|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 275, Issue 18, 13827-13834, May 5, 2000
From the Locus control regions (LCRs) are capable of
activating target genes over substantial distances and establishing
autonomously regulated chromatin domains. The basis for this action is
poorly defined. Human growth hormone gene (hGH-N)
expression is activated by an LCR marked by a series of DNase
I-hypersensitive sites (HSI-III and HSV) in pituitary chromatin. These
HSs are located between The majority of DNA in the eukaryotic nucleus is packaged into a
compact chromatin conformation. For a gene to be expressed, this
chromatin structure must be disrupted to accommodate transcription factor binding and RNA polymerase assembly and passage (1, 2). A
specific set of distal regulatory elements termed locus control regions
(LCRs)1 are postulated to
function in promoting changes in chromatin structure conducive to gene
activation. Critical determinants that constitute an LCR co-map to one
or more DNase I-hypersensitive sites (HSs) flanking
LCR-dependent genes in the chromatin of expressing cells
(3). The functions of such LCR determinants are operationally defined
by their ability to establish autonomously functioning transgene
chromatin domains that are independent of the site of integration
within the host genome (4, 5). The biochemical mechanism(s) by which
LCRs activate their target genes has not been determined.
Histone-modifying enzymes are directly involved in modulating chromatin
structures relevant to gene transcription (6-11). The acetylation of
nucleosomal histones at the promoters of certain genes promotes
chromatin disruption (12-15) and facilitates transcriptional activation (16-18). Histone deacetylase activity has the opposite effect, resulting in gene silencing (19, 20). The linkage between
histone acetylation and transcriptional activation was strongly
supported by the discovery that a number of transcriptional coactivators (GCN5, CBP/p300, SRC1, TAF11250, and PCAF)
possess histone acetyltransferase activity (7, 21-25), whereas a
number of transcriptional repressors (Rpd3, HDAC1, and HDAC2) associate with histone deacetylases (26-31). Collectively, these data provide strong evidence that acetylases and deacetylases activate or repress gene expression by being recruited to specific promoters and/or proximal enhancer elements. The role of such histone acetylation and
deacetylation in LCR function remains to be explored.
The human growth hormone gene (hGH) cluster comprises five
closely linked genes:
5'-hGH-N/hCS-L/hCS-A/hGH-V/hCS-B-3'.
Expression of hGH-N is limited to the somatotrope and
somatolactotrope cells of the anterior pituitary, whereas expression of
the remaining four genes is restricted to the syncytiotrophoblast layer
of the placental villi (32, 33). A set of tissue-specific DNase
I-hypersensitive sites located between A central question raised by studies of LCR function concerns the
mechanism by which LCRs selectively alter chromatin structures and
establish transcriptionally productive chromatin environments in
specific tissues or cell types. Given the effects of histone modification on modulating chromatin structure, we have investigated the potential association of histone acetylation with LCR function. The
data support a role for LCR-mediated histone acetylation in the
establishment of a pituitary-specific, transcriptionally active chromatin environment required for hGH-N gene activation.
Tissue Culture and Primary Cells--
Mouse GHFT1 presomatotrope
cells (37) and human K562 erythroid cells were maintained in
Dulbecco's modified Eagle's medium and RPMI 1640 medium supplemented
with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin. The GHFT1/HSIII cell line was generated by transfection
of GHFT1 cells with the HSIII/hGHneor
construct.2 A portion of a
surgically removed GH-secreting human pituitary adenoma was utilized.
Mouse pituitaries and livers were isolated from the indicated
transgenic lines. Pituitary and liver samples were dissociated in cell
dissociation buffer (Life Technologies, Inc.). Nuclei were isolated
from the dissociated pituitary and liver cells and cultured GHFT1/HSIII
and K562 cells by hypotonic lysis in the presence of mild detergent as
described previously (38).
Transgenic Mice--
Mice carrying the hGH/P1
transgene were generated by microinjection of a linearized P1 plasmid
carrying the hGH/P1 clone (encompassing the entire
hGH LCR and the first four genes of the cluster) into fertilized mouse oocytes to establish founder lines (39). Frozen embryos carrying the human growth hormone-releasing factor
(GRF) transgene were a kind gift from R. Brinster
(University of Pennsylvania). To generate hGH/P1×GRF
compound transgenic mice, an hGH/P1 transgenic line was
crossed to the GRF line. Doubly positive transgenic mice were identified by dot blot analysis of tail DNA using the probes described below.
Preparation of Unfixed Chromatin--
Nuclei were resuspended in
1.0 ml of digestion buffer (50 mM NaCl, 20 mM
Tris-HCl (pH 7.5), 3.0 mM MgCl2, 1.0 mM CaCl2, 10 mM sodium butyrate,
and 0.1 mM phenylmethylsulfonyl fluoride) at a
concentration of 0.3 mg/ml. They were digested with 25 units of
micrococcal nuclease (Amersham Pharmacia Biotech) for 6 min at
37 °C. The digestion was terminated by the addition of EDTA to 0.5 mM, and the sample was centrifuged at 12,000 × g in a microcentrifuge for 10 min at 4 °C to generate
supernatant S1. The pellet was resuspended in 300 µl of low salt
lysis buffer (10 mM Tris-HCl (pH 7.5), 10 mM
sodium butyrate, 0.25 mM EDTA, and 0.1 mM
phenylmethylsulfonyl fluoride), incubated on ice for 2 min, and then
centrifuged as before. The resulting supernatant, S2, was combined with
S1. The soluble chromatin was concentrated using Microcon centrifugal filters (Amicon, Inc., Bedford, MA).
Immunoprecipitation of Unfixed Chromatin--
The chromatin
immunoprecipitation (ChIP) procedure was carried out according to
previously reported methods (40, 41) with minor modifications. Antisera
specific to the acetylated lysine residues of histone H3 or H4 were
diluted 1:100 and used in a 1:1 mixture. Alternatively, 15 µl of
antiserum to unacetylated histone H3 was used. Each antiserum was
kindly provided by C. D. Allis (University of Virginia).
Immunoprecipitations contained 250-µg aliquots of chromatin DNA in a
total volume of 500 µl. Protein A-Sepharose (Amersham Pharmacia
Biotech) precipitates were generated and washed, and DNA and proteins
were harvested from pellets and supernatants according to published
protocols (40). Input, unbound, and bound DNA samples were each
analyzed by electrophoresis on 1% agarose gels stained with ethidium
bromide prior to use to ensure the quality of oligonucleosome
preparations. Southern hybridization of micrococcal nuclease-digested
input DNA using sheared 32P-labeled total genomic DNA as a
probe demonstrated that the majority of DNA ranged in size from ~0.16
to 1 kb.
Proteins from bound and unbound fractions were obtained from the first
phenol/chloroform phase to which 8 µg of bovine serum albumin was
added as carrier. HCl was then added to 0.1 M, followed by
precipitation with 12 volumes of acetone as described previously (40,
41). Proteins were analyzed by electrophoresis on 15% SDS-polyacrylamide gels and then transferred to nitrocellulose filters
and separately incubated with anti-acetylated H4 and anti-acetylated H3
(both at 1:1000 dilution) and anti-unacetylated H3 (1:250
dilution) antibodies. The blots were developed by incubation with
horseradish peroxidase-conjugated goat anti-rabbit IgG (used at 1:3000
dilution; Roche Molecular Biochemicals), and immune complexes were
visualized by the Lumi-Light Western blotting substrate system (Roche
Molecular Biochemicals).
Slot Blots and PhosphorImager Quantification--
Equal masses
of DNA (1.0 µg) from input (unfractionated chromatin),
antibody-bound, and unbound fractions were loaded onto Zetabind
membranes (Cuno, Inc., Meriden, CT) using a slot-blot manifold. The
blots were incubated overnight at 65 °C with hybridization solution
containing 1-2 × 106 cpm/ml random primer-labeled
probe. Subsequent washes were at 60 °C in 0.1% SDS and 0.5× SSC.
Signals were quantified by PhosphorImager (Molecular Dynamics, Inc.,
Sunnyvale, CA) using ImageQuant software, and the ratios between bound
and unbound DNA fractions were calculated for each probe used. To
correct for potentially unequal slot-blot loadings, each blot was
rehybridized with random primer-labeled sheared genomic DNA (see
below). All ratios are reported after normalization to this loading control.
Preparation of DNA Probes--
The majority of DNA fragments
used as hybridization probes were generated by polymerase chain
reaction using AmpliTaq DNA polymerase (Perkin-Elmer). The template for
the polymerase chain reaction was a P1 clone encompassing the
hGH LCR and the first four of the five clustered
hGH genes (39). The primer sets are as shown in Table
I. Probe p9 was a subcloned 263-bp
EcoRI fragment of a repeated element ("P-element") (42)
located 2 kb 5' to hCS-L, hCS-A,
hGH-V, and hCS-B. Probe p10 was an amplified
fragment of a repeated element ("enhancer element") (43) located 2 kb 3' to hCS-L, hCS-A, and hCS-B. Due
to the high sequence homology between each of the hGH
promoters, primers specific for the hGH-N promoter were
designed to amplify a 200-bp region Specificity of the Chromatin Immunoprecipitation Procedure--
A
set of studies were designed to establish the specificity and accuracy
of the ChIP approach used in this report. Salt-soluble, unfixed
oligonucleosome preparations from each cell or tissue type studied
(K562 erythroid cells, GHFT1/HSIII presomatotrope cells, human
pituitary tumor tissue, and P1/GRF transgenic mouse pituitary and liver
tissue) were generated by micrococcal nuclease digestion of purified
nuclei. To ensure the quality of the chromatin preparations and to
determine the size distribution of the resulting oligonucleosomes for
each sample, the DNA from the digested chromatin preparation was
analyzed by gel electrophoresis. In each case, this analysis revealed a
typical oligonucleosome ladder; the majority of DNA ranged from ~160
bp (mononucleosomes) to 1 kb, and minimal DNA could be visualized above
2 kb (Fig. 1A).
To establish the specificity of the antibodies for acetylated H3 and H4
under the experimental ChIP conditions used for our studies, soluble
oligonucleosomes prepared from the K562 erythroleukemia cell line (Fig.
1A) were immunoprecipitated with a 1:1 mixture of antibodies
specific to acetylated histones H3 and H4. DNA and proteins were
separately isolated from the total input chromatin, the unacetylated
(unbound) chromatin, and the acetylated (bound) chromatin. DNA from
each fraction was applied via a slot-blot manifold to nylon membranes
for hybridization analysis (see below). In parallel, equal amounts of
core histones isolated from each fraction were analyzed by
SDS-polyacrylamide gel electrophoresis (Fig. 1B)
and then Western-blotted and immunostained with antibodies specific to
acetylated H3 or H4 (Fig. 1C). These results demonstrated
that the immunoprecipitates were enriched for acetylated histones H3
and H4 compared with the unbound fractions. The specificity of the
immunoprecipitation was further validated by demonstrating a reciprocal
enrichment of unacetylated histone H3 in the unbound fraction (Fig.
1C, lower panel). As expected, prolonged exposure of the blot revealed a small amount of residual unacetylated H3 in the
bound fraction as well (not shown). These protein studies demonstrated
that this ChIP procedure enriched chromatin fractions for acetylated
histones H3 and H4.
K562 DNA isolated by the ChIP procedure was analyzed by hybridization
using probes corresponding to promoter sequences of actively
transcribed HSIII of the hGH LCR Is Acetylated when Integrated into the
Chromatin of a Pituitary Presomatotrope Cell Line--
The association
of HS formation with localized histone acetylation was initially tested
in a cell culture setting. HSIII of the hGH LCR (Fig.
2A) was used for these studies
because the DNase I-hypersensitive structure of this chromatin element
could be reproduced in stably transfected pituitary cells.2
A 3150-kb fragment encompassing HSIII was linked to 500 bp of the
hGH-N promoter region driving a neomycin resistance
(neor) cassette (HSIII/hGHneor) (Fig.
2B, bottom). Following stable transfection into
GHFT1 presomatotrope cells, a neor cell line (GHFT1/HSIII)
was obtained. A DNase I-hypersensitive site formed in the integrated
3150-bp HSIII segment at a position corresponding precisely to that of
native HSIII in primary pituitary and placental tissue (data not
shown). Cell nuclei isolated from these neor cells were
digested with micrococcal nuclease, and the resulting soluble
oligonucleosomes were immunoprecipitated with a 1:1 mixture of
antibodies specific to acetylated histones H3 and H4 using the ChIP
procedure (40, 41). Equal amounts of intact DNA extracted from the
input, unbound, and bound chromatin fractions were sequentially probed
for specific DNA sequence content as described above. The normalized
ratio of HSIII sequences in the bound versus unbound chromatin fractions was 3.3, and that for the hGH-N promoter
was 2.8. In contrast, the ratios for the inactive Each HS of the hGH LCR Is Enriched in the Acetylated Chromatin
Fraction of a GH-secreting Human Pituitary Adenoma--
Analysis of
hGH LCR chromatin acetylation was next expanded to an
in vivo setting in which the entire hGH cluster
and adjacent sequences could be studied in their native setting. The
chromatin of a primary GH-secreting human pituitary adenoma was
analyzed using a set of probes corresponding to the hGH-N
promoter, each of the hGH LCR HSs, and two sites located 5'
to the LCR. Tissue specificity of the acetylation map was established
by comparing these results with those obtained from analysis of
chromatin isolated from the human K562 erythroid cell line. The
positions of the probes in relation to the hGH cluster, the
closely linked CD79b (the B lymphocyte-specific
immunoglobulin receptor subunit gene encoding Ig Histone Acetylation of the hGH LCR Is Recapitulated and Enhanced in
Somatotrope-enriched Transgenic Mouse Pituitaries--
The pituitary
is made up of six differentiated hormone-secreting cell types: the
somatotropes (secreting GH), somatolactotropes (GH and prolactin),
lactotropes (prolactin), thyrotropes (thyrotropin-releasing hormone),
gonadotropes (luteinizing hormone and follicle-stimulating hormone),
and corticotropes (ACTH). The representation of somatotropes in the
surgical human pituitary tumor sample was difficult to quantify, and
the scarcity of the material limited the number of chromatin regions
that could be experimentally assessed. Therefore, a surrogate mouse
model with pituitary somatotrope hyperplasia was established to extend
the chromatin analysis. A transgenic mouse line was generated carrying
an extensive human genomic DNA insert isolated from a P1 bacteriophage
library that encompassed the entire hGH LCR and first four
genes of the cluster ("P1 clone") (Fig.
4A). Lines containing this
hGH/P1 transgene expressed the hGH-N gene in a
physiologically appropriate manner in the pituitary (39). A second
mouse line was obtained that was transgenic for an overexpressing human
GRF transgene. This GRF overexpression stimulates
hyperproliferation of pituitary somatotropes (47). Crossing the
hGH/P1 and GRF lines generated compound
transgenic mice (hGH/P1×GRF) expressing high levels of
mouse and human GH. Such mice were 1.5-2 times greater than normal
size by 3 months of age, and their pituitaries were 5-10 times normal
size, composed predominantly of somatotropes and containing HSI-III
and HSV.3
Pooled pituitaries from hGH/P1×GRF adult mice were isolated
and analyzed by the ChIP assay (Fig. 4B). Livers of the same
hGH/P1×GRF mice served as a source of chromatin from a
non-expressing tissue. The hGH LCR in hGH/P1×GRF
chromatin displayed a pattern of acetylation enrichment paralleling
that seen in primary human pituitary chromatin (compare Figs.
3B and 4B). As in the primary human pituitary, each of the HSs and the adjacent hGH-N promoter were
enriched in the acetylated chromatin fraction, whereas regions
immediately 5' to HSV were unacetylated. However, when compared with
the primary human pituitary sample, the overall level of modification
was significantly greater in the hGH/P1×GRF transgenic
mouse pituitaries. This difference was particularly marked toward the
central region of the LCR, with the enrichment of HSIV, HSIII, and
HSI/II in the acetylated chromatin fraction being 2-2.5-fold higher in
this tissue than that seen in the human pituitary adenoma. Thus, the acetylation peak at HSI/II in the hGH/P1×GRF pituitaries
was accentuated to 8.8-fold above control levels and was 3.5-fold
higher than in the active hGH-N promoter region. The
acetylation enrichment of the LCR chromatin domain seen in the
pituitaries of hGH/P1×GRF mice was specifically absent in
the livers of the same animals (Fig. 4C). These data
demonstrated a localized peak of modification of HSI/II and supported
the conclusion that the acetylation enrichment of the hGH
LCR was somatotrope cell-specific, thus confirming and extending the
observations obtained using primary human pituitary tissue.
Acetylation of the hGH LCR Chromatin Domain Extends beyond the HSs
and Reveals Defined 5'- and 3'-Borders--
Additional probes were
generated to determine the extent of histone acetylation in pituitary
chromatin segments between and surrounding the HSs (Fig.
4A). Probes located between the hGH-N gene and
HSI/II (probes p1, p2, and p3, located at Histone acetyltransferases modulate chromatin structure by
acetylation of specific basic lysine residues present on the N-terminal tails of histones. This modification neutralizes the positively charged
lysines, thereby weakening interactions between neighboring nucleosomes. This effect promotes the destabilization of higher order
chromatin structure, thus facilitating the transcription process
(49-51). In vitro studies suggest that acetylation of
histones increases the accessibility of transcription factors for
nucleosomal DNA (52) and facilitates recruitment of SWI/SNF-like
chromatin-remodeling factors (53). The work presented here suggests
that chromatin acetylation may be a critical step in LCR function in
addition to its more established correlation with enhancer
activation (54, 55).
Previous studies in yeast strongly support a mechanism by which highly
localized, promoter-targeted histone-modifying activities lead to
selective effects on transcriptional regulation (Refs. 17, 20, and 31;
reviewed in Ref. 2). More recently, Parekh and Maniatis (56)
demonstrated that promoter-localized histone acetylation is also
required for metazoan gene activation. This work demonstrated that
transcriptional activation of the virally induced human interferon- The observation that the entire domain of the hGH LCR is
hyperacetylated needs to be compared with prior observations of
acetylation of the domain spanning the chicken How might histone acetyltransferase activity be recruited to the HSI/II
LCR element specifically in pituitary chromatin? Recent studies have
demonstrated that certain transcriptional cofactors such as CBP/p300
and PCAF possess histone acetyltransferase activities (22, 24),
acetylate histones H3 and H4 in vitro (59), and associate
with numerous tissue-specific transcription factors (60-64). In this
regard, Pit-1, a pituitary-specific DNA-binding factor shown to be
central to the regulation of somatotrope-restricted hGH
expression, was recently shown to associate with CBP in the pituitary
cell (65). Additionally, we have recently demonstrated that HSI/II
contains an array of functional Pit-1-binding sites critical for
activation of hGH transgene expression in the somatotrope cells of the pituitary (36). These observations suggest a model in
which CBP/p300 is specifically recruited to HSI/II through its
interaction with Pit-1. The HSI/II-bound complex containing DNA-binding
factors and associated histone acetyltransferase coactivators might
then "track" via small steps along the LCR, concomitantly modifying
chromatin structure (66). Alternatively, further acetylation of the
domain might reflect recruitment of histone acetyltransferase coactivators by each of the pituitary-specific HSs that subsequently spread the modification throughout the LCR from these multiple target
sites. Subsequent studies can now be designed to further refine this model.
We gratefully acknowledge Yuhua Su for the
hGH/P1 transgenic mouse line, Idriss Bennani-Baiti and Anita
Abu-Daya for establishing and DNase I-mapping the GHFT1/HSIII cell
culture line, Brian M. Shewchuk and Yugong Ho for critically reading
the manuscript, and Jessie Harper for excellent secretarial support.
*
This work was supported by National Institutes of Health
Grant HD25147 (to N. E. C. and S. A. L.).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.
¶
To whom correspondence should be addressed: Depts. of Genetics
and Medicine, University of Pennsylvania School of Medicine, Rm. 428, Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104. Tel.:
215-898-7834; Fax: 215-573-5157; E-mail:
liebhaber@mail.med.upenn.edu.
2
I. Bennani-Baiti, A. Abu-Daya, N. E. Cooke,
and S. A. Liebhaber, unpublished data.
3
F. Elefant, A. Zhang, N. E. Cooke, and S. A. Liebhaber, unpublished data.
The abbreviations used are:
LCRs, locus control
regions;
HS, DNase I-hypersensitive site;
CBP, cAMP-responsive
element-binding protein-binding protein;
hGH, human growth hormone;
GH, growth hormone;
kb, kilobase(s);
bp, base pair(s);
GRF, growth
hormone-releasing factor;
ChIP, chromatin immunoprecipitation;
ACTH, adrenocorticotropic hormone.
Targeted Recruitment of Histone Acetyltransferase Activity to a
Locus Control Region*
§,§¶
Howard Hughes Medical Institute and the
§ Departments of Genetics and Medicine, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
15 and 32 kilobases (kb) relative to the
hGH transcription start site. To establish a mechanistic
basis for hGH LCR function, we carried out acetylation
mapping of core histones H3 and H4 in chromatin encompassing the
hGH cluster. These studies revealed that the entire LCR was
selectively enriched for acetylation in chromatin isolated from a human
pituitary somatotrope adenoma and in pituitaries of mice transgenic for
the hGH locus, but not in hepatic or erythroid cells.
Quantification of histone modification in the pituitary revealed a
dramatic peak at HSI/II, the major pituitary-specific hGH
LCR determinant (15 kb), with gradually decreasing levels of
modification extending from this site in both 5'- and 3'-directions.
The 5'-border of the acetylated domain coincided with the 5' most
hGH LCR element, HSV (34 kb); and the 3'-border included
the expressed hGH-N gene, but did not extend farther 3'
into the placenta-specific region of the gene cluster. These data
support a model of LCR function involving targeted recruitment and
subsequent spreading of histone acetyltransferase activity to encompass
and activate a remote target gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
15 and 32 kb upstream of the
hGH gene have been identified and shown to be required for
appropriate tissue-specific expression of the hGH gene
cluster in transgenic mice. Pituitary chromatin contains a subset of
four HSs (HSI-III and HSV), and chromatin isolated from placental
syncytiotrophoblasts contains a partially overlapping set of three HSs
(HSIII-V). The full set of HSs renders expression of hGH-N
transgenes reproducibly copy number-dependent and site of
integration-independent in the mouse pituitary (34). HSI and HSII,
which are closely linked and thus considered as a single determinant,
are located 15 kb 5' to the hGH gene and are unique to the
pituitary (34). They are fully sufficient to confer high level,
developmentally appropriate, somatotrope-specific and
position-independent expression on a linked hGH-N transgene
(35). As such, HSI/II constitutes the major element of the
hGH LCR in the pituitary. Critical cis-acting determinants that bind the pituitary-specific POU homeodomain trans-factor Pit-1 have recently been identified within
HSI/II (36). These sites are necessary for HSI/II function in
vivo, but the mechanistic basis for their action is not defined.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0.8 kb to 1 kb upstream of the
hGH-N transcription start site. The mouse 
-globin probe
was a 1.3-kb BamHI fragment encompassing the mouse
-globin coding region. The human -globin probe was a 1.8-kb
BglII fragment encompassing the human -globin coding
region. The probe used as a loading control was generated by random
primer labeling of sonicated mouse or human total genomic DNA.
Transgenic genotypes were determined by tail blots using two probes:
the GRF probe was a 1.7-kb
BglII/HindIII fragment encompassing the human
GRF coding region, and the hGH probe was a 500-bp
EcoRI/BamHI fragment encompassing the
hGH-N promoter.
Amplification primer sets
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (49K):
[in a new window]
Fig. 1.
Analysis of proteins and DNA by the ChIP
procedure. A, size distribution of oligonucleosomes used for
ChIP analysis. Nuclei were isolated from the following cell and tissue
types: K562 erythroid cells, GHFT1/HSIII presomatotrope cells,
GH-secreting human pituitary tumor, P1/GRF transgenic mouse liver, and
P1/GRF transgenic mouse pituitary tissue. The nuclei were subjected to
micrococcal nuclease digestion to yield a soluble oligonucleosome
fraction. DNA from each preparation was resolved on a 1% agarose gel
and stained with ethidium bromide. Size markers are shown for each of
the two panels (derived from two independently run gels), and the
markers on the right panel are labeled according to size.
The predicted positions of the mono-, di-, and oligonucleosomes (as
inferred from the molecular size markers) are indicated on the
left panel. B, SDS-polyacrylamide gel
electrophoresis of proteins isolated after the ChIP procedure using
oligonucleosomes obtained from K562 cells. Proteins were isolated from
input, unbound, and bound fractions following immunoprecipitation with
a 1:1 mixture of antibodies specific to acetylated histones H3 and H4.
Core histones from each fraction were resolved by 15%
SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue.
The positions of each of the histone proteins as well as the
immunoglobulin subunits (heavy (H) and light (L))
and the bovine serum albumin (BSA; present as carrier in the
precipitation procedure) are indicated to the right. C,
Western analysis of core histones isolated by the ChIP procedure. Equal
amounts of core histones isolated from input, unbound, and bound
fractions (as estimated from B above) were resolved by 15%
SDS-polyacrylamide gel electrophoresis, Western-blotted, and
immunostained with antibodies to acetylated H3, acetylated H4, or
unacetylated H3. The identity of each of the antiserum used is
indicated to the right of the respective panels. D,
acetylation status of the chromatin encompassing the active human
-globin (h-Globin) promoter and the inactive
hGH-N promoter (hGHp) in K562 erythroid cells.
The human erythroleukemia cell line K562 expresses the human embryonic
-globin gene. Nuclear chromatin from these cells was
subjected to ChIP using a 1:1 mixture of antisera against acetylated
histones H3 and H4. Equal amounts of DNA purified from starting
chromatin (Input DNA), unacetylated (Unbound
DNA), and acetylated antibody-bound (Bound DNA)
fractions were slot-blotted onto nylon membranes and then hybridized
sequentially with probes to total genomic DNA, inactive
hGH-N promoter, and active human -globin promoter
sequences. The normalized ratios (bound/unbound) are indicated below
each hybridization panel (autoradiograph) and are summarized in the
histogram. As a specificity control, antiserum specific to unacetylated
histone H3 was used in the ChIP procedure and hybridized with the human
-globin promoter. The mean value from at least three determinations
from individual ChIP assays is shown; error bars indicate
S.E. All ratios were normalized to the ratio obtained using a probe for
total genomic DNA as a loading control. The ratio of acetylation of
chromatin encompassing the human -globin gene was significantly
greater that that of the hGH promoter (p < 0.001).
-globin and unexpressed hGH (Fig.
1D). Equal amounts of intact DNA extracted from input,
unacetylated (unbound), and acetylated (bound) chromatin were applied
to membranes via a slot-blot manifold and sequentially probed for
specific DNA sequence content. Hybridization signal intensities in each
of these three fractions were normalized for minor differences in DNA
loading by directly quantifying the DNA in each slot using a labeled
total genomic DNA probe. As expected from previous acetylation studies
of active human globin genes (44), the chromatin encompassing the
expressed -globin promoter was enriched for acetylation (bound versus unbound ratio of 3.7). The acetylation of the
chromatin encompassing the silent hGH promoter was at
background levels (ratio of 1.0). The difference between these two
ratios was highly significant (p < 0.001). In five
independent ChIP experiments using K562 chromatin, the ratios of the
-globin promoter ranged between 3.5 and 3.8. The specificity of the
immunoprecipitation was further validated by demonstrating that K562
nuclear chromatin immunoprecipitated with antibodies to unacetylated
histone H3 was not enriched for -globin promoter sequences. These
DNA hybridization studies demonstrated the specificity and
reproducibility of the ChIP procedure that was used in the studies that follow.
-fetoprotein and -globin genes in the same chromatin samples were 1.1 and 1.2, respectively (Fig. 2B). In four independent experiments
using GHFT1/HSIII chromatin, the ratios of HSIII ranged between 3.2 and
3.5. Specificity was validated by demonstrating that antibodies to
unacetylated H3 failed to enrich for HSIII sequences (Fig. 2B). These data further established the specificity and
reproducibility of this ChIP procedure and demonstrated the enrichment
of HSIII in the acetylated chromatin fraction of a stably transfected, pituitary-derived cell line.
View larger version (23K):
[in a new window]
Fig. 2.
HSIII of the hGH
LCR is hyperacetylated in stably transfected GHFT1 cells.
A, diagram of the hGH gene cluster and adjacent
LCR. The black boxes are expressed genes, and the
white boxes are silent genes. The top line
represents the status of the cluster in pituitary somatotropes, and the
bottom line represents the status of the cluster in
placental syncytiotrophoblasts. HSs common to both tissues are
indicated by the elongated shaded ovals (HSIII and HSV); HSs
specific to the pituitary (HSI/II) or placenta (HSIV) are indicated
separately. B, acetylation status of the hGH
promoter (hGHp) and HSIII in GHFT1 presomatotrope cells
stably transfected with the HSIII/hGHneor construct.
The HSIII/hGHneor construct, shown at the bottom,
contains a 3.15-kb genomic fragment encompassing HSIII linked to the
hGH-N promoter and neor coding region.
After transfection of GHFT1 presomatotrope cells with this
neor vector, cells were selected; nuclei were isolated; and
soluble chromatin was immunoprecipitated with antisera against
acetylated H3 and H4 or, as a specificity control, antiserum
recognizing unacetylated H3. Equal amounts of DNA purified from input
chromatin, acetylated antibody-bound, and unacetylated unbound
fractions were slot-blotted as described for Fig. 1D and
hybridized sequentially with probes to total genomic DNA, HSIII, and
hGH-N promoter sequences in the integrated
HSIII/hGHneor construct and the endogenous mouse
-globin (m-Globin) and mouse -fetoprotein
(mAFP) loci. All ratios were normalized to the ratio
obtained using a probe for total genomic DNA as a loading control. The
ratios of signals in the bound versus unbound chromatin are
shown below each slot-blot panel and are summarized in the histogram.
The mean value from at least three individual ChIP assays is shown;
error bars indicate S.E. The ratios of HSIII and the hGH
promoter were not significantly different (p > 0.1),
and both were significantly greater than ratios detected with the other
probes (p < 0.001).
) (45), and
SCN4A (the striated muscle-specific sodium channel gene)
(46) are shown in Fig. 3A.
ChIP assays of the human pituitary adenoma chromatin revealed that the
segments encompassing each of the five HSs as well as the adjacent
hGH-N promoter were all enriched for acetylation (all
ratios = 2.0) (Fig. 3B). The most highly modified
region coincided with HSI/II (3.4-fold enrichment). Acetylation at the
two sites upstream of the LCR (probes p7 and p8) (Fig. 3B)
was insignificant (ratios of 1.4). There was virtually no acetylation
enrichment at any of these sites in erythroid (K562) cell chromatin.
These data demonstrated tissue-specific enrichment of acetylation at
all HSs in the chromatin of this human pituitary cell line enriched for
somatotropes. Furthermore, they identified a 5'-boundary to the LCR
modification in the pituitary just upstream of HSV and suggested that
HSI/II was the most highly modified among the HSs. Of additional note
was the acetylation of HSIV. Because HSIV does not form in the
pituitary, its modification in this tissue suggested that acetylation
of the hGH locus in pituitary chromatin might not be limited
to the immediate locale of the active HSs, but might instead be
generally distributed throughout the LCR domain.
View larger version (37K):
[in a new window]
Fig. 3.
Each HS of the hGH LCR is
enriched in the acetylated chromatin fraction isolated from a
GH-secreting human pituitary adenoma. A, schematic
representation of the hGH LCR and hGH gene
cluster contiguous with the two linked upstream genes, CD79b
and SCN4A. Each of the genes is shown as a shaded
box, and the dark vertical lines indicate exons. Probes
used for acetylation mapping are underlined, and their
coordinates relative to the hGH-N transcription start site
are indicated below. B, acetylation status of each HS in
human pituitary adenoma and K562 erythroid chromatin. Soluble chromatin
samples isolated from each of the indicated sources were
immunoprecipitated with anti-acetylated H3 and H4 antibodies. Equal
amounts of DNA purified from input, antibody-bound, and unbound
fractions were slot-blotted onto nylon membranes and then sequentially
hybridized with the indicated probes (underlined). Ratios
(bound/unbound) are shown beneath each blot. All ratios were normalized
to the ratio obtained using a probe for total human genomic DNA as a
loading control (shown to the right).
View larger version (46K):
[in a new window]
Fig. 4.
The chromatin domain encompassing the
hGH locus transgene in the mouse pituitary
recapitulates a peak of acetylation at HSI/II; the modified domain
encompasses all HSs and intervening regions and extends to the
hGH-N promoter. A, shown is a schematic
representation of the hGH/P1 transgene. The extent of the
transgene (P1 clone) relative to the positions of the hGH
locus is defined by the double-headed arrow (coordinates
45 to +40.8). The positions of the HSs and the probes used in the
study (underlined) are shown below the diagram. B
and C, autoradiographs using hybridization probes
corresponding to the HSs and the hGH-N promoter in
hGH/P1×GRF transgenic mouse pituitary
(B) and liver chromatin
(C) are double-boxed.
Autoradiographs using hybridization probes corresponding to sites lying
upstream of the LCR (p7 and p8), between the HSs (p6, p5, and p4),
between the HSs and the hGH-N promoter (p3, p2, and p1), or
3' to hGH-N (p9 and p10) are single-boxed.
Soluble chromatin samples isolated from hGH/P1×GRF compound
transgenic mouse pituitaries (enriched for somatotropes) and liver
tissue from the same animals were immunoprecipitated with
anti-acetylated H3 and H4 antibodies and analyzed as described for Fig.
2. All ratios were normalized to the ratio obtained using a probe for
total genomic mouse DNA as a loading control (shown on the right). The
ratios of signals in bound versus unbound chromatin were
determined as described for Fig. 1 and are shown below each
autoradiograph. The ratios are summarized in the histograms.
Black bars (ratios > 2.0) are considered to be
enriched for acetylation, and shaded bars (ratios < 2.0) are considered to represent background.
5, 9, and 12 kb,
respectively) and between HSI/II and HSIII (probes p4, p5, and p6,
located at 17, 21, and 25 kb, respectively) were used in the ChIP
assay. These data demonstrated that all regions tested between HSV and
the hGH-N promoter were highly enriched in the acetylated
chromatin fraction (Fig. 4B, black bars). The levels of acetylation at each of the sites flanking the HSI/II peak
decreased in a graded fashion, culminating at discrete 5'- and
3'-borders. The 5'-border was deemed to be coincident with HSV because
the upstream probes p7 and p8 were unacetylated both in the human
pituitary and in the hGH/P1×GRF transgenic pituitary (Figs.
3B and 4B). The 3'-border of the acetylated
domain was sought by mapping internal regions of the hGH
transgene cluster using probes that recognize two elements flanking
hCS-L (as well as hCS-A) and hGH-V
(probes p9 and p10). These two elements, generated by localized
duplication events during the evolution of the hGH cluster
(48) and not represented elsewhere in the genome (data not shown), were
both unacetylated (Fig. 4B). Thus, the acetylation present
in the LCR encompassed pituitary-expressed hGH-N, but did
not extend farther 3' into the cluster. These data suggested that the
acetylation activity, once recruited to the LCR, modified a 32-kb
domain extending from HSV through the activated hGH-N locus.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gene is associated with promoter-localized hyperacetylated histones H3
and H4. Our data would suggest that LCR elements are similarly
recruiting histone acetylation activity in a targeted manner. However,
in clear contrast to the highly restricted promoter-localized histone
acetylation observed for inducible genes, acetylase activity, once
targeted by the hGH LCR, appeared to extend throughout the
extensive LCR chromatin domain and encompass the hGH-N
promoter. The capacity for acetylation spreading was initially
suggested by the extensive acetylation of an episomal Epstein-Barr
virus-derived plasmid containing the IgH LCR (57). The
acetylase activity may be targeted by a single component of the LCR
such as HSI/II or by several of the LCR HS elements at differing
levels. In either case, the general modification between these elements
suggests a subsequent spreading mechanism.
-globin gene cluster
and its 5'-LCR (58). The pattern of modification for the hGH
LCR demonstrates a distinct central peak corresponding to the major
pituitary HS. Its extension into the hGH cluster is limited
to the 5' most (expressed) hGH-N gene (Figs. 3 and
4). This pattern suggests a mechanism involving targeted
recruitment and spreading of a histone acetyltransferase activity by
the LCR. In contrast, the chicken -globin LCR modification is
uniform throughout the domain and encompasses the entire cluster. This
lack of localized peaks suggests that, in this system, the modification
may not be reflecting specific targeting to HSs. A strict comparison
between these two systems is, however, complicated by the fact that the
differences may partially reflect the use of antibodies recognizing the
N-acetyllysine on all histones in the former study (58)
rather than the specific acetyllysine H3 and H4 antibodies used in the
present study. Thus, generalizations on the basis of comparisons
between these two systems may be premature at this point.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Felsenfeld, G.
(1996)
Cell
86,
13-19[CrossRef][Medline]
[Order article via Infotrieve]
2.
Workman, J. L.,
and Kingston, R. E.
(1998)
Annu. Rev. Biochem.
67,
545-579[CrossRef][Medline]
[Order article via Infotrieve]
3.
Boyes, J.,
and Felsenfeld, G.
(1996)
EMBO J.
15,
2496-2507[Medline]
[Order article via Infotrieve]
4.
Grosveld, F.,
Blom van Assendelft, G.,
Greaves, D. R.,
and Kollias, G.
(1987)
Cell
51,
975-985[CrossRef][Medline]
[Order article via Infotrieve]
5.
Kioussis, D.,
and Festenstein, R.
(1997)
Curr. Opin. Genet. Dev.
7,
614-619[CrossRef][Medline]
[Order article via Infotrieve]
6.
Wolffe, A. P.
(1994)
Cell
77,
13-16[CrossRef][Medline]
[Order article via Infotrieve]
7.
Brownell, J. E.,
Zhou, J.,
Ranalli, T.,
Kobayashi, R.,
Edmondson, D. G.,
Roth, S. Y.,
and Allis, C. D.
(1996)
Cell
84,
843-851[CrossRef][Medline]
[Order article via Infotrieve]
8.
Grunstein, M.
(1997)
Nature
389,
349-352[CrossRef][Medline]
[Order article via Infotrieve]
9.
Hampsey, M.
(1997)
Trends Genet.
13,
427-429[CrossRef][Medline]
[Order article via Infotrieve]
10.
Wade, P. A.,
Pruss, D.,
and Wolffe, A. P.
(1997)
Trends Biochem. Sci.
22,
128-132[CrossRef][Medline]
[Order article via Infotrieve]
11.
Struhl, K.
(1998)
Genes Dev.
12,
599-606 12.
Wolffe, A. P.,
and Pruss, D.
(1996)
Cell
84,
817-819[CrossRef][Medline]
[Order article via Infotrieve]
13.
Gregory, P. D.,
Schmid, A.,
Zavari, M.,
Lui, L.,
Berger, S. L.,
and Horz, W.
(1998)
Mol. Cell
1,
495-505[CrossRef][Medline]
[Order article via Infotrieve]
14.
Nightingale, K. P.,
Wellinger, R. E.,
Sogo, J. M.,
and Becker, P. B.
(1998)
EMBO J.
17,
2865-2876[CrossRef][Medline]
[Order article via Infotrieve]
15.
Steger, D. J.,
Eberharter, A.,
John, S.,
Grant, P. A.,
and Workman, J. L.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
12924-12929 16.
Brownell, J. E.,
and Allis, C. D.
(1996)
Curr. Opin. Genet. Dev.
6,
176-184[CrossRef][Medline]
[Order article via Infotrieve]
17.
Kuo, M.-H.,
Zhou, J.,
Jambeck, P.,
Churchill, M. E. A.,
and Allis, C. D.
(1998)
Genes Dev.
12,
627-639 18.
Wang, L.,
Liu, L.,
and Berger, S. L.
(1998)
Genes Dev.
12,
640-653 19.
Nagy, L.,
Kao, H.-Y.,
Chakravarti, D.,
Lin, R. J.,
Hassig, C. A.,
Ayer, D. E.,
Schreiber, S. L.,
and Evans, R. M.
(1997)
Cell
89,
373-380[CrossRef][Medline]
[Order article via Infotrieve]
20.
Kadosh, D.,
and Struhl, K.
(1998)
Mol. Cell. Biol.
18,
5121-5127 21.
Mizzen, C. A.,
Yang, X.-J.,
Kokubo, T.,
Brownell, J. E.,
Bannister, A. J.,
Owen-Hughes, T.,
Workman, J.,
Wang, L.,
Berger, S. L.,
Kouzarides, T.,
Nakatani, Y.,
and Allis, C. D.
(1996)
Cell
87,
1261-1270[CrossRef][Medline]
[Order article via Infotrieve]
22.
Shikama, N.,
Lyon, J.,
and La Thangue, N. B.
(1997)
Trends Cell Biol.
7,
230-236
23.
Spencer, T. E.,
Jenster, G.,
Burcin, M. M.,
Allis, C. D.,
Zhou, J.,
Mizzen, C. A.,
McKenna, N. J.,
Onate, S. A.,
Tsai, S. Y.,
Tsai, M.-J.,
and O'Malley, B. W.
(1997)
Nature
389,
194-198[CrossRef][Medline]
[Order article via Infotrieve]
24.
Ogryzko, V. V.,
Kotani, T.,
Zhang, X.,
Schlita, R. L.,
Howard, T.,
Yang, X.-J.,
Howard, B. H.,
Qin, J.,
and Nakatani, Y.
(1998)
Cell
94,
35-44[CrossRef][Medline]
[Order article via Infotrieve]
25.
Korzus, E.,
Torchia, J.,
Rose, D. W.,
Xu, L.,
Kurokawa, R.,
McInerney, E. M.,
Mullen, T. M.,
Glass, C. K.,
and Rosenfeld, M. G.
(1998)
Science
279,
703-706 26.
Alland, L.,
Muhle, R.,
Hou, H. J.,
Potes, J.,
Chin, L.,
Schriber-Agus, N.,
and DePinho, R. A.
(1997)
Nature
387,
49-55[CrossRef][Medline]
[Order article via Infotrieve]
27.
Brehm, A.,
Miska, E. A.,
McCance, D. J.,
Reid, J. L.,
Bannister, A. J.,
and Kouzarides, T.
(1998)
Nature
391,
597-601[CrossRef][Medline]
[Order article via Infotrieve]
28.
Kadosh, D.,
and Struhl, K.
(1998)
Genes Dev.
12,
797-805 29.
Kao, H.-Y.,
Ordentlich, P.,
Koyano-Nakagawa, N.,
Tang, Z.,
Downes, M.,
Kintner, C. R.,
Evans, R. M.,
and Kadesch, T.
(1998)
Genes Dev.
12,
2269-2277 30.
Magnaghi-Jaulin, L.,
Groisman, R.,
Naguibneva, I.,
Robin, P.,
Lorain, S.,
LeVillain, J. P.,
Troalen, F.,
Trouche, D.,
and Harel-Bellan, A.
(1998)
Nature
391,
601-605[CrossRef][Medline]
[Order article via Infotrieve]
31.
Rundlett, S. E.,
Carmen, A. A.,
Suka, N.,
Turner, B. M.,
and Grunstein, M.
(1998)
Nature
392,
831-835[CrossRef][Medline]
[Order article via Infotrieve]
32.
Liebhaber, S. A.,
Urbanek, M.,
Ray, J.,
Tuan, R. S.,
and Cooke, N. E.
(1989)
J. Clin. Invest.
83,
1985-1991
33.
McWilliams, D.,
and Boime, I.
(1980)
Endocrinology
107,
761-765 34.
Jones, B. K.,
Monks, B. R.,
Liebhaber, S. A.,
and Cooke, N. E.
(1995)
Mol. Cell. Biol.
15,
7010-7021[Abstract]
35.
Bennani-Baiti, I. M.,
Asa, S. L.,
Song, D.,
Iratni, R.,
Liebhaber, S. A.,
and Cooke, N. E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
10655-10660 36.
Shewchuk, B. M.,
Asa, S. L.,
Cooke, N. E.,
and Liebhaber, S. A.
(1999)
J. Biol. Chem.
274,
35725-35733 37.
Lew, D.,
Brady, H.,
Klausing, K.,
Yaginuma, K.,
Theill, L. E.,
Stauber, C.,
Karin, M.,
and Mellon, P. L.
(1993)
Genes Dev.
7,
683-693 38.
Ginder, G.
(1989)
Methods Hematol.
20,
111-123
39.
Su, Y.,
Liebhaber, S. A.,
and Cooke, N. E.
(2000)
J. Biol. Chem.
275,
7902-7909 40.
O'Neill, L.,
and Turner, B. M.
(1996)
Methods Enzymol.
274,
189-203[Medline]
[Order article via Infotrieve]
41.
Crane-Robinson, C.,
Hebbes, T. R.,
Clayton, A. L.,
and Thorne, A. W.
(1997)
Methods
12,
48-56[CrossRef][Medline]
[Order article via Infotrieve]
42.
Nachtigal, M. W.,
Nickel, B. E.,
and Cattini, P. A.
(1993)
J. Biol. Chem.
268,
8473-8479 43.
Jiang, S.-W.,
Trujillo, M. A.,
and Eberhardt, N. L.
(1997)
Mol. Endocrinol.
11,
1233-1244 44.
Clayton, A. L.,
Hebbes, T. R.,
Thorne, A. W.,
and Crane-Robinson, C.
(1993)
FEBS Lett.
336,
23-26[CrossRef][Medline]
[Order article via Infotrieve]
45.
Bennani-Baiti, I. M.,
Cooke, N. E.,
and Liebhaber, S. A.
(1998)
Genomics
48,
258-264[CrossRef][Medline]
[Order article via Infotrieve]
46.
Bennani-Baiti, I. M.,
Jones, B. K.,
Liebhaber, S. A.,
and Cooke, N. E.
(1995)
Genomics
29,
647-652[CrossRef][Medline]
[Order article via Infotrieve]
47.
Mayo, K. E.,
Hammer, R. E.,
Swanson, L. W.,
Brinster, R. L.,
Rosenfeld, M. G.,
and Evans, R. M.
(1988)
Mol. Endocrinol.
2,
606-612 48.
Chen, E. Y.,
Liao, Y.-C.,
Smith, D. H.,
Barrera-Saldana, H. A.,
Gelinas, R. E.,
and Seeburg, P.
(1989)
Genomics
4,
479-497[CrossRef][Medline]
[Order article via Infotrieve]
49.
Luger, K.,
Mader, A. W.,
Richmond, R. K.,
Sargent, D. F.,
and Richmond, T. J.
(1997)
Nature
389,
251-260[CrossRef][Medline]
[Order article via Infotrieve]
50.
Rhodes, D.
(1997)
Nature
389,
231-233[CrossRef][Medline]
[Order article via Infotrieve]
51.
Luger, K.,
and Richmond, T. J.
(1998)
Curr. Opin. Genet. Dev.
8,
140-146[CrossRef][Medline]
[Order article via Infotrieve]
52.
Vettese-Dadey, M.,
Grant, P. A.,
Hebbes, T. R.,
Crane-Robinson, C.,
and Allis, C. D.
(1996)
EMBO J.
15,
2508-2518[Medline]
[Order article via Infotrieve]
53.
Armstrong, J. A.,
Bieker, J. J.,
and Emerson, B. M.
(1998)
Cell
95,
93-104[CrossRef][Medline]
[Order article via Infotrieve]
54.
Sheridan, P. L.,
Mayall, T. P.,
Verdin, E.,
and Jones, K. A.
(1997)
Genes Dev.
11,
3327-3340 55.
Krumm, A.,
Madisen, L.,
Yang, X.-J.,
Goodman, R.,
Nakatani, Y.,
and Groudine, M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
13501-13506 56.
Parekh, B. S.,
and Maniatis, T.
(1999)
Mol. Cell
3,
125-129[CrossRef][Medline]
[Order article via Infotrieve]
57.
Madisen, L.,
Krumm, A.,
Hebbes, T. R.,
and Groudine, M.
(1998)
Mol. Cell. Biol.
18,
6281-6292 58.
Hebbes, T. R.,
Clayton, A. L.,
Thorne, A. W.,
and Crane-Robinson, C.
(1994)
EMBO J.
13,
1823-1830[Medline]
[Order article via Infotrieve]
59.
Schlitz, R. L.,
Mizzen, C. A.,
Vassiliv, A.,
Cook, R. G.,
Allis, C. D.,
and Nakatani, Y.
(1999)
J. Biol. Chem.
274,
1189-1192 60.
Puri, P. L.,
Satorelli, V.,
Yang, X.-J.,
Hamamori, Y.,
Ogryzko, V. V.,
Howard, B. H.,
Kedes, L.,
Wang, J. Y. J.,
Graessmann, A.,
Nakatani, Y.,
and Levrero, M.
(1997)
Mol. Cell
1,
35-45[CrossRef][Medline]
[Order article via Infotrieve]
61.
Blobel, G. A.,
Nakajima, T.,
Eckner, R.,
Montminy, M.,
and Orkin, S. H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2061-2066 62.
Mutoh, H.,
Naya, F. J.,
Tsai, M.-J.,
and Leiter, A. B.
(1998)
Genes Dev.
12,
820-830 63.
Topper, J. N.,
DiChiara, M. R.,
Brown, J. D.,
Williams, A. J.,
Falb, D.,
Collins, T.,
and Gimbrone, J., M. A.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9506-9511 64.
Yang, C.,
Shapiro, L. H.,
Rivera, M.,
Kumar, A.,
and Brindle, P. K.
(1998)
Mol. Cell. Biol.
18,
2218-2229 65.
Xu, L.,
Lavinsky, R. M.,
Dasen, J. S.,
Flynn, S. E.,
McInerney, E. M.,
Mullen, T. M.,
Heinzel, T.,
Szeto, D.,
Korzus, E.,
Kurokawa, R.,
Aggarwal, A. K.,
Rose, D. W.,
Glass, C. K.,
and Rosenfeld, M. G.
(1998)
Nature
395,
301-306[CrossRef][Medline]
[Order article via Infotrieve]
66.
Blackwood, E. M.,
and Kadonaga, J. T.
(1998)
Science
281,
60-63
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  Y.-J. Li, Y.-S. Wei, X.-H. Fu, D.-L. Hao, Z. Xue, H. Gong, Z.-Q. Zhang, D.-P. Liu, and C.-C. Liang The Apolipoprotein CIII Enhancer Regulates Both Extensive Histone Modification and Intergenic Transcription of Human Apolipoprotien AI/CIII/AIV Genes but Not Apolipoprotein AV J. Biol. Chem., October 17, 2008; 283(42): 28436 - 28444. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P. Kimura, D. Sizova, S. Handwerger, N. E. Cooke, and S. A. Liebhaber Epigenetic Activation of the Human Growth Hormone Gene Cluster during Placental Cytotrophoblast Differentiation Mol. Cell. Biol., September 15, 2007; 27(18): 6555 - 6568. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kim, H. Zhao, I. Ifrim, and A. Dean {beta}-Globin Intergenic Transcription and Histone Acetylation Dependent on an Enhancer Mol. Cell. Biol., April 15, 2007; 27(8): 2980 - 2986. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Zhu, N. Singh, C. Donnelly, P. Boimel, and F. Elefant The Cloning and Characterization of the Histone Acetyltransferase Human Homolog Dmel\TIP60 in Drosophila melanogaster: Dmel\TIP60 Is Essential for Multicellular Development Genetics, March 1, 2007; 175(3): 1229 - 1240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhao, A. Kim, S.-h. Song, and A. Dean Enhancer Blocking by Chicken beta-Globin 5'-HS4: ROLE OF ENHANCER STRENGTH AND INSULATOR NUCLEOSOME DEPLETION J. Biol. Chem., October 13, 2006; 281(41): 30573 - 30580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Trujillo, M. Sakagashira, and N. L. Eberhardt The Human Growth Hormone Gene Contains a Silencer Embedded within an Alu Repeat in the 3'-Flanking Region Mol. Endocrinol., October 1, 2006; 20(10): 2559 - 2575. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. M. Shewchuk, Y. Ho, S. A. Liebhaber, and N. E. Cooke A Single Base Difference between Pit-1 Binding Sites at the hGH Promoter and Locus Control Region Specifies Distinct Pit-1 Conformations and Functions. Mol. Cell. Biol., September 1, 2006; 26(17): 6535 - 6546. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. J. Yoo, I. Cajiao, J.-S. Kim, A. P. Kimura, A. Zhang, N. E. Cooke, and S. A. Liebhaber Tissue-Specific Chromatin Modifications at a Multigene Locus Generate Asymmetric Transcriptional Interactions Mol. Cell. Biol., August 1, 2006; 26(15): 5569 - 5579. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Hiroki, Y.-H. Song, S. A. Liebhaber, and N. E. Cooke The human vitamin D-binding protein gene contains locus control determinants sufficient for autonomous activation in hepatic chromatin. Nucleic Acids Res., January 1, 2006; 34(8): 2154 - 2165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bulger Hyperacetylated Chromatin Domains: Lessons from Heterochromatin J. Biol. Chem., June 10, 2005; 280(23): 21689 - 21692. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. I. Kalmykova, D. I. Nurminsky, D. V. Ryzhov, and Y. Y. Shevelyov Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster Nucleic Acids Res., March 8, 2005; 33(5): 1435 - 1444. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Krawczyk, N. Peyraud, N. Rybtsova, K. Masternak, P. Bucher, E. Barras, and W. Reith Long Distance Control of MHC Class II Expression by Multiple Distal Enhancers Regulated by Regulatory Factor X Complex and CIITA J. Immunol., November 15, 2004; 173(10): 6200 - 6210. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Guyot, V. Valverde-Garduno, C. Porcher, and P. Vyas Deletion of the major GATA1 enhancer HS 1 does not affect eosinophil GATA1 expression and eosinophil differentiation Blood, July 1, 2004; 104(1): 89 - 91. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Kimura, S. A. Liebhaber, and N. E. Cooke Epigenetic Modifications at the Human Growth Hormone Locus Predict Distinct Roles for Histone Acetylation and Methylation in Placental Gene Activation Mol. Endocrinol., April 1, 2004; 18(4): 1018 - 1032. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Jin, L. D. Norquay, X. Yang, S. Gregoire, and P. A. Cattini Binding of AP-2 and ETS-Domain Family Members Is Associated with Enhancer Activity in the Hypersensitive Site III Region of the Human Growth Hormone/Chorionic Somatomammotropin Locus Mol. Endocrinol., March 1, 2004; 18(3): 574 - 587. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Gerrish, J. C. Van Velkinburgh, and R. Stein Conserved Transcriptional Regulatory Domains of the pdx-1 Gene Mol. Endocrinol., March 1, 2004; 18(3): 533 - 548. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Zhou, S. Chang, and T. M. Aune From the Cover: Long-range histone acetylation of the Ifng gene is an essential feature of T cell differentiation PNAS, February 24, 2004; 101(8): 2440 - 2445. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kim and A. Dean A Human Globin Enhancer Causes both Discrete and Widespread Alterations in Chromatin Structure Mol. Cell. Biol., November 15, 2003; 23(22): 8099 - 8109. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. A. Myers, W. Chong, D. R. Evans, A. W. Thorne, and C. Crane-Robinson Acetylation of Histone H2B Mirrors that of H4 and H3 at the Chicken {beta}-Globin Locus but Not at Housekeeping Genes J. Biol. Chem., September 19, 2003; 278(38): 36315 - 36322. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-y. Gui and A. Dean A major role for the TATA box in recruitment of chromatin modifying complexes to a globin gene promoter PNAS, June 10, 2003; 100(12): 7009 - 7014. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. D. Norquay, X. Yang, P. Sheppard, S. Gregoire, J. G. Dodd, W. Reith, and P. A. Cattini RFX1 and NF-1 Associate with P Sequences of the Human Growth Hormone Locus in Pituitary Chromatin Mol. Endocrinol., June 1, 2003; 17(6): 1027 - 1038. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Li, K. R. Peterson, X. Fang, and G. Stamatoyannopoulos Locus control regions Blood, October 16, 2002; 100(9): 3077 - 3086. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. M. Shewchuk, S. A. Liebhaber, and N. E. Cooke Specification of unique Pit-1 activity in the hGH locus control region PNAS, September 3, 2002; 99(18): 11784 - 11789. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Anguita, C. A. Johnson, W. G. Wood, B. M. Turner, and D. R. Higgs Identification of a conserved erythroid specific domain of histone acetylation across the alpha -globin gene cluster PNAS, September 26, 2001; (2001) 201413098. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. M. Shewchuk, N. E. Cooke, and S. A. Liebhaber The human growth hormone locus control region mediates long-distance transcriptional activation independent of nuclear matrix attachment regions Nucleic Acids Res., August 15, 2001; 29(16): 3356 - 3361. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. C. Forsberg, K. M. Downs, H. M. Christensen, H. Im, P. A. Nuzzi, and E. H. Bresnick Developmentally dynamic histone acetylation pattern of a tissue-specific chromatin domain PNAS, December 19, 2000; 97(26): 14494 - 14499. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Scully, E. M. Jacobson, K. Jepsen, V. Lunyak, H. Viadiu, C. Carrière, D. W. Rose, F. Hooshmand, A. K. Aggarwal, and M. G. Rosenfeld Allosteric Effects of Pit-1 DNA Sites on Long-Term Repression in Cell Type Specification Science, November 10, 2000; 290(5494): 1127 - 1131. [Abstract] [Full Text]
|
|
|
|
|
|
E. R. Smith, C. D. Allis, and J. C. Lucchesi Linking Global Histone Acetylation to the Transcription Enhancement of X-chromosomal Genes in Drosophila Males J. Biol. Chem., August 17, 2001; 276(34): 31483 - 31486. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Hebbes and S. C. H. Allen Multiple Histone Acetyltransferases Are Associated with a Chicken Erythrocyte Chromatin Fraction Enriched in Active Genes J. Biol. Chem., September 29, 2000; 275(40): 31347 - 31352. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. A. Myers, D. R. Evans, A. L. Clayton, A. W. Thorne, and C. Crane-Robinson Targeted and Extended Acetylation of Histones H4 and H3 at Active and Inactive Genes in Chicken Embryo Erythrocytes J. Biol. Chem., June 1, 2001; 276(23): 20197 - 20205. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Anguita, C. A. Johnson, W. G. Wood, B. M. Turner, and D. R. Higgs Identification of a conserved erythroid specific domain of histone acetylation across the alpha -globin gene cluster PNAS, October 9, 2001; 98(21): 12114 - 12119. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |