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J Biol Chem, Vol. 273, Issue 23, 14516-14522, June 5, 1998


Histone Acetylation Is Required to Maintain the Unfolded Nucleosome Structure Associated with Transcribing DNA*

Harminder Walia, Hou Yu Chen, Jian-Min Sun, Laurel T. Holth, and James R. DavieDagger

From the Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E OW3, Canada

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Nucleosomes associated with transcribing chromatin of mammalian cells have an unfolded structure in which the normally buried cysteinyl-thiol group of histone H3 is exposed. In this study we analyzed transcriptionally active/competent DNA-enriched chromatin fractions from chicken mature and immature erythrocytes for the presence of thiol-reactive nucleosomes using organomercury-agarose column chromatography and hydroxylapatite dissociation chromatography of chromatin fractions labeled with [3H]iodoacetate. In mature and immature erythrocytes, the active DNA-enriched chromatin fractions are associated with histones that are rapidly highly acetylated and rapidly deacetylated. When histone deacetylation was prevented by incubating cells with histone deacetylase inhibitors, sodium butyrate or trichostatin A, thiol-reactive H3 of unfolded nucleosomes was detected in the soluble chromatin and nuclear skeleton-associated chromatin of immature, but not mature, erythrocytes. We did not find thiol-reactive nucleosomes in active DNA-enriched chromatin fractions of untreated immature erythrocytes that had low levels of highly acetylated histones H3 and H4 or in chromatin of immature cells incubated with inhibitors of transcription elongation. This study shows that transcription elongation is required to form, and histone acetylation is needed to maintain, the unfolded structure of transcribing nucleosomes.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Acetylation of the core histones (H2A, H2B, H3, and H4) is a dynamic process catalyzed by histone acetyltransferases and histone deacetylases (1, 2). In chicken immature erythrocytes, 4% of the modifiable lysine sites participate in dynamic histone acetylation. These core histones are rapidly acetylated (t1/2 = 12 min for monoacetylated H4) and rapidly deacetylated (t1/2 = 5 min for the tetraacetylated isoform of H4) (3, 4). Histones undergoing rapid acetylation and deacetylation are associated with transcriptionally active chromatin (5-7). The recent findings that histone acetyltransferases and deacetylases are transcriptional coactivators and corepressors have increased our understanding of how the process of dynamic histone acetylation is established on transcriptionally active chromatin (2, 8).

Transcriptionally active chromatin has a soluble and insoluble nature (9). Transcribed DNA is found in chromatin fragments that are soluble in 0.15 M NaCl and/or 2 mM MgCl2 and in chromatin fragments associated with the low salt-insoluble residual nuclear material (nuclear skeletons) (for review, see Davie (10)). Chromatin engaged in transcription is thought to be retained by the nuclear skeleton by multiple dynamic attachments between the nuclear matrix and transcribed chromatin; hence rendering the transcribing chromatin insoluble (11, 12). As histone acetyltransferase and deacetylase activities are associated with the nuclear matrix (7, 13), we proposed that these nuclear matrix-bound enzymes may mediate some of the dynamic attachments between active chromatin and nuclear matrix (13, 14).

Most information on the structure and composition of transcriptionally active nucleosomes is from studies that analyze soluble transcriptionally active chromatin. However, most of the transcribed chromatin fragments partition with the low salt-insoluble nuclear material (nuclear skeleton) (7, 15, 16). We presented evidence that dynamically acetylated histones are associated with the nuclear matrix-bound transcriptionally active chromatin (7). Otherwise, little is known about the structure and composition of transcribing nucleosomes attached to the nuclear skeleton.

Allfrey and co-workers demonstrated that nucleosomes in the transcribed regions of soluble chromatin of mammalian cells unfold exposing the cysteinyl-thiol groups of histone H3 (17, 18). The unfolding of the nucleosome was dependent upon ongoing transcription. Exploiting this feature of transcribing nucleosomes, a procedure to isolate soluble transcriptionally active chromatin by organomercury-agarose affinity chromatography was developed. The transcribing chromatin was associated with highly acetylated histones (18-20). However, current evidence argues that histone acetylation is not involved in the generation of the unfolded nucleosome. Reconstitution of nucleosomes with highly acetylated histones did not result in the formation of a thiol-reactive nucleosome (21). Further, treating mammalian cells with the histone deacetylase inhibitor, sodium butyrate, did not increase the level of thiol-reactive nucleosomes (6).

Analysis of chicken mature erythrocyte salt-soluble polynucleosomes highly enriched in transcriptionally competent DNA and highly acetylated histones (22, 23) showed that this chromatin fraction lacked thiol-reactive nucleosomes.1 To address the question of whether unfolded nucleosomes exist in chicken erythrocytes, we investigated the H3 thiol reactivity of salt-soluble and low salt-insoluble (nuclear skeleton-associated) chromatin from mature (transcriptionally silent) and immature (transcriptionally active) chicken erythrocytes. We report that the thiol-reactive, unfolded nucleosome exists in immature, but not mature, erythrocyte salt-soluble chromatin fragments and chromatin fragments associated with the nuclear skeleton. However, histone deacetylase activity had to be suppressed to detect thiol-reactive nucleosomes in immature erythrocyte chromatin. These studies show that highly acetylated histones maintain the unfolded nucleosome structure formed by transcriptional elongation.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Isolation and Treatments of Immature and Mature Chicken Erythrocytes-- Mature and immature erythrocytes were isolated from normal and anemic young adult White Leghorn chickens, respectively, as described previously (24). Immature and mature erythrocytes were collected in 75 mM NaCl and 25 mM EDTA, while cells to be incubated with sodium butyrate or trichostatin A were collected in 75 mM NaCl, 25 mM EDTA, and 30 mM sodium butyrate. Cells were incubated at 37 °C in Swims S-77 medium (Sigma) with 30 mM sodium butyrate or 100 ng/ml trichostatin A for 90 min to prevent the deacetylation of highly acetylated histones. To inhibit transcription elongation, cells were incubated at 37 °C for 90 min with transcription inhibitors actinomycin D (0.04 µg/ml) (24), 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole (DRB)2 (75 µM) (24, 25), or camptothecin (20 µM) (26) followed by a 90-min incubation with or without 30 mM sodium butyrate.

Erythrocyte Chromatin Fractionation-- The fractionation of chromatin was done as described previously (24). All buffers contained 1 mM phenylmethylsulfonyl fluoride. Briefly, nuclei (50 A260 units/ml) were digested with micrococcal nuclease (15 A260 units/ml for 25 min at 37 °C), collected by centrifugation, and then resuspended in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA. Following centrifugation the soluble chromatin fraction (fraction SE) and low salt insoluble chromatin fraction (nuclear skeleton, PE fraction) were isolated. The chromatin fragments of fraction SE were further fractionated by the addition of NaCl to 150 mM. Following centrifugation, chromatin fractions P150 (pellet) and S150 (salt-soluble chromatin) were isolated.

Organomercury Column Affinity Chromatography-- Chicken erythrocyte chromatin fraction S150 was dialyzed against buffer A (10 mM Tris-HCl, pH 7.5, 25 mM KCl, 25 mM NaCl, 5 mM sodium butyrate, 0.1 mM phenylmethylsulfonyl fluoride, and 5 mM EDTA, pH 7.5) and then applied to an organomercurial column (Affi-Gel 501; Bio-Rad) that was pre-equilibrated with buffer A. The column (1.5 × 8 cm) was then washed with buffer A (flow rate, 60 ml/h) to remove unbound chromatin fragments until the absorbance at 260 nm returned to base line. This was followed by washing the column with 0.5 M NaCl in buffer A (buffer B) until the absorbance at 260 nm returned to base line. The bound nucleosomes were eluted by 10 mM DTT in buffer A. The released material was monitored by measuring absorbance at 260 nm (17). The fractions containing the unbound material and 0.5 M NaCl eluted material were pooled; the fractions eluted with DTT were pooled. For analysis of histones in the unbound and bound fractions, the fractions were acid-extracted by the addition of 4 N sulfuric acid to a final concentration of 0.4 N. Before lyophilization, the supernatants were dialyzed overnight against 0.1 M acetic acid and then against two changes of double-distilled H2O.

Reaction of Nucleosomes with [3H]Iodoacetic Acid-- Ten µl/ml [3H]iodoacetic acid (NEN Life Science Products, 204.9 mCi/mmol) containing 2.50 µCi was added to the chromatin fraction SE, S150, P150, and PE (2 A260/ml) in buffer E (10 mM Tris-HCl, pH 8.2, 1 mM EDTA) and allowed to incubate at room temperature for 1 h in the dark. The chromatin fraction was applied directly to a hydroxylapatite column. The histones were isolated by acid extraction as described above. Histones were electrophoretically resolved on SDS-polyacrylamide gel electrophoresis. Following staining with Coomassie Blue, the gel pieces containing a histone band were disrupted in hydrogen peroxide and then counted in 5 ml of scintillation fluid.

Hydroxylapatite Chromatography-- The chromatin fraction was mixed with hydroxylapatite HTP gel powder (Bio-Rad) at a ratio of 1 mg of DNA to 0.25 g of hydroxylapatite as described previously (27). The column was washed with 0.63 M NaCl in 0.1 M potassium phosphate buffer, pH 6.7, to remove histone H1, H5, and non-histone chromosomal proteins before applying a linear gradient of NaCl (0.63 to 2 M NaCl in 0.1 M potassium phosphate buffer, pH 6.7) at a flow rate of 35 ml/h as described previously (27).

DNA Preparation and Southern Blot Hybridization-- DNA was prepared from the different chromatin fractions as described previously (24). For electrophoresis, equal amounts of DNA were dissolved in DNA sample loading buffer, and the samples were loaded onto 1% agarose minigels containing 0.5 µg of ethidium bromide/ml. The DNA was transferred to Hybond-N+ nylon transfer membrane and hybridized to radiolabeled probes as described previously (28). The cloned probes used were pCBG 14.4, a unique intronic sequence of chicken beta  globin gene; pChV2.5B/H, which contains the gene coding for chicken histone H5 and flanking sequences; and pVTG412, that recognizes the 5' region of the chicken vitellogenin gene (24).

Polyacrylamide Gel Electrophoresis and Western Blotting-- AUT (acetic acid, 6.7 M urea, 0.375% (w/v) Triton X-100) and SDS-15% polyacrylamide gel electrophoresis were performed as described elsewhere (24). Antiacetylated H3 and antiacetylated H4 antibodies generously supplied by Dr. D. Allis were used to detect acetylated species of H3 and H4 (29-31) in Western blot experiments using a protocol described previously (32).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

State of Acetylation of Dynamically Acetylated Histones in Chicken Immature and Mature Erythrocytes-- Histone acetylation is rapidly reversible in immature erythrocytes (3, 4, 7). To find the steady state of acetylated H3 and H4, histones of immature and mature erythrocyte chromatin fractions S150 and PE were electrophoretically resolved on AUT-polyacrylamide gels, transferred to nitrocellulose, and immunochemically stained with either antiacetylated H4 antibodies or antiacetylated H3 antibodies. Both antibodies detect the multiacetylated forms of H4 and H3, with the antibody to acetylated H3 showing a strong preference for the highest acetylated isoforms of H3 (30). We have shown previously that highly acetylated histones are found in chromatin fractions S150 and PE, but not in fraction P150 (7, 24). Thus, this latter fraction was not analyzed. Fig. 1 shows that the steady state levels of highly acetylated H3 and H4 isoforms were low in fractions S150 and PE from immature erythrocytes. However, the steady state levels of the highly acetylated H3 and H4 isoforms in fractions S150 and PE were markedly increased when immature erythrocytes were incubated in the presence of sodium butyrate, a histone deacetylase inhibitor, for 60 min (Fig. 1). These results suggest that the rate of deacetylation is so rapid in immature erythrocytes that, once dynamically acetylated H3 and H4 reach a highly acetylated state, they are rapidly deacetylated (4); the net result is a low steady state level of highly acetylated histone isoforms in untreated immature cells.


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  		Fig. 1.   Level of acetylated H3 and H4 histones in immature and mature erythrocyte chromatin fractions S150 and PE. A, histones were acid-extracted from chromatin fractions S150 (S) or PE (P) of immature (IE) or mature (ME) cells that were either untreated (-) or incubated in the presence of sodium butyrate for 1 h (+). The histones (9 µg) were electrophoretically resolved on an AUT-15% polyacrylamide gel. The gel was stained with Coomassie Blue. B and C, the proteins were electrophoretically transferred to nitrocellulose and immunochemically stained with antiacetylated H3 (B) or antiacetylated H4 (C) antibodies. The mono-, di-, tri-, and tetra-acetylated isoforms of H4 are marked as 1, 2, 3, and 4, respectively. Note that the content of H1 and H5 in fraction S150 is typically lower than that of the other chromatin fractions (65).

The steady state of acetylated H3 and H4 in mature erythrocyte chromatin fractions was higher than that of H3 and H4 in the corresponding fractions of immature erythrocytes (Fig. 1). Incubation of mature cells with sodium butyrate elevated the level of hyperacetylated H3 and H4 isoforms in chromatin fraction S150. The level of highly acetylated H3 and, to a lesser extent, highly acetylated H4 in fraction PE was also increased.

Mercury-Agarose Column Fractionation of Salt-soluble Immature Erythrocyte Chromatin-- The chromatin fraction S150 was isolated from chicken immature erythrocytes that were untreated or incubated with sodium butyrate for 90 min. The S150 chromatin fractions were applied to mercury-agarose columns (Fig. 2). Analysis of the proteins released from the mercury column with DTT showed that histones were present in the S150 chromatin fraction from butyrate incubated cells (Fig. 2C), but absent in the S150 fraction from untreated cells (Fig. 2B). The major proteins in the latter fraction were the cysteine-containing high mobility group proteins 1 and 2 (Fig. 2B). These results suggested that unfolded nucleosomes were absent or at very low levels in the S150 chromatin fraction from untreated immature erythrocytes. However, the unfolded nucleosome appeared to be present in immature erythroid cells that were incubated with butyrate. This result suggested that when deacetylation of dynamically acetylated histones was halted, the transcriptionally active nucleosomes was prevented from reverting to a thiol-nonreactive state.


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  		Fig. 2.   Fractionation of S150 fraction of immature erythrocyte chromatin by mercury affinity chromatography. S150 chromatin fragments from immature erythrocytes or immature erythrocytes incubated with sodium butyrate were applied to a mercury-agarose column. Following the elution of unbound chromatin fragments, the column was washed with 0.5 M NaCl containing buffer. The mercury-bound chromatin fragments and proteins were released from the column by the addition of 10 mM DTT. The absorbance at 260 nm was monitored. Panel A shows the chromatograph of S150 (125 A260 units applied; 3-ml fractions collected) of butyrate-incubated cells. Panels B and C, the proteins (UB, 10 µg) of the unbound fractions including the 0.5 M NaCl wash fractions and the proteins (B, 10 µg) in the DTT-released fractions were electrophoretically resolved on AUT-15% polyacrylamide gels. The gels were stained with Coomassie Blue.

To find if the mercury-agarose-bound nucleosomes from fraction S150 of butyrate-incubated immature erythrocytes had hyperacetylated histones, histones from S150, mercury-agarose-bound, and mercury-agarose-unbound chromatin fractions were analyzed in Western blot experiments with antiacetylated H3 and H4 antibodies. Fig. 3 shows that bound nucleosomes were enriched in hyperacetylated H3 and H4 isoforms.


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  		Fig. 3.   Analysis of acetylated H3 and H4 isoforms of immature erythrocyte chromatin fractionated by mercury affinity chromatography. A, chromatin fraction S150 isolated from immature erythrocytes incubated with sodium butyrate was fractionated by mercury affinity chromatography. The proteins (15 µg) of fraction S150 (T), unbound (UB), and bound (B) fractions were electrophoretically resolved on an AUT-15% polyacrylamide gel. The gel was stained with Coomassie Blue. The protein indicated by the arrow comigrates with H1 on SDS-polyacrylamide gels. B and C, the proteins were electrophoretically transferred to nitrocellulose and immunochemically stained with antiacetylated H3 (B) or antiacetylated H4 (C) antibodies. The mono-, di-, tri-, and tetra-acetylated isoforms of H4 are marked as 1, 2, 3, and 4, respectively.

DNA isolated from the S150, unbound and bound mercury-agarose chromatin fractions was analyzed by Southern blotting with probes containing DNA sequences to genes that were either expressed or repressed in immature erythrocytes. The chromatin fraction bound to mercury-agarose contained transcriptionally active histone H5 and beta -globin (not shown) DNA, but not repressed vitellogenin DNA (Fig. 4). These results show that mercury-bound nucleosomes were associated with transcriptionally active DNA.


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  		Fig. 4.   Analysis of DNA sequences of immature erythrocyte chromatin fractionated by mercury affinity chromatography. Chromatin fraction S150 isolated from immature erythrocytes incubated with sodium butyrate was fractionated by mercury affinity chromatography. The DNA (10 µg) of chromatin fractions SE, S150, and mercury column unbound (U) and bound (B) fractions were electrophoretically resolved on a 1% agarose gel. The gel (DNA) was stained with ethidium bromide. The DNA was transferred to membranes and probed with either chicken vitellogenin (repressed) or histone H5 (active) DNA sequences.

Hydroxylapatite Dissociation Chromatography Analysis of Immature Erythrocyte-soluble and Nuclear Skeleton-associated Chromatin Labeled with [3H]Iodoacetic Acid-- The characterization of the thiol-reactive, unfolded nucleosome of transcribing chromatin described by Allfrey and colleagues has been done solely with soluble chromatin fragments. However, most transcribing chromatin is associated with the residual nuclear material (fraction PE), the nuclear skeleton. In immature erythrocytes approximately 75% of the transcribed DNA sequences are associated with the nuclear skeleton (7). To test the reactivity of the thiol group (Cys-110) of H3 in chromatin from butyrate-incubated immature erythrocytes, fraction SE and PE chromatin fragments were incubated with [3H]iodoacetic acid. Fig. 5, B and C, shows that H3 was labeled in SE and PE chromatin. In contrast to the results obtained with fraction SE and PE chromatin, the H3 of chromatin fraction P150, which contained repressed DNA, was not labeled (data not shown).


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  		Fig. 5.   Detection of thiol-reactive H3 in SE and PE chromatin. Chromatin fractions SE and PE were isolated from butyrate-incubated immature erythrocytes. The chromatin fraction was incubated with [3H]iodoacetic acid. Histones (10 µg) acid extracted from SE and PE chromatin were electrophoretically resolved on a SDS-15% polyacrylamide gel. Panel A shows the Coomassie Blue-stained gel. The stained bands were excised and counted (panel B, fraction SE histones; panel C, fraction PE histones).

To monitor the labeling of H3 in the chromatin fractions, hydroxylapatite dissociation chromatography was applied (27). Hydroxylapatite was added to fraction SE or PE in 0.63 M NaCl, removing non-histone chromosomal proteins and H1 histones from the hydroxylapatite-bound chromatin (27). Increasing concentrations of NaCl were then applied to the hydroxylapatite column, resulting first in the dissociation of H2A-H2B dimers followed by H3-H4 tetramers from the hydroxylapatite-bound chromatin (Fig. 6, A and C) (27). The interpeak fractions contained H2A, H2B, H3, and H4 (Fig. 6C, lane b). The concentration of NaCl required to dissociate the H2A-H2B dimer or H3-H4 tetramer from the nucleosomal DNA provides a measure of the strength of the interaction between the dimer or tetramer and DNA (27). For example, highly acetylated H3-H4 tetramers dissociate from hydroxylapatite-bound nucleosomal DNA at a lower ionic strength than do unmodified H3-H4 tetramers (27, 33).


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  		Fig. 6.   Hydroxylapatite dissociation chromatography of fraction SE and PE chromatin fragments of chicken immature erythrocytes. Chromatin fraction SE (160 A260 units) and PE (226 A260 units) isolated from immature erythrocytes incubated with sodium butyrate (+ Na Butyrate) were incubated with [3H]iodoacetate and then added to hydroxylapatite as described under "Experimental Procedures." For the chromatograms shown in panels A and B, fractions of 2 and 5 ml were collected, respectively. Panel C, chromatin fraction SE was applied to hydroxylapatite column, and fractions equivalent to fractions 8-17, 18-28, and 29-42 shown in panel A were pooled, dialyzed against water, and then lyophilized. The proteins (4 µg) from the three fractions (lanes a, b, and c, respectively) were electrophoretically resolved in a SDS-15% polyacrylamide gel. The gel was stained with Coomassie Blue.

Chromatin fragments of fractions SE and PE isolated from butyrate-incubated immature erythrocytes were incubated with [3H]iodoacetate and then subjected to hydroxylapatite dissociation chromatography. Fig. 6, A and B, shows that labeled H3 dissociated from the hydroxylapatite-bound chromatin after the H2A-H2B dimers but before the bulk of the H3-H4 tetramers.

To determine whether incubation of immature erythrocytes with histone deacetylase inhibitors was required to detect the thiol-reactive H3 in nucleosomes, chromatin fractions SE and S150 were isolated from cells that were untreated or incubated with sodium butyrate. Following incubation with [3H]iodoacetate, the chromatin fractions were applied to hydroxylapatite. Labeled H3 was detected only in fraction SE and S150 when cells were incubated with sodium butyrate (compare Fig. 6A with 7C and Fig. 7, A with B). Immature erythrocytes were also incubated with trichostatin A, a specific histone deacetylase inhibitor, instead of sodium butyrate. The dissociation profiles of hydroxylapatite bound SE chromatin fragments from trichostatin A-treated immature erythrocytes were similar to those from butyrate-treated cells (compare Fig. 7D with 6A).


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  		Fig. 7.   Hydroxylapatite dissociation chromatography of salt-soluble chromatin fragments of chicken immature erythrocytes. Panels A and B, chromatin fraction S150 isolated from immature erythrocytes untreated (panel A, 160 A260 units) or incubated with sodium butyrate (panel B, 40 A260 units) was incubated with [3H]iodoacetate and then added to hydroxylapatite as described under "Experimental Procedures." Panels A and B, 2- and 4-ml fractions were collected, respectively. Panels C and D, chromatin fraction SE isolated from immature erythrocytes untreated (panel C, 160 A260 units) or incubated with trichostatin A (panel D, 168 A260 units) was incubated with [3H]iodoacetate and then applied to hydroxylapatite as described under "Experimental Procedures." Two-ml fractions were collected.

Hydroxylapatite dissociation chromatography with PE chromatin was problematic as the addition of insoluble nuclear skeletons to hydroxylapatite reduced the flow rate appreciably. For the following experiments with the PE fraction, the suspension of nuclear skeletons was incubated with [3H]iodoacetate, and then the acid-extracted histones were separated by SDS-gel electrophoresis. The H3 band was excised and counted (see Fig. 5). In accordance with the results with fraction SE and S150, H3 of fraction PE was thiol-reactive in chromatin fragments from butyrate-incubated, but not untreated, immature erythrocytes (Fig. 8). These observations suggest that the thiol reactivity of H3 in immature erythrocyte chromatin fractions SE, S150, and PE is dependent upon the acetylation states of the nucleosomal histones.


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  		Fig. 8.   Thiol reactivity of H3 of chromatin fragments associated with the residual insoluble erythroid nuclear skeleton. Chromatin fraction PE isolated from or immature erythrocytes (IE) or mature erythrocytes (ME) untreated (-B) or incubated with transcriptional inhibitors (DRB; camptothecin, Cam) and sodium butyrate (+B) was incubated with [3H]iodoacetate. Acid-extracted histones were electrophoretically resolved on SDS-15% polyacrylamide gels. Histone H3 was excised and counted.

Hydroxylapatite Dissociation Chromatography Analysis of Mature Erythrocyte Chromatin Labeled with [3H]Iodoacetic Acid-- Transcriptional elongation is arrested in mature erythrocytes. To find if hyperacetylating histones associated with transcriptionally competent chromatin (3, 34) was sufficient to observe a thiol-reactive H3, chromatin fractions S150 and PE from mature cells untreated or incubated with sodium butyrate were labeled with [3H]iodoacetate. Figs. 8 and 9 show that labeled H3 was not observed in the mature erythrocyte S150 and PE chromatin fractions. These results and those with immature erythrocyte chromatin suggest that histone acetylation is required but not sufficient for formation and/or stabilization of nucleosomes with thiol-reactive H3.


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  		Fig. 9.   Hydroxylapatite dissociation chromatography of salt-soluble chromatin fragments of chicken mature erythrocytes. Chromatin fraction S150 isolated from mature erythrocytes untreated (panel A, - Na Butyrate) or incubated with sodium butyrate (panel B, + Na Butyrate) was incubated with [3H]iodoacetate and then added to hydroxylapatite as described under "Experimental Procedures." Panels A and B, 160 A260 units of chromatin were added to hydroxylapatite; 2-ml fractions were collected.

Effect of Inhibitors of Transcription on the Thiol Reactivity of H3 in Nucleosomes-- The absence of thiol-reactive nucleosomes in butyrate-treated mature erythrocytes indicates that both transcription elongation and highly acetylated histones are required to form and maintain the unfolded nucleosome conformation. To test whether inhibition of transcription elongation has an effect on the H3 thiol reactivity of immature erythrocyte nucleosomes, immature erythrocytes were incubated with inhibitors of transcription before the addition of sodium butyrate. Camptothecin is an inhibitor of topoisomerase I and has been reported to stimulate initiation but inhibit elongation by RNA polymerase II (26, 35, 36). A thiol-reactive H3 was not detected in the SE and PE chromatin of immature cells treated with DRB or camptothecin followed by butyrate (Figs. 8 and 10) (25). These results show that both highly acetylated histones and elongation are needed to detect the thiol-reactive H3 in immature erythrocyte chromatin.


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  		Fig. 10.   Hydroxylapatite dissociation chromatography of fraction SE chromatin fragments of chicken immature erythrocytes incubated with inhibitors of transcription elongation. Panel A, and B, chromatin fractions SE were isolated from immature erythrocytes incubated with either DRB or camptothecin and then with sodium butyrate. The chromatin fractions were incubated with [3H]iodoacetate and then added to hydroxylapatite as described under "Experimental Procedures." Panel A, 160 A260 units of chromatin applied; 2-ml fractions collected. Panel B, 60 A260 units of chromatin applied; 5-ml fractions collected.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We show that highly acetylated histones are required to maintain the unfolded, thiol-reactive structure of transcribing nucleosomes. The thiol-reactive nucleosome is not detected in the chromatin of transcriptionally active immature erythrocytes where the steady state level of highly acetylated histones is low. But when deacetylation of the highly acetylated histones is prevented by incubating immature erythrocytes with histone deacetylase inhibitors, the thiol-reactive nucleosome is detected. The rate of deacetylation in immature erythrocytes is such that the highly acetylated H3 and H4 isoforms are short lived (4). In contrast, the thiol-reactive nucleosome is detected in the chromatin of mammalian cells without the use of histone deacetylase inhibitors (37) (unpublished observations). These observations suggest that the net activities of the histone acetyltransferases and deacetylases decide the longevity of the unfolded nucleosome. In transcribing regions where the rate of histone deacetylation exceeds the rate of acetylation, the unfolded nucleosome structure will be short lived and will rapidly revert to a thiol nonreactive state following passage of the RNA polymerase. In yeast the converse is the case. The unfolded nucleosome structure associated with a specific gene persists well after the transcription of that gene has been arrested (38). The rates of histone acetylation and deacetylation are very slow in yeast, but the high steady state of highly acetylated histones argues that the rate of histone acetylation exceeds the rate of deacetylation (39, 40).

Histone acetylation, however, is not sufficient to generate the unfolded nucleosome structure; transcription elongation is required. Thiol-reactive nucleosomes could not be found in the chromatin of chicken mature erythrocytes. Transcription may be initiated in these mature cells, but RNA polymerases are paused at the 5' end of the transcribed genes (41, 42). The treatment of immature erythrocytes with camptothecin may mimic the mature erythrocyte situation, that is, transcription initiation occurs but elongation does not (26). Thus, although dynamic histone acetylation and initiation are occurring in these cells at transcriptionally competent/active loci, without elongation the thiol-reactive nucleosome is not formed.

Our results suggest that unfolded transcribing nucleosomes are associated with highly acetylated H3-H4 tetramers. In agreement with the studies of Allfrey and colleagues (19), we found that immature erythrocyte chromatin fragments bound to mercury agarose are enriched in highly acetylated H3 and H4. Furthermore, Sterner et al. (20) showed that the thiol-reactive H3 of unfolded mammalian nucleosomes is hyperacetylated. Acetylation of H3 and H4 may maintain the unfolded nucleosome conformation by breaking interactions between the histone N-terminal tail and nucleosomal DNA. The N-terminal tail of H4 is not mobile in nucleosomes, and there is evidence that the H4 N-terminal tail makes intranucleosomal contacts (43). Indeed, His-18 in the N-terminal region of H4 cross-links to nucleotides 57, 66, and 93 from the 5' end of nucleosomal DNA (44, 45). This position in the nucleosomal DNA corresponds to where the nucleosomal DNA is sharply bent or kinked. In active gene chromatin and in chromosomal domains containing hyperacetylated histones, the cross-linking between His-18 of H4 and nucleosomal DNA in situ is greatly diminished (44, 46, 47). Moreover, site 60 from the end of nucleosomal DNA of hyperacetylated nucleosomes has an increased susceptibility to DNase I (48). These observations strongly suggest that acetylation at lysines located in N-terminal tail of H4 may have important functions in altering histone-DNA contacts and nucleosome structure. Further, hyperacetylation of the H3-H4 tetramer reduces the linking number change per nucleosome, that is, negative DNA supercoils constrained in unmodified nucleosomes are partially released in nucleosomes with hyperacetylated histones (49, 50).

The destabilizing effect that histone acetylation has on H3-H4 tetramer-DNA interactions in transcribing nucleosomes is seen in hydroxylapatite dissociation chromatography. The H3 of thiol-reactive nucleosomes dissociated from hydroxylapatite bound S150 or SE chromatin after the dissociation of the H2A-H2B dimers but before the bulk of the H3-H4 tetramers. In a previous study we monitored the dissociation of labeled ([3H]acetate) dynamically acetylated histones from hydroxylapatite-bound chromatin of immature erythrocytes. The dissociation of the labeled ([3H]acetate) highly acetylated H3-H4 tetramers coincided exactly with that of labeled ([3H]iodoacetate) H3 (27). These observations with SE chromatin from chicken immature erythrocytes suggest that the interaction between highly acetylated H3-H4 tetramer and DNA of transcribing nucleosomes is weaker than that of typical nucleosomes. Analysis of mercury-agarose bound nucleosomes by electron spectroscopic imaging also indicated that the H3-H4 tetramer of unfolded nucleosomes is disrupted (18). The disruption of the tetramer in transcribing nucleosomes may facilitate subsequent rounds of elongation.

Transcribing chromatin is associated with the insoluble residual nuclear material (fraction PE) which contains the nuclear matrix. The PE chromatin from butyrate treated immature erythrocytes had 76% of the active DNA and 74% of the acetate-labeled tetraacetylated H4 (7). Further, the PE fraction retained 75-85% of the nuclear histone acetyltransferase and histone deacetylase activity (7, 13). The thiol-reactive nucleosome was detected in PE chromatin of butyrate-treated immature erythrocytes, but not in the PE chromatin of untreated immature erythrocytes or mature erythrocytes. Further, inhibition of transcription with camptothecin or DRB prevented the detection of the unfolded nucleosome. Thus, the results obtained with PE chromatin were similar to those observed with S150 or SE chromatin; both hyperacetylated histones and elongation are required to detect the unfolded nucleosome in fraction PE.

There is increasing evidence that the transcription machinery is associated with the nuclear matrix and that for chromatin to be transcribed it is spooled through the anchored large RNA polymerase complex (51-54). We have proposed that histone acetyltransferase and deacetylase are localized in these transcription foci (13, 14). Recent studies show that coactivators (CBP/p300, ACTR, and GCN5) and proteins associated with TATA-binding protein (TAFII250) have histone acetyltransferase activity (55-60). Histone deacetylases (HDAC-1 and HDAC-2) are associated with corepressors (mSin3A and N-CoR) and the nuclear matrix bound transcription factor YY1 (61-64). These studies suggest that the basal transcription machinery and transcription factors recruit histone acetyltransferases and histone deacetylases to sites of transcription at the nuclear matrix. Nucleosome structure will be perturbed when the chromatin fiber is passed through the fixed RNA polymerase (transcriptosome) (53). While in a highly acetylated state, the unfolded nucleosome structure will persist, helping subsequent rounds of transcription. Our results are consistent with the idea that the nucleosome is a dynamic structure conforming its structure to facilitate movement of chromatin through the RNA polymerase II elongation complex, with dynamic histone acetylation having a major role in modulating the unfolded structure of transcribing nucleosomes.

    FOOTNOTES

* This work was supported in part by the Medical Research Council of Canada (MRC) Grant MT-9186 (to J. R. D.), a United States Army Breast Cancer Postdoctoral Research Fellowship DAM17-96-1-6269 (to L. T. H.), and a MRC Senior Scientist Award (to J. R. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Ave., Winnipeg, Manitoba R3E 0W3, Canada. Tel.: 204-789-3215; Fax: 204-789-3900; E-mail: Davie{at}cc.umanitoba.ca.

1 J. A. Ridsdale, P. Fredette, and J. R. Davie, unpublished observations.

2 The abbreviations used are: DRB, 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole; DTT, dithiothreitol.

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References

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