MOLECULAR AND CELLULAR BIOLOGY, July 2005, p. 5648–5663
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 13
Arginine Methylation Provides Epigenetic Transcription Memory for
Retinoid-Induced Differentiation in Myeloid Cells
Balint L. Balint,1Attila Szanto,1Andras Madi,1,5Uta-Maria Bauer,2Petra Gabor,1Szilvia Benko,1†
Laszlo G. Puska ´s,3Peter J. A. Davies,4and Laszlo Nagy1*
Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, University of Debrecen, Medical
and Health Science Center,1and Signaling and Apoptosis Research Group of the Hungarian Academy of Sciences,5
Nagyerdei krt. 98, Debrecen H-4012, Hungary; Institute for Molecular Biology and Tumor Research,
Philipps-University Marburg, Emil-Mannkopff-Str. 2, 35033 Marburg, Germany2; Laboratory of
Functional Genomics, Biological Research Center of the Hungarian Academy of Sciences,
Temesva ´ri krt. 62, Szeged H-6726, Hungary3; and Department of Integrative Biology
and Pharmacology, Medical School, University of Texas—Houston,
6431 Fannin, Houston, Texas 770304
Received 16 August 2004/Returned for modification 15 September 2004/Accepted 31 March 2005
Cellular differentiation is governed by changes in gene expression, but at the same time, a cell’s identity
needs to be maintained through multiple cell divisions during maturation. In myeloid cell lines, retinoids
induce gene expression and a well-characterized two-step lineage-specific differentiation. To identify mecha-
nisms that contribute to cellular transcriptional memory, we analyzed the epigenetic changes taking place on
regulatory regions of tissue transglutaminase, a gene whose expression is tightly linked to retinoid-induced
differentiation. Here we report that the induction of an intermediary or “primed” state of myeloid differenti-
ation is associated with increased H4 arginine 3 and decreased H3 lysine 4 methylation. These modifications
occur before transcription and appear to prime the chromatin for subsequent hormone-regulated transcrip-
tion. Moreover, inhibition of methyltransferase activity, preacetylation, or activation of the enzyme PAD4
attenuated retinoid-regulated gene expression, while overexpression of PRMT1, a methyltransferase, enhanced
retinoid responsiveness. Taken together, our results suggest that H4 arginine 3 methylation is a bona fide
positive epigenetic marker and regulator of transcriptional responsiveness as well as a signal integration
mechanism during cell differentiation and, as such, may provide epigenetic memory.
The HL-60 cell line is a well-characterized M2 myeloid leu-
kemia cell line (6) that can be induced to undergo differenti-
ation along different pathways, myeloid versus monocytic, in
response to a variety of physiological and pharmacological
stimuli. The process of myeloid differentiation itself involves
two distinct and sequential steps. The first is an identifiable
intermediate state termed the precommitment or primed state,
and the second is a series of late events that lead to the onset
of lineage-specific terminal differentiation (55–58). Primed
cells are characterized by altered nuclear structure and feature
retention of phenotype, a form of cellular memory that can last
for several cell cycles. Most significantly, primed cells require
only an abbreviated exposure to an appropriate inducer, such
as all-trans-retinoic acid, for onset of G1/0-specific growth ar-
rest and phenotypic differentiation (55). For example, a short
exposure (16 to 24 h) to dimethyl sulfoxide (DMSO) or vitamin
D induces a state of precommitment in HL-60 cells. Subse-
quent exposure of these precommitted cells to another in-
ducer, such as all-trans-retinoic acid, results in the onset of
growth arrest and differentiation that is much more rapid (?24
h) than for cells that have not been subject to precommitment
(?48 h). In this example, DMSO exposure results in the ac-
quisition of a precommitment memory, a state which can be
sustained for more than one cell cycle (55). These experimen-
tal observations have been explained by suggesting a two-step
model for induction of terminal differentiation in the cells. In
this model, early events preceding precommitment regulate
growth arrest, and late events that occur after precommitment
regulate the choice of a specific differentiation lineage. Inter-
estingly, both events occur while the cells are still proliferating
(57). Very little is known about the molecular characteristics of
the precommitment or primed state, and in particular, very
little is known about how these molecular changes impact the
regulation of gene expression.
One mechanism that is an obvious candidate to explain the
generation of a primed state of transcriptional memory is the
alteration in chromatin structure. The structure of the chro-
matin plays a fundamental role in regulating gene expression
by controlling the access of transcription factors to the regu-
latory regions of genes. Two classes of enzymes are known to
play a role in regulating chromatin structure. The ATP-depen-
dent chromatin-remodeling enzymes act by the sliding of DNA
and the triggering of conformational changes of the nucleo-
somes (20). The other class of chromatin-modifying enzymes is
responsible for the posttranslational modification of histone
tails. According to the “histone code” hypothesis, the covalent
modifications of the histone tails are responsible for the sus-
tained maintenance and modulation of patterns of gene ex-
* Corresponding author. Mailing address: Department of Biochem-
istry and Molecular Biology, Research Center for Molecular Medicine,
University of Debrecen, Medical and Health Science Center, Nagyer-
dei krt. 98, Debrecen H-4012, Hungary. Phone: 36 52 416 432. Fax: 36
52 314 989. E-mail: email@example.com.
† Present address: Department of Immunology, University of Deb-
recen, Medical and Health Science Center, Nagyerdei krt. 98, Debre-
cen H-4012, Hungary.
pression (25, 45, 49, 50). Modified histone tails have been
reported to form binding sites of specific classes of proteins:
bromodomain-containing proteins bind to histone tails with
acetylated lysine residues, while chromodomain-containing
proteins bind to methylated histone lysine residues. It is thought
that these histone binding proteins form a functional link be-
tween the covalently modified histone tails and the effectors of
transcription initiation (32).
To investigate the molecular mechanisms that may be asso-
ciated with the priming of HL-60 cells and the establishment of
a precommitted state, we have characterized the covalent mod-
ification of the tails of histones specifically associated with the
promoter of tissue transglutaminase, a gene that undergoes
marked up-regulation during retinoid-induced myeloid cell dif-
We used chromatin immunoprecipitation combined with re-
al-time PCR to quantify the level of histone acetylation and
methylation on the nucleosomes associated with the proximal
regions of the tissue transglutaminase promoter. We found
that acetylation of histone 4 (H4) is correlated with increases
in gene expression. Changes in methylation on histone H3
lysine 4 (H3K4) and histone H4 arginine 3 (H4R3) are linked
to the presence of the primed state of differentiation and
increased hormonal responsiveness. In an attempt to establish
more-mechanistic links between H4R3 methylation and retin-
oid responsiveness, we evaluated the role of an enzyme pair
shown to be responsible for the methylation and demethylation
(citrullination) of H4R3 (53). We found that inhibition of
methylation by preacetylation or activation of PAD4 by Ca2?
ionophores attenuated retinoid responsiveness and overex-
pression of PRMT1 enhanced it.
MATERIALS AND METHODS
Cells and materials. HL-60/CDM-1 cells, a kind gift of Diane Lucas (Walter
Reed Army Medical Center, Washington, D.C.), were cultured in suspension in
RPMI 1640 medium supplemented with ITS (I1884; Sigma) using standard cell
culture conditions. Cells were treated with 9-cis retinoic acid at a concentration
of 1 ?M dissolved in ethanol-DMSO. Priming of cells was done with 1.25%
DMSO for 16 h. Blocking of methyltransferases was achieved by treatment with
10 ?M adenosine dialdehyde (ADOX, A7154;Sigma) for the same period. After
pretreatment, medium was replaced with fresh medium and retinoic treatment
was carried out. If not specified, all materials were purchased from Sigma.
Antibodies for flow cytometric analysis, if not otherwise mentioned, were pur-
chased from DAKO A/S, as were the appropriate control antibodies.
Plasmids. Full-length wild-type pcDNA3.1 human PRMT1 was cloned from
the pGEX-HRMT1L2 (v2) (43) by PCR introducing a BamHI site at the 5? end
and an EcoRI site at the 3? end of the HMRT1L2 cDNA (deleting the stop at the
same time). Subsequently, the HMRT1L2 cDNA was subcloned with BamHI
and EcoRI into pcDNA3.1-B (Invitrogen), generating a C-terminal myc/His tag.
The point mutations S69A, G70A, and T71A were introduced into pcDNA3.1
human PRMT1 via site-directed mutagenesis using the QuikChange kit (Strat-
agene) to give rise to the pcDNA3.1 PRMT1 full-length catalytic mutant. All
constructs were verified by DNA sequencing. Mammalian expression vectors for
PAD4 and the PAD4 C645S mutant were received from Yanming Wang and
described previously (53). All other plasmids used were described previously (4).
Transfection. HL-60 cells were transfected with the AMAXA electroporator
system using 1 ?g of plasmid for 2 million cells according to the manufacturer’s
instructions (electroporation solution V and program V01). 293T cells were
transfected with jetPEI reagent (Qbiogene) according to the manufacturer’s
instructions. The ?RARE thymidine kinase Luc reporter plasmid was cotrans-
fected with full-length pCMV retinoic acid receptor ? (RAR?), full-length
pCMV retinoic X receptor ? (RXR?), and the ?-galactosidase plasmid. Wild-
type PRMT1, mutant PRMT1, wild-type PAD4, and mutant PAD4 were added
to the reporter plasmids.
Extraction of total RNA. Total RNA was extracted by using Trizol reagent
(Invitrogen) according to the manufacturer’s instructions.
Real-time QPCR. Real-time quantitative PCR (QPCR) was carried out on
ABI7700 and ABI7900 real-time sequence detection systems. QPCR measure-
ments were performed as described previously (51). For the TGM2 promoter
assays, the reactions were carried out similarly without the reverse transcription
step. For standard calibration, DNA from the bacterial artificial chromosome
clone RP5-1054A22 was used which contained the whole promoter of TGM2 and
was received from the Sanger Centre, Clone Resources Group, Hinxton, United
Chromatin immunoprecipitation. Chromatin immunoprecipitation was car-
ried out as described by Kuo and Allis (29) with modifications. Briefly, cells were
fixed with 1% formaldehyde for 10 min at room temperature. Fixation was
stopped by adding chilled glycine to a final concentration of 150 mM. Cells were
scraped and washed twice with ice-cold phosphate-buffered saline (PBS) that
contained proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 ?g/?l
aprotinin, and 1 ?g/?l pepstatin A). Nuclei were prepared by incubation for 10
min on ice in a buffer containing 5 mM piperazine-N,N?-bis(2-ethanesulfonic
acid) (PIPES), pH 8, 85 mM KCl, 0.5% NP-40, and proteinase inhibitors. After
centrifugation at 3,000 ? g for 10 min at 4°C, nuclei were resuspended in
sonication buffer (1% sodium dodecyl sulfate [SDS], 0.1 M NaHCO3, and pro-
teinase inhibitors), lysed on ice for 10 min, and sonicated on ice to an average
fragment size of 300 base pairs. Cell debris was pelleted twice by centrifugation
at 10,000 ? g for 30 min at 4°C in a bench-top centrifuge. Soluble chromatin was
aliquoted, frozen in liquid nitrogen, and stored at ?70°C. For immunoprecipi-
tation, chromatin was diluted 10-fold in an immunoprecipitation (IP) buffer
(0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris, pH 8.1, 16.7 mM
NaCl, and proteinase inhibitors). One milliliter of diluted chromatin was pre-
cleared twice with 40 ?l and blocked with protein A-Sepharose beads. Immu-
noprecipitation was carried out with specific antibodies purchased from Upstate
Biotech and Abcam against modified histones as follows: from Upstate, anti-
acetyl H4 (2 ?l/IP, catalog no. 06-866), anti-dimethyl H4 Arg 3 (6 ?l/IP, catalog
no. 07-213), anti-dimethyl H3 Lys 4 (5 ?l/IP, catalog no. 07-030), anti-dimethyl
H3 Lys 9 (5 ?l/IP, catalog no. 07-212); from Abcam, H4 methyl R3 antibody (5
?l/IP, catalog no. ab5823), pan dimethyl arginine (5 ?l/IP, catalog no. 413-200).
Incubation with the antibodies was carried out overnight on a rotating plate at
4°C. Complexes were collected with 40 ?l blocked protein A-agarose (catalog no.
16-157; Upstate). An aliquot of the no-antibody control supernatant was used to
measure and calculate the input DNA. Beads were pelleted and washed twice
with each of the following buffers: buffer A (low salt) (0.1% SDS, 1% Triton
X-100, 2 mM EDTA, 20 mM Tris, pH 8.1, 150 mM NaCl, and propidium iodide
[PI]), buffer B (high salt) (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM
Tris, pH 8.1, 500 mM NaCl, and PI), buffer C (0.25 M LiCl, 1% NP-40, 1%
sodium deoxycholate, 1 mM EDTA, 10 mM Tris, pH 8.1, and PI), and TE buffer
(10 mM Tris, 10 mM EDTA, pH 8, and PI). Immunoprecipitated nucleosomes
were eluted twice from beads with elution buffer (1% SDS, 0.1 M NaHCO3), and
eluates were combined. Cross-links were reversed by incubation for 6 h at 65°C
after the addition of 20 ?l 5 M NaCl. The eluate was combined with 10 ?l of 0.5
M EDTA, 20 ?l 1 M Tris, pH 6.5, and 2 ?g proteinase K and incubated for 1 h
at 45°C. DNA was recovered after phenol-chloroform extraction and ethanol
precipitation using 20 ?g of glycogen as a carrier. DNA was resuspended in 50
?l of 50 ng/?l yeast tRNA (catalog no. 15401-011; Invitrogen). Two microliters
of this solution was used for real-time QPCR in a 25-?l reaction mixture. All
measurements were done in triplicate. All chromatin results were verified from
independent chromatin preps.
Cell cycle analysis. Cell cycle analysis was performed as follows: cells were
washed in PBS and fixed in 70% ethanol overnight. Briefly, cells were washed in
PBS and fixed in 70% ethanol overnight. Fixed cells were then washed twice and
resuspended in PI working solution (50 ?g of propidium iodide, 20 ?g/ml RNase,
and 0.5% Tween-20 in PBS). After a 15-min incubation at 37°C, cells were
analyzed on a Coulter flow cytometer and data were analyzed with WinMDI
Intracellular staining and flow cytometry. Cells were fixed (2% paraformal-
dehyde, 0.1% sodium azide, 1% heat-inactivated filtered fetal bovine serum) at
room temperature for 20 min. After washing, cells were permeabilized with
buffer P (PBS, 2% fetal bovine serum, 0.2% sodium azide, 0.5% saponin) for 10
min on ice. Immunostaining was carried out with a mouse monoclonal antibody
which specifically recognizes the TGM2 protein (a kind gift of Laszlo Fesus,
University of Debrecen) and an isotype control antibody. As a secondary anti-
body, fluorescein isothiocyanate-conjugated anti-mouse antibody was used. All
staining procedures were done in saponin-containing buffers. Flow cytometry
analysis was done in buffer P as described above.
VOL. 25, 2005H4R3 METHYLATION AND EPIGENETIC MEMORY5649
Microarray analysis of gene expression patterns. Total RNA was extracted as
described above. RNA was labeled with Cy3 and Cy5 and hybridized to a
microarray containing 3,200 human genes as described previously (42). Data
processing and statistical analysis were done as described previously (41).
Protein expression pattern analysis. Protein expression pattern analysis was
performed as described previously (31). Briefly, 150 ?g of total protein was run
on parallel gels. After isoelectric focusing between pH 3 and 10, samples were
run on an 11% SDS gel. Gels were silver stained and analyzed as described
previously (31) using Phoretix 5.01 software (NonLinear Dynamics Ltd., New-
castle upon Tyne, United Kingdom).
Quantification of DNase I sensitivity. Quantification of DNase I sensitivity was
performed as described previously (33) with modifications. Briefly, cells were
washed with ice-cold PBS resuspended in lysis buffer (50 mM Tris-Cl pH 7.9, 100
KCl, 5 mM MgCl2, 0.05% saponin, 50% glycerol, 200 mM ?-mercaptoethanol)
and incubated on ice for 10 min. Nuclei were recovered by centrifugation at 1,300
? g for 15 min at 4°C and resuspended in buffer A (50 mM Tris-Cl, pH 7.9, 100
mM NaCl, 3 mM MgCl21 mM dithiothreitol, and proteinase inhibitors). After
centrifugation at 1,300 ? g for 15 min at 4°C, nuclei were resuspended in buffer
A and divided into several aliquots. After this step, nuclei were treated with
different concentrations of DNase I for 20 min at 37°C. The reaction was stopped
with a 1/10 volume of 0.5 M EDTA. After RNase and proteinase K digestion,
DNA was extracted with phenol-chloroform and precipitated with absolute eth-
anol. Extracted DNA was treated with EcoRI, purified with PCR purification
columns (QIAGEN), and measured by QPCR for the specific promoter regions.
Values were normalized to the total DNA concentration in each sample as
measured with a photometer (A260and A280).
Retinoid regulation of tissue transglutaminase gene expres-
sion in naive and primed myeloid leukemia cells. The expres-
sion of tissue transglutaminase type 2 (TGM2) is very tightly
regulated in myeloid leukemia cells. In HL-60 cells, in the
absence of exposure to retinoids, the level of TGM2 mRNA is
below the limits of detection of a sensitive real-time reverse
transcription (RT)-QPCR assay (less than 10 copies per nano-
gram of total RNA). Exposure of cells to either natural or
synthetic retinoid receptor agonists increases transglutaminase
gene expression markedly (17). Priming of the cells by pre-
treatment with differentiating agents such as vitamin D or the
polar-planar solvent DMSO increases retinoid-induced TGM2
expression. This experimental system (Fig. 1A) has allowed us
to study the effects of priming on the expression of a specific
gene, the induction of which is increased by the process of
precommitment. We will refer to the unprimed and uncom-
mitted cells as “naive” and those that advanced to precommit-
ment as “primed.”
Priming enhances retinoid response in HL-60 cells. To
study the effect of priming on the differentiation of HL-60 cells,
we analyzed the expression of a set of differentiation-linked
surface markers. Cells were primed with DMSO or vitamin D.
The priming agent was washed out, and cells were treated with
9-cis retinoic acid. The cell surface expression of integrin mol-
ecules CD18 or integrin ?2 and its heterodimeric partners
CD11b and CD11c or integrin alpha M and integrin alpha X
showed an increased expression after retinoid treatment in
primed cells if compared to naive cells (Fig. 1B, C, and D). An
increased expression could be detected in the case of all three
markers with DMSO and for CD11b with vitamin D priming.
The priming effect of both DMSO and vitamin D pretreat-
ment was detectable on the retinoid response of tissue trans-
glutaminase type 2, a molecular marker of myeloid cell differ-
entiation and a direct target of retinoid receptors (37). As seen
on Fig. 1E, priming with DMSO produced a marked increase
in the expression of TGM2. To determine whether priming
conferred some sort of transcriptional memory, we analyzed
the expression of tissue transglutaminase type 2 after the prim-
ing agent was washed out and the cells were treated with 9-cis
retinoic acid after 1, 2, or 3 days. The priming effect of DMSO
proved to be transient and declined rapidly (Fig. 1E). Similarly,
in the case of vitamin D priming, an increase in the expression
of TGM2 was detected but with a smaller amplitude (Fig. 1F).
This effect decreased in 2 days to the level of naive cells. These
experiments showed that the priming effect is transient, lasting
for 24 to 48 h. Since both of these agents, DMSO and vitamin
D, produced similar effects, we decided to carry out our ex-
periments with the more potent priming agent available,
To provide a baseline for our further studies we determined
the time course of mRNA induction. Exposure of “naive”
HL-60 cells to retinoids (9-cis retinoic acid) results in a very
rapid (?2 h) increase in transglutaminase gene expression
(Fig. 2A) that reached a plateau after 12 h (data not shown).
This induction is strikingly enhanced (approximately 100-fold)
upon DMSO priming (Fig. 2A and B). DMSO alone does not
increase TGM2 expression (Fig. 2A and B). It was apparent by
comparing the induction in the naive and primed cells that,
while the magnitude of the induction was very different (Fig.
2B), the kinetics was very similar whether or not the cells had
been primed (Fig. 2A). Thus, priming resulted in a state char-
acterized by greater induction of the target gene without an
alteration in the time course of the transcriptional response.
A key question is whether increased induction of transglu-
taminase expression in the primed cells is a result of a higher
mRNA expression level in each individual cell or is due to an
increase in the fraction of the cells responding to the inducer
(retinoid). Since it has already been demonstrated that there is
a correlation between TGM2 protein and mRNA levels in
HL-60 cells (9, 10, 14–16), we addressed the issue of the in-
duction of the enzyme by using a coupled immunohistochem-
ical/flow cytometric analysis to evaluate the levels of TGM2 in
individual cells prior to and following retinoid treatment. Us-
ing these techniques, the basal level of TGM2 was undetect-
able in both untreated HL-60 cells and HL-60 cells treated
with DMSO alone (data not shown). In naive cells (as shown in
Fig. 2C) 9-cis retinoic acid induced detectable levels of TGM2
in 19.7% of the cells. The level of TGM2 was normally distrib-
uted in the population of cells with a mean fluorescence inten-
sity of 305.05 arbitrary units (AU). When DMSO-primed cells
were treated with 9-cis retinoic acid, a much greater fraction of
the population responded to the retinoid stimulus than did the
naive, retinoid-treated cells. Among primed cells, 63.7% had
detectable levels of TGM2 protein (Fig. 2D) and this also
showed a normal distribution. The mean fluorescence intensity
in primed, retinoid-treated cells was not different (327.8 AU)
from the level of fluorescence intensity in retinoid-treated na-
ive cells (305.0 AU). The mean fluorescence intensity of the
entire cell population increased from 39.6 to 179.43 AU after
DMSO priming and retinoid induction. The most likely expla-
nation for these findings is that the maximal level of TGM2
expression by individual cells did not change after DMSO
priming, but more cells gained competence to respond to reti-
noids with increased TGM2 expression. It appears that priming
is likely to lower the threshold for induction of gene expression
5650 BALINT ET AL.MOL. CELL. BIOL.
FIG. 1. Priming enhances retinoid response in HL-60 cells. (A) Experiment outline. HL-60/CDM-1 cells were primed with 1.25% DMSO
overnight. The control (naive) cells received no priming. After overnight incubation, cells were washed, resuspended in fresh medium, and treated
with 1 ?M 9-cis retinoic acid. (B) Priming with DMSO or vitamin D enhances 9-cis retinoic acid-dependent expression of integrin CD11b. Cells
were primed with 1.25% DMSO or 100 nM vitamin D. After overnight incubation and washing out of the priming agent, cells were treated with
1 ?M 9-cis retinoic acid and analyzed on a flow cytometer at the indicated time points as described in Materials and Methods. Isotype controls
had no change (not shown). (C) Priming with DMSO or, to a lesser extent, with vitamin D enhances 9-cis retinoic acid-dependent expression of
integrin CD11c. Cells were primed with 1.25% DMSO or 100 nM vitamin D. After overnight incubation and washing out of the priming agent,
cells were treated with 1 ?M 9-cis retinoic acid and analyzed with a flow cytometer at the indicated time points as described in Materials and
Methods. Isotype controls had no change (not shown). (D) Priming with DMSO but not with vitamin D enhances 9-cis retinoic acid-dependent
expression of integrin CD18. Cells were primed with 1.25% DMSO or 100 nM vitamin D. After overnight incubation and washing out of the
priming agent, cells were treated with 1 ?M 9-cis retinoic acid and analyzed with a flow cytometer at the indicated time points as described in
Materials and Methods. Isotype controls had no change (not shown). (E) Relative increase of TGM2 mRNA induction compared to that of naive,
retinoid-treated cells. Assessment of the maintenance in time of the priming effect: TGM2 mRNA was measured by real-time QPCR after priming
with 1.25% DMSO and compared to naive, retinoid-treated cells. After overnight incubation and washing out of the priming agent, cells were
treated with 1 ?M 9-cis retinoic acid at different time points after the priming, as indicated. The retinoid treatment was 12 h long in all cases. Values
are the means of the results from three independent QPCR measurements ? standard deviations. (F) Relative increase of TGM2 induction
compared to that of naive, retinoid-treated cells. Assessment of the maintenance in time of the priming effect: TGM2 mRNA was measured by
real-time QPCR after priming with 100 nM vitamin D and compared to naive, retinoid-treated cells. After overnight incubation and washing out
of the priming agent, cells were treated with 1 ?M 9-cis retinoic acid at different time points after the priming, as indicated. The retinoid treatment
was 12 h long in all cases. Values are the means of the results from three independent QPCR measurements ? standard deviations.
VOL. 25, 2005 H4R3 METHYLATION AND EPIGENETIC MEMORY5651
and differentiation, resulting in a larger portion of cells able to
respond. This is consistent with our previous findings shown in
Fig. 2A, namely that priming affects the amplitude but not the
dynamics of the response to retinoic acid.
Role of receptor levels and cell cycle distribution of cells in
enhanced retinoid response. To investigate possible explana-
tions for the increased frequency of response by primed cells,
we tested two obvious hypotheses. The first is that the effect of
priming is to increase the level of expression of retinoid recep-
tors in individual cells. To address this hypothesis, we mea-
sured the levels of transcripts of RAR?, -?, and -? and RXR?
and ? in naive and DMSO-primed HL-60 cells. There was no
significant change in the expression level of RAR? and there
was only a slight increase (less than twofold, as measured by
real-time RT-QPCR assay) in the level of RXR? mRNA as the
result of DMSO priming (data not shown). There was also no
change in the levels of RAR? and RXR? mRNA (data not
shown). The second is that differentiation is linked to cell cycle
FIG. 2. TGM2 expression is enhanced by DMSO priming at both the RNA and protein levels, and this effect is not cell type specific. (A) 9-cis
retinoic acid induction of TGM2 mRNA as measured by real-time QPCR. The expression of TGM2 mRNA in primed and naive cells is shown
on the same graph with two different y axes with a difference of 2 orders of magnitude of the scales. Transcript copy numbers were normalized
to 36B4 transcript levels. Values are the means of the results from three independent QPCR measurements ? standard deviations. (B) Relative
difference in gene expression in naive and primed cells. Fold changes are indicated above the bars. Copy numbers of TGM2 mRNA are determined
by real-time QPCR and normalized to 36B4 transcript levels. Values are the means of the results from three independent QPCR measurements
? standard deviations. (C) Intracellular immunostaining and flow cytometric analysis of retinoid-treated HL-60 cells as described in Materials and
Methods. The expression of TGM2 protein in naive cells (shaded) and its isotype control (white) after retinoid induction. The percentage of cells
expressing TGM2 protein is shown on the graph along with the mean fluorescence intensity of the positive cells. Values are expressed in arbitrary
units (AU). (D) Intracellular immunostaining and flow cytometric analysis of retinoid-treated HL-60 cells as described in Materials and Methods.
TGM2 expression in DMSO-primed cells (shaded) along with its isotype control (white). The percentage of cells expressing TGM2 is shown on
the graph along with the mean fluorescence intensity of the positive cells, expressed in arbitrary units (AU). At least three independent
determinations have been carried out. Data from a representative experiment are shown. (E) Effect of priming upon TGM2 induction by retinoids
in MonoMac6 (MM6) cells. TGM2 copy numbers were normalized to cyclophilin transcript levels. Values are the means of the results from three
independent QPCR measurements ? standard deviations. (F) TGM2 induction in NB4 and RAR? mutant NB4R2 cells in naive and primed states.
The mRNA copy numbers were normalized to cyclophilin transcript levels. Values are the means of the results from three parallel QPCR
measurements ? standard deviations, and results were confirmed from at least three independent biological samples. ?, present; ?, absent.
5652 BALINT ET AL.MOL. CELL. BIOL.
arrest. We considered it possible that the change in the fraction
of cells responding to retinoids following DMSO priming was
linked to alterations in the distribution of cells in different
stages of the cell cycle. It has previously been shown that
priming HL-60 cells with DMSO for a period shorter than 24 h
does not significantly change its cell cycle distribution (58), but
to be sure that something different was not happening in our
cell population, we compared the distribution of cells in naive
and DMSO-primed populations. Our results confirmed the
previously reported findings, DMSO priming had only a minor
effect on the distribution of cells: there was a slight increase in
the number of cells in G1(51% to 63%) that was compensated
for by slight decreases in the fraction of cells in the S and M
phases (from 27% to 20% and from 22% to 17%, respectively)
(data not shown).
To test whether the effect of priming on retinoid-regulated
gene expression was restricted to HL-60 cells, we carried out
comparable studies in a human macrophage-like cell line
(monocytic leukemia cell line, FAB M5/MonoMac6 or MM6)
and in the well-characterized NB4 and NB4R2 promyelocytic
leukemia (FAB M3) cell lines. The two NB4 cell lines also
allowed us to address the issue of retinoid receptor depen-
dence. As shown in Fig. 2E, we have found that retinoid-
induced expression of TGM2 was increased in DMSO-primed
MM6 cells compared to unprimed cells. In the NB4 cells,
DMSO priming also increased retinoid-induced TGM2 expres-
sion (Fig. 2F). The NB4R2 cells have a mutation in the ligand
binding domain of RAR? that disrupts retinoid signaling (18).
In this cell line, TGM2 could not be induced after retinoid
treatment either in naive or in DMSO-primed cells (Fig. 2F).
These findings demonstrated that the effects of priming on
retinoid-regulated gene expression are not unique to HL-60
cells but represent a generalized phenomenon at least among
human myeloid leukemia cell lines.
Alterations in chromatin modifications in naive and primed
cells upon retinoid treatment. A precommitment state of
HL-60 cells can be generated by short treatment (?24 h) with
several differentiating agents (57). This transient primed state
is not associated with growth arrest but is characterized by lack
of lineage commitment, by altered nuclear structure, and by
the retention of a cellular memory that lasts for at least three
rounds of cell division (55, 56). Changes in chromatin structure
are major contributors to the regulation of transcriptional ac-
tivity that can, through epigenetic modifications, provide tran-
scriptional memory. We therefore investigated the question of
whether DMSO priming had any demonstrable effects on ei-
ther the DNA or histone components of chromatin associated
with retinoid-regulated genes such as tissue transglutaminase.
Previous studies have identified an 1,800-bp fragment in the
5?-flanking DNA of the human tissue transglutaminase gene as
containing the core promoter and the HR1 enhancer (37). We
therefore focused our attention on the effects of DMSO prim-
ing on the chromatin and covalent modification of histones on
this key regulatory sequence.
To find out whether priming is producing changes on the
chromatin level at the regulatory regions of this gene, we
performed a DNase I hypersensitivity analysis of the promoter
of TGM2. The promoter of RAR? that is not inducible in this
cell line by retinoid treatment and priming did not change this
property of the gene (data not shown); DNase I hypersensitiv-
ity was not induced by priming (Fig. 3A). On the other hand,
as shown on Fig. 3B, the core promoter of the TGM2 gene
became more sensitive to DNase I solely by DMSO priming,
suggesting that priming induces changes at the chromatin level
The next step in our studies was to characterize the effects of
DMSO priming on the posttranslational modifications of his-
tones associated with regions of the tissue transglutaminase
gene promoter. We have used chromatin immunoprecipitation
in combination with QPCR (real-time QPCR with TaqMan
probes) to obtain accurate quantitation of the level of post-
translational modification of specific histone tails in chromatin
isolated from naive and DMSO-primed HL-60 cells. We de-
signed five promoter-specific probe sets spanning the 1,800-bp
fragment from the HR1 enhancer to the core promoter (Fig.
4). The AG- and TG-rich tandem repeat regions embedded in
the promoter were not covered in this analysis.
We first examined the effect of retinoid treatment on H4
acetylation in naive and DMSO-primed cells. As shown on Fig.
3C, in naive cells, retinoid treatment produced little change in
the level of acetylation of H4 histones associated with the HR1
region of the transglutaminase promoter. In primed cells, on
the other hand, retinoid treatment for 2 h results in increased
levels of H4 acetylation. There is a similar and significant
increase in H3 acetylation on the core promoter region in
retinoid-treated primed cells and no detectable change in the
level of acetylation of histones in this enhancer region HR1 in
the retinoid-treated naive cells (data not shown). These find-
ings demonstrate that after retinoid induction of transcription,
histones associated with both the HR1 enhancer and the core
promoter of TGM2 gene become acetylated in primed cells but
do so to a lesser extent in naive cells. Due to the fact that this
method is not suitable for analysis of individual cells, we can
only state that, after retinoid treatment of DMSO-primed
HL-60 cells, the acetylation level of the TGM2 promoter of the
entire cell population is much higher than in naive retinoid-
treated cells. The level of acetylation of the TGM2 promoter in
naive cells after retinoid treatment is below the levels of de-
tection by this method. Whether there is a small fraction of
cells in which this region is acetylated or not cannot be as-
Based on these findings, we carried out a more comprehen-
sive analysis of the effects of DMSO priming and retinoid
treatment of primed cells on histone modifications (acetylation
of H3 and H4 and methylation of H3K4, H3K9, and H4R3)
(Fig. 4). The upper two panels of Fig. 4 show the changes in
histone acetylation that occurred in response to retinoid treat-
ment. In the case of H4 acetylation, retinoid treatment of
primed cells results in a significant and uniform increase in the
level of acetylation of this histone H4 in all five regions of the
promoter. This increase in acetylation starts within 2 h of the
addition of the retinoid and reaches a plateau in 6 to 8 h.
Retinoid treatment also results in increased acetylation of H3,
but unlike H4 acetylation, this effect is not global but is re-
stricted to histones associated with the core promoter.
The lower three panels of Fig. 4 profile the effects of retinoid
treatment on the pattern of histone tail methylation. In the
case of H3K4, DMSO priming results in a marked decrease in
the level of methylation of histones on the core promoter
region, as seen in the left bar graph of Fig. 4 (for other regions,
VOL. 25, 2005H4R3 METHYLATION AND EPIGENETIC MEMORY5653
H3K4 levels were lower by at least 1 order of magnitude [data
not shown]). DMSO priming does not have any marked effect
on the K9 methylation of H3 in any region of the TGM2
promoter. Retinoid treatment does induce transient changes in
the methylation of this histone in most regions of the pro-
moter, and these changes are most prominent in histones as-
sociated with the distal enhancer (HR1) region of the pro-
moter. While DMSO priming decreased methylation of the H3
histone side chains (K4), it increases the level of methylation
on the H4 histone (R3). The effect is selective, being most
marked on histones associated with the distal regions of the
promoter, particularly the HR1 enhancer, and less marked for
the histones associated with the proximal regions and the core
of the promoter. Retinoid treatment induces a rapid increase
in H4R3 methylation that peaks at about 2 h and then rapidly
returns to the baseline.
These results demonstrate that the priming of HL-60 cells
results in a coordinated set of histone modifications that are
likely to be linked to alterations in chromatin structure. Among
the most prominent of these effects are the suppression of
H3K4 methylation at the core promoter and the increased
methylation of the R3 residue of H4 on histones associated
with the distal regulatory regions of the promoter. Further-
more, although DMSO priming itself has little effect on either
H3 or H4 acetylation, retinoid treatment results in a marked
and generalized increase in H4 acetylation and more localized
increase in H3 acetylation at the core promoter region.
Role of H4R3 methylation and H3K4 demethylation. The
comparison of histone modifications between naive and DMSO-
primed HL-60 cells indicated that reciprocal changes in side
chain modifications might be linked to the altered activation of
gene expression associated with the precommitment process.
To better understand the types of processes that might be
involved in the observed alterations in histone methylation, we
examined the time course for the changes in H4R3 and H3K4
methylation that followed the initiation of priming with
DMSO. Naive cells were treated with DMSO for 4 and 12 h,
and the chromatin immunoprecipitation (ChIP) analysis was
carried out to determine the levels of H4R3 methylation of
histones bound to the HR1 enhancer and H3K4 methylation of
histones bound to the core promoter (Fig. 5). There is a strik-
ing similarity between the reciprocal changes of H4R3 and
H3K4 methylation. Both are substantially changed within 4 h
FIG. 3. Priming produces changes at the chromatin level on the promoter of TGM2. (A) DNase I sensitivity of the RAR? promoter in naive
and primed cells. DNase I hypersensitivity was measured as described in Materials and Methods. Values are the means of three independent
QPCR measurements ? standard deviations of a representative experiment. (B) DNase I sensitivity of the TGM2 promoter in naive and primed
cells. DNase I hypersensitivity was measured as described in Materials and Methods. Values are the means of three independent QPCR
measurements ? standard deviations of a representative experiment. (C) Chromatin immunoprecipitation analysis of HR1 enhancer element of
TGM2. H4 acetylation was measured as described in Materials and Methods. The acetylation level is expressed as a percentage of input DNA. All
no-antibody controls were lower than 0.2% of input DNA. Values are the means of three independent QPCR measurements of a representative
experiment. Chromatin immunoprecipitation results were confirmed in at least three independent chromatin preps.
5654 BALINT ET AL.MOL. CELL. BIOL.
of the initiation of DMSO priming, and these changes re-
mained until the end of the monitored period.
To test whether there is a functional link between the
changes in H4R3 methylation induced by priming and altered
gene expression, we used ADOX, an inhibitor of methyltrans-
ferases (38, 48) to suppress methylation. Cotreatment of
HL-60 cells with ADOX and DMSO eliminated H4R3 meth-
ylation (Fig. 6A) and also reduced arginine methylation in
general on the studied enhancer element, as measured by ChIP
QPCR analyses with an anti-Pan-methylated arginine antibody
(Fig. 6B). The decrease in H3K4 methylation induced by
DMSO priming was not blocked with ADOX (Fig. 6C). Inhi-
bition of methylation with ADOX also blocked retinoid-in-
duced acetylation of H4 histones (Fig. 6D). In parallel with the
inhibition of H4R3 methylation and H4 acetylation, there was
marked, but not complete, inhibition in the retinoic acid-in-
duced expression of tissue transglutaminase (Fig. 6E). It is
important to note that simultaneous treatment of HL-60 cells
with ADOX and DMSO reduced but did not completely block
the induction of the transglutaminase gene. Collectively, these
data suggest that the inhibition of methyltransferases by
ADOX leads to the inhibition of H4 arginine 3 methylation
and a concomitant decrease in retinoid-induced transglutami-
nase promoter activation. We speculate that the decrease in
acetylation of H4 histones associated with the transglutami-
nase promoter is the consequence of the decrease in the reti-
noid-dependent activation of transcription of this gene. More-
over, the results we have obtained point to a strong correlation
between H4 arginine 3 methylation and the precommitment of
HL-60 cells to terminal differentiation.
There were two main concerns with the results obtained in
the previous studies and these pertained to how widespread
the priming effect was (i.e., was it limited to the tissue trans-
glutaminase gene or were other retinoid-regulated genes also
involved?) and whether DMSO induced a general induction in
gene expression rather than affecting only a subset of genes
during priming. To address the issue of whether DMSO prim-
ing was limited to only one gene, we compared the induction of
FIG. 4. Detailed map of histone tail modifications on the promoter/enhancer of TGM2. The bar graphs on the left show the effect of priming
on the histone tails bound to the enhancer region HR1 (except H3K4, which is shown on the core promoter). The line graphs show the changes
in histone tail modifications along the 1.8-kb studied fragment of the promoter/enhancer in primed cells after 9-cis retinoic acid treatment for the
indicated period (in hours). Copy numbers are expressed as percentages of input. All no-antibody controls were lower than 0.2% of input DNA.
Values are the means of three independent QPCR measurements of a representative experiment. Chromatin immunoprecipitation results were
confirmed in at least three independent chromatin preps. The scheme of the 1.8-kb human TGM2 promoter and the location of the various QPCR
assays are shown at the bottom.
VOL. 25, 2005 H4R3 METHYLATION AND EPIGENETIC MEMORY 5655
tissue transglutaminase to the induction of two other genes
(CD38 and CYP27) regulated by retinoids in myeloid leukemia
cells (27, 47). The expression of both CD38 and CYP27 was
increased by 9-cis retinoic acid, and this induction is much
greater in cells that had been primed by pretreatment with
DMSO (Fig. 6F). Cotreatment of the cells with different con-
centrations of ADOX and DMSO resulted in a dose-depen-
dent suppression of retinoid-induced expression of both of
To address the issue of a potential general effect on transcrip-
tion by DMSO and ADOX on primed gene expression, we
used a general expression profiling approach. HL-60 cells were
primed with DMSOADOX, or DMSO and ADOX for 16 h
and subsequently treated with 9-cis retinoic acid for 6 h. RNA
was then prepared from these cells, and global expression
profiles were determined using a 3,200-feature human cDNA
microarray. There were approximately the same number of
genes (104 for DMSO and 111 for ADOX) induced ?1.5-fold
in the DMSO- and ADOX-treated cultures (http://
www.biochem.dote.hu/nagylab/suppdata.htm). These results
confirmed that treatment of HL-60 cells with ADOX does not
result in a generalized perturbation of the preexisting patterns
of gene expression.
To address the question of the more general effects of DMSO
priming and ADOX on retinoid-regulated gene expression, we
compared the profiles of retinoid-induced genes in RNA from
control naive cells and retinoid-treated cells that had been
primed with either DMSO alone or DMSO and ADOX. We
detected 38 genes with expression that was upregulated more
than 1.5-fold by the treatment of naive HL-60 cells with reti-
noids for 6 h. Exposure of similar cells to retinoids following
DMSO priming resulted in a substantially larger pool of genes
with expression that was changed (for 104 genes, the expres-
sion was increased more than 1.5-fold compared to the
DMSO-primed cells). Of the 75 retinoid-induced genes with
expression which was selectively increased in DMSO-primed
cells, the induction of 62 was blocked by coadministration of
DMSO plus ADOX (http://www.biochem.dote.hu/nagylab
/suppdata.htm). These experiments suggested that neither
DMSO nor ADOX had a widespread effect on transcription in
these cells. Since methylation is involved not only in RNA
processing but also in protein synthesis, we asked whether a
general inhibitor of methyltransferases like ADOX would pro-
duce a major rearrangement in the proteins of the studied
cells. For this, we analyzed, with two-dimensional electro-
phoresis and silver staining, the proteins of HL-60 cells and the
ways ADOX is changing this protein expression pattern. We
could not detect any substantial change in the protein compo-
sition of HL-60 cells after ADOX treatment with this method.
In fact, there was less than 5% change in the number of spots
detected. These data strongly suggest that ADOX is not a general
inhibitor of RNA and protein synthesis under the conditions
used (http://www.biochem.dote.hu/nagylab/suppdata.htm). To
gain a more mechanistic insight into this process and also to
take advantage of recent developments in the field, we have
evaluated the role of the enzymes proposed to be responsible
for H4R3 methylation. These are PRMT1, a methyltransferase,
and PAD4, a peptidyl arginine deiminase recently identified as
the enzyme responsible for methyl arginine’s conversion into
citrulline and thereby removing the methyl group (13, 23, 53).
We used gene-specific TaqMan assays and carried out real-
time RT-QPCR analysis to determine the expression level of
PRMT1 and PAD4 in the myeloid cell lines used in our studies
in the absence and presence of 9-cis retinoic acid. As shown in
Fig. 7A, the expression level of PRMT1 was determined. All
cell lines expressed an appreciable level of the PRMT1
mRNA, and retinoid treatment did not appear to significantly
change the expression level. PAD4 is expressed at low or not
detectable levels in the six cell lines examined but induced to
high levels upon 2 days of retinoid treatment in NB4 and
HL-60 cells and to a lesser degree in KG1 and PLB cells (Fig.
7B). Finally, we examined the expression level of PAD4 during
priming and the subsequent retinoid response in HL-60 cells.
As shown in Fig. 7C, PAD4 is induced by priming itself and
further induced during retinoid treatment.
These data established that both PRMT1 and PAD4 are
present in HL-60 cells and, while PRMT1’s expression level
appears to be constant, PAD4 is dynamically changing during
priming and retinoid stimulation, in agreement with previous
findings (39). If one assumes that H4R3 methylation is the
cause of the priming effect, a few predictions can be put for-
ward and tested. One is that preacetylation of chromatin in-
terferes with H4R3 methylation (52); therefore, the priming is
likely to be attenuated. We tested this by using trichostatin A
(TSA), a histone deacetylase inhibitor. TSA alone had no
effect on TGM2 expression (Fig. 8A). Increasing the amount of
TSA potentiated the retinoid’s effect as anticipated and as was
shown by us previously (36). Importantly, the DMSO priming
effect was completely abolished if cells were pretreated with
TSA, suggesting that acetylation of histones interfering with
H4R3 methylation eliminates the priming effect. These find-
FIG. 5. Histone tail modifications during priming. (A) H4 arginine
3 methylation levels detected by chromatin immunoprecipitation on
the enhancer element (HR1) during priming with DMSO. (B) H3
lysine 4 methylation levels detected by chromatin immunoprecipitation
on the core promoter during priming with DMSO. Values are the
means of three independent QPCR measurements of a representative
experiment. Chromatin immunoprecipitation results were confirmed
in at least three independent chromatin preps.
5656BALINT ET AL.MOL. CELL. BIOL.
ings confirm previous reports on the interference of acetylation
and H4R3 methylation (52). Another prediction is that activa-
tion of PAD4, the enzyme converting methyl arginine to cit-
rulline, also attenuates the priming effect. To test this predic-
tion, we used calcium ionophores to activate PAD4 in HL-60
cells similar to that reported by Wang and coworkers (53).
Cells, after priming and prior to retinoid induction, were ex-
posed to a short 15-min treatment of 1 ?M A23187 calcium
ionophore. After this treatment, cells were washed extensively
and treated with 9-cis retinoic acid. The presence of A23187
FIG. 6. Epigenetic changes on TGM2 promoter and mRNA expression upon priming in methylation mute cells (methyltransferases were
blocked with 10 ?M ADOX for 16 h as described in Materials and Methods). Values are the means of three parallel QPCR measurements ?
standard deviations, and results were confirmed from at least three independent biological samples. All chromatin immunoprecipitation values are
the means of three independent QPCR measurements of a representative experiment. Chromatin immunoprecipitation results were confirmed in
at least three independent chromatin preps. (A) H4 arginine 3 methylation levels determined by chromatin immunoprecipitation on the HR1
enhancer element. (B) Arginine methylation levels determined by chromatin immunoprecipitation on the HR1 enhancer element with an anti-Pan
methyl arginine antibody. (C) H3 lysine 4 methylation studied by chromatin immunoprecipitation on the core promoter. (D) Chromatin
immunoprecipitation analysis of H4 acetylation levels in naive, primed, and primed methylation mute cells on the enhancer element HR1.
(E) TGM2 mRNA levels normalized to 36B4 copy numbers in naive, primed, and primed methylation mute cells (the structure of ADOX is shown
in the inset). (F) TGM2, CD38, and CYP27 transcript levels after blocking methylation with ADOX in naive and primed cells. ADOX was used
in increasing concentrations as shown. The mRNA copy numbers were normalized to 36B4 transcript levels. Values are the means of three parallel
QPCR measurements ? standard deviations, and results were confirmed from at least three independent biological samples.
VOL. 25, 2005 H4R3 METHYLATION AND EPIGENETIC MEMORY5657
reduced retinoid responsiveness and priming, as shown in Fig.
8B. The interpretation of these data is corroborated by chro-
matin immunoprecipitation results. As shown in Fig. 8C, both
TSA pretreatment and PAD4 activation prevented/eliminated
H4R3 methylation, respectively. Moreover, comparison of H4
acetylation and H4R3 methylation revealed that TSA treat-
ment enhanced acetylation while preventing H4R3 methyl-
ation (Fig. 8D and E). The third prediction we tested was that
the increased level of PRMT1, the methylase responsible for
H4R3 methylation, would lead to increased priming. On one
hand, as shown in Fig. 9A, in the absence of retinoid treatment,
transfection of PRMT1 does not induce gene expression. On
the other hand, it can further induce the priming effect. It is
noteworthy that under the conditions used, increased PRMT1
expression does not substitute for priming. Finally, we have
evaluated the combined effect of transfected wild-type or mu-
tant PRMT1 and PAD4 on the retinoid-regulated expression
of TGM2 and also that of a retinoid-inducible reporter gene.
As shown in Fig. 9B and C, PRMT1’s enzymatic activity is
required for its coactivator activity. PAD4 does not act as a
corepressor, but its enzymatically inactive mutant synergizes
with PRMT1 in enhancing transcription (Fig. 9B). These data,
if put together, make it very likely that the priming effect
involves H4R3 methylation and that the level of H3R4 meth-
ylation is regulated by the activity of PRMT1 and PAD4.
The profile of gene expression in cells, both differentiated
and in their precursors, is often viewed from the perspective of
a static “snapshot” rather than as parts of a dynamic process
akin to the many individual frames that when combined con-
stitute a movie. For cells undergoing differentiation, the phe-
notypic identity of a cell, as defined by its distinctive pattern of
gene expression, has to be maintained through multiple cycles
of DNA replication, chromatin assembly, and repackaging. It
has been suggested that there has to be some sort of cellular
memory that provides a cell with an epigenetically coded iden-
tity that can be preserved during differentiation (50). One form
of cellular memory, provided by gene “silencing,” has been
studied in detail (22, 40). However, the mechanisms that allow
for either persistent activation of gene expression or a presen-
sitization to expression of specific genes has not been well
characterized. An understanding of these mechanisms will con-
tribute a significant new level of insight to the regulation of
Epigenetic changes that affect the structure of chromatin
associated with regulated genes and gene networks are obvious
candidates to serve as components of the postulated cellular
memory of gene expression. Methylation of CpG islands and
deacetylation of lysines have been clearly linked to the induc-
tion of a silenced state of chromatin (26). On the other hand,
acetylation of histones H3 and H4 have been linked to the
activation of gene expression (1). In the studies reported here,
we have set out to address a complex issue, namely to investi-
gate the changes in epigenetic markers that are linked to the
regulation of gene expression during retinoid-induced myeloid
cell differentiation. We have chosen to work with the HL-60
cell line because previous work from several laboratories has
established that differentiation of these cells involves a two-
step process that can be separated pharmacologically (11, 55–
57). The first step, termed precommitment or priming, involves
a persistent state of presensitization that constitutes a well-
defined model of cellular memory. The second step, which is
also well characterized, involves selective activation of gene
expression by inducers of terminal differentiation for this lin-
FIG. 7. Analysis of PRMT1 and PAD4 mRNA levels in different
cell lines upon retinoid treatment. (A) PRMT1 mRNA copy numbers
were normalized to cyclophilin transcript levels. Values are the means
of three independent QPCR measurements ? standard deviations.
(B) PAD4 mRNA copy numbers were normalized to cyclophilin tran-
script levels. Values are the means of three independent QPCR mea-
surements ? standard deviations. (C) mRNA levels of PAD4 in 9-cis
retinoic acid-treated naive and DMSO-primed HL-60 cells. Relative
increases of mRNA levels during priming or retinoid treatment of
primed cells are shown on the arrows. PAD4 mRNA copy numbers
were normalized to cyclophilin transcript levels. Values are the means
of three independent QPCR measurements ? standard deviations.
5658BALINT ET AL.MOL. CELL. BIOL.
eage, such as retinoids. We have then used the promoter of a
well-characterized retinoid-regulated gene in this pathway, tis-
sue transglutaminase, a key marker of retinoid response, to
investigate the role of epigenetic modification of chromatin
components in the establishment of the precommitted state of
differentiation of these cells. We have carried out a detailed
analysis of the covalent modifications of histones bound to
different regions of the transglutaminase promoter during
three distinct states of differentiation: the naive state that oc-
curs prior to the initiation of differentiation, the primed or
precommitted state that is induced by brief exposure of the
naive cells to vitamin D or DMSO, and the differentiated state
FIG. 8. Modulation of TGM2 expression levels in HL-60 cells. (A) TSA pretreatment for 1 h of HL-60 cells enhances the retinoid response
in naive cells (50, 100, and 150 nM TSA was used). TSA pretreatment for 1 h of HL-60 cells before DMSO priming reduces the effect of priming
on TGM2 induction (50, 100, and 150 nM TSA was used). Arrows indicate the effect of pretreatment with 150 nM TSA on primed versus naive
retinoid-treated cells. mRNA copy numbers were normalized to 36B4 transcript levels. Values are the means of three independent QPCR
measurements ? standard deviations. (B) Activation of PAD4 after priming by a 15-min treatment with 1 ?M calcium ionophore A23187 reduces
the priming effect. mRNA copy numbers were normalized to cyclophilin transcript levels. Values are the means of three independent QPCR
measurements ? standard deviations. (C) H4R3 methylation levels are changed during priming and modulated by pretreatment with 100 nM TSA
for 1 hour or a 15-min treatment with 1 ?M calcium ionophore A23187. H4R3 methylation was determined by chromatin immunoprecipitation
on the HR1 enhancer element. Copy numbers are expressed as percentages of the input. (D) H4 acetylation levels in primed, retinoid-treated cells
and TSA-pretreated, primed retinoid-treated cells. Cells were pretreated with 100 nM TSA for 1 hour, and H4 acetylation levels were determined
by chromatin immunoprecipitation on the HR1 enhancer element. Copy numbers are expressed as percentages of the input. (E) H4R3 methylation
levels in primed, retinoid-treated cells and TSA-pretreated, primed retinoid-treated cells. Cells were pretreated with 100 nM TSA for 1 hour, and
H4R3 methylation levels were determined by chromatin immunoprecipitation on the HR1 enhancer element. Copy numbers are expressed as
percentages of the input. The values of the no-antibody controls were subtracted. Values are the means of three independent QPCR measurements
of a representative experiment. Chromatin immunoprecipitation results were confirmed in two independent chromatin preps with two independent
immunoprecipitations, respectively. ?, present; ?, absent.
VOL. 25, 2005 H4R3 METHYLATION AND EPIGENETIC MEMORY5659
that occurs following the addition of a retinoid to the primed
cells. In characterizing the precommitted state, we found that
it was a “threshold phenomenon.” The increased induction of
gene expression in primed versus naive cells was not due to an
increase in the transcriptional activation of individual cells but
rather to an increase in the fraction of the population of cells
that was able to respond to retinoids with activation of gene
expression (Fig. 2). This result is entirely consistent with a
model that suggests that the development of epigenetic mem-
ory entails a persistent “marking” of those cells that have been
exposed to the memory inducer (in this case, vitamin D or
The analysis of histone tail modifications during both prim-
ing and transcriptional activation revealed distinct mechanisms
that mark both of these processes. Priming itself appears to be
linked to major changes in histone side chain methylation. In
particular, methylation of K4 on histone H3 (H3K4) associated
with the core promoter is rapidly decreased after the initiation
of priming (Fig. 4 and 5). This decrease in H3K4 methylation
level was not blocked by the methyltransferase inhibitor
ADOX (Fig. 6C), suggesting that a non-methyltransferase-
dependent pathway might be responsible for this effect. This
observation is in agreement with the recent identification of
the enzymes responsible for demethylation (44). According to
Shi et al., demethylation is mediated by LSD1, a member of the
amine oxidase enzyme family. Priming increases the methyl-
ation of arginine on histone H4 (H4R3) specifically on histones
associated with a prominent enhancer element in the transglu-
taminase promoter. Arginine methylation of histones seems
particularly important to the induction of the primed state,
since its elimination with a pharmacologic inhibitor of meth-
yltransferases resulted in the loss of priming for the induction
of tissue transglutaminase and several other retinoid-regulated
genes (Fig. 6).
Cross talk between enhancer and core promoter. There is an
apparent discrepancy between the epigenetic changes occur-
ring on the HR1 enhancer and the core promoter, as revealed
by our studies. While histone methylation of H4R3 on the
enhancer element appears to play an important role in the
establishment of the precommitted state, histone acetylation
seems to contribute to the activation of transcription. Retin-
oid-activated transcription of the primed cells resulted in a
marked increase in H4 acetylation, particularly in regions of
the enhancer element, and also a lesser increase in H3 acety-
lation of histones associated with the core promoter. There
could be numerous reasons for this discrepancy. For example,
acetylation and arginine methylation were shown by An and
colleagues to be detectable on a larger promoter fragments
than the regions in the proximity of acetyltransferases or meth-
yltransferases (2). The activation of transcription of the trans-
glutaminase gene in addition to the changes in acetylation
results in increased H3 methylation (H3K4) of the core pro-
moter and, to a lesser and more transient extent, H3 methyl-
ation (H3K9) of the enhancer. H3K4 methylation was reported
as being localized to core promoters and transcribed regions of
active genes if studied by the ChIP technique on larger
genomic regions (5). If studied on individual promoters, the
location of increased H3K4 methylation seems also to corre-
late with core promoters (2, 24). We have also found a clear
and marked difference in the H3K4 levels (more than 50 times
FIG. 9. Effect of PRMT1 and PAD4 on retinoid-regulated gene
expression. (A) Transfection of PRMT1 expression vector into HL-60
cells increases the TGM2 induction after retinoid treatment in primed
cells, while the catalytic mutant has no effect on retinoid responsive-
ness. Cells were treated with 1 ?M 9-cis retinoic acid for 10 h. mRNA
copy numbers were normalized to cyclophilin transcript levels. Values
are the means of three independent QPCR measurements ? standard
deviations. (B) Cotransfection of PAD4 and PRMT1 expression vec-
tors and their catalytic mutants into HL-60 cells modulates TGM2
induction. Cells were treated with 1 ?M 9-cis retinoic acid for 24 h.
mRNA copy numbers were normalized for cyclophilin transcript levels.
Values are the means of two independent biological replicates and
three independent QPCR measurements for each sample ? standard
deviations. (C) Cotransfection of PAD4 and PRMT1 expression vec-
tors and their catalytic mutants into 293T cells modulates the luciferase
expression on a ?RARE tkLuc reporter system. Values were normal-
ized to measured ?-galactosidase values and expressed in arbitrary
units (AU) as described in Materials and Methods. Values are the
mean of three independent biological replicates ? standard deviations.
?, present; ?, absent.
5660 BALINT ET AL.MOL. CELL. BIOL.
higher levels on the core promoter than on the enhancer
HR1). Taken together, these results suggest a cross talk be-
tween the promoter and enhancer of TGM2, with the activa-
tion of distinct enzymatic pathways both during priming and
retinoid activation. This so far unsubstantiated cross talk sug-
gests a physical interaction between these regulatory DNA
elements, an interaction that needs to be investigated further.
Nuclear receptor complexes induce histone tail modifica-
tions. The epigenetic changes associated with both the priming
and transcriptional activation that occur during retinoid-in-
duced differentiation are clearly linked to the activity of reti-
noid receptors. In the absence of a ligand, RAR-RXR het-
erodimers, the mediators of the effects of retinoids on myeloid
cell differentiation, are believed to bind to their cognate re-
sponse elements and repress transcription. Liganding of these
receptors results both in the loss of this repressive effect and in
the induction of transcriptional activity. While little detail is
known about the molecular steps involved in the activation of
transcription by retinoid receptors, significantly more is known
about the activity of other members of the nuclear receptor
superfamily. A general model of transcriptional activation, de-
veloped primarily from studies on estrogen and glucocorticoid
receptor-regulated genes (3, 34) suggests that liganding of the
receptors results in a sequential recruitment of proteins in-
volved in transcriptional activation. According to the present
concept, the sequential recruitment of cofactors may be differ-
ent from gene to gene (reviewed by M. P. Cosma) (12) but, in
all cases, results in an orderly process of covalent modifications
of the tails of histone proteins associated with the promoter
and enhancer elements of the target gene. Our data on the
effects of retinoids on the covalent modifications of histone
proteins associated with the transglutaminase promoter are in
agreement with this general model. We have demonstrated
that induced transcription of the transglutaminase gene in
these myeloid cells is paralleled by a wave of histone tail
acetylation that is most likely due to the recruitment of histone
acetyltransferase (HAT)-containing complexes to the pro-
moter. Our studies have demonstrated, however, that activa-
tion of transcription is not only linked to histone acetylation
but also to some very selective effects on histone methylation.
H3K4 methylation has been characterized in several systems
and has generally been found to oppose H4K9 methylation and
to mark areas of increased gene expression (19, 21). We have
found that this methylation pattern is concentrated in the core
promoter and is reduced by priming and increased by retinoid
treatment. These data are in agreement with a purported role
of H3K4 as a marker of positive gene expression.
A role for histone 4 arginine 3 methylation in nuclear re-
ceptor signaling. H4 arginine 3 methylation is one of the least-
characterized histone tail modifications. Arginine methylation
on both H4 and H3 tails has been shown to be related to
nuclear receptor coactivation in several experimental systems.
The family of p160 transcriptional coactivators (e.g., SRC1,
GRIP1, ACTR) binds two members of the arginine methyl-
transferase family, PRMT1 and CARM1 (also called PRMT4).
Both these transferases have been implicated in the activation
of nuclear receptor-dependent genes (7, 46). The arginine
methyltransferases modify histone tails and arginine side
chains on other proteins as well (e.g., CBP, STAT) (8, 35, 54).
The PRMT1 enzyme has been shown in cotransfection studies
to be a cofactor of nuclear receptor-activated gene expression
(28). In vitro studies have demonstrated that PRMT1 methy-
lates H4 arginine 3 and that, once methylated, H4 is a better
substrate for HAT-s and vice versa, once acetylated, the his-
tone tails lose their ability to become methylated by PRMT1
(52). The other factor implicated in the regulation of arginine
methylation is the enzyme called peptidyl arginine deiminase,
or PAD4. In HL-60 cells, PAD4 was shown to be regulated by
DMSO, vitamin D, and retinoic acid (39). All of these agents
have the potential to prime HL-60 cells (56, 57). Recently, this
gene was found to be responsible for the removal of the methyl
mark on H4R3 by converting it to a citrullinated histone H4
(53). These observations provide a plausible link between our
observations that the precommitment state of HL-60 cells in-
volves increased H4R3 methylation and increased retinoid-
activated gene expression. In parallel with the increase in
H4R3 methylation, the gene responsible for the removal of the
methyl mark is also induced. Our transfection data (Fig. 9) also
FIG. 10. Proposed model of gene expression modulation by changes in H4 arginine 3 methylation. PRMT1 and PAD4 regulate arginine 3
methylation. The naive state is characterized by lack of H4 arginine methylation on the enhancer region. Priming results in increased H4 R3
methylation, while retinoid treatment leads to decreased H4R3 methylation and to the indicated changes in lysine acetylation. Preacetylation of
histone tails prior to priming by TSA is likely to reduce the affinity of histone tails toward PRMT1 and, by this, prevents the enhanced retinoid
responsiveness caused by priming. The activation of PAD4 after priming by calcium ionophore treatment leads to the removal of the methyl mark
from H4R3 and abolition of the enhanced retinoid responsiveness caused by priming.
VOL. 25, 2005 H4R3 METHYLATION AND EPIGENETIC MEMORY5661
suggest that PAD4 may not act as a transcriptional repressor
per se, but some of its activity it is required along with PRMT1
for synergistic/cooperative activation of retinoid-mediated
transcription. This activity does not require but is inhibited by
its peptidyl arginine deiminase activity. The complex role of
PAD4 in the regulation of transcription is further supported by
recent work of Lee and colleagues (30). According to their
data, demethylimination of p300 by PAD4 is changing its ac-
tivity to an activated or even hyperactive form. This change in
p300 activity is modulated via the regulation of p300-GRIP1
interaction. We propose that priming-induced modifications of
H4 arginine 3 could increase retinoid-induced gene activation
by potentiating HAT activity on histones. This in turn would
result in increased histone acetylation and increased transcrip-
tional activation. Our data also suggest that the full activation
can be achieved in two distinct ways. The first is by switching off
histone deacetylase activity with a histone deacetylase inhibitor
such as TSA. In this case, the response to a retinoid signal will
increase. The other way is by transforming the histone tails to
become better substrates of HAT-s. Arginine methylation on
H4R3 is making such a change. The two mechanisms are mu-
tually exclusive and antagonistic. By this, the two parallel path-
ways provide a signal integration mechanism for the cell.
The most detailed characterization of the dynamics of nu-
clear receptor-mediated transcriptional activation has been
carried out by Metivier and colleagues (34). By analyzing the
estrogen receptor-induced transcription on the pS2 gene, they
described the ordered and sequential recruitment of specific
cofactors and the resultant ordered sequence of histone tail
modifications. According to their data, in the first, “unproduc-
tive,” cycle, binding of PRMT1 to the receptor complex and
subsequent H4R3 methylation occur. In the second, “produc-
tive,” cycle, recruitment of PRMT1 is followed by binding of
HAT-s and extensive acetylation of the histones H3 and H4.
We propose that the two phases of gene activation proposed by
Metivier and colleagues (34) may be dissociated in HL-60 cell
differentiation. Our results suggest that the transition to the
precommitted state (i.e., the priming of the cells) induces an
increase in H4R3 methylation but no change in H4 acetylation
and no activation of transcription. This first cycle of HL-60
differentiation does not require liganding of the nuclear recep-
tors, it occurs via an independent and uncharacterized vitamin
D- or DMSO-dependent step. This step provides a further
signal integration node for the cell by integrating two indepen-
dent signals in time and space.
We propose a model for the epigenetic regulation of retin-
oid response and differentiation competence in myeloid leu-
kemia cells (Fig. 10). In this study, we were able to dissect some
of the epigenetic changes taking place on a retinoid-regulated
gene. We found that H4R3 methylation takes place prior to
gene activation and is a hallmark of the primed cell state. This
modification represents a transcriptionally silent (unproduc-
tive) but primed state (34) which marks key histones and
makes them better substrates for receptor-bound acetyltrans-
ferases (HAT-s). We propose that this mechanism accounts for
the increased susceptibility of the cell to respond to terminal
differentiating agents, such as a retinoid, with increased gene
expression and an increased potential for phenotypic differen-
tiation. Preacetylated histones are refractory to this mecha-
nism. This model is consistent with the proposal that histone
tail modifications function as the physical mediators of cellular
memory. By providing docking sites for transcription factors
and marking histones for subsequent covalent modifications,
these methylation reactions serve as silent switches of gene
expression. Our findings suggest an active and physiological
role for arginine 3 methylation on H4 tails in retinoid response
and provide a model amenable to further investigation and
potentially to pharmacological exploitation.
We thank Ibolya Furtos and Marta Beladi for excellent technical
assistance and members of the Nagy laboratory and Laszlo Tora, Mate
Demeny, Zsuzsanna Nagy, and Beata Scholtz for suggestions and com-
ments on the manuscript.
This work was supported by grant FP5-RTN from the EU (to L.N.),
FIRCA award 5 RO3 TW 01146-02 (to P.J.A.D and L.N.), and Hun-
garian Scientific Research Fund (OTKA) T034434 (to L.N.). L.N. is an
International Scholar of HHMI and an EMBO Young Investigator
and holds a Wellcome Trust Senior Research Fellowship in Biomed-
ical Sciences in Central Europe. B.L.B. is a Young Researcher of the
EU NUC REC NET (an EU FP5 training network).
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