Targeted and Extended Acetylation of Histones H4 and H3 at Active and Inactive Genes in Chicken Embryo Erythrocytes*

Fiona A. Myers, Dain R. Evans, Alison L. ClaytonDagger, Alan W. Thorne, and Colyn Crane-Robinson§

From the Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, Faculty of Science, University of Portsmouth, Portsmouth PO1 2DT, United Kingdom

Received for publication, October 17, 2000, and in revised form, March 22, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Affinity-purified polyclonal antibodies recognizing the most highly acetylated forms of histones H3 and H4 were used in immunoprecipitation assays with chromatin fragments derived from 15-day chicken embryo erythrocytes by micrococcal nuclease digestion. The distribution of hyperacetylated H4 and H3 was mapped at the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the tissue-specific gene, carbonic anhydrase (CA). H3 and H4 acetylation was found targeted to the CpG island region at the 5' end of both these genes, falling off in the downstream direction. In contrast, at the beta A-globin gene, both H3 and H4 are highly acetylated throughout the gene and at the downstream enhancer, with a maximum at the promoter. Low level acetylation was observed at the 5' end of the inactive ovalbumin gene. Run-on assays to measure ongoing transcription showed that the GAPDH and CA genes are transcribed at a much lower rate than the adult beta A-globin gene. The extensive high level acetylation at the beta A-globin gene correlates most simply with its high rate of transcription. The targeted acetylation of histones H3 and H4 at the GAPDH and CA genes is consistent with a role in transcriptional initiation and implies that transcriptional elongation does not necessarily require hyperacetylation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The association of core histone acetylation, particularly of H3 and H4, with transcriptionally active genes is by now a familiar story (1-9). However, apparent inconsistencies have arisen between promoter/enhancer-specific acetylation and more widespread acetylation. Mapping the modification at the chicken beta -globin locus in 15-day erythrocytes showed 33 kb1 of acetylated chromatin, having boundaries coincident with the limits of open chromatin (10). Furthermore, the inactive embryonic epsilon -globin was hyperacetylated as well as the active adult beta A-globin gene (11). This implied that core histone acetylation was a precondition for transcription, a view supported by the observation that acetylation at the inducible PDGFbeta gene was not enhanced upon induction (12) and the modification might therefore be related to the formation or maintenance of accessible chromatin. In a study analyzing the basis of aberrant transcription of c-myc genes linked to an enhancer-LCR from the 3' end of the human immunoglobulin heavy chain (IgH) locus (modeling a well known translocation in Burkitt's lymphoma), widespread hyperacetylation was observed both upstream and downstream of the transcriptional start site, with little concentration in the region of the c-myc promoter (13). More recently, analysis of H3 and H4 acetylation at the human beta -globin locus in MEL cells containing a complete human chromosome 11, has also shown locus-wide acetylation, particularly of H4. Hyperacetylation of H3 was also widespread but more concentrated at Dnase I-hypersensitive sites and at the active beta -gene (14). The possibility of direct linkage between core histone acetylation and the passage of RNA polymerase II, thereby generating widespread modification, was raised by the observation that the elongator complex contains a subunit having HAT activity (15). Tracking-mediated chromatin modification has recently been discussed (16).

Such widespread acetylation contrasts with the "directed" or "targeted" acetylation implied by observations that gene activation is often accompanied by the recruitment to promoters/enhancers of protein complexes that include subunits having histone acetyltransferase (HAT) activity (17-21); gene repression frequently results in the recruitment to promoters/enhancers of protein complexes containing components with histone deacetylase activity (22-29). Several papers provide experimental support for targeted acetylation. Using a serum response factor-controlled reporter gene construct in mouse NIH3T3 cells, it was shown that extracellular stimulation of gene activation induced rapid acetylation of H4, but not H3, in the region of the serum response element (30). In Saccharomyces cerevisiae, promoter-specific hyperacetylation of histone H3 was observed in GCN5-mediated transcription (31), whereas promoter-specific hypoacetylation of histone H4 was found for Sin3/Rpd3-mediated repression (32). A concentration of H4 and H3 acetylation covering only about 3 nucleosomes in the region of the enhanceosome is induced by viral induction of the human interferon-beta gene in HeLa cells (33). Mapping of this induced acetylation showed it to extend only marginally into the (rather short) coding sequences. Hyperacetylation of histones H4 and H3 was also noted at the hormone response elements of several estrogen receptor target genes in human MCF-7 cells following induction with hormone (34) and at the LCR of the human growth hormone locus (35). Acetylation of histone H4 at K16 by MOF, a Drosophila dosage compensation protein, has been shown to activate transcription when targeted to a his3 reporter gene promoter in yeast (36). In a recent in vitro assay using purified components, SAGA and NuA4 HAT complexes targeted by Gal4-VP16 produced a more restricted region of H3 acetylation than that of H4 (37).

Two mechanisms have been proposed for the consequences of acetylation in the tail regions of core histones. 1) The modification disrupts inter-nucleosomal interactions mediated by core histone tails, thereby opening up higher order structure and rendering the chromatin accessible to the transcriptional apparatus. This mechanism can be fitted logically to the widespread acetylation (often of H4) found, for example, at globin genes (10, 14). It has recently been proposed that transcriptional elongation is required to form, and core histone acetylation to maintain, the open chromatin structure (38). 2) The modification has also been shown to facilitate the access of transcription factors to their DNA recognition sequences in individual nucleosomes at promoters/enhancers/LCRs (39-41). Although this mechanism can provide an explanation for the concentration of acetylation (often of H3) in promoter regions, HAT-containing complexes are generally assumed to be recruited by already bound primary transcription factors. Defining the order of binding events is thus crucial to understanding the role of promoter/enhancer/LCR-specific acetylation (42).

To further explore the distinction between localized and locus-wide histone acetylation, we have mapped the acetylation of histones H3 and H4 at a housekeeping gene (GAPDH) and a tissue-specific gene (CA) in the same cells as used for acetylation mapping at the beta -globin locus, i.e. 15-day chicken embryo erythrocytes. The mapping of acetylated histones at housekeeping genes was in part provoked by the observation of Tazi and Bird (43) that chromatin derived from CpG islands is highly enriched in hyperacetylated histone H4, indicating a concentration of the modification in such regions. At the beta -globin locus, where the adult gene does not have a CpG island but the embryonic rho -gene does, no correlation of acetylation with the presence or absence of a CpG island was observed (10). However, all housekeeping genes and about one-half of tissue-specific genes have CpG islands; therefore, the enrichment seen by Tazi and Bird (43) could be predominantly from housekeeping genes. The present results show a concentration of H4 and H3 acetylation in the upstream CpG island regions of both the GAPDH and CA genes, in contrast to the beta A-globin gene for which the modification extends throughout the gene and into the 3' enhancer.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation and Affinity Purification of Antibodies-- Anti-hyperacetylated histone H4 serum was prepared by immunizing rabbits with chemically acetylated H4, and antibodies were affinity-purified over a column of immunogen, immobilized on agarose beads (2, 10, 44, 45). The anti-acetylated histone H3 peptide serum was obtained by immunizing rabbits with peptide 1-27 of H3, acetylated at residues 9, 14, 18, and 23, chemically synthesized as multiple antigenic peptides (MAPs). Antibodies were affinity-purified over a column of the same H3 peptide immobilized on controlled-pore glass beads (Alta Bioscience) using elution conditions as detailed in Ref. 44.

Western Blotting-- Acid-extracted histones from butyrate-treated HeLa cells were resolved by 15% AUT-polyacrylamide gel electrophoresis (50) and after equilibration in transfer buffer (15 mM glycine, 20 mM Tris, 0.1% SDS, and 20% methanol) were electrophoretically transferred to nitrocellulose using a Bio-Rad transblot apparatus (400 mA, 90 min at 4 °C). Membranes were blocked in 5% (w/v) Marvel in 1× PBS for 1 h, washed in 1× PBS, 0.1% (v/v) Tween 20, and incubated with 1:2000 diluted serum for 1 h. After further washing with 1× PBS, 0.1% Tween, chemiluminescent detection was performed using an ECL kit (Amersham Pharmacia Biotech).

Preparation of Nucleosomes-- Salt-soluble chromatin from 15-day chicken embryo erythrocytes was prepared essentially as described in Ref. 2. In brief, a 5-mg DNA/ml suspension of nuclei was digested with MNase at 37 °C for 10 min in digestion buffer (10 mM Tris-HCl, pH 7.4, 10 mM butyrate, 3 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine). Digestion was terminated by the addition of EDTA to a final concentration of 10 mM. Released chromatin was recovered from supernatant S1 after centrifugation (13000 × g, 1 min). The pellet was resuspended in lysis buffer (0.25 mM EDTA, 10 mM Tris-HCl, 10 mM sodium butyrate) to release further material. which was recovered in supernatant S2 after centrifugation. S1 and S2 were pooled, and H1/H5-containing chromatin was precipitated by the addition of NaCl to 100 mM. Following centrifugation, the supernatant was layered onto 5-30% exponential sucrose gradients in lysis buffer. Di- and tri-nucleosomal fractions were pooled and used as the input chromatin for the experiments because probing with beta A-globin and housekeeping sequences showed them to be well represented in this chromatin size class.

Immunoselection of Chromatin-- ChIP assays were performed as described in Ref. 10. Typically, 400 µg of input chromatin (as DNA) was mixed with 100 µg of affinity-purified antibody in immunoprecipitation (IP) buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 10 mM butyrate, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine) and incubated for 2 h at 4 °C with constant agitation. Immunocomplexes were immobilized using 50 mg of protein A-Sepharose equilibrated in IP buffer, and the suspension was incubated for an additional hour at 4 °C. Unbound chromatin was recovered from the filtrate by centrifugation (6000 rpm, 30 s) through a 0.45-µm Spin-X filter (Sigma). After a repeat washing with IP buffer, the resin pellet was resuspended in 150 µl of IP buffer containing 1.5% SDS, incubated for 15 min at room temperature, and centrifuged to release antibody-bound chromatin in the filtrate. The resin pellet was resuspended in 150 µl of IP buffer containing 0.5% SDS and re-centrifuged, and the two "Bound" filtrates pooled. The histones and DNA from the input, unbound, and bound fractions were recovered as described in Ref. 10.

Quantitative PCR-- Input, unbound, and bound DNA samples were subjected to PCR amplification in the presence of 5 µCi of [32P]dCTP and the appropriate primers. Template concentrations and numbers of cycles were determined for each primer pair so that the products fell within the exponential phase of amplification. Typically, 26-28 cycles were used with templates serially diluted from 8.0 to 0.5 ng. Amplification conditions were: 2-min denaturation at 94 °C followed by n cycles of: denaturation (94 °C for 1 min), annealing (temperature and time optimized for each primer pair), and extension (72 °C for 1 min). Products were analyzed on 6% native acrylamide gels and quantitated using a PhosphorImager. The signal from the correctly sized product derived from the input (I) and bound (B) samples were plotted as a function of template concentration to check for linearity and the B/I value determined as the ratio of the slopes of the two plots in the linear region (data not shown). Comparing the bound signal to the input normalizes for variations in the input signal that arise from differing susceptibilities to MNase at different points in the genome (or within a single gene). B/I ratios >1 represent "fold enrichments" achieved by the immunoprecipitation. B/I values of <1 represent depletions in the bound DNA and are plotted as I/B, "fold depletions."

Primer Pairs-- The following primer pairs were used: A1, GTATGGCGCACTCTGGTATAGA, and A2, GAGCGGCCGTCTGTGTC, 304-bp product; A3, ACCTTCTCCCAACTGTCC, and A4, ATTCCTTTCTCACTATGCT, 258-bp product; A5, AAGCCTAGGAATGTTTCC, and A6, TTAGTGGTACTTGCGAGC, 224-bp product; A7, ACAAAGTGAAGGCTTTAATC, and A8, TTTTAGTTCCAGAACATCATT, 254-bp product; Ga, GCTCTTTGTCCCGCCC, and Gb, CGGGGCGATGCGGCTG, 100-bp product; G1, TCTCGCGCAGGACCGCGTGG, and G2, GTGTTCCTGCGGGGAGAGACCG, 244-bp product; G3, ACCTTTGTGGTGTGGGTGCC, and G4, GCCAGAGAGGACGGCAGCCC, 246-bp product; Gc, GAGTCCACTGGTGTCTTCAC, and Gd, GAGATGATAACACGCTTAGC, 250-bp product; CA1, TCAGTGCGGACACAGAGGAGCATT, and CA2, AGTTGAATCACCACTCCCACGGCT, 273-bp product; CA3, TACGCCAGCCACAACGGTGA, and CA4, CTCAGGCCTGGCATCTCAAGGT, 145-bp product; CA5, ACTGCCTTCTCCAGACACTGC, and CA6, TTTCCAGCACCATTCCCTAAGT, 100-bp product; CA7, CTGGATGGAGTCTACAGG, CA8, GCAAAGCACATCATACCTCTGC, 118-bp product; CA9, CAGCGATGAGTGTGTTAGAA, and CA10, TGTCAGTCGCAGTAAGT, 249-bp product; OvA, TTGTTCTCACTTATGTCCTGCC, and OvB, TTCAGTTACAACCAGATAATGG, 201-bp product; Ov1, ACAGCACCAGGACACAGATAA, and Ov2, AAGTCTACTGGCAAGGCTGAA, 175-bp product; Ov3, AACTCATGGATGAAGGCTTAAGG, and Ov4, TTGTCAGCATAGGAATGGTTGG, 220-bp product.

Nuclear Run-on Analysis-- The run-on analysis was performed essentially as described in Refs. 51 and 52. 15-day chicken embryo erythrocyte nuclei were prepared as follows. Blood was collected into 1× PBS, 10 mM sodium butyrate, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 mM benzamidine and filtered through sterile gauze. Erythrocytes were pelleted at 2200 × g for 5 min at 4 °C and washed and pelleted in the same buffer an additional two times. The final pellet was resuspended in ice-cold RSB buffer (10 mM Tris-HCl pH 7.4, 1.5 mM MgCl2 10 mM KCl, 0.5% Nonidet P-40) and incubated for 5 min on ice with vigorous agitation to lyse the cells. Nuclei were pelleted at 2000 × g for 5 min at 4 °C and the supernatant removed by aspiration. Pellets were resuspended in glutamate run-on buffer (125 mM potassium glutamate, 10 mM HEPES, pH 8.0, 5 mM MgCl2, 2 mM dithiothreitol, 1 mM EGTA, 40% glycerol), snap-frozen in liquid nitrogen, and then stored at -80 °C. Nuclei for analysis (100 µg of DNA) were thawed quickly and placed on ice, and then 1.0 µl of 1 M creatine phosphate in 10 mM HEPES, pH 8.0, 2.4 µl of 2 mg/ml creatine kinase, 1 µl of 100 mM ATP, 1 µl of AGC (25 mM of each of GTP, ATP, and CTP), 8 µl of [32P]UTP (160 µCi at 800 Ci/mmol) and 50 units of RNAsin (Promega) were added followed by incubation at 37 °C for 15 min. CaCl2 was added to a final concentration of 10 mM together with 50 units of RNase-free Dnase 1 followed by incubation at 37 °C for 20 min. 5 µl of 10× SET buffer (10% SDS, 100 mM Tris-HCl, pH 7.5, 50 mM EDTA) and 150 µl of 1× SET buffer were added along with 10 µl of 10 mg/ml proteinase K and incubated at 37 °C for 45 min. RNA was extracted using phenol-chloroform and precipitated with isopropanol. The RNA pellet was resuspended in 90 µl of 1 mM EDTA, 0.5% SDS, and 15 µl of 2 M NaOH was added followed by incubation on ice for 10 min to partially fragment the RNA. Samples were neutralized by adding 0.48 M HEPES and heated to 100 °C for 5 min before applying to the filter. 5 µg of both sense and antisense single-stranded DNA was slot-blotted onto a Biodyne B membrane for each of the GAPDH, CA, ovalbumin, and beta A-globin genes. The hybridization buffer was 50% deionized formamide, 6× SSPE buffer (saline/sodium phosphate/EDTA), 0.1% SDS, and 100 µg/ml tRNA. Prehybridization was for 2 h at 42 °C with hybridization overnight at 42 °C. Washing was for 10 min in 2× SSPE, 0.1% SDS at room temperature and 20 min in 0.2× SSPE, 0.1% SDS at 68 °C.

RT-PCR-- Total RNA was extracted from 15-day chicken erythrocytes using an RNAqueous kit (Ambion) and DNase 1-treated (1 unit/µg, 30 min, 37 °C). First strand cDNA was prepared from 2-µg aliquots using random hexamer primers (Promega) and Superscript II enzyme (Life Technologies, Inc.) under the following conditions: 90 °C for 3 min, add 300 units of Superscript II, 37 °C for 60 min, 95 °C for 3 min. Products were amplified by PCR (94 °C for 2 min; followed by 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min, for 28 cycles; finally, 72 °C for 7 min) with gene-specific primers that where designed to reveal the presence of contaminating genomic DNA in the RNA preparations.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of Antibodies-- The two immunogens used to raise the antibodies utilized in the ChIP assays were chemically acetylated histone H4, in which essentially all of the lysine residues of H4 become modified (2, 10, 44, 45) and the peptide 1-27 of histone H3 acetylated at residues 9, 14, 18, and 23 chemically synthesized as multiple antigenic peptides (Alta Bioscience). Western blots were conducted to characterize the specificity of the sera, using histones extracted from butyrate-treated HeLa cells; in each case a Coomassie-stained marker lane of the these histones is shown for comparison. Fig. 1 shows the results for each serum using both SDS gels and acetic acid/urea (AU) or acetic acid/urea/Triton (AUT) gels. The SDS gels in Fig. 1, A and C, show that both sera are highly specific for the histone used as immunogen, although careful inspection shows that the specificities are not absolute. For example, the anti-acetylated H4 serum shows a weak recognition of H3 at the higher loading that amounts to about 10% of the activity against H4, whereas the anti-acetylated H3 peptide serum shows a very weak recognition of H4 and H2B histones. This weak cross-reactivity is almost certainly due to the presence of anti-acetyl lysine activity in the sera, as previously documented for sera derived from chemically acetylated H4 (44). When the acetylated subspecies are spread out using AUT or AU gels the specificity is more precisely revealed. For histone H4, about half of the activity is directed at the tetra (fully)-acetylated species, with the remainder against Ac3 and Ac2, whereas for histone H3 the activity is about equally directed against the Ac4 and Ac3 species (although a low activity against Ac2 can also be detected). These antisera can most simply be described as being against hyperacetylated H4 and H3. Antibodies from both sera were then affinity-purified using columns carrying the immobilized immunogens, i.e. chemically acetylated histone H4 and the acetylated H3 peptide. All of the ChIP experiments described below were carried out using these affinity-purified antibodies.


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Fig. 1.   Characterization of the antibodies by Western blotting using SDS-, AUT-, and AU-polyacrylamide gel electrophoresis. The marker lanes (M) represent Coomassie-stained tracks of the HeLa butyrate histone mixture used for all panels. Panel A, SDS gel and Western blot (loadings ×1 and ×4) using serum from a rabbit immunized with chemically acetylated histone H4 and detected using a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody with a chemiluminescent substrate (ECL). The bulk of the activity is against histone H4 and ~10% against histone H3. Panel B, AUT gel and Western blot using serum from a rabbit immunized with chemically acetylated histone H4 and detected using a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody with a chemiluminescent substrate (ECL). The activity is against tetra-, tri-, and di-acetylated H4 with very weak activity against the histone H3 subfractions 2 and 3 detected on very long exposures (data not shown). Panel C, SDS gel and Western blot (loadings ×1 and ×2) using serum from a rabbit immunized with the acetylated histone H3 N-terminal peptide and detected using a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody with a chemiluminescent substrate (ECL). The bulk of the activity is against histone H3 together with very weak recognition of histones H2B and H4. Panel D, AU gel and Western blot using serum from a rabbit immunized with the acetylated histone H3 N-terminal peptide and detected using a goat-anti-rabbit horseradish peroxidase-conjugated secondary antibody with a chemiluminescent substrate (ECL). The activity is against tetra-, tri-, and at a much lower level, di-acetylated H3 with little evidence of activity against other histones.

Chromatin Immunoprecipitation Assays-- Nucleosomal fragments from 15-day chicken embryo erythrocyte nuclei were prepared by micrococcal nuclease digestion and fractionated on a sucrose gradient. The di- and tri-nucleosomal components were pooled and used as "input" chromatin for ChIP assays. In a typical assay, 400 µg of input chromatin (as DNA) was mixed with 100 µg of affinity-purified antibodies (see "Experimental Procedures"). Immunocomplexes were immobilized on protein A-Sepharose and washed to remove the "unbound" chromatin, and the "bound" chromatin was released by a SDS-containing buffer. Typically about 5-10 µg of DNA was recovered from the bound fraction, i.e. about 1.25-2.5% of the input. The content of acetylated histones in the two precipitated chromatin fractions was assessed by comparing the "input" (I) and "bound" (B) samples on stained AUT gels (Fig. 2). When the anti-acetylated H4 antibodies were used, as expected, the bound chromatin was indeed much enriched in multiply acetylated H4 species as compared with the input chromatin. Close inspection also showed some increase in multiply acetylated H3 species. In the experiment using the anti-acetylated H3 antibodies, an enrichment in multiply acetylated H3 species could be seen, as expected, but in addition there was a considerable rise in the acetylation level of the histone H4 present. The tight histone specificity of the anti-acetylated H3 peptide antibodies (Fig. 1) means that the co-isolation of other multiply acetylated histone species must be because of their presence in chromatin fragments selected by virtue of their acetylated H3 content, i.e. there is co-habitation of hyperacetylated H4 with hyperacetylated H3 at the di/tri-nucleosomal level.


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Fig. 2.   AUT analysis of histones in the input (I) di/tri-nucleosomal chromatin used for the immunoprecipitations and the histones isolated from the antibody-bound (B) chromatin. Panel A shows the proteins from the anti-hyperacetylated H4 immunoprecipitation stained with Coomassie, and Panel B shows the proteins from the anti-hyperacetylated H3 immunoprecipitation stained with silver.

The adult beta A-Globin Gene and Its 3' Enhancer-- DNA from the ChIP assays using both anti-acetylated H3 and H4 antibodies was analyzed by quantitative PCR for chicken beta A-globin gene sequences. The gene has 3 exons and is about 2.0 kb in length, and the enhancer is located just 3' of the end of the major transcript. The amplicons are: (a) at the promoter (primers A1-A2), (b) spanning the boundary between exon II and the large intron (primers A3-A4), and (c) at exon III (primers A5-A6). The fourth amplicon is in the enhancer region, ~360 bp downstream of the transcription termination site, primers A7-A8, (see Fig. 3). In the exponential phase of PCR amplification, the ratio of the amount of product from antibody-bound to input DNA (the B/I ratio), gives a measure of the enrichment achieved, i.e. the relative acetylation level at the different regions of the gene (see "Experimental Procedures"). The promoter region shows a 9.5-fold enrichment using the anti-acetyl H4 antibodies and an 8.1-fold for H3 acetylation. In the transcribed region, both primer pairs showed enrichments of 4-6-fold with both antibodies, and similar enrichments were observed at the enhancer. There is thus a high level of H4 and H3 acetylation throughout the chicken beta A-globin gene, including its enhancer, but the promoter exhibits higher levels of modification. Strikingly, levels of H4 and H3 acetylation follow a similar distribution, in accord with the generalized observation from polyacrylamide gel electrophoresis analysis of cohabitation of the two modified histones (Fig. 2).


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Fig. 3.   Distribution of H4 and H3 acetylation at the chicken beta -adult beta A-globin gene and its 3' enhancer using four primer pairs A1-A2, A3-A4, A5-A6, and A7-A8 with positions in the gene indicated by the bold lines. Quantitative PCR analysis was used of DNA taken from input and antibody-bound fractions obtained from ChIP assays that used affinity-purified anti-acetyl H4 (AcH4) and H3 (AcH3) antibodies. PCR products were quantitated using a PhosphorImager, and the signals from the correctly sized product from input and bound template samples were plotted as a function of template concentration. Values of B/I were determined as the ratio of the slopes of the two plots and are given in the bottom panel as enrichments for each set of primer pairs.

The Glyceraldehyde 3-Phosphate Dehydrogenase Gene-- The chicken housekeeping gene GAPDH contains 12 exons, is about 4.5 kb long and has a CpG island of about 1.5 kb in length at its 5' end (46). The primers pairs chosen for analysis (Fig. 4) were: (a) Ga-Gb, located in the promoter just upstream of the TATA box; (b) G1-G2, spanning the translational start, i.e. largely in the 5'-untranslated region and within the CpG island; (c) G3-G4, covering all of exon III and surrounding intron sequences; and (d) Gc-Gd, covering exons VI and surrounding intron sequences. The two most 3' amplicons are outside of the CpG island, whereas the other two pairs are within it. Quantitative PCR analysis showed a 4-fold enrichment for H4 acetylation at the promoter but only half of this for H3 acetylation (B/I ~ 2). About 600 bp into the transcribed region (G1-G2), the H4 acetylation has dropped markedly (B/I ~ 2), whereas a 2-fold depletion in the bound DNA (B/I ~ 0.5) is observed using the anti-acetylated H3 antibodies. Further into the transcribed region, (G3-G4 and Gc-Gd), depletions are observed using both antibodies, and the effect is more pronounced in regard to H3 acetylation, reaching a 4-fold depletion at only about 1.2 kb from the transcriptional start (G3-G4). The distribution of acetylation is thus different from that at the beta A-globin gene in that although the promoter is acetylated on both H3 and H4, the transcribed region essentially lacks acetylated H3 and H4, with the exception of modest H4 acetylation at the most 5' end (G1-G2).


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Fig. 4.   Distribution of H4 and H3 acetylation at the chicken GAPDH gene. Four primers pairs, Ga-Gb, G1-G2, G3-G4, and Gc-Gd, were used in quantitative PCR analysis, the same as for the beta A-globin gene in Fig. 3. Measured B/I values greater than 1 are given as Fold Enrichment (as in Fig. 3), and values less than 1 are given as Fold Depletion, i.e. I/B.

The Carbonic Anhydrase Gene-- The distribution of acetylated H3 and H4 histones was also monitored across the chicken carbonic anhydrase gene. This is an active tissue-specific gene in 15-day erythrocytes that possesses a CpG island. The gene covers about 18 kb and comprises 7 exons (Fig. 5, Ref. 47). The amplicons used were in the promoter region just upstream of the TATA box (primers CA1-CA2) and located just upstream of the CpG island. These primers gave a 2.0-fold enrichment using both the anti-acetylated H4 and H3 antibodies. The second amplicon (primers CA3-CA4) is within the CpG island covering the first intron and a small amount of exon II; this gave a 16.5-fold enrichment using the anti-acetylated H4 antibodies and an ~7-fold enrichment using anti-acetylated H3 antibodies. The third amplicon (primers CA5-CA6) is located ~1.5 kb into the transcribed region, and both antibodies gave an ~2.4-fold enrichment. This is in marked contrast to the GAPDH gene for which, at a similar distance from the transcriptional start (primers G3-G4), there is a 1.5-fold depletion using the anti-acetylated H4 antibodies and a 4-fold depletion using anti-acetylated H3 antibodies. The fourth amplicon (primers CA7-CA8) covers exon III, and the most 3' amplicon (primers CA9-CA10) is within the final exon (VII). For both of these amplicons, a low enrichment was observed using the anti-acetylated H4 antibodies, which although not high is still 1.5-fold at exon VII, 16 kb downstream of the transcriptional start. For the anti-acetylated H3 antibodies, modest depletions (1.2- and 1.3-fold) are observed at exon III and exon VII of the CA gene; this is again very different from the GAPDH gene for which a 2.1-fold depletion is observed only 600 bp into the gene (primers G1-G2).


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Fig. 5.   Distribution of H4 and H3 acetylation at the chicken carbonic anhydrase gene. Four primer pairs, CA1-CA2, CA3-CA4, CA5-CA6, and CA7-CA8, were used in quantitative PCR analysis, the same as for the beta A-globin gene in Fig. 3. Measured B/I values greater than 1 are given as Fold Enrichment (as in Fig. 3), and values less than 1 are given as Fold Depletion, i.e. I/B.

The Ovalbumin Gene-- The 9.5-kb ovalbumin gene is inactive in this tissue, does not have a CpG island, and was chosen as a negative control for these experiments. The three amplicons used, primers pairs OvA-OvB, Ov1-Ov2, and Ov3-Ov4, are located at the promoter, in the 5'-untranslated region, and at exon VII of the gene, respectively. For both the anti-H4 and H3 antibodies, increasing depletions are observed in a downstream direction, considerably more so for H3 than for H4 (Fig. 6). At the promoter, a B/I value of close to unity is observed using the anti-acetylated H4 antibodies; this implies a low but non-zero level of hyperacetylation at this point, which decreases to a 2.1-fold depletion near the end of the gene (Ov3-Ov4). As regards H3 acetylation, a significant depletion is observed at the promoter, but this becomes more pronounced in a 3' direction, reaching a 5.3-fold depletion at exon VII. Levels of H4 and H3 acetylation on the ovalbumin gene are clearly very low, but the considerable changes in depletion measured along the gene suggest that there may be some acetyl groups at the 5' end of the gene.


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Fig. 6.   Distribution of H4 and H3 acetylation at the chicken ovalbumin gene. Three primer pairs, OvA-OvB, Ov1-Ov2, and Ov3-Ov4, were used in quantitative PCR analysis, the same as for the beta A-globin gene in Fig. 3. Measured B/I values greater than 1 are given as Fold Enrichment (as in Fig. 3), and values less than 1 are given as Fold Depletion, i.e. I/B.

The pattern of histone acetylation at the beta A-globin, GAPDH, CA, and ovalbumin genes is different in each case: throughout for beta A-globin gene, very concentrated at the promoter of the GAPDH gene, and while maximal at the 5'-end of the CA gene, not falling off so sharply in a 3' direction as at the GAPDH gene. A very low level of H3 and H4 acetylation is found at the 5' end of the silent ovalbumin gene. Both the GAPDH and CA genes have CpG islands, whereas the beta A-globin and ovalbumin genes do not. The differences between the acetylation patterns prompted us to determine to what extent the acetylation pattern observed reflects the transcriptional status of the genes in 15-day chicken erythrocytes.

The Transcriptional Status of the Studied Genes-- Total RNA was extracted from 15-day erythrocytes, and RT-PCR was used with primer pairs specific for the studied genes to establish the amount of each mRNA present, i.e. the pool levels of the four messages in 15-day erythrocytes. Fig. 7A shows ethidium bromide-stained agarose gels of the RT-PCR products with multiple loadings of the beta A-globin and CA products. Qualitatively, the 460-bp beta A-globin product is very much stronger (>50-fold) than the 420-bp product from the GAPDH housekeeping gene. The 368-bp product from the tissue-specific CA gene is about 20-fold weaker than the beta A-globin product. As expected, there is no evidence for the presence of any ovalbumin mRNA. Although the PCR reactions used were not accurately quantified, the amplified sequences were all about 50% GC, the amplicon lengths were similar to each other, and a limited number of cycles (33) was used; product intensities should therefore approximately reflect mRNA pool sizes.


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Fig. 7.   Panel A, RT-PCR detection of mRNA pool sizes in 15-day chicken erythrocyte nuclei. Relative loadings are given below each lane: ×1 = 5 µl from a 50-µl PCR amplification. Product sizes correspond to those expected: beta A-globin (beta A), 460 bp; GAPDH, 420 bp; ovalbumin, 488 bp; CA, 368 bp. The intensity of the ethidium staining indicates a much greater pool size for beta A-globin than for the other genes. M, marker lanes. Panel B, nuclear run-on experiments to assess relative rates of transcription at the studied genes. 32P-labeled run-on transcripts were used to probe 5 µg of antisense and sense single-stranded DNA fragments from the studied genes, immobilized on Biodyne B membrane. There is no evidence of transcription from the ovalbumin gene, while the housekeeping gene GAPDH and the tissue-specific gene CA give a weak signal. The signal from beta A-globin is more than 2 orders of magnitude greater.

Variable rates of mRNA degradation mean that pool sizes cannot be equated with actual rates of transcription. In vitro run-on assays were therefore used as a direct approach to defining the actual levels of transcription at the different genes. Preparation of nuclei results in losses of nucleotide precursors and stalling of engaged transcription complexes; on incubation of nuclei in the presence of radiolabeled UTP and unlabeled other NTPs, these complexes complete transcription of the gene but do not re-initiate (48). The labeled transcripts were then used to probe immobilized gene sequences, in this case single-stranded DNA corresponding to the antisense sequence (complementary to mRNA), with sense sequences also dotted as controls. Fig. 7B shows the results for all the four studied genes. The very strong signal from beta A-globin sequences demonstrates the high level of transcription on this gene. In marked contrast, very weak but above background levels of CA and GAPDH transcripts can be detected, whereas no signal at all is detected for ovalbumin transcription, as expected. Thus transcription from the tissue-specific beta A-globin gene is at a very much greater rate than from the tissue-specific CA gene, and transcription from the housekeeping GAPDH gene is at similar levels to that from the CA gene.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The nuclei used in these experiments were made from 15-day embryo erythrocytes directly after blood collection and then used to prepare chromatin for ChIP experiments. The observations should therefore relate to the natural state of the chromatin at the four single copy genes in these cells and not be subject to any of the caveats that inevitably apply to tissue culture cells, transgenic cells, and transfected cells. Nevertheless, in considering the detail of the acetylation mapping results, it is important to bear in mind the spatial resolution of the experiments. Having in mind that di/tri-nucleosomes were antibody-selected and that amplicons were typically of about mononucleosomal length, the "worst case scenario" is that the amplification comes from the next but one nucleosome to the acetylated nucleosome that gave rise to the immunoprecipitation. The centers of these two nucleosomes would be about 400 bp apart, and this could be considered as the resolution of the experiments. In the case of the primer pairs Ga-Gb and G1-G2 at the GAPDH gene, the centers of the two amplicons are about 500 bp apart; it is noteworthy that with the anti-acetylated H3 antibodies the former gave a 2-fold enrichment but the later a 2-fold depletion. Therefore the spatial resolution is empirically seen to be better than 500 bp. If shorter chromatin fragments are used in ChIP assays to improve the resolution and, necessarily, reduced DNA lengths are amplified, then the spatial modulation of acetylation levels (the observed enrichments B/I) would probably be enhanced. However, the resolution in the present experiments allows very large differences in B/I ratios to be detected over quite short distances, e.g. comparing the H4 acetylation mapped with the CA1-CA2 and the CA3-CA4 primer pairs at the carbonic anhydrase gene that are centered about 1 kb (5 nucleosomes) apart.

The enrichment (B/I) or depletion (I/B) values obtained from a single immunoprecipitation experiment represent relative levels of acetylation with a good degree of accuracy, both within a gene and between genes. Indeed, for the present experiments all the data on the distribution of H4 acetylation were obtained from a single immunoprecipitation, and the same is true for the H3 acetylation data. If a ChIP experiment is repeated to test the overall reproducibility of the modulation of the enrichments, then variations in the batch of antibody, the chromatin preparation, and the efficiency of the actual immunoprecipitations can lead to significant variations in the measured B/I values. However, the relative values at different points along a gene or locus remain essentially the same.2 For this reason we present B/I values obtained from a single IP. To assess the repeatability of the enrichments, multiple PCR amplifications were performed using template DNAs from the same immunoprecipitation, repeating the dilutions as well as the amplifications. For the beta A-globin promoter amplicon (A1-A2) in the anti-acetylated H4 immunoprecipitation, this gave a B/I value of 9.5 with a root-mean-square deviation of 1.2, which indicates the repeatability of B/I measurements from a single IP, i.e. ±15%.

However, caution must be exercised when using B/I values to compare levels of H4 acetylation with levels of H3 acetylation because the efficiency of immunoprecipitating modified nucleosomes may be very different for the two antibodies, and even for a given antibody there are variations between individual immunoprecipitations. Thus although the enrichments found using the anti-acetylated H3 antibodies are frequently less than for the anti-acetylated H4 antibodies (although not at the beta A-globin gene), this does not necessarily mean that there is more H4 than H3 acetylation, e.g. at the GAPDH promoter (Ga-Gb) or at the first intron of the CA gene (CA3-CA4). It is clear however that the H4 acetylation at the first intron of the CA gene is about 8-fold more than at the promoter, whereas the H3 acetylation is only 3-4-fold more than at the promoter.

It is important to note the practical advantages of using quantitative PCR for sequence content analysis, rather than hybridization, in particular for detecting depletion of signals in the bound DNA. Slot blots frequently show (variable) backgrounds even in the absence of any sample DNA, making it difficult to measure depletions with any accuracy. However, when linear relationships between amplicon intensity and template concentration are obtained in quantitative PCR, there is no background problem. Therefore a depletion (I/B) of, for example, 5.3-fold is readily distinguishable from a depletion of 2.7- and 1.2-fold, to quote the values observed for H3 acetylation at the ovalbumin gene. It is also important to note that an enrichment of 1.04-fold is experimentally fairly close (~20%) to a depletion of 1.2-fold, the results for H4 and H3 at the OvA-OvB amplicon, but very distinct from the 5.3-fold depletion for H3 at the Ov3-Ov4 amplicon.

Because B/I values give the change in concentration (representation) of a given sequence between the input and the bound DNA samples, it is worth noting that with a range of reliably measured B/I ratios from substantially >1 to significantly <1 a value of unity has no special significance. This is because although the representation of a sequence in the input sample is roughly the same as in total genomic DNA, the bound DNA sample is a completely different collection of sequences, selected for their characteristic and variable acetylation status.

With these considerations in mind, several clear observations stand out. There is a high level of both H3 and H4 acetylation throughout the beta A-globin gene, including its downstream enhancer, with an approximately 2-fold increase at the promoter, but this is not a characteristic feature of all tissue specific genes because it is not observed for the CA gene. The continuous acetylation is certainly not an obligatory feature of CpG island-containing genes, because the beta A-globin gene does not have an island. The continuous and extensive acetylation of the beta A-globin gene does appear to correlate with its high level of transcription in 15-day embryos, as seen in the run-on experiments. However, the silent embryonic beta rho -globin gene is also highly acetylated, as seen from experiments using a hybridization probe (P5) that covers most of the beta rho transcribed sequences (10), so there is no simple correlation between hyperacetylation and a high level of transcription over transcribed sequences. At the GAPDH and CA genes, the H4 and H3 hyperacetylation is concentrated within the CpG island region at the 5' ends of these genes, and it is striking that at the CA gene a peak of very intense acetylation is observed not at the promoter (as for the GAPDH gene) but in the middle of the CpG island part of the transcribed region. At the GAPDH gene the H3 acetylation falls off more rapidly than the H4, as seen particularly from the B/I values at the G1-G2 amplicon (located in the 5'-untranslated region) for which a 2-fold enrichment is observed using the anti-acetylated H4 antibodies (but a 2-fold depletion is seen using anti-acetylated H3 antibodies); this is in contrast to the promoter for which enrichments are found in both H3 and H4 immunoprecipitations. We have previously reported that H4 acetylation is also concentrated in the region of the CpG island for the housekeeping gene thymidine kinase (TK) in the same cells as used here, falling off rapidly in a 3' direction, as for the GAPDH gene (49). The contrast between the GAPDH and CA genes is considerable in that although both exhibit a falling off of acetylation in a 3' direction, the overall acetylation patterns are rather different. The CA gene is very much longer than the GAPDH gene, yet there is still a small (×1.5) but significant enrichment for H4 at exon VII of the CA gene (~16 kb from the transcriptional start), whereas only 1.2 kb from the transcriptional start of the GAPDH gene there is a 1.5-fold depletion for H4 acetylation. To decide whether this is a distinguishing difference between tissue-specific and housekeeping genes will require the study of several more genes in both categories.

The results for the ovalbumin gene demonstrate very low levels of both H4 and H3 acetylation, as expected for an inactive gene and as shown by our earlier results using hybridization, (2, 10, 11). Importantly however, it is clear that acetylation is not completely absent, because not only is there a B/I value of >1 for H4 at the promoter but the changing levels of depletion in a 3' direction with both antibodies are well outside the experimental error. In fact, the pattern of decreasing H4 and H3 acetylation in a 5' to 3' direction on the ovalbumin gene looks similar to that at the GAPDH gene, with the difference that the levels of acetylation are everywhere much lower at the ovalbumin gene.

The results overall suggest that acetylation, particularly of H4, is concentrated at the CpG island regions of active genes, as suggested by the observations of Tazi and Bird (43), rather than just being targeted at promoters. In the case of the CA gene, a peak of hyperacetylation is seen within the CpG island (CA3-CA4) and only modest enrichments (2-fold) are observed in the promoter region (CA1-CA2). For this gene the promoter amplicon is located outside of the CpG island. At the GAPDH gene, where the promoter is within the CpG island, there is acetylation of both H3 and H4 in the region of the promoter. In this particular case, therefore, H3 acetylation appears to be restricted to the promoter, whereas H4 acetylation is somewhat more extensive, a situation not dissimilar to that at the human beta -globin locus (14) and that resulting from Gal4-VP16 recruitment (37). So, although both the GAPDH and CA genes exhibit hyperacetylation of H3 and H4 only at the 5'-end of the genes, in contrast to the beta A-globin gene, it is not at present possible to conclude that this is a promoter-specific effect, particularly as regards histone H4. In fact, the data obtained for GAPDH suggest that the H4 hyperacetylation could be a CpG island phenomenon, whereas H3 hyperacetylation is a feature of active promoter/enhancers.

The correlation of the modification with chromatin structure at the chicken beta -globin locus (10) suggested a direct relationship of H4 acetylation to either the formation or maintenance of the open conformation of the chromatin. Assuming that an open chromatin structure over the whole of a gene is required for transcription, then the spatially restricted H4 and H3 acetylation observed for the housekeeping gene GAPDH and the tissue-specific gene CA must have another function. The observed localization of hyperacetylated H4 and H3 at the 5' end of the GAPDH and CA genes, as well as the TK gene (49), is qualitatively similar to that mapped at the promoter regions of genes for which the recruitment of acetyltransferase-containing complexes on induction is well documented and the consequent targeted H3 acetylation (31), or H4 and H3 acetylation (33), has been demonstrated. This is in marked contrast to the extensive high level H4 and H3 acetylation found for the beta A-globin gene, which has the appearance of a promoter-targeted component lying on top of a high level continuum of acetylation throughout the gene and into the enhancer. Of the genes studied here, only the beta -globin transcripts produce a strong signal in a run-on assay, while the housekeeping gene GAPDH and the other tissue-specific gene, CA, are detected only weakly. Similarly, previous transcription through these genes, as seen from the transcript pool size (RT-PCR, Fig. 7A), also shows a much stronger signal for the beta A-globin gene than for the GAPDH or CA genes. Clearly in the 15-day erythrocytes the predominant active transcription is that from the beta A-globin gene and this may be related to the high levels of acetylation.

The observations of targeted acetylation suggest that when gene transcription first starts, hyperacetylation of H4 and H3 occurs in the promoter or CpG island region and persists as long as the gene is active. A possible explanation for this persistence would be that hyperacetylation of histones H4 and H3 is required for every re-initiation of a transcribed gene. Such a model is not the same as assuming that H4/H3 hyperacetylation plays the role of rendering the chromatin permissive for the binding of gene-specific primary transcription factors when transcription is first initiated (as implied by in vitro nucleosome binding assays (39-41)), because such factors presumably remain promoter-bound on active genes or at least are not required to re-bind at every re-initiation. It is quite possible that hyperacetylation of H4/H3 is important in both contexts, i.e. it is required for the initial binding of transcription factors and must also be maintained for subsequent re-initiations. Further experiments to determine the timing of acetylation during the assembly of transcriptionally competent complexes at promoters/enhancers/LCRs will be necessary to define the role of hyperacetylation at different stages of gene induction and continuing transcription.

    FOOTNOTES

* This work was made possible by the generous support of the Wellcome Trust.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 Present address: Dept. of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

§ To whom correspondence should be addressed. Tel.: 44-23-92842055, Fax: 44-23-92842053; E-mail: colyn.crane-robinson@port.ac.uk.

Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M009472200

2 F. A. Myers, D. R. Evans, A. L. Clayton, A. W. Thorne, and C. Crane-Robinson, our unpublished observations.

    ABBREVIATIONS

The abbreviations used are: kb, kilobase pair(s); bp, base pair(s); LCR, locus control region; HAT, histone acetyltransferase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase gene; CA, carbonic anhydrase gene; AUT, acetic acid/urea/Triton; IP, immunoprecipitation; ChIP, chromatin immunoprecipitation; RT-PCR, reverse transcriptase-polymerase chain reaction; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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