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
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ABSTRACT |
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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 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 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- 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 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 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 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.
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.
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.
The adult 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 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 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.
The pattern of histone acetylation at the 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
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 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 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 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
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 The correlation of the modification with chromatin structure at the
chicken 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.
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
A-globin gene. The extensive
high level acetylation at the
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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
globin
was hyperacetylated as well as the active adult
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 PDGF
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
-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
-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).
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).
-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
-globin locus, where the adult
gene does not have a CpG island but the embryonic
-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
A-globin gene for which the modification extends
throughout the gene and into the 3' enhancer.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A-globin and
housekeeping sequences showed them to be well represented in this
chromatin size class.
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
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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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.
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
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
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 -adult
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.
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 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.
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 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.
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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
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.
A-globin,
GAPDH, CA, and ovalbumin genes is different in
each case: throughout for
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
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.
A-globin and CA products. Qualitatively,
the 460-bp
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
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:
A-globin (
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
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
A-globin is more than 2 orders
of magnitude greater.
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
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
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%.
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.
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
A-globin gene does not have an island. The
continuous and extensive acetylation of the
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
-globin gene is also highly acetylated, as
seen from experiments using a hybridization probe (P5) that covers most
of the
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.
-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
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.
-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
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
-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
A-globin gene than for the
GAPDH or CA genes. Clearly in the 15-day erythrocytes the predominant active transcription is that from the
A-globin gene and this may be related to the high levels
of acetylation.
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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.
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.
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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.
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