multistate model of chromatin folding (Hansen, J. C., and Ausio, J.(1992) TIBS 197, 187-191) as well as its biological relevance are discussed. A homogeneous oligonucleosome complex was prepared by reconstitution of highly hyperacetylated histone octamers onto a linear DNA template consisting of 12 tandemly arranged 208-base pair fragments of the 5 S rRNA gene from the sea urchin Lytechinus variegatus. The ionic strength-dependent folding of this oligonucleosome assembly was monitored by sedimentation velocity and electron microscopy. Both types of analysis indicate that under ionic conditions resembling those found in the physiological range and in the absence of histone H1, the acetylated oligonucleosome complexes remain in an extended conformation in contrast to their nonacetylated counterparts. The implications of this finding in the context of a multistate model of chromatin folding (Hansen, J. C., and Ausio, J.(1992) TIBS 197, 187-191) as well as its biological relevance are discussed. In our opinion, one of the important landmarks in the chromatin field during the last two decades has been the realization that histones are not merely passive structural players but have an important functional role in the modulation of genomic expression (2Grunstein M. Annu. Rev. Cell Biol. 1990; 6: 643-678Google Scholar, 3Grunstein M. TIG (Trends Genet.). 1990; 6: 395-400Google Scholar, 4Wolfe A.P. FASEB J. 1992; 6: 3354-3361Google Scholar). In other words, they have both a structural and a functional role. At the structural level, histones provide the protein blocks that allow for the hierarchical folding of DNA in nucleosomes, higher order, and chromosomal structures. At the highest level of compaction the structures arising from such organization (chromosomes) may be essential to prevent the shearing of long eukaryotic DNA molecules during the transmission of the genetic material from one cell to another in the course of cell division (5Cavalier-Smith T. Soc. Gen. Microbiol. 1981; 32: 33-84Google Scholar, 6Cavalier-Smith T. BioEssays. 1988; 9: 72-78Google Scholar) in eukaryotes. At the lower levels, chromatin structure and nucleosomes may have also been selected during the evolutionary transition from prokaryotes to eukaryotes, as the scaffold that provides support and modulates the fine tuning of the more complex functional mechanisms of transcription and replication. All these levels of folding must be reversible in order to accommodate the different functional needs during the cell cycle. The dynamic aspects of this chromatin folding and its implications both at the structural and functional level are still poorly understood. During the cell cycle, histones undergo several chemical post-translational modifications that could presumably be involved in the modulation of chromatin folding. One of the more extensively characterized of such modifications has been histone acetylation. Histone acetylation is a dynamic post-translational metabolic modification (7Davie J.R. Hendzel M.J. J. Cell. Biochem. 1994; 55: 98-195Google Scholar) that has been strongly correlated with transcriptional activity and with the processes of histone deposition (such as during replication) or histone displacement (such as during spermatogenesis) (for detailed reviews see (8Csordas A. Biochem. J. 1990; 265: 23-38Google Scholar, 9Turner B.M. J. Cell Sci. 1991; 99: 13-20Google Scholar, 10Ausio J. J. Cell Sci. 1992; 102: 1-5Google Scholar, 11Turner B.M. Cell. 1993; 75: 5-8Google Scholar)). At the structural level, histone acetylation is a lysine amidation reaction catalyzed by acetyltransferases. By virtue of its chemical nature, it alters the net positive charge balance of the N-terminal regions (tails) of the core histones (histones H2A, H2B, H3, and H4), and as such, it has been long hypothesized to weaken the histone-DNA interactions involved at the different levels of chromatin folding. A disruption of the protein-DNA interactions would be expected to lead to the loss of stability (folding) of both the chromatin fiber and its constitutive subunits, the nucleosomes. Nevertheless, the experimental effort to provide support to this hypothesis has led in the past, quite often, to conflicting results. At its best it has provided evidence for only minor changes in chromatin folding. The structural effect of histone acetylation on the conformation of the nucleosome core particle (12Bauer W.R. Hayes J.J. White J.H. Wolffe A.P. J. Mol. Biol. 1994; 236: 685-690Google Scholar) (consisting of a double set of each H2A, H2B, H3, and H4 histones and 146-base pair DNA) has been found to be small(12Bauer W.R. Hayes J.J. White J.H. Wolffe A.P. J. Mol. Biol. 1994; 236: 685-690Google Scholar, 13Simpson R.T. Cell. 1978; 13: 691-699Google Scholar, 14Ausio J. van Holde K.E. Biochemistry. 1986; 25: 1421-1428Google Scholar, 15Imai B.S. Yau P. Baldwin J.P. Ibel K. May R.P. Bradbury E.M. J. Biol. Chem. 1986; 261: 8784-8792Google Scholar). A larger effect was observed in the case of nucleosomes consisting of longer DNA(16Bode J.H. Gomez-Lira M. Schrter H. Eur. J. Biochem. 1983; 130: 437-445Google Scholar). Very small charges were also reported at the higher order structure level of chromatin folding(17McGhee J.D. Nickol J.M. Felsenfeld G. Ran D.C. Nucleic Acids Res. 1983; 11: 4065-4075Google Scholar, 18Dimitrov S.I. Makarov V. Apostolova T. Pashev I. FEBS Lett. 1986; 197: 217-221Google Scholar). Despite all this, there is increasing evidence that the major effect of the histone tails (and hence acetylation) on chromatin folding (19Garcia-Ramirez M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar) arises from their interactions with the linker DNA region connecting adjacent nucleosomes in the chromatin fiber. We have recently proposed a coupled multistate model of chromatin dynamics to account for the contribution of chromatin folding in the modulation of genetic activity (1Hansen J.C. Ausio J. Trends Biochem. Sci. 1992; 17: 187-191Google Scholar). The results that follow, while providing experimental support for this model, also point to the importance of acetylation in the mechanisms of chromatin folding and dynamics. Chicken blood was obtained from commercial slaughterhouses in Victoria and processed immediately after collection. The 208-12 DNA template consisting of 12 tandem repeats of a 208-base pair fragment of the 5 S rRNA gene from the sea urchin Lytechinus variegatus(25Simpson R.T. Thoma F. Brubaker J.M. Cell. 1985; 42: 799-808Google Scholar) was a generous gift of Dr. Robert Simpson. HeLa (S3 strain) cells were purchased from ATCC (American Type Culture Collection; Rockville, MD). Sephacryl S-1000 was obtained from Pharmacia Biotech Inc., and Hydroxylapatite BioGel HTP was from Bio-Rad. HeLa cells (S3 strain) were grown in spinner culture at 37°C in the presence or absence of sodium butyrate as described elsewhere(14Ausio J. van Holde K.E. Biochemistry. 1986; 25: 1421-1428Google Scholar). Chicken erythrocyte histone octamers were obtained as described elsewhere(20Hansen J.C. Ausio J. Stanik V.H. van Holde K.E. Biochemistry. 1989; 28: 9129-9136Google Scholar). Control and highly hyperacetylated histone octamers from HeLa cells were prepared from chromatin fractions isolated as described previously(14Ausio J. van Holde K.E. Biochemistry. 1986; 25: 1421-1428Google Scholar). Only fraction a (14Ausio J. van Holde K.E. Biochemistry. 1986; 25: 1421-1428Google Scholar) was used as a source of hyperacetylated histones. The chromatin fractions (~6 mg) were then dialyzed against 0.633 M NaCl, 0.1 M potassium phosphate, 1 mM dithiothreitol (pH 6.7) with or without 5 mM sodium butyrate and loaded onto a hydroxylapatite column (1.5 × 15 cm at a flow rate of 16 ml/8 fractions/h) previously equilibrated with the same buffer. After elution of the linker histones under these conditions (about 100 ml) the eluting buffer was changed to 1 M NaCl in the same buffer to elute the histone octamers. The histone octamers obtained in this way were concentrated in a centricon 10 and used immediately thereafter or kept frozen at −60°C. The 208-12 DNA template used in the oligonucleosome reconstitutions was prepared as described in (19Garcia-Ramirez M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar). This procedure was carried out as described previously(19Garcia-Ramirez M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar). SDS-15% polyacrylamide gel electrophoresis was carried out according to Laemmli(21Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar). Acetic acid, 8 M urea, 0.5% Triton X-100, polyacrylamide gel electrophoresis was carried out according to (22Bonner W.M. West M.H.P. Stedman J.D. Eur. J. Biochem. 1980; 109: 17-23Google Scholar) with a few minor modifications. The acrylamide:bisacrylamide ratio was 30:1 (separating gel) and 20:1 (stacking gel). The sample buffer was 10 M urea, 5% acetic acid, 5%β-mercaptoethanol, and 0.3% Pyronine Y. 1.2% agarose gels were prepared in TBE buffer (25 mM Tris borate, 0.5 mM EDTA, pH 8.0), according to Maniatis et al.(23Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). Preparation of the samples for electron microscopy was carried out according to Labhart and Koller (24Labhart P. Koller T. Eur. J. Cell Biol. 1981; 24: 309-316Google Scholar) as described in (19Garcia-Ramirez M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar). Micrographs were taken with a Philips 301 electron microscope. Sedimentation velocity and sedimentation equilibrium analysis were performed on a Beckman model E analytical ultracentrifuge with a computer-interfaced UV scanner (Ultrascan Interface and Data Analysis Program version 1.70; Borries Demeler (Missoula, MT). Experimental conditions and analysis of the runs were as described previously (19Garcia-Ramirez M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar) or as indicated in the figure legends. We have characterized in the past (19Garcia-Ramirez M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar, 20Hansen J.C. Ausio J. Stanik V.H. van Holde K.E. Biochemistry. 1989; 28: 9129-9136Google Scholar) the folding behavior of reconstituted oligonucleosome complexes using a DNA template consisting of 12 tandemly arranged copies of a 208-base pair fragment of 5 S rRNA gene from the sea urchin L. variegatus and chicken erythrocyte core histones. In the present work we have reconstituted 208-12 oligonucleosome complexes with the same DNA template but using highly acetylated core histones obtained from HeLa cells grown in the presence of sodium butyrate (to inhibit the cell deacetylases) and core histones obtained from HeLa cells grown in the absence of butyrate (used as a control). To this purpose, the chromatin from the butyrate-treated cells was fractionated based on its differential solubility in the presence of divalent cations to yield fractions highly enriched in highly hyperacetylated core histones(14Ausio J. van Holde K.E. Biochemistry. 1986; 25: 1421-1428Google Scholar). Upon depletion of histone H1 the core histones from both butyrate (acetylated) and non-butyrate (control) treated cells were purified under nondenaturing conditions using hydroxylapatite as described under “Experimental Procedures.” Fig. 1 shows the electrophoretic analysis of the core histones (panels A and B) as well as the DNA template and the resulting reconstituted oligonucleosome complexes (panel C). The acetylated histones used in this work (Fig. 1A, lanes 1) correspond to fraction a of the chromatin fractionation procedure (14Ausio J. van Holde K.E. Biochemistry. 1986; 25: 1421-1428Google Scholar) and contain an average of ~17 acetyl groups/histone octamer(14Ausio J. van Holde K.E. Biochemistry. 1986; 25: 1421-1428Google Scholar). The core histones obtained from non-butyrate-treated HeLa cells are shown in Fig. 1A, lanes 2, and in Fig. 1B, lane 2. Fig. 1 also shows a native agarose gel of the oligonucleosome complexes reconstituted with hyperacetylated histones (panel C, lane A) and histones of low level acetylation (Fig. 1C, lane N) in comparison with the 208-12 DNA used as a template (Fig. 1C, lane D). In agreement with previous observations(19Garcia-Ramirez M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar, 25Simpson R.T. Thoma F. Brubaker J.M. Cell. 1985; 42: 799-808Google Scholar), the electrophoretic mobility of the 208-12 oligonucleosome complexes reconstituted with nonacetylated core histones is very similar to that of their 208-12 DNA template. Under the electrophoretic conditions used here (see “Experimental Procedures”) the acetylated oligonucleosome 12-mers also exhibit an almost identical mobility (Fig. 1C, lane A). Sedimentation equilibrium analysis carried out with both samples (see also Fig. 2) provided linear plots of log (absorbance) versus the square of the radial distance for the concentration gradient at equilibrium. The molecular weight established from these plots Mr= 2.92 × 106 (nonacetylated oligonucleosome complexes) and Mr = 2.93 × 106 (acetylated oligonucleosome complexes) is fully consistent with the presence of 12 nucleosomes/molecule of DNA template(19Garcia-Ramirez M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar). The salt-dependent hydrodynamic behavior of the control and hyperacetylated oligonucleosome complexes is shown in Fig. 3 in comparison with the hydrodynamic data obtained from oligonucleosomes reconstituted with chicken erythrocyte histones. As can be seen in Fig. 3A, both the HeLa control oligonucleosomes (reconstituted with histones from non-butyrate-treated HeLa cells) and the oligonucleosomes reconstituted with chicken erythrocyte histones behave very similarly. As has already been exhaustively discussed(19Garcia-Ramirez M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar), this dependence of the sedimentation coefficient within this ionic strength range (0-100 mM NaCl) corresponds to a folding pattern of the oligonucleosome structure such as that shown in Fig. 5B. Unfortunately, our results do not allow us to establish whether this folding is due to the bending of the internucleosomal DNA linker regions(26Yao J. Lowary P.T. Widom J. Proc. Natl. Acad. Sci. U. A. 1990; Scholar), to an of these DNA regions the histone or to a of both it is that the in the within the mM range provides charge of the in the DNA that for adjacent nucleosomes to In oligonucleosomes reconstituted with histones from butyrate-treated HeLa cells exhibit a different At low mM 0.1 mM EDTA, 3 mM sodium pH the sedimentation coefficient of these complexes is lower that of the control for HeLa histone oligonucleosomes or S for chicken using the J. Sci. Scholar) (see also (19Garcia-Ramirez M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar) for a detailed that such a in S can be for by an of the oligonucleosome fiber as a of a of the DNA at the of the acetylated nucleosome The model is fully consistent with data on the of hyperacetylated nucleosome core J. van Holde K.E. Biochemistry. 1986; 25: 1421-1428Google Scholar, J. K. Eur. J. Biochem. 1980; Scholar). was shown that hyperacetylated nucleosome core a in the (14Ausio J. van Holde K.E. Biochemistry. 1986; 25: 1421-1428Google Scholar) to the DNA regions at the of the DNA K. van Holde K.E. H. Nucleic Acids Res. 1978; Scholar, R.T. J. Biol. Chem. Scholar). a of the DNA within these regions has been more recently proposed B.S. Yau P. Bradbury E.M. Cell. 1989; Scholar). As the concentration the sedimentation coefficient of the hyperacetylated oligonucleosome complexes also a at 100 mM NaCl with an = S (Fig. This is very similar to the sedimentation coefficient of the control oligonucleosomes at low (see Fig. in the linker DNA regions are in a extended hydrodynamic data indicate that under ionic strength conditions similar to those found under physiological conditions and in the absence of histone H1 acetylated oligonucleosome an extended A similar in conformation would also the observed in Fig. for the to chicken erythrocyte oligonucleosomes and to hyperacetylated histone oligonucleosomes due to the in their levels of acetylation acetyl H4 for chicken and acetyl H4 in HeLa cells L. Nucleic Acids Res. 1990; Scholar)). experimental evidence in support of this from the electron microscopy analysis shown in Fig. In contrast to to nonacetylated oligonucleosome M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar), hyperacetylated oligonucleosomes remain in an conformation within the range of At low ionic strength (see Fig. the exhibit an extended conformation that is of that by oligonucleosomes (19Garcia-Ramirez M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar) and well with the hydrodynamic The results in the previous that histone acetylation has a positive role in the folding dynamics of the chromatin fiber. In the absence of linker histones under ionic strength similar to those of the physiological acetylated remain in an extended conformation in contrast to their nonacetylated results at the chromatin level, the major structural effect of the acetylation of the N-terminal regions of the histones is on the linker DNA connecting adjacent nucleosome This is fully consistent with the finding that histone acetylation the nucleosome core particle previously reported by et B.S. Yau P. Bradbury E.M. Cell. 1989; Scholar). is also in agreement with our that the linker region is more by in hyperacetylated Ausio J. van Holde K.E. J. 1988; Scholar). Nevertheless, all these data are in with the results recently obtained with from isolated from butyrate-treated L. Mol. Cell. Biol. 1992; Scholar). the histones of the a of in the level of could be L. Mol. Cell. Biol. 1992; Scholar). In an to these experimental it has been L. Mol. Cell. Biol. 1992; Scholar) that in the in histone acetylation in nucleosomes that are already in the in oligonucleosome used by et B.S. Yau P. Bradbury E.M. Cell. 1989; Scholar) oligonucleosomes were from histones that were already this very for the 1) previously reported of the linker regions Ausio J. van Holde K.E. J. 1988; Scholar) was observed in a native 2) As be of the structural of histone acetylation on chromatin folding can be (to a by histone of the histone N-terminal by was in this that the same structural can be observed of whether in oligonucleosomes or oligonucleosomes reconstituted from core histones were M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar). was also shown that the reconstituted 208-12 oligonucleosome has a folding behavior identical to that of oligonucleosomes isolated from chicken erythrocyte M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar). This also against DNA as L. Mol. Cell. Biol. 1992; Scholar) for the results with the hyperacetylated oligonucleosome used by et B.S. Yau P. Bradbury E.M. Cell. 1989; Scholar) and is very similar to that used in this The effect of histone acetylation on the linker DNA region also the larger conformation effect observed at the nucleosome level J.H. Gomez-Lira M. Schrter H. Eur. J. Biochem. 1983; 130: 437-445Google Scholar, E.M. M. Gomez-Lira M. J. Biochem. Scholar) with the core J. van Holde K.E. Biochemistry. 1986; 25: 1421-1428Google Scholar). The results obtained here with the hyperacetylated oligonucleosome complexes with our with native and reconstituted oligonucleosomes provide experimental support to the multistate model of chromatin folding (1Hansen J.C. Ausio J. Trends Biochem. Sci. 1992; 17: 187-191Google Scholar) (see Fig. the role of the core histone tails in chromatin has been long J. D.C. H. J. Cell Biol. Scholar, J. Cell Biol. Scholar), these regions to a more important role in the dynamic modulation of this folding was In the absence of H1, the interactions of the core histone tails with the linker DNA regions provide the charge to under physiological the bending the histone of this DNA This adjacent nucleosome in a order used order order order is from all our that under ionic strength conditions similar to those found in the cell chromatin of linker histones not exhibit an extended conformation the histones are we have this of folding as one in nucleosomes are arranged in a is consistent with hydrodynamic M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Google Scholar, 20Hansen J.C. Ausio J. Stanik V.H. van Holde K.E. Biochemistry. 1989; 28: 9129-9136Google Scholar). the of more complex arising from of folding be G. Robert van Holde K.E. J. Proc. Natl. Acad. Sci. U. A. 1994; Scholar). order higher order structure low order This structure is not to J.C. Wolffe A.P. Biochemistry. 1992; Scholar). may be transition to by histone acetylation. In both transcription and by have been shown to be by the J.C. Wolffe A.P. Biochemistry. 1992; Scholar). In the presence of linker histones the of compaction of the chromatin fiber to the higher order structure on the effect of histone acetylation on the of the chromatin fiber an almost J.D. Nickol J.M. Felsenfeld G. Ran D.C. Nucleic Acids Res. 1983; 11: 4065-4075Google Scholar, 18Dimitrov S.I. Makarov V. Apostolova T. Pashev I. FEBS Lett. 1986; 197: 217-221Google Scholar). the biological relevance of our finding has to do with the still of the linker histone in chromatin J. van Holde K.E. J. Cell Sci. 1992; Scholar) with whether or not the are or not in this of the there is increasing evidence in support of an histones of the linker and acetylated Hendzel M.J. J.R. J. Biol. Chem. 1990; 265: Scholar, Cell. Res. 1991; Scholar). Upon displacement of histone H1 from its native of histone H1 from the linker DNA region mechanisms not quite the major role of histone acetylation could be that of the chromatin fiber in a an would allow the to the regions of the gene during transcriptional with this model are the experimental that core histone with the in the chicken J. 1994; 13: Scholar). has also been shown that chromatin has a linker Nucleic Acids Res. 1990; Scholar). a positive structural role for histone acetylation that transcription to DNA has been Hayes J.J. Wolffe A.P. Cell. 1993; the of acetylated with the of the chicken against an role of histone acetylation. A of histone acetylation may an important role in the the dynamic equilibrium mechanisms described (see Fig. Despite the transcription and histone Proc. Natl. Acad. Sci. U. A. Scholar, Proc. Natl. Acad. Sci. U. A. 55: Scholar), the structural both has for As the of this to the results of the structural such as those reported for the to hyperacetylated chromatin complexes in of the in to the molecular mechanisms involved in the eukaryotic transcription of R.T. Annu. Rev. Biochem. 1994; Scholar). We are very to Dr. T. at the of for providing the 208-12 We are very to J. A. and to Dr. M. T. from the for us to their electron We are also to Dr. for us to the electron microscopy of the Molecular We Dr. A. from the for generous support for of the by M. during this we for of the and for of the