Dynamically Acetylated Histone Association with Transcriptionally Active and Competent Genes in the Avian Adult β-Globin Gene Domain

In chicken immature erythrocytes, class 1 acetylated histones are rapidly tri- and tetra-acetylated and rapidly deacetylated. Class 2 acetylated H3 and H4 are rapidly acetylated to mono- and di-acetylated isoforms and slowly deacetylated. Our previous studies suggested that class 1 acetylated histones were primarily associated with transcriptionally active DNA (βA-globin) but not competent DNA (ε-globin). Chromatin salt solubility (chromatin fiber oligomerization) is directly influenced by hyperacetylation. In this study we investigated the association of class 1 histones with βA- and ε-globin DNA by measuring their loss of solubility rates in 150 mmNaCl and 3 mm MgCl2 as a function of hyperacetylated histone deacetylation. Expressed and competent chromatin was associated with class 1 acetylated histones. As most active chromatin and hyperacetylated histones are associated with the low salt-insoluble residual nuclear material containing the nuclear matrix, we investigated whether hyperacetylated histones are bound to the βA- and ε-globin DNA in this fraction. In chromatin immunoprecipitation assays, we found that the βA- and ε-globin coding regions are bound to hyperacetylated H3 and H4. Our observations are consistent with a model in which nuclear matrix-associated histone acetyltransferases and deacetylases mediate a dynamic attachment between active and competent chromatin and the nuclear matrix. In chicken immature erythrocytes, class 1 acetylated histones are rapidly tri- and tetra-acetylated and rapidly deacetylated. Class 2 acetylated H3 and H4 are rapidly acetylated to mono- and di-acetylated isoforms and slowly deacetylated. Our previous studies suggested that class 1 acetylated histones were primarily associated with transcriptionally active DNA (βA-globin) but not competent DNA (ε-globin). Chromatin salt solubility (chromatin fiber oligomerization) is directly influenced by hyperacetylation. In this study we investigated the association of class 1 histones with βA- and ε-globin DNA by measuring their loss of solubility rates in 150 mmNaCl and 3 mm MgCl2 as a function of hyperacetylated histone deacetylation. Expressed and competent chromatin was associated with class 1 acetylated histones. As most active chromatin and hyperacetylated histones are associated with the low salt-insoluble residual nuclear material containing the nuclear matrix, we investigated whether hyperacetylated histones are bound to the βA- and ε-globin DNA in this fraction. In chromatin immunoprecipitation assays, we found that the βA- and ε-globin coding regions are bound to hyperacetylated H3 and H4. Our observations are consistent with a model in which nuclear matrix-associated histone acetyltransferases and deacetylases mediate a dynamic attachment between active and competent chromatin and the nuclear matrix. phenylmethylsulfonyl fluoride supernatant low salt-insoluble nuclear fraction chromatin immunoprecipitation Histone acetylation is a dynamic process catalyzed by histone acetyltransferases and histone deacetylases. Transcriptionally active chromatin is thought to be associated with histones that are rapidly acetylated and deacetylated, whereas histones situated along transcriptionally inactive DNA are either unacetylated or statically mono- or di-acetylated (1Hebbes T.R. Clayton A.L. Thorne A.W. Crane-Robinson C. EMBO J. 1994; 13: 1823-1830Crossref PubMed Scopus (481) Google Scholar). In chicken immature erythrocytes, 4% of the modifiable lysine residues located within the N-terminal tails of core histones become dynamically acetylated and deacetylated (2Zhang D.-E. Nelson D.A. Biochem. J. 1986; 240: 857-862Crossref PubMed Scopus (14) Google Scholar). The core histones within these cells display a similar rate of acetylation (t½ = 12 min for mono-acetylated H4) (3Zhang D.-E. Nelson D.A. Biochem. J. 1988; 250: 233-240Crossref PubMed Scopus (54) Google Scholar). However, these histones can be divided into two classes based on the extent of dynamic acetylation along their N-terminal tails and the rate at which the N-terminal acetylated lysine residues become deacetylated. Class 1 acetylated histones become tri- or tetra-acetylated when exposed to sodium butyrate, a histone deacetylase inhibitor. When the inhibitor is removed, these hyperacetylated histones are rapidly deacetylated (t½ = 5 min for tetra-acetylated H4) (4Zhang D.-E. Nelson D.A. Biochem. J. 1988; 250: 241-245Crossref PubMed Scopus (40) Google Scholar, 5Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1991; 266: 21936-21942Abstract Full Text PDF PubMed Google Scholar). Class 2 acetylated H3 and H4 histones become mono- or di-acetylated in the presence of sodium butyrate at the same rate as class 1 histones and then are slowly deacetylated (t½ = 90 min for H4 when mono-acetylated) once this inhibitor is removed. Chromatin fractionation studies have shown that chicken immature erythrocyte chromatin fragments soluble in 3 mmMgCl2 or 0.15 m NaCl are enriched in transcriptionally active DNA sequences and class 1, dynamically hyperacetylated histones (5Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1991; 266: 21936-21942Abstract Full Text PDF PubMed Google Scholar, 6Ferenz C.R. Nelson D.A. Nucleic Acids Res. 1985; 13: 1977-1995Crossref PubMed Scopus (25) Google Scholar). In reconstitution experiments, chromatin fragments containing transcriptionally active/competent DNA sequences are more resistant to 0.15 m NaCl precipitation caused by the addition of exogenously added H1 histones (7Ridsdale J.A. Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1990; 265: 5150-5156Abstract Full Text PDF PubMed Google Scholar). (Transcriptionally competent chromatin is sensitive to DNase I digestion but transcriptionally silent.) Further, the degree of salt solubility of the chromatin fragments containing the transcriptionally active/competent DNA sequences in 150 mm NaCl correlates with the level of histone hyperacetylation (7Ridsdale J.A. Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1990; 265: 5150-5156Abstract Full Text PDF PubMed Google Scholar). In fact, the level of histone acetylation was shown to be the primary determinant for the resistance of transcriptionally active/competent DNA fragments to H1-induced salt precipitation. In support of these findings, the treatment of mouse fibroblast cells with trichostatin A, a histone deacetylase inhibitor, induces histone hyperacetylation and increases the rate of exchange of a mobile fraction of H1 (8Misteli T. Gunjan A. Hock R. Bustin M. Brown D.T. Nature. 2000; 408: 877-881Crossref PubMed Scopus (517) Google Scholar). Histone acetylation also has a profound effect on higher order compaction of chromatin. Acetylating core histones past a threshold level of 12 acetates/octamer disrupts higher order folding and oligomerization of chromatin fibers (9Tse C. Sera T. Wolffe A.P. Hansen J.C. Mol. Cell. Biol. 1998; 18: 4629-4638Crossref PubMed Scopus (480) Google Scholar). Thus, in addition to interfering with chromatin fiber-fiber interactions (9Tse C. Sera T. Wolffe A.P. Hansen J.C. Mol. Cell. Biol. 1998; 18: 4629-4638Crossref PubMed Scopus (480) Google Scholar, 10Davie J.R. Spencer V.A. J. Cell. Biochem. 1999; S32–33: 141-148Crossref Google Scholar), histone acetylation enhances the 0.15m NaCl solubility of chromatin fragments by altering H1-mediated condensation of transcriptionally active/competent DNA. In addition to being salt-soluble, transcriptionally active/competent DNA fractionates with the insoluble nuclear material that remains following low ionic extraction of chromatin fragments from micrococcal nuclease-digested nuclei (11Delcuve G.P. Davie J.R. Biochem. J. 1989; 263: 179-186Crossref PubMed Scopus (49) Google Scholar). Approximately 76% of the transcriptionally active histone H5 and βA-globin DNA sequences and 30.5% of the transcriptionally competent ε-globin DNA sequences are located with the low salt-insoluble nuclear material, which includes chromatin fragments associated with the nuclear matrix of chicken immature erythrocytes (5Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1991; 266: 21936-21942Abstract Full Text PDF PubMed Google Scholar). The low salt-insoluble nuclear material of butyrate-treated immature erythrocytes contains 74% of class 1, tetra-acetylated H4 and 26.5% of class 2, mono- and di-acetylated H4 along with 75–80% of the nuclear histone deacetylase and acetyltransferase activities (12Hendzel M.J. Sun J.-M. Chen H.Y. Rattner J.B. Davie J.R. J. Biol. Chem. 1994; 269: 22894-22901Abstract Full Text PDF PubMed Google Scholar). The co-enrichment of transcriptionally active DNA sequences and class 1 tetra-acetylated H4 in the low salt-insoluble nuclear material suggests that the histones associated with transcriptionally active DNA sequences bound to the nuclear matrix are class 1 dynamically and rapidly acetylated and deacetylated. Crane-Robinson and co-workers mapped the distribution of proteins containing acetylated lysine residues along the entire β-globin chromatin domain and in regions adjacent to this domain (1Hebbes T.R. Clayton A.L. Thorne A.W. Crane-Robinson C. EMBO J. 1994; 13: 1823-1830Crossref PubMed Scopus (481) Google Scholar). Their study showed that the core histones situated along the entire β-globin chromatin domain are acetylated, whereas the histones located in the DNase I-insensitive regions outside the domain are hypoacetylated. However, this study analyzed only the steady state levels of acetylated core histones along β-globin domain DNA sequences in soluble chromatin fragments; low salt-insoluble chromatin fragments, which contain most of the dynamically acetylated histones and transcriptionally active β-globin DNA sequences, were excluded from analysis. Further, the antibody used to map the distribution of acetylated histones recognized acetylated histone and acetylated non-histone chromosomal proteins (13Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64: 435-459Crossref PubMed Scopus (1403) Google Scholar). Whether the dynamics of histone acetylation varies between transcriptionally active and competent DNA sequences within the β-globin domain remains to be determined. In addition, little is known about the distribution of acetylated histones along the sections of the β-globin domain that are associated with the nuclear matrix. In this study we determined whether class 1, dynamically acetylated histones are associated with the transcriptionally active adult βA globin and transcriptionally competent ε-globin DNA sequences of salt-soluble chromatin fragments (chromatin fibers unable to oligomerize at the ionic conditions tested) and low salt-insoluble chromatin fragments. Immature erythrocytes were isolated from anemic, young adult White Leghorn chickens as previously described (7Ridsdale J.A. Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1990; 265: 5150-5156Abstract Full Text PDF PubMed Google Scholar). Immature erythrocytes were collected in an ice-cold buffer containing 75 mm NaCl, 25 mm EDTA, and 25 mmTris-HCl (pH 7.5). Cells were resuspended in an equal volume of Swims S-77 medium (Sigma) and then incubated in the presence or absence of 10 mm sodium butyrate for 60 min at 37 °C. The erythrocytes were then washed three times in ice-cold Swim's media, resuspended in fresh Swims media prewarmed to 37 °C, and incubated for 0, 5, 10, 15, and 30 min at 37 °C. Following treatment, the erythrocytes were immediately resuspended in ice-cold Swim's media, collected by centrifugation, and stored at −80 °C. Three different preparations were analyzed in this study. Chromatin fragments soluble in 150 mmNaCl because of their inability to oligomerize were isolated from chicken immature erythrocytes as previously described (7Ridsdale J.A. Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1990; 265: 5150-5156Abstract Full Text PDF PubMed Google Scholar). All buffers contained 1 mm phenylmethylsulfonyl fluoride (PMSF).1 In brief, nuclei from immature erythrocytes were suspended to 50A260 units/ml in W & S buffer (1 mhexylene glycol, 10 mm Pipes, pH 7.0, 1% thiodiglycol, 30 mm sodium butyrate) containing 2 mmMgCl2 and 1 mm CaCl2, digested with 15 units of micrococcal nuclease (Worthington Biochemical Corporation, Freehold, NJ)/mg of total DNA for 5 min at 37 °C and then collected by centrifugation (9000 × g, 10 min, 4 °C). The addition of EGTA to 10 mm stopped the reaction. The nuclei were then resuspended in 10 mm EDTA, pH 8.0, and incubated on ice for 30 min. The suspension was centrifuged at 9000 ×g for 10 min at 4 °C, yielding the supernatant (SE) and the pellet (PE, low salt-insoluble nuclear fraction). SE fraction was made to 150 mm NaCl. The salt-soluble fraction (S150) was separated from the salt-insoluble fraction (P150) by centrifugation. Chromatin fragments soluble in 3 mm MgCl2 were isolated from chicken immature erythrocytes as previously described (6Ferenz C.R. Nelson D.A. Nucleic Acids Res. 1985; 13: 1977-1995Crossref PubMed Scopus (25) Google Scholar). All buffers contained 1 mm PMSF. In brief, immature erythrocyte nuclei were suspended to 70 A260units/ml in a digestion buffer (0.25 m sucrose, 60 mm KCl, 15 mm NaCl, 10 mm sodium butyrate, 15 mm PIPES, pH 6.6) containing 3 mmMgCl2 and 1 mm CaCl2. The nuclei were then digested with 1 unit of micrococcal nuclease/50 μg of total DNA for 5 min at 37 °C and centrifuged at 9000 × gfor 10 min at 4 °C. The addition of EGTA to 20 mmterminated the reaction. The supernatant containing the salt-soluble chromatin fragments was isolated. DNA from S150, MgCl2-soluble and MgCl2-insoluble chromatin fractions was extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1). The resulting DNA fragments were precipitated with sodium acetate and ethanol, resuspended in Tris-EDTA buffer (pH 8), quantified by UV spectrophotometry, and then either slot blotted using a Schleicher and Schuell slot blotting manifold or Southern blotted onto Hybond N+-charged as previously described (11Delcuve G.P. Davie J.R. Biochem. J. 1989; 263: 179-186Crossref PubMed Scopus (49) Google Scholar). the slot an of DNA was to slot that the between the and the of DNA was Thus, the from slot was directly to the of DNA The slot or Southern was then at °C to × of DNA with a of × DNA. Following the slot or Southern was washed to bound In the the of to slot was quantified by a DNA the ε-globin and were used (11Delcuve G.P. Davie J.R. Biochem. J. 1989; 263: 179-186Crossref PubMed Scopus (49) Google Scholar). The βA-globin and ε-globin DNA the of the βA- and ε-globin were in with the βA- and ε-globin being and from the J.B. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. J.B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The DNA is in and the of the were isolated from nuclei and chromatin preparations by extraction with as previously described (5Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1991; 266: 21936-21942Abstract Full Text PDF PubMed Google Scholar). were determined using the and of the proteins to were as previously described (11Delcuve G.P. Davie J.R. Biochem. J. 1989; 263: 179-186Crossref PubMed Scopus (49) Google Scholar). isoforms of H3 and H4 were by the with to di-acetylated H3 and H4 was isolated as previously described (7Ridsdale J.A. Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1990; 265: 5150-5156Abstract Full Text PDF PubMed Google with the that the nuclei were digested with 15 units of micrococcal of total DNA for 10 min at 37 °C. The fraction was resuspended in buffer mm Pipes, pH mm sucrose, mm KCl, 3 mm 1 mm to was added to the suspension to a of 1% for 10 min on and the was by the addition of (pH to a of The suspension was at 4 °C and mm and then to using to the suspension was NaCl, EDTA, and to 25 mm (pH 5 and The DNA within the suspension was to fragments by on ice for a total of 4 min at The of was divided into with on ice in between The suspension was then to units/ml and centrifuged for 10 min at 9000 × to insoluble The resulting suspension was made to 1 mm and volume of of antibody to di-acetylated H3 or H4 was added to of the and the was incubated at 4 °C. The suspension was then incubated for 3 at 4 °C on an with 20 of a that at 4 °C with of DNA and 1 of for of DNA to of the suspension was incubated for 3 at 4 °C with 20 of the in the absence of primary The of was then centrifuged at × for 30 and washed with 1 of mm NaCl, mm pH 8.0, sodium 1 of salt buffer mm NaCl, mm pH 8.0, 1 mm 1 of buffer sodium 1 mm pH and two times with 1 of buffer (pH volume of of buffer (pH was added to the along with and mm NaCl. The was incubated at 37 °C, and then at °C for The was centrifuged at × for 30 and the supernatant was extracted once with an equal volume of phenol/chloroform/isoamyl (25:24:1). The DNA in the supernatant was precipitated with 20 of the volume of 3 m sodium pH and 3 of The DNA was then resuspended in quantified by on to a Hybond N+-charged and to the previously and Immature erythrocytes were incubated with 10 mm sodium butyrate for 60 min to a state of histone hyperacetylation. The erythrocytes were then incubated in the absence of sodium butyrate for min to the hyperacetylated histones. were extracted from the nuclei of cells collected at following butyrate and to and The resulting was with to acetylated H3 and H4 of the total nuclear histone showed a in the levels of H3 within the 5 min of in the absence of sodium 10 min the levels of H3 at 30 min. in the levels of tetra-acetylated H3 was also at 5 min. 10 min the level of tetra-acetylated H3 at a low of H3 not but at 10 min a in the levels of this acetylated H3 was 5, 10, and 15 min following butyrate the levels of mono- and di-acetylated H3 but at a rate the acetylated H3 the with H4 that the levels of tetra-acetylated H4 rapidly at 10 min levels the 20 min. the levels of H4 also at 10 min, this was not as as that for the tetra-acetylated H4 The mono- and di-acetylated H4 isoforms at 15 and 30 min In the that class 1 acetylated H3 and H4 isoforms to low levels by 10 min following of cells in media Our previous studies showed that the solubility of active/competent chromatin fragments in 0.15 m NaCl is on the level of acetylated histone (7Ridsdale J.A. Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1990; 265: 5150-5156Abstract Full Text PDF PubMed Google Scholar). histone hyperacetylation with the of chromatin fibers to (9Tse C. Sera T. Wolffe A.P. Hansen J.C. Mol. Cell. Biol. 1998; 18: 4629-4638Crossref PubMed Scopus (480) Google Scholar). the inability of chromatin fragments to oligomerize in 0.15 m NaCl is histone acetylation the rate of of histones associated with transcriptionally active and competent chromatin fragments can be determined by their rate of loss of 0.15 m NaCl solubility and of to chromatin fragments were isolated from nuclei of cells incubated for times 5, 10, 15, and 30 following the of of material was into this fraction. The SE chromatin fraction was made 0.15 m in NaCl, and the salt-soluble chromatin fragments were isolated. The DNA fragments were analyzed by slot with to the and DNA 2 that of cells in the absence of butyrate in a in the of βA-globin and ε-globin DNA sequences in the 0.15 m chromatin fragments. The of DNA sequences in the salt-soluble chromatin fraction was not the The in salt solubility and in of the βA-globin and ε-globin chromatin fragments to oligomerize suggests that the rates of the histones associated with these chromatin fragments are from micrococcal nuclease-digested erythrocyte nuclei into buffers containing 3 mm MgCl2 are enriched in the transcriptionally active βA-globin DNA sequences and in inactive DNA histone increases the of active from the nuclease-digested nuclei (6Ferenz C.R. Nelson D.A. Nucleic Acids Res. 1985; 13: 1977-1995Crossref PubMed Scopus (25) Google Scholar). Thus, solubility of transcriptionally active from nuclease-digested nuclei is a of histone acetylation D.E. Nelson D.A. Nucleic Acids Res. PubMed Scopus Google Scholar). whether the of dynamically acetylated histones the of competent ε-globin from nuclease-digested nuclei isolated from cells incubated in the absence or presence of butyrate for 60 min were digested with micrococcal and the chromatin fragments digestion and with the nuclei were The of chromatin from the nuclease-digested nuclei was similar for to βA-globin DNA sequences were enriched in the fraction from the nuclease-digested nuclei of butyrate-treated immature However, this was not with isolated from cells incubated in the absence of butyrate not observations are to the of and Nelson (4Zhang D.-E. Nelson D.A. Biochem. J. 1988; 250: 241-245Crossref PubMed Scopus (40) Google Scholar). The of ε-globin DNA sequences in the fraction was from nuclei isolated from cells incubated from butyrate with that from nuclei of cells incubated in the absence of butyrate The of ε-globin DNA sequences in the 3 was that by the β-globin DNA In the that histone hyperacetylation increases the of ε-globin from nuclease-digested hyperacetylation directly the of transcriptionally active and competent we the of these sequences in the fraction as a function of which hyperacetylated histones were deacetylated. As with the previous cells were incubated with butyrate to the acetylation state of class 1 histones by of the cells for times in the absence of butyrate to the of the hyperacetylated class 1 histones. The DNA from the 3 was onto and to DNA βA-globin and ε-globin regions and the of the of the following of butyrate showed that the of βA-globin and ε-globin was by 5 min by a more In to the βA-globin and ε-globin the of hyperacetylated histones not the of from the nuclease-digested The in the of βA-globin and ε-globin within the 5 min of in the absence of butyrate the of class 1 hyperacetylated histone that of H3 In the in the of βA-globin and ε-globin from nuclease-digested nuclei the of the hyperacetylated class 1 histones. studies have directly that acetylated histones are associated with the transcriptionally active βA-globin and transcriptionally competent ε-globin in However, these used soluble chromatin fragments. acetylated histones and transcriptionally active βA-globin DNA sequences are associated with fraction the low salt-insoluble residual nuclear material chromatin associated with the nuclear matrix (5Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1991; 266: 21936-21942Abstract Full Text PDF PubMed Google Scholar). studies have determined transcriptionally active chromatin bound to the nuclear matrix is associated with acetylated histones. this chromatin fragments associated with the low salt-insoluble nuclear material of butyrate-treated immature chicken erythrocytes were incubated with In addition to histones to chromatin fragments from the nuclear matrix J.A. A. PubMed Scopus Google Scholar). The chromatin fragments bound to hyperacetylated H3 and H4 were isolated by in we showed that the used in the recognized acetylated isoforms of H3 or H4 Chen H.Y. Sun J.-M. Davie J.R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the H3 antibody and was more for the acetylated H3 isoforms was the H4 antibody for the acetylated H4 The DNA sequences bound to hyperacetylated H3 and H4 were isolated and analyzed by slot using DNA to the regions of the βA-globin and ε-globin and to the of the of the of the three in the and acetylated DNA fractions showed that hyperacetylated H3 was bound to βA-globin and ε-globin DNA but not to DNA. 5 that acetylated H4 was bound to βA-globin and ε-globin DNA DNA was also bound to acetylated which we is the mono- and di-acetylated In that hyperacetylated H3 and H4 are bound to the βA-globin and ε-globin DNA sequences associated with the insoluble residual nuclear of histone acetylation and are determined in in which is with (4Zhang D.-E. Nelson D.A. Biochem. J. 1988; 250: 241-245Crossref PubMed Scopus (40) Google Scholar). Our with H3 and H4 that the rates of of hyperacetylated H3 and H4 were with in that not the between histone acetyltransferase and histone deacetylase in chicken immature Our that class 1 which are rapidly acetylated and deacetylated, are bound to transcriptionally active βA-globin and transcriptionally competent ε-globin In βA-globin and ε-globin chromatin fragments the to oligomerize in 150 mm NaCl as of the hyperacetylated H3 and H4 isoforms Further, the of hyperacetylated H3 isoforms to a rapidly solubility in 3 mmMgCl2 of βA-globin and ε-globin from nuclease-digested The loss of the hyperacetylated H3 histones the of higher order globin chromatin the of from the globin chromatin domain (9Tse C. Sera T. Wolffe A.P. Hansen J.C. Mol. Cell. Biol. 1998; 18: 4629-4638Crossref PubMed Scopus (480) Google Scholar, C. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, the extent of MgCl2 solubility loss of the βA-globin was more that of the ε-globin chromatin fragments. and studies that a of βA-globin with ε-globin chromatin is soluble in 150 mm NaCl or 3 mm MgCl2 (11Delcuve G.P. Davie J.R. Biochem. J. 1989; 263: 179-186Crossref PubMed Scopus (49) Google Scholar). these studies to that active coding regions of the βA-globin are associated with class 1 acetylated whereas the competent ε-globin is a of class 1 and class 2 acetylated histones. in previous study the of βA-globin DNA sequences that of the hyperacetylated H4 whereas competent ε-globin DNA sequences not (5Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1991; 266: 21936-21942Abstract Full Text PDF PubMed Google Scholar). Crane-Robinson and co-workers have shown that the entire β-globin domain is associated with acetylated histones in soluble chromatin fragments (1Hebbes T.R. Clayton A.L. Thorne A.W. Crane-Robinson C. EMBO J. 1994; 13: 1823-1830Crossref PubMed Scopus (481) Google Scholar). The low salt-insoluble chromatin which contains the of the acetylated histones and transcriptionally active was excluded from their Our for the that βA- and ε-globin DNA sequences associated with the residual insoluble nuclear material are bound to acetylated H3 and H4. most of the histone acetyltransferase and deacetylase Further, histone acetyltransferase and histone deacetylase activities are associated with the nuclear matrix (12Hendzel M.J. Sun J.-M. Chen H.Y. Rattner J.B. Davie J.R. J. Biol. Chem. 1994; 269: 22894-22901Abstract Full Text PDF PubMed Google Scholar). Our observations are consistent with a model in which nuclear matrix-associated histone acetyltransferases and deacetylases mediate a dynamic attachment between transcriptionally active chromatin and the nuclear matrix. In the of the β-globin these dynamic interactions are not to the but also the coding regions of and competent Our studies that βA- and ε-globin DNA sequences are associated with class 1 dynamically acetylated with βA-globin DNA sequences a higher of this class of acetylated histones that associated with the ε-globin DNA The acetylation and of the class 1 histones bound to the βA-globin DNA sequences suggests that the core histone tails bound to the βA-globin be in with nuclear histone acetyltransferases and deacetylases. The between these and the competent ε-globin chromatin be these dynamic interactions with the βA-globin this at nuclear matrix that are in

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