From the Molecular and Cellular Pharmacology Program,
Department of Pharmacology, University of Wisconsin Medical School,
Madison, Wisconsin 53706, the § Department of Pathology
and Immunology and Developmental Biology Program, Washington University
School of Medicine, St. Louis, Missouri 63110, and the
¶ Lineberger Comprehensive Cancer Center, Department of
Biochemistry and Biophysics, University of North Carolina,
Chapel Hill, North Carolina 27599
Received for publication, January 27, 2003, and in revised form, February 24, 2003
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ABSTRACT |
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Post-translational modifications of individual
lysine residues of core histones can exert unique functional
consequences. For example, methylation of histone H3 at lysine 79 (H3-meK79) has been implicated recently in gene silencing in
Saccharomyces cerevisiae. However, the distribution and
function of H3-meK79 in mammalian chromatin are not known. We found
that H3-meK79 has a variable distribution within the murine Post-translational modification of core histones in chromatin
represents a common epigenetic mechanism that controls diverse nuclear
processes. An almost endless number of histone modifications has been
documented, and it has been hypothesized that a histone code exists in
which unique combinations of histone modifications confer distinct
functional consequences (1). Acetylation of core histones enhances
factor access to the chromatin template by increasing accessibility of
nucleosomal DNA (2, 3). Increased accessibility does not appear to be
accompanied by a major change in nucleosome structure in
vitro (4, 5). However, a 3-fold increase in acetylation strongly
inhibits folding of chromatin fibers into higher order structures (6).
Furthermore, the acetylated amino-terminal tails of core histones are
recognized by bromodomains (7, 8), which are often present in
coactivators that mediate transcriptional activation. Although the
structure of acetylated chromatin and the functional implications of
acetylation have been studied for many years (9), considerably less is
known about other histone modifications such as methylation.
The concept of a histone code is illustrated by methylation of histone
H3 at amino acids 4 and 9. Histone H3 methylated at lysine 9 (H3-meK9),1 but not at lysine
4 (H3-meK4), is selectively recognized by heterochromatin protein 1 (HP1) (10-14). HP1 serves an important role in the assembly of
repressive chromatin structures (15), thus linking a site-specific histone modification to heterochromatin formation. By contrast, H3-meK4
is preferentially enriched in transcriptionally active chromatin
(16-18), including transcriptional regulatory elements such as the
murine In addition to acetylation and methylation of Lys-4 and Lys-9 within
the amino terminus of histone H3, H3 is modified in the central
globular domain by methylation at Lys-79 (26-28). Lys-79 methylation
is catalyzed by the histone methyltransferase disruptor of telomeric
silencing 1 (DOT1) in yeast (26-28) and DOT1-like protein (DOT1L) in
humans (29). Methylation of H3 at Lys-79 requires Rad6-mediated
ubiquitination of Lys-123 of H2B (H2B-K123) (30, 31), analogous to the
coupling of H2B ubiquitination and methylation of H3 at Lys-4 (32).
DOT1 was identified by a genetic screen in yeast to isolate genes that
disrupt telomeric silencing (33). DOT1 was also identified as a
regulator of the pachytene checkpoint, which ensures proper chromosome
segregation during the meiotic cell cycle (34). Mutation of Lys-79 and
mutations that abrogate DOT1 catalytic activity impair telomeric
silencing (26, 27), indicating that DOT1 mediates telomeric silencing via methylation of Lys-79. Taken together with results that H3-meK79 is
abundant in bulk yeast histones and is deficient from telomeres, it
has been proposed that H3-meK79 excludes silent information regulator
(SIR) complexes (27). SIR complexes assemble at telomeres and mediate
heterochromatin formation (35). The model assumes that H3-meK79
excludes SIR proteins from the bulk of genomic DNA, thus yielding
sufficient amounts of SIR proteins to assemble repressive complexes at
heterochromatic sites.
The studies described above have led to a compelling model to explain
the functional consequences of H3-meK79 in yeast. However, given the
considerably larger size of mammalian genomes versus the
yeast genome, it would be striking if all active chromatin regions
require H3-meK79 to prevent SIR-mediated heterochromatization. The
distribution of H3-meK79 in mammalian chromatin has not been determined. Thus, we developed a quantitative chromatin
immunoprecipitation (ChIP) assay to measure H3-meK79 levels at a
mammalian chromatin domain, the erythroid-specific murine Cell Culture--
MEL (36) and p45/NF-E2-null CB3 (37) cells
were maintained in Dulbecco's modified Eagle's medium (Biofluids)
containing 1% antibiotic/antimycotic (Invitrogen) and 5% calf serum,
5% fetal calf serum (MEL), or 10% fetal calf serum (CB3). Stably
transfected clones of CB3 cells expressing p45/NF-E2 (WT) (38) were
selected and maintained in 1 mg/ml G418 sulfate (Calbiochem). Cells
were grown in a humidified incubator at 37 °C in the presence of 5% carbon dioxide.
Chromatin Immunoprecipitation Assay--
ChIP was performed as
described (18, 19, 39). Erythroid maturation of MEL cells was induced
by treatment of 0.5 × 105 cells/ml with 1.5%
Me2SO (Sigma) for 4 days. Aliquots of immunoprecipitated chromatin (1.5 µl) were analyzed by real time PCR as described (18,
19, 39). Primers were designed by Primer Express R 1.0 software (PE
Applied Biosystems) to amplify 50-150-bp products. Samples from at
least two independent immunoprecipitations were analyzed.
ChIP primer pairs to amplify Antibodies--
Anti-acetylated histone H3 (06-599) and
anti-H3-meK4 (07-030) antibodies were obtained from Upstate
Biotechnology, Inc. Anti-H3 dimethyl Lys-79 was described previously
(26). Preimmune rabbit serum served as a control for the specific antibodies.
RT-PCR Analysis--
Total RNA was prepared from the same CB3
and WT cells used for ChIP with Trizol (Invitrogen). cDNA was
synthesized by annealing RNA (1 µg) with 250 ng of a 5:1 mixture of
random and oligo(dT) primers by heating at 68 °C for 10 min. After
denaturation, samples were incubated with reverse transcriptase
(Superscript II, Invitrogen) combined with 10 mM
dithiothreitol, RNasin (Promega), and 0.5 mM dNTPs for
1 h at 42 °C. The reaction mixture was diluted to a final
volume of 150 µl and heat-inactivated at 95-99 °C for 5 min.
Quantitative real time RT-PCRs (25 µl) contained 2.5 µl of
cDNA, 12.5 µl of SYBR Green (Applied Biosystems), and the
appropriate primers. Product accumulation was monitored by SYBR Green
fluorescence. Relative expression levels were determined from a
standard curve of serial dilutions of MEL cDNA samples. Forward and
reverse primers for real time RT-PCR (5'-3') are as follows:
Protein Analysis--
Lysates were prepared by boiling cells for
5 min in SDS sample buffer (50 mM Tris (pH 6.8), 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10%
glycerol). Extracts from 1 × 105 cells were resolved
on SDS-polyacrylamide gels, transferred to Immobilon P membranes
(Millipore), and analyzed by Western blotting as described (18). Blots
were incubated with anti-p45/NF-E2 antibody (40), followed by protein
A-peroxidase (Sigma) and detected via chemiluminescence with an
ECL-plus kit (Amersham Biosciences).
Differentiation of Murine Embryonic Stem Cells into Primitive
Erythroid Colonies--
Murine ES cells were differentiated into EryP
as described (41, 42). Briefly, day 4 embryoid bodies were collected,
dissociated with trypsin, and replated at 1 × 105/ml
in methylcellulose (Sigma) containing 10% plasma-derived serum (Antech, Tyler, TX), 5% protein-free hybridoma medium (PFHM2, Invitrogen), L-glutamine (2 mM), transferrin
(300 µg/ml; Roche Molecular Biochemicals), and monothioglycerol
(4.5 × 10 Quantitative Analysis of H3-meK79 Levels in Mammalian
Chromatin--
A quantitative ChIP assay was developed to measure
H3-meK79 levels in mammalian chromatin (Fig. 2). We initially analyzed the distribution of H3-meK79 within the erythroid-specific
Analysis of H3-meK79 at functionally distinct sites of the
To gain a broader perspective of how H3-meK79 is distributed in
mammalian chromatin, we measured H3-meK79 at other active and inactive
genes (Fig. 4). H3-meK79 levels were high
at both the promoter and exon 2 of the broadly expressed cad
gene. By contrast, upstream sequences, promoters, and exonic sequences from the inactive neuronal-specific necdin gene and the
muscle-specific MyoD1 gene had very low levels of H3-meK79.
Thus, taken together with the Dynamic Regulation of H3-meK79 at the Endogenous Murine
Similar to the
We have shown that acetylated H3, H3-meK4, and to a lesser extent,
acetylated H4 are induced by p45/NF-E2 at the
Because H3-meK79 was present at HS2 in induced MEL cells, albeit at low
levels (Fig. 3), we also tested whether p45/NF-E2 is important for
establishing H3-meK79 at HS2. However, H3-meK79 at HS2 was insensitive
to p45/NF-E2. Similarly, p45/NF-E2 only slightly affected the levels of
H3-meK4 and acetylated H3 at HS2, consistent with our previous results
(18, 45). Taken together with the results of Fig. 5, H3-meK79 levels
are highly regulated at the Mechanistic Insights Arising from the H3-meK79 Pattern in Mammalian
Chromatin--
The distribution of H3-meK79 in mammalian chromatin had
not been measured previously. Our results on the distribution of
H3-meK79 at functionally distinct sites of mammalian chromatin are
summarized in Fig. 7. Results described
herein support a model in which H3-meK79 is highly enriched in
mammalian chromatin at active genes. Conversely, inactive genes are
characterized by low level or no detectable H3-meK79. Because our
results demonstrate that H3-meK79 is dynamically regulated, resembling
the other functionally important modifications H3-meK4 and acetylated
H3, we propose that H3-meK79 and, importantly, enzymes that establish
H3-meK79, regulate
It is instructive to relate our results to the current model for the
function of H3-meK79 derived from studies in yeast (27). Genetic and
biochemical analysis provided evidence that DOT1 methylates H3 at
Lys-79 and is required for telomeric silencing (26-28). As summarized
in the Introduction, it was proposed that H3-meK79 excludes SIR
silencing complexes, which are critical for heterochromatin formation
(27). Our results showing that inactive genes have low or undetectable
levels of H3-meK79 are consistent with this model. However, the
variable distribution of H3-meK79 throughout the mammalian
Why would the central subdomain and HS2 have such low levels of
H3-meK79 in induced MEL cells? The central subdomain also has low
levels of acetylated histones H3 and H4 (19, 20) and H3-meK4 (18), is
inaccessible to restriction endonucleases, and contains sites highly
modified by CpG dinucleotide methylation (19). The histone acetylation
pattern of MEL cells is physiologically relevant, because it is similar
to that of normal adult erythroid cells of the 14.5-day post-conception
murine fetal liver (20). It is unknown whether SIR proteins are
components of the repressive chromatin structure of the central
subdomain in erythroid cells. The low level H3-meK79 at the central
subdomain might allow SIR proteins to localize to the central
subdomain, thereby facilitating assembly of repressive complexes to
establish and maintain the silent state of the embryonic
Based on the recent cloning of human DOT1L, which catalyzes methylation
of histone H3 at Lys-79 (29), and the dynamic regulation of H3-meK79 at
the
In summary, we have described the first analysis of the distribution of
H3-meK79 in mammalian chromatin. This analysis in adult and embryonic
erythroid cells yielded patterns that were not predictable from prior
studies in yeast. H3-meK79 was considerably more enriched at the
-globin
locus in adult erythroid cells, being preferentially enriched at the
active
major gene. By contrast, acetylated H3 and H4 and
H3 methylated at lysine 4 were enriched both at
major
and at the upstream locus control region. H3-meK79 was also enriched at
the active cad gene, whereas the transcriptionally inactive
loci necdin and MyoD1 contained very little
H3-meK79. As the pattern of H3-meK79 at the
-globin locus differed
between adult and embryonic erythroid cells, establishment and/or
maintenance of H3-meK79 was developmentally dynamic. Genetic complementation analysis in null cells lacking the erythroid and megakaryocyte-specific transcription factor p45/NF-E2 showed that p45/NF-E2 preferentially establishes H3-meK79 at the
major promoter. These results support a model in which
H3-meK79 is strongly enriched in mammalian chromatin at active genes
but not uniformly throughout active chromatin domains. As H3-meK79 is
highly regulated at the
-globin locus, we propose that the murine
ortholog of Disruptor of Telomeric Silencing-1-like (mDOT1L)
methyltransferase, which synthesizes H3-meK79, regulates
-globin transcription.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-globin locus control region (LCR) (Fig. 1) (18, 19).
However, H3-meK4 is not uniformly present throughout the active
-globin locus in adult murine erythroid cells (18). The central
portion of the locus, which contains the Ey and
H1 embryonic
-globin genes and is characterized by
broad, low level acetylation (19-21), also has low level H3-meK4. H3
dimethylated at Lys-4 is enriched within coding regions of transcribed
genes in yeast, and this modification is established by the
methyltransferase Set1 (22). A more recent analysis in yeast revealed
that H3 dimethylated at Lys-4 was enriched at active and inactive
genes, whereas H3 trimethylated at Lys-4 marked only active genes (23). The functional consequences of H3-meK4 include inhibiting interaction of the nucleosome remodeling and deacetylase repressor complex with the
amino terminus of H3; H3-meK9 does not prevent nucleosome remodeling
and deacetylase repressor complex binding (24, 25).
-globin
locus, and at other functionally distinct sites of mammalian chromatin.
Although the distribution of H3-meK79 at these sites is consistent with
the model in which H3-meK79 is enriched at active promoters,
surprisingly, H3-meK79 levels were highly variable throughout the
active
-globin domain in adult erythroid cells. As H3-meK79 levels
were dynamically regulated by the hematopoietic activator p45/NF-E2 and
differed between adult and embryonic erythroid cells, H3-meK79 is
likely to be an important determinant of
-globin gene expression.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-globin locus sequences were based on
Hbbd haplotype sequences (GenBankTM accession
numbers Z13985, X14061, AF128269, and AF133300). Real time PCR primers
(5'-3') are as follows: HS2, AGTCAATTCTCTACTCCCCACCCT and
ACTGCTGTGCTCAAGCCTGAT; IVR3, TGTGCTAGCCTCAAGCTCACA and
TCCCAGCACTCAGAAGAAGGA;
major, CAGGGAGAAATATGCTTGTCATCA
and GTGAGCAGATTGGCCCTTACC;
major I2,
CTTCTCTCTCTCCTCTCTCTTTCTCTAATC and AATGAACTGAGGGAAAGGAAAGG; necdin 5', TTCAGTAGCTGATGCCCAGGT and
GGGAGGATACCAGAGATGGGA; necdin promoter, GGTCCTGCTCTGATCCGAAG
and GGGTCGCTCAGGTCCTTACTT; necdin 3',
TTCAACTGGCACAGGAAGCA, AACAGTCCAGTTCAAATCAGTCCAT; MyoD1
5', CCAGATCTCAGTGCTGCAGG and CCGCTTGCATAGCATAACCAG;
MyoD1 promoter, GGGTAGAGGACAGCCGGTGT and
GTACAATGACAAAGGTTCTGTGGGT; MyoD1 3', TACCCAAGGTGGAGATCCTGC and GCAGACCTTCGATGTAGCGGAT; cad,
TTCTAACTTGACCGGCTGGTTT and GGACCATAGGATGGTTCCACAG; and cad
exon2, GCCCTCACTGACCCTTCCTAC and TTGCCGATGAGAGGATACGTT.
-globin, CAGCCTCAGTGAGCTCCACTG and GATCATATTGCCCAGGAGCC; and
glyceraldehyde-3-phosphate dehydrogenase, GAAGGTACGGAGTCAACGGATTT and GAATTTGACCATGGGTGGAAT.
4 M), together with 2 units/ml
erythropoietin (Amgen, Thousand Oaks, CA). Primitive erythroid colonies
were collected 4 days later via digestion with cellulase (1 unit/ml)
for 20 min at 37 °C. Cells were resuspended in culture media,
cross-linked with 0.4% formaldehyde for 10 min at room temperature,
and analyzed by ChIP as described above with the cell lines.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-globin locus (Fig. 1) in
Me2SO-induced MEL cells, which express high levels of the
adult
-globin gene
major. The cross-linked chromatin fragments used for ChIP had an average size of 400-500 bp (Fig. 2A), and assays were conducted
under linear conditions (Fig. 2B). Representative
amplification curves for the detection of H3-meK79 at HS2 of the LCR
and at the
major promoter are shown in Fig. 2C. Thermal dissociation of the PCR products revealed a
homogenous dissociation curve (Fig. 2D), indicative of a
single major PCR product for each primer set.
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Fig. 1.
Organization of the murine
Hbbd -globin locus. The
murine
-globin locus contains embryonic (Ey and
H1) and adult (
major and
minor)
-globin genes.
-Globin genes are shown as
boxes and HSs as spheres. HS, DNase
I-hypersensitive site. The lower bars indicate H3- and
H4-hyperacetylated zones.
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Fig. 2.
Quantitative ChIP analysis of H3-meK79
at the murine -globin locus.
A, agarose gel analysis of sonicated, deproteinized
chromatin used in the ChIP assay. Chromatin fragments smaller than 1 kb, averaging ~400-500 bp, were visualized by ethidium bromide
staining. B, standard curves of SYBR Green fluorescence
signals obtained from dilutions of input DNA from induced MEL cells
using HS2 and
major promoter specific primer pairs.
C, representative amplification plot of H3-meK79 at HS2
and
major promoter in induced MEL cells; PI,
preimmune. D, dissociation plot obtained from amplicon
shown in C. The single peak indicates the generation of
single amplicon for each primer pair.
-globin
locus (Fig. 3A) revealed
strong enrichments at the active
major promoter and
within the
major gene at intron 2 (Fig. 3B). By contrast, H3-meK79 levels were low at HS2, at the inactive
H1 promoter, and at the representative intergenic site
IVR3. IVR3 does not contain known functionally relevant sequences.
Acetylated H3 was high at HS2, at the
major promoter, and
at
major intron 2. The fact that H3-meK79 was
considerably lower at HS2 versus the
major
promoter was surprising, because we showed previously (18-20) that
other histone modifications, notably acetylated H3, acetylated H4, and
H3-meK4, are strongly enriched at both the HS2 and the
major promoter. Based on the fact that ~90% of yeast histone H3 is methylated at Lys-79, and based on the SIR localization model (27), we reasoned that H3-meK79 might be uniformly enriched throughout the active
-globin domain. Rather, H3-meK79 had a highly
variable distribution, paralleling the distribution of acetylated H3,
with the exception that H3-meK79 was low at HS2 (Fig.
3B).
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Fig. 3.
Acetylated histone H3 and H3-meK79 have
distinct patterns at the -globin locus of
Me2SO-induced MEL cells. A,
organization of the murine
-globin locus. B,
cross-linked chromatin was isolated from Me2SO-induced MEL
cells. The relative levels of H3-meK79 and H3 acetylation were measured
by quantitative ChIP analysis at various sites of the endogenous murine
-globin locus (mean ± S.E., at least three independent
experiments). Note that the first four graphs have a common
y axis with the units indicated on the left, and
the graph depicting
maj I2 data has a distinct
y axis. Pro, promoter; I2, intron 2;
PI, preimmune; acH3, acetylated H3.
-globin results of Fig. 3, H3-meK79
levels are highest at active genes.
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Fig. 4.
H3-meK79 is preferentially enriched at
transcriptionally active genes. Cross-linked chromatin was
isolated from Me2SO-induced MEL cells. The relative levels
of H3-meK79 and H3 acetylation were measured by quantitative ChIP
analysis at cad promoter, cad exon 2, upstream
from necdin (necdin 5'),
necdin promoter, necdin exon 1, upstream of
MyoD1 (MyoD1 5'), MyoD1 promoter, and
MyoD1 exon 1. Mean ± S.E. of at least three
independent experiments. Pro, promoter; E1, exon
1; E2, exon 2; 5', 5' upstream; PI,
preimmune; acH3, acetylated H3.
-Globin
Locus--
We showed previously (20) that the central region of the
murine
-globin locus, characterized by low level histone acetylation in adult erythroid cells, is highly acetylated in embryonic erythroid cells of a 10.5-day post-conception murine yolk sac. This result indicated that the acetylation pattern of the
-globin locus is developmentally dynamic. Accordingly, we hypothesized that
establishment of broad low level acetylation at the central subdomain
of the
-globin locus, which contains the embryonic Ey and
H1 genes, was required for silencing of the embryonic
-globin genes. Based on the low level H3-meK79 at the
H1 promoter and at IVR3, both residing within the central
subdomain, we asked whether establishment and/or maintenance of
H3-meK79 is developmentally dynamic. Rather than using yolk sac as in
our previous analysis, we used primitive erythroid cells derived from
murine ES cells via in vitro differentiation (Fig.
5A). This in vitro
differentiation system recapitulates regulatory events occurring during
normal erythropoiesis (41-44). As large numbers of embryonic
erythroid cell colonies, EryP, can be readily differentiated from ES
cells, and it is difficult to obtain large numbers of primary embryonic
erythroid cells from murine yolk sac, the ES cell system has major
advantages for analyzing the chromatin structure of the
-globin
locus during embryonic erythropoiesis. No ChIP analyses have been
described using specific cell types differentiated from ES cells.
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Fig. 5.
In contrast to Me2SO-induced MEL
cells, H3-meK79 is broadly distributed throughout the
-globin domain in primitive erythroid cells derived
from murine embryonic stem cells. A, EryP cells
were generated via in vitro differentiation of murine ES
cells. B, cross-linked chromatin was isolated from
EryP, and the relative level of H3-meK79 was measured by quantitative
ChIP analysis (mean ± S.E. of two independent experiments).
Pro, promoter; PI, preimmune.
-globin locus of induced MEL cells (Fig. 3), H3-meK79
was enriched at the
major promoter in EryP cells (Fig. 5B). Despite the fact that these cells are embryonic, EryP
express low levels of
major (data not shown). By contrast
to the results with induced MEL cells (Fig. 3), H3-meK79 was high at
IVR3 and the active
H1 promoter. Only low levels of
H3-meK79 were apparent at the inactive necdin promoter,
whereas H3-meK79 was enriched at the active cad promoter,
comparable to induced MEL cells. Thus, H3-meK79 was developmentally
dynamic at IVR3 and the
H1 promoter, being high in EryP (embryonic
erythroid cells) and low in induced MEL cells (adult erythroid cells).
The developmentally specific H3-meK79 patterns suggest an important
regulatory role for H3-meK79 in controlling
-globin expression
during erythropoiesis. H3-meK79 levels at HS2 were 2.8-fold greater in
EryP versus induced MEL cells. However, H3-meK79 at HS2 in
EryP was much lower than at the active
H1 promoter,
analogous to the result in induced MEL cells, in which H3-meK79 was
much lower at HS2 versus the active
major promoter.
major
promoter in CB3 erythroleukemia cells, which lack p45/NF-E2 (18, 45). Given the developmental modulation of H3-meK79, we tested whether establishment of H3-meK79 at the
major promoter is
p45/NF-E2-dependent (Fig. 6).
H3-meK79 levels were compared in CB3 cells lacking or stably expressing
physiological levels of the two p45/NF-E2 isoforms (Fig.
6A). Expression of p45/NF-E2 in CB3 cells strongly activated
-globin transcription as expected (Fig. 6B). ChIP
analysis revealed that H3-meK79 was only high at the
major promoter when p45/NF-E2 was expressed (Fig.
6C). Similarly, establishment of H3-meK4 and acetylated H3
at the promoter was p45/NF-E2-dependent, consistent with
our previous results (18, 45).
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Fig. 6.
p45/NF-E2-dependent establishment
of H3-meK79 at the major promoter but not at
HS2. A, Western blot analysis of p45/NF-E2
expression in total cell lysates from CB3, CB3-WT21, and
Me2SO-induced MEL cells. The arrows depict two
p45/NF-E2 isoforms, which result from usage of distinct translation
start sites. No functional differences have been ascribed to the two
isoforms. B, real time RT-PCR analysis of
-globin
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA
expression in CB3 and CB3-WT21 cells. C, relative
levels of H3-meK79, H3-meK4, and H3 acetylation were measured by
quantitative ChIP analysis at HS2, IVR3,
H1 promoter,
major promoter, and MyoD1 promoter in CB3 and
CB3-WT21 (mean ± S.E. of three independent experiments, except
for H3-meK4 levels at HS2 in CB3-WT21, which represent duplicate
values). Pro, promoter; PI, preimmune;
acH3, acetylated H3.
-globin locus via developmental signals
and by the hematopoietic-specific activator p45/NF-E2.
-globin expression. As multiple histone
modifications of the
-globin locus are insensitive to
transcriptional inhibition by the elongation inhibitor
5,6-dichloro-1-
-D-ribofuranosyl-benzimidazole (data not
shown), it is highly unlikely that dynamic changes in these
modifications result from altered transcriptional activity. Rather,
changes in histone modifications at the
-globin locus are likely to
regulate transcription.
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Fig. 7.
Summary of relative levels of H3-meK79 at
selected loci in Me2SO-induced MEL, EryP, CB3, and CB3-WT21
cells. Averaged data obtained from quantitative ChIP analysis in
Me2SO-induced MEL, EryP, CB3, and CB3-WT21. Values were
assigned units of , +, ++, and +++ based on the scale shown on the
bottom. N.D., not determined.
-globin
locus in adult erythroid cells, being almost entirely absent from sites
within the central subdomain and at HS2, was not predictable from the
yeast model.
-globin
genes. Alternatively, other factors might preclude the need to utilize
H3-meK79 to exclude SIR proteins from the central subdomain.
-globin locus, we propose that mDOT1L is an important regulator
of
-globin gene transcription. Whereas acetylated H3 and H4 and
H3-meK4, which are hallmarks of active chromatin, are highly enriched
at the LCR and the active promoters (18, 20), H3-meK79 is uniquely high
at active genes in induced MEL cells (Fig. 3). The variable
distribution of H3-meK79 within the
-globin locus in MEL cells is
consistent with an important regulatory function of H3-meK79 locally at
the active adult
-globin genes. By contrast, H3-meK79 had a broader
distribution throughout the locus in EryP, and notably the intergenic
site IVR3 of the central subdomain had high levels of H3-meK79. The
EryP results indicate that H3-meK79 does not reside exclusively at
active promoters and genes.
major promoter versus HS2, and H3-meK79 represents the only histone modification detected to date that is
preferentially enriched at
major. Based on these
findings, it will be important to determine how mDOT1L expression and
activity are regulated during erythropoiesis and whether hematopoietic specific activators differentially recruit mDOT1L to the
-globin locus in embryonic and adult erythroid cells.
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ACKNOWLEDGEMENT |
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We thank Soumen Paul for a critical review of this manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DK50107 and DK55700 (to E. H. B.).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.
Supported by National Research Service Award T32 HL07936 from
the National Institutes of Health, and by the University of Wisconsin-Madison Cardiovascular Research Center.
** Kimmel Scholar and supported by National Institutes of Health Grant GM63067 and American Cancer Society Grant RSG-00-351-010GMC.
Scholar of the Leukemia Society of America, a Romnes Faculty
Scholar, and a Shaw Scientist. To whom correspondence should be
addressed: Dept. of Pharmacology, University of Wisconsin Medical School, 1300 University Ave., 383 Medical Sciences Center, Madison, WI
53706. Tel.: 608-265-6446; Fax: 608-262-1257; E-mail:
ehbresni@facstaff.wisc.edu.
Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M300890200
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ABBREVIATIONS |
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The abbreviations used are: H3-meK9, methylation of histone H3 at lysine 9; cad, carbamoyl-phosphate synthase/aspartate transcarbamoylase/dihydroorotase; ChIP, chromatin immunoprecipitation; Me2SO, dimethyl sulfoxide; DOT1, disruptor of telomeric silencing-1; DOT1L, disruptor of telomeric silencing-1-like protein; EryP, primitive erythroid cell colony derived from murine ES cells; ES, embryonic stem cell; H3-meK4, methylation of histone H3 at lysine 4; H3-meK79, methylation of histone H3 at lysine 79; HP1, heterochromatin protein 1; HS, DNase I-hypersensitive site; IVR, intervening region; LCR, locus control region; MEL, mouse erythroleukemia cell; mDOT1L, murine DOT1L; RT, reverse transcriptase; SIR, silent information regulator.
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