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Nucleic Acids Research, Vol. 20, No. 5 1017-1022. Histone acetylation and globin gene switching. Tim R.Hebbes, Alan W.Thorne, Alison L.Clayton and Colyn ...
Nucleic Acids Research, Vol. 20, No. 5 1017-1022

Histone acetylation and globin

gene

switching

Tim R.Hebbes, Alan W.Thorne, Alison L.Clayton and Colyn Crane-Robinson* Portsmouth Polytechnic, Biophysics Laboratories, St Michaels Building, White Swan Road, Portsmouth P01 2DT, UK Received December 30, 1991; Revised and Accepted February 10, 1992

ABSTRACT An affinity-purified antibody that recognises the epitope E-acetyl lysine has been used to fractionate chicken erythrocyte mononucleosomes obtained from 5 and 15 day embryos. The antibody bound chromatin was enriched in multiply acetylated forms of the core histones H3, H4 and H2B, but not in ubiquitinated H2A. The DNA of these modified nucleosomes was probed with genomic sequences from the embryonic flQ gene (active at 5 days) and from the adult AA gene (active at 15 days). Both genes were found to be highly enriched in the acetylated nucleosomes fractionated from both 5 day and from 15 day erythrocytes. We conclude that globin switching is not linked to a change in acetylation status of the genes and that a 'poised' gene carries histones acetylated to a similar level as a transcriptionally active gene. Core histone acetylation is not therefore a direct consequence of the transcriptional process and might operate at the level of the globin locus as a general enabling step for transcription. INTRODUCTION The acetylation of specific lysine residues in the N-terminal domains of core histones has long been correlated with transcriptional activity (for reviews see references 1-3). High levels of this modification have recently been observed in CpG island chromatin (4). There is little known about the structural aspects of histone acetylation i.e. it is unclear whether the major consequences stem from the change in net charge resulting from acetylation or whether there are specific structural effects associated with acetylation at different lysine residues, as suggested by the non-random utilisation of the acetylation sites (5,6). A partial unwinding of the DNA superhelix in the core particle has however been documented when such particles are reconstituted using highly acetylated core histones (7,8). The functional role of histone acetylation has been studied using genetic experiments in the yeast Saccharomyces cerevisiae. It has been demonstrated that deletions of the amino terminal domain of the H4 gene prevent silencing of the yeast mating type genes (9) and that this domain is required for promotor activation of the GALl and PHOS genes in vivo (10). This region of H4

*

To whom correspondence should be addressed

contains the four lysines that are reversibly acetylated and their replacement with other amino acids resulted in activation being repressed as much, or in some cases even more, than observed following deletion of the whole N-terminal domain (10). A direct link between this post-translational modification and a gene in the process of active transcription was established for the chicken aD globin gene by selecting chromatin containing acetylated histones using an antibody recognising the epitope Eacetyl lysine (11) and then demonstrating the enrichment of a D globin sequences in the antibody bound chromatin (12). Core histone acetylation is one of a number of biochemical features of active chromatin (13). Other features include: 1) Generalised sensitivity to nuclease digestion, in particular DNase I (14). 2) The appearance of short regions hypersensitive to DNase I digestion (I5). 3) The demethylation of specific CpG dinucleotides (16). 4) Reduced levels (17) and altered binding of histone HI (18,19). 5) The presence of HMG 14/17 (20-22), ubiquitinated H2A (23) and ubiquitinated H2B (24). 6) The formation of nucleosomes with an altered crosslinking pattern (18) or an open conformation (25,26). Some of these features are closely linked to the transcriptional event, for example the transient opening of the nucleosome on induction of transcription (26). Generalised DNase I sensitivity however is observed well beyond the boundaries of the transcribed region and can include genes not in the process of actual transcription (27) but capable of being transcribed in the tissue concerned. Such genes are referred to as 'poised' (28), a category which can include those that were previously transcribed but are no longer engaged in active transcription. Examples of these are the genes in the chicken a and ,B globin loci, for which a switch in transcription from embryonic to adult genes occurs. In the present work we have used the chicken a globin locus to ask whether core histone acetylation is closely linked to developmentally regulated transcription, or whether it is also a feature of a poised gene. The experimental approach was to use an affinity purified antibody that recognises acetylated histones in order to select modified nucleosomes from the chromatin of both 5 day and 15 day chicken embryo erythrocytes and then probe the DNA of these two selected fractions with sequences from both the embryonic (Q gene and the adult ,8A gene. The data show that a poised gene carries acetylated nucleosomes in the same way as a gene in active transcription.

1018 Nucleic Acids Research, Vol. 20, No. 5

MATERIALS AND METHODS Preparation of antigen Chemically acetylated pig thymus histone H4 complexed with tRNA was used as immunogen. Histone H4 was prepared from 0.4 M HCl extracts of nuclei by gel filtration in Bio-gel P60 as described by Thorne et al., (29). Acetylation of lysine residues was achieved by treatment with acetic anhydride: H4 was dissolved at 0.5 mg/ml in 50 mM sodium bicarbonate pH 8.0 and acetylated with 2 mM acetic anhydride for 30 min on ice. The pH was reduced to 2.0 using HCI and the products lyophilised.

Antibody generation and purification Chemically acetylated histone H4 was complexed with tRNA (3:1 w/w) in 0.25 ml saline according to the method of Stollar and Ward (30). New Zealand White rabbits were inoculated with 1 mg of the antigen emulsified with an equal volume of a mixture of Freunds complete:incomplete adjuvant (8:2). This was injected into eight subcutaneous sites on the back of the animals. Booster injections were carried out in a similar way utilising incomplete adjuvant. The total Ig fraction of the rabbit serum was obtained by ammonium sulphate precipitation followed by DE 52 anion exchange chromatography (31). Affinity chromatography using chemically acetylated H4 coupled to Sepharose CL 4B was then used to select the anti-acetylated-H4 antibodies from the Ig population, as previously described (11).

Preparation of nuclei Blood was collected from 5 and 15 day old chicken embryo erythrocytes by vein puncture into phosphate buffered saline (PBS), 10 mM Na butyrate, 5 mM Na3 EDTA, 0.1 mM PMSF, 0.1 mM benzamidine. After washing in the above buffer omitting the Na3 EDTA, erythrocytes were isolated by centrifugation through lymphoprep (Nyegaard) cushions at 1000 r.p.m. for 40 min at 4°C. The erythrocyte cell pellet was resuspended in wash buffer: 80 mM NaCl, 10 mM Tris-HCl pH 7.5, 10 mM Na butyrate, 6 mM MgCl2, 0.1 mM PMSF, 0.1 mM benzamidine. Cells were lysed in 10 volumes of wash buffer supplemented with 0.1 % Triton X-100. Nuclei were pelleted at 3000 r.p.m. for 5 min, resuspended in wash buffer and purified by centrifuging through a 30% sucrose cushion in the same buffer at 4000 r.p.m for 5 min at 4°C. Preparation of mononucleosomes Nuclei were resuspended in digestion buffer: 10 mM NaCl, 10 mM Tris-HCl pH 7.5, 10 mM Na butyrate, 3 mM MgCl2, 1 mM CaCl2, 0.1 mM PMSF, 0.1 mM benzamidine, at a concentration of 5 mg/ml DNA and digested with 200 U/ml micrococcal nuclease (Porton) for 10 min at 37°C. The digestion was terminated with 5 mM Na3EDTA and the suspension chilled and immediately centrifuged at 13000 g for 20 seconds, retaining the supematant S1. The pellet was resuspended in lysis buffer: 10 mM Tris-HCl pH 7.5, 10 mM Na butyrate, 0.25 mM Na3 EDTA, 0.1 mM PMSF, 0.1 mM benzamidine, incubated on ice for 2 min and recentrifuged as above. The supematant S2 was combined with S1. The chromatin in these supernatants was depleted of HI by incubating with 30 mg/mil of Sephadex CM 25 and the addition of NaCl to a final concentration of 50 mM (32) for 1.5 hrs at 4°C under constant agitation. Following removal of resin/H 1 complexes, mononucleosomes were purified

by centrifuging the HI depleted chromatin through 14 ml 5-30% exponential sucrose gradients containing 10 mM Tris-HCI pH 7.5, 10 mM Na butyrate, 0.25 mM Na3 EDTA, 100 mM NaCl, 0.1 mM PMSF, 0.1 mM benzamidine at 40 k r.p.m. (Beckman SW40) for 18 hrs at 4°C. This nucleosomal preparation procedure typically releases greater than 80% of the nuclear DNA for the HI depletion step and 25% of this is recovered from the sucrose gradients as the monomer fraction.

Immunofractionation of mononucleosomes Immunofractionations were performed as previously described (12). Mononucleosomes containing 400 yg of DNA were incubated with 100 ,tg of affinity purified anti-acetylated-H4 antibody in 750 1l of incubation buffer: 50 mM NaCl, 10 mM Tris-HCl pH 7.5, 10 mM Na butyrate, 1 mM Na3 EDTA, 0.1 mM PMSF, 0.1 mM benzamidine, for 2 hrs at 4°C under constant agitation. To this was added 200 ,ul of a washed 10% w/v suspension of formalin fixed S. aureus cells (Gibco Immunoprecipitin). The suspension was incubated for 1 hr at 4°C under constant agitation. The immunocomplexes were collected by centrifugation (13000 g for 2 min) and the unbound fraction in the supernatant retained. Pellets were washed 5 times with 1 ml of incubation buffer by repeated centrifugation and resuspension. Chromatin and antibody bound to the S. aureus cells were released by resuspending the pellet in 150 1, of incubation buffer containing 1.5% SDS, followed by incubation at room temperature for 15 min S. aureus cells were removed by centrifugation and the released material (the antibody 'bound' fraction) retained. The cell pellet was washed with a further 150 yl of the incubation buffer containing 0.5% SDS. The supernatant obtained after centrifugation (12000 g for 2 min) was combined with the first. DNA from all chromatin fractions was obtained by two phenol/chloroform and one chloroform extraction followed by ethanol precipitation. The DNA was dissolved and treated with 50 itg/ml RNase A and subsequently with 50 Ag/ml proteinase K before repeating the extraction procedures. Proteins from all chromatin fractions were isolated from the phenol/chloroform phase of the initial phenol/chloroform extraction (33), by the addition of 1/100th the volume of 10 M HCl followed by precipitation with 12 volumes of acetone. Precipitates were washed twice with acidified acetone (6:1, acetone :100 mM HCl) and finally three times with dry acetone before drying under vacuum. Slot blots DNA samples, quantified by uv spectrophotometry, were denatured in 500 mM NaOH, 1.5 M NaCl for 10 min at 37°C and 1 minute at 100°C. Samples were then applied to HybondN filters (Amersham) using a slot blot manifold (Bio-Rad) using 0.5 ,tg DNA/slot. The filters were immersed in 500 mM NaOH, 1.5 M NaCl for 5 min and then in 0.5 M Tris-HCl pH 7.2, 1.5 M NaCl, 10 mM Na3EDTA for 30 seconds before blotting dry and crosslinking the DNA to the filters by exposure to uv irradiation.

Southern blots Chicken genomic DNA was restricted with either Bam HI or Bam 1tg per track of restricted DNA electrophoresed HI/Hind m andlO a I % through agarose gel. Following electrophoresis the gel was

Nucleic Acids Research, Vol. 20, No. 5 1019

incubated in 0.5 M NaOH, 1.5 M NaCl for 30 min and the DNA transferred to nylon membranes in 20 x SSC. After transfer the membranes were rinsed in 2 x SSC, blotted dry and the DNA crosslinked to the filters by exposure to uv irradiation.

and the histone to be extracted. The histones were analysed by electrophoresis in acetic acid/urea/Triton polyacrylamide gels (AUT PAGE) and the DNA was fixed onto nylon membranes for hybridisation analysis.

Hybridisations Filters were probed with sequences of either a 1.1 kb Sma IHind III genomic fragment of gA-globin (adult), a 1.35 kb Hpa II genomic fragment of (e-globin (embryonic) or a 850 bp cDNA sequence of ovalbumin. Probes were labelled by random priming to specific activities of 4-8 x 108 cpm./g. The filters were prehybridised for 16 hrs at 65°C in 6 x SSC, 5 x Denhardts solution, 0.1% SDS, 0.1% Na pyrophosphate, 5% dextran sulphate. Hybridisation was performed using 50 ng of labelled probe at a concentration of 10 ng/ml in 6 x SSC, 5 x Denhardts solution, 0.1 % SDS, 0.1% Na pyrophosphate, 5% dextran sulphate, 10 mM Na3EDTA at 65°C for 24 hrs. Alternatively filters were prehybridised for 30 mins and hybridised for 2 hours, using Quick Hyb solution (Stratagene) at 68°C. Following hybridisation, filters were washed as follows: twice in 2 x SSC, 0.1% SDS for 10 min at 42°C, once with 2xSSC, 0.1% SDS for 30 min at 65°C, once with 0.2 x SSC, 0. 1 % SDS for 30 min at 65°C and finally twice with 2xSSC, 0.1% SDS for 10 min at 42°C. All hybridisation and washing procedures were performed in bottles in a hybridisation oven (Hybaid). Filters were blotted dry and autoradiographed. Enrichment factors were obtained by flying spot densitometry of the exposed film.

Analysis of immunofractionated chromatin The immunofractionation procedure generates 8-20ig of antibody bound mononucleosomes. This represents 2-5% of the input chromatin, the remaining 95-98% being recovered in the unbound fraction. Histones. The proteins recovered from an immunofractionation of 15 day erythrocyte mononucleosomes are shown in Figure 1. Proteins in the nucleosomes selected by the antibody (Bound) are compared with those in the initial chromatin sample (Input) and those not selected by the antibody (Unbound). A high level of histone acetylation can be seen in the bound fraction, most obviously in histones H4 and H3, demonstrating that the antibody is indeed selecting highly-modified nucleosomes. In addition to the multiply acetylated forms of the two arginine-rich histones, the gel also shows a high level of acetylation of histone H2B. More than half of the histone H2B is spread approximately evenly over the mono, di, tri and tetra acetylated states of the histone, whilst the remainder is unmodified. This strikingly high level of H2B acetylation has not previously been noted in chromatin

Ig

Electrophoresis Proteins were analysed on 12% polyacrylamide/acetic acid/urea /Triton (AUT) gels as described by Bonner et al. (34) and stained with 0.1 % Coomassie Page 83 in 40% methanol, 10% acetic acid and destained in 7% acetic acid.

uH2A H2A

RESULTS The developmentally regulated switch in 3-globin gene synthesis during chicken embryogenesis has been studied in some detail (35 -37). The switch of expression from the embryonic to adult globin genes is coincident with a changeover in erythroid lineages. Primitive red blood cells predominate during the first week of development and these cells transcribe only the embryonic (Q and j,e genes and not the adult OA and OH (35). Between the 5th and 12th day the main transition occurs and several cell lineages may be present. After day 12 the erythrocyte population is dominated by the definitive lineage which transcribes the adult OA and OH genes and not the embryonic (3Q or 03E. At the time of hatching (22days) a secondary switch occurs when the transcription of OH is completely replaced by OA. In order to study the role of histone acetylation in the developmental expression of the (3-globin genes we therefore isolated erythrocyte nuclei from both 5 and 15 day embryos, i.e. from the primitive and definitive lineages respectively. Chromatin particles were prepared by micrococcal nuclease digestion and depleted of histone H1. Sucrose gradient purified mononucleosomes were mixed with affinity-purified antibody to allow the formation of immunocomplexes. Antibody/chromatin complexes were then immobilised by the addition of formalin-fixed S. aureus cells. After pelleting these cells, antibody bound chromatin was released with a solution containing SDS, thereby allowing both the DNA

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Figure 1. Acetic acid/urea/triton gel of proteins in chromatin samples from 15 day erythrocytes, before and after antibody fractionation. Input=histones in the input chromatin, Unbound=histones in the unbound supernatant and Bound=histones in the antibody bound fraction. A high level of acetylation is observed in histones H4, H3 and unexpectedly, H2B. There is no evidence of enrichment of uH2A in the antibody-bound chromatin. * indicates the multiply acetylated forms of the replacement histone H2A.Z overlayed on uH2B.

1020 Nucleic Acids Research, Vol. 20, No. S fractions enriched in transcriptionally active genes. It shows a somewhat different distribution amongst sub-species to that of histones H4 and H3, for which the more highly acetylated species predominate and there is very little unmodified histone. Histone H2A is about 50% mono-acetylated (data not shown). It is also apparent that the antibody bound material is not enriched in uH2A, an observation confirmed by silver staining of the gels (data not shown). Since we know that the antibody bound chromatin includes that from actively transcribing genes (12), this result indicates that transcribed chromatin is not enriched in ubiquitinated histone H2A. This accords with the findings of Dawson et al., (38) who detected no differential distribution of uH2A between the DNase I sensitive and bulk chromatin fractions. Multiple bands in the antibody bound fraction are also observed in the region between histones H2A and H3 (marked with an asterisk * in Figure 1). These are more clearly visible in the silver stained tracks of both 5 and 15 day antibody bound material (data not shown). By comparison with the observations of Ridsdale and Davie (39) on salt fractionated chromatin we assign these bands to multiply acetylated forms of the H2A.Z variant, overlayed on top of uH2B. This variant of H2A is unusual in having not one but several sites of acetylation (40). DNA analysis. The sequence content of the DNA isolated from the input, unbound and antibody bound fractions from 5 and 15 day cells was assessed by hybridisation analysis with two ,B-globin genes, OA (adult) and fe (embryonic) and with the inactive ovalbumin gene. For analyses, 0.5,4g aliquots of each of the DNAs from both 5 and 15 day erythrocytes were applied to nylon membranes using a slot blot manifold. If a gene carries highly acetylated histones it will be selected by the antibody and so the DNA in the bound fraction will produce a more intense signal

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than that from in the input DNA. A test of the fractionation procedure is the noticeable depletion observed for the unbound DNA fractions, relative to the input, for genes selected by the antibody. The signal ratio between the unbound and input DNA samples represents the proportion of the gene sequences which remain unselected by the antibody. In these experiments this averages about 20%, a figure which is to be regarded as an overestimate since background signal from 0.5itg of salmon sperm DNA is significant when compared with that from the unbound DNA. The ratio of signal obtained between the bound and input DNA (B/I) is a measure of the enrichment for that particular gene. This ratio is determined both by the proportion of that gene selected and also by the total sum of all genes selected, since a fixed amount of DNA is loaded in each slot. At 5 days the embryonic 13Q gene is active and on the basis of our earlier results with the active a!D adult gene (12) we expected to find an enrichment of the transcribed sequences in the acetylated chromatin. The hybridisation results using the embryonic flQ-globin as probe are shown in the left hand panel of Figure 2A. High enrichments ( =40 fold) were obtained for the DNA of the antibody bound chromatin from 5 day erythrocytes as expected. In 15 day erythrocytes however, the flQ gene is silent and yet the hybridisation analysis demonstrates a comparable enrichment ( = 30 fold) showing that the chromatin containing this gene is also acetylated. When an identical membrane was probed with the adult OA gene (right hand panel of Fig 2A), a similar result was obtained, i.e. the chromatin selected from the 15 day erythrocytes (in which the gene is active) is again enriched (estimated at 20-30 fold) as expected. In addition, the chromatin from the 5 day erythrocytes also shows a high enrichment ( = 50 fold) with the OA probe. Identical results were obtained on reprobing these same filters with the opposite probe (data not shown).

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Figure 2. Hybridisation analysis of mononucleosomal DNA from chicken erythrocyte chromatins. Equal quantities (0.5Ag) of DNA were immobilised in each slot. Probes were labelled with 32P by random priming. Input chromatin 'I' is that prepared from nuclei using micrococcal nuclease. 'I' is fractionated into the Bound component 'B' (about 3-5% of 'I') and the Unbound supernatant 'U'. Total 'T' is genomic DNA reduced by sonication to about 400 bp length. Antibody generated enrichments of the sequences probed for can be seen as B to I intensity ratios. Concomitant depletion of the supernatants 'U' is also evident for genes carrying acetylated histones. PROBES: Fig 2A. Left hand panel, chicken flQ globin, genomic, Hpa II fragment 1.35 kb. Right hand panel, chicken OA globin, genomic, Sma I-Hind III fragment, 1. 1kb. Shaded boxes represent exons. Fig 2B: chicken ovalbumin cDNA, 850bp fragment. High enrichments of both (LQ and 13A sequences are seen in the antibody bound chromatin at both 5 and 15 days, relative to the input. No enrichment of ovalbumin sequences is seen in the antibody bound chromatin.

Nucleic Acids Research, Vol. 20, No. 5 1021 In contrast, when DNA fractions from both 5 and 15 day chromatins were probed with the ovalbumin gene (permanently repressed in this tissue) no enrichments in the antibody bound fractions were observed (Fig. 2B). Figure 2 also shows the hybridisation signals from 0.5Ag of total (T) chicken genomic DNA, sonicated to reduce it to about 400 bp. The somewhat lower signal often obtained from the input (I), when compared with total (T), is partially due to the shorter DNA length of mononucleosomes, but also due variations in micrococcal nuclease digestions. We note that significant differences in the input signal can be obtained from different micrococcal nuclease digests. Samples showing a higher hybridisation signal from the input material produce significantly lower enrichments (B/I) on antibody fractionation, as might be expected. This phenomenon underlies the higher enrichments found for the 5 day chromatin when probed with either of the globin genes. Since there is a significant level of homology between the coding regions of the four ,B globin genes the specificity of the OA and (e probes was tested by southern blot hybridisations, the results of which are presented in Figure 3. Using a single Bam HI digest only one of the four possible restriction fragments shows strong hybridisation with each probe. Lack of cross hybridisation between genes active at 5 days (fQ and (3E) and those active at 15 days (H and A) iS confirmed with the double digest using Bam HI and Hind III. These data clearly show that in erythrocytes the chromatin of the OA and 3Q-globin genes contains highly acetylated histones at both stages of development, irrespective of their transcriptional status. In 5 day erythrocytes the adult 1A gene is non-transcribed and furthermore since the clone of cells is different from those dominating at 15 days, the jA gene will never be transcribed in the 5 day cells. Nevertheless, it carries acetylated histones. The 3Q gene in 15 day erythrocytes could be considered as in the 'previously transcribed' category, although the clone of cells dominating at 15 days is different from those dominant at 5 days (35). This means that in the erythrocytes taken from 15 day embryos, the fQ gene probably has never been transcribed. Nevertheless it is observed to carry acetylated histones. We

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therefore conclude that histone acetylation is established in 'poised' as well as in actively transcribing chromatin. The simplest interpretation of these results would be to suppose that the whole of the 13-globin locus contains acetylated histones in terminally differentiated erythrocytes, both of the primitive and definitive cell lines.

DISCUSSION The chromatin selected on the basis of its histone acetylation is shown to include that of both transcriptionally active and poised genes. The simplest conclusion is to assume that both these classes of 'active' chromatin contain highly acetylated histones H3, H4 and H2B, (with histone H2A being 50% acetylated at its single modified site). Whilst it may indeed be that highly modified forms of all four core histones are present in active chromatin, it is formally possible that since the chromatin was selected on the basis of its acetylation, only a subset of this contains the 'active' sequences. For example, the 'active' chromatin might carry only acetylated histones H3 and H4, whilst a different chromatin subset might include the acetylated histones H2B and H2A. Without a more selective antibody however, it is not yet possible to draw such distinctions. Since the erythrocytes from both 5 and 15 day embryos are terminally differentiated, it seems unlikely that any of the acetylation in the chromatin selected by the antibody is linked to replication, i.e. is deposition related acetylation. The enrichment of the replacement histone H2A.Z in the antibody bound fraction is fully consistent with the expectation that replacement core histones accumulate within transcriptionally active regions and therefore acquire high levels of acetylation. As regards the level of uH2A in the antibody selected chromatin, the straightforward conclusion to be drawn is that uH2A is not enriched in active gene loci. However, we cannot exclude the possibility that within an active gene locus there are nucleosomes that carry a uH2A molecule but which are not acetylated and therefore not selected by the antibody. This seems unlikely however, bearing in mind that acetylation appears to be rather widely spread over the globin locus in erythrocytes, rather than concentrated at particular points. Although the four enrichments measured for the 5 and 15 day chromatins with the (BQ and OA probes are not the same, the experimental variation in hybridisation signals that occur between experiments means that we cannot reliably distinguish the enrichments achieved for active genes ((Q and OA probes with 5 and 15 day erythrocytes, respectively) from those for poised genes ((IQ and OA probes with 15 and 5 day erythrocytes, respectively). We therefore conclude at present that the level of core histone acetylation in the (Q and OA globin genes is similar, that this acetylation is already present at 5 days and does not change by 15 days. Since there are different clones of cells predominating at 5 and 15 days (the primitive and definitive lines) then both clones must have a similar level of acetylation at both genes. It follows that the switch in transcription from (Q to OA is not a result of a switch in the acetylation status of these genes. One may also conclude that acetylation is not a direct consequence of active transcription through a gene, since the adult OA gene is acetylated in the 5 day embryos in which it has never been transcribed. It must be noted however, that for this conclusion to be drawn with complete certainty, one must be sure that not a single polymerase has passed through the OA gene in the 5 day erythrocytes, a point difficult to demonstrate experimentally.

1022 Nucleic Acids Research, Vol. 20, No. 5 We have considered the possibility that the presence of the enhancer, close to the 3' end of the A gene, which is active at both stages of development (41,42) may be responsible for the acetylation of nucleosomes in the immediately adjacent region. This might give rise to the observed acetylation of the adult ,B gene at both stages of development. However, the OlA probe terminates within the second intron at a distance of =700 bp from the enhancer. Furthermore one would not expect such an effect on chromatin structure to extend as far as the embryonic 13Q gene = 9 Kbp upstream. Therefore this is unlikely to be the cause of the observed acetylation of the (Q gene in the erythrocytes of 15 day embryos. If both the flQ and (A genes are acetylated in both the 5 and 15 day erythrocytes, it might therefore be that core histone acetylation exists over the whole of the globin locus in erythrocytes, i.e. the acetylation of histones at the globin locus represents a marker of an erythroid cell. Since general DNase I sensitivity covers a wide region of the globin locus in erythroid cells and is also characteristic of poised as well as active genes, it might be that these two features of active loci are linked. More detailed mapping of acetylation will be needed to establish whether such a relationship exists. Monitoring of : globin expression in chicken erythrocytes has led to the conclusion that cells first become committed to expression of the globin gene families and only later select specific globin genes for expression (35).Core histone acetylation would appear to be linked to establishing the overall transcriptional competence rather than mediating the switching of the individual genes.

ACKNOWLEDGEMENTS We acknowledge the financial support of the Cancer Research Campaign of Great Britain during the course of this work.

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