Estradiol differentially induces progesterone receptor isoforms expression through alternative promoter regulation in a mouse embryonic hypothalamic cell line

Abstract

Progesterone receptor (PR) presents two main isoforms (PR-A and PR-B) that are regulated by two specific promoters and transcribed from alternative transcriptional start sites. The molecular regulation of PR isoforms expression in embryonic hypothalamus is poorly understood. The aim of the present study was to assess estradiol regulation of PR isoforms in a mouse embryonic hypothalamic cell line (mHypoE-N42), as well as the transcriptional status of their promoters. MHypoE-N42 cells were treated with estradiol for 6 and 12 h. Then, Western blot, real-time quantitative reverse transcription polymerase chain reaction, and chromatin and DNA immunoprecipitation experiments were performed. PR-B expression was transiently induced by estradiol after 6 h of treatment in an estrogen receptor alpha (ERα)-dependent manner. This induction was associated with an increase in ERα phosphorylation (serine 118) and its recruitment to PR-B promoter. After 12 h of estradiol exposure, a downregulation of this PR isoform was associated with a decrease of specific protein 1, histone 3 lysine 4 trimethylation, and RNA polymerase II occupancy on PR-B promoter, without changes in DNA methylation and hydroxymethylation. In contrast, there were no estradiol-dependent changes in PR-A expression that could be related with the epigenetic marks or the transcription factors evaluated. We demonstrate that PR isoforms are differentially regulated by estradiol and that the induction of PR-B expression is associated to specific transcription factors interactions and epigenetic changes in its promoter in embryonic hypothalamic cells.

Full text

ORIGINAL ARTICLE
Estradiol differentially induces progesterone receptor isoforms
expression through alternative promoter regulation in a mouse
embryonic hypothalamic cell line
Edgar Ricardo Va
´zquez-Martı
´nez
1
•Ignacio Camacho-Arroyo
1
•Angel Zarain-Herzberg
2
•
Marı
´a Carmen Rodrı
´guez
3
•Luciano Mendoza-Garce
´s
4
•Patricia Ostrosky-Wegman
5
•
Marco Cerbo
´n
1
Received: 5 August 2015 / Accepted: 29 November 2015
ÓSpringer Science+Business Media New York 2015
Abstract Progesterone receptor (PR) presents two main
isoforms (PR-A and PR-B) that are regulated by two
specific promoters and transcribed from alternative tran-
scriptional start sites. The molecular regulation of PR
isoforms expression in embryonic hypothalamus is poorly
understood. The aim of the present study was to assess
estradiol regulation of PR isoforms in a mouse embryonic
hypothalamic cell line (mHypoE-N42), as well as the
transcriptional status of their promoters. MHypoE-N42
cells were treated with estradiol for 6 and 12 h. Then,
Western blot, real-time quantitative reverse transcription
polymerase chain reaction, and chromatin and DNA
immunoprecipitation experiments were performed. PR-B
expression was transiently induced by estradiol after 6 h of
treatment in an estrogen receptor alpha (ERa)-dependent
manner. This induction was associated with an increase in
ERaphosphorylation (serine 118) and its recruitment to
PR-B promoter. After 12 h of estradiol exposure, a
downregulation of this PR isoform was associated with a
decrease of specific protein 1, histone 3 lysine 4
trimethylation, and RNA polymerase II occupancy on PR-
B promoter, without changes in DNA methylation and
hydroxymethylation. In contrast, there were no estradiol-
dependent changes in PR-A expression that could be
related with the epigenetic marks or the transcription fac-
tors evaluated. We demonstrate that PR isoforms are dif-
ferentially regulated by estradiol and that the induction of
PR-B expression is associated to specific transcription
factors interactions and epigenetic changes in its promoter
in embryonic hypothalamic cells.
Keywords Progesterone receptor isoforms Estradiol 
Epigenetic marks Transcription factors Hypothalamus 
Histone acetylation
Introduction
Progesterone receptor (PR) is a member of the nuclear
receptor superfamily that regulates several reproductive
and nonreproductive functions [1]. PR gene encodes two
main isoforms, PR-A and PR-B, whose transcription is
regulated by two specific promoters and alternative tran-
scription start sites (TSS) [2]. PR-A differs from PR-B by
lacking 164 amino acids at the amino terminus of the
protein. PR-A usually functions as a transcriptional inhi-
bitor of PR-B-regulated genes [3]. However, it has been
also demonstrated that both PR isoforms act as transcrip-
tional activators of different target genes in the same cell
line [4]. Therefore, it has been proposed that PR function
results from the counterbalancing proportion of both iso-
forms when they are expressed in the same cell type [5,6].
It has been reported that there is a specific variation in PR
&Marco Cerbo
´n
mcerbon85@yahoo.com.mx
1
Unidad de Investigacio
´n en Reproduccio
´n Humana, Instituto
Nacional de Perinatologı
´a-Facultad de Quı
´mica, Universidad
Nacional Auto
´noma de Me
´xico (UNAM), Ciudad
Universitaria, Av. Universidad 3000, Coyoaca
´n,
04510 Mexico, DF, Mexico
2
Departamento de Bioquı
´mica, Facultad de Medicina, UNAM,
Mexico, Mexico
3
Instituto Nacional de Salud Pu
´blica, SSA, Cuernavaca,
Mexico
4
Instituto Nacional de Geriatrı
´a, SSA, Cuernavaca, DF,
Mexico
5
Departamento de Medicina Geno
´mica y Toxicologı
´a
Ambiental, Instituto de Investigaciones Biome
´dicas, UNAM,
Mexico, Mexico
123
Endocrine
DOI 10.1007/s12020-015-0825-1

isoforms expression during the estrous cycle and under
estradiol and progesterone treatments in the rodent
hypothalamus. Particularly, PR-B isoform is induced by
estradiol to a greater extent than PR-A in the hypothalamus
and preoptic area of the female rat, and this differential
regulation has been associated with the display of sexual
behavior [7–12]. It has been reported that the expression of
PR-A is higher than that of PR-B in the immortalized rat
embryonic hypothalamic cell line, D12, and that the
expression of both PR isoforms was induced by estradiol
[13]. Moreover, it has been demonstrated that PR expres-
sion regulation by estradiol is sexually dimorphic during
development in several brain regions, particularly in those
that control sexual differentiation and reproductive behav-
ior during adulthood, such as medial preoptic nucleus and
the ventromedial nucleus of hypothalamus [14–16].
Expression of PR isoforms is mainly regulated by
estrogen receptor alpha (ERa), despite the lack of con-
sensus estrogen responsive elements (ERE) in the PR gene
promoter [17]. ERais recruited to the PR gene promoter
through interactions with specific protein 1 (SP1) and
activator protein-1 (AP1) in MCF7 cells [18,19]. In turn,
ERarecruits transcriptional coregulators that induce
changes in chromatin in order to activate transcription [20].
Moreover, both DNA methylation and chromatin basal
states in the PR promoter region also influence the
expression of PR isoforms, highlighting the importance of
epigenetic processes in the regulation of this gene [21,22].
We recently reported a transient and differential DNA
methylation pattern of PR gene isoform promoters during
the evening of proestrus in rat hypothalamus [10]. It has
also been reported that estradiol induced a differential
DNA methylation pattern of PR-A promoter in the med-
iobasal hypothalamus of developing rats [23].
It has been described a specific and a transient induction
pattern of transcriptionally active and repressive histone
marks in mouse hypothalamus after 6 h of estradiol treat-
ment [24]. Furthermore, the authors found a specific
enrichment of H3Ac and H3K4me3 histone marks on four
different regions upstream of the TSS of PR-A which
included PR-A promoter and other three regions near PR-A
and PR-B promoters. Interestingly, this histone marks
enrichment was dependent on the estradiol exposure time
and the hypothalamic region. The authors also reported that
ERawas not recruited on any of the four PR gene studied
regions [24].
Although the regulation of PR isoforms expression by
estradiol has been extensively reported in adult hypotha-
lamic cells, there is lacking information about their regu-
lation in embryonic hypothalamic cells, as well as the
molecular mechanisms involved in such regulation. In this
study, we used the estradiol responsive mHypoE-N42
mouse embryonic hypothalamic cell line as a model for
exploring PR isoforms regulation [25]. This cell line
expresses PR-A and PR-B, and it has been previously used
for analyzing the transcriptional regulation of the aro-
matase gene by estradiol [26].
The aim of this study was to assess the regulation of PR
isoforms expression by estradiol in the mHypoE-N42 cell
line, as well as the epigenetic changes and interaction of
transcription factors on PR-A and PR-B promoters asso-
ciated to such regulation.
Materials and methods
Cell culture and treatments
Mouse hypothalamic mHypoE-N42 cells were purchased
from Cellutions Biosystems
Ò
(Toronto, Canada) and are
derived from mouse embryonic hypothalamic primary
cultures (from days 15, 17, and 18) although the sex of
embryos was not determined. Cells were maintained in
high-glucose DMEM medium (Life Technologies
Ò
, Cali-
fornia, USA) supplemented with 10 % of fetal bovine
serum (FBS). Cells were used for experiments at passages
24–26. Before treatments, 2 910
6
cells were maintained
in DMEM medium without phenol red and charcoal-
stripped FBS until they reached a confluence of 80–90 %,
and then cells were serum starved in the same medium
overnight. Cells were treated with different concentrations
of estradiol (Sigma
Ò
, Missouri, USA) or 0.002 % of
ethanol (vehicle) for 6 and 12 h. In order to confirm that
the estradiol regulation of gene expression was mediated
by ERaand not by other estrogen receptor subtypes, the
effect of a highly selective ERaantagonist, 1,3-bis(4-hy-
droxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-
1H-pyrazole dihydrochloride (MPP, Tocris Bioscience,
Bristol, UK), was evaluated. 5 nM of MPP was adminis-
tered alone or in combination with 100 nM of estradiol.
Subsequent methodological approaches and the corre-
sponding experiments were performed from whole cell
preparations.
RNA isolation and real-time RT-qPCR
Total RNA was isolated with TRIzol
Ò
reagent (Life
Technologies
Ò
, California, USA) and RT-qPCR was car-
ried out using the SuperScript
Ò
III First-Strand Synthesis
SuperMix
Ò
(Life Technologies
Ò
, California, USA), as
specified by the distributor. Total RNA isolated was
quantified by the Quant-iT
Ò
RiboGreen
Ò
RNA Assay Kit
(Life Technologies
Ò
, California, USA). 100 ng of cDNA
were amplified using the 7500 real-time PCR system (Life
Technologies
Ò
, California, USA). 1 ll of RT reaction was
subjected to PCR in order to simultaneously amplify a gene
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123

fragment of PR-B isoform, total PR (PR-B ?PR-A), and
GAPDH. This was used as an internal control and was
obtained from Life Technologies
Ò
, California, USA (Assay
ID: Mm99999915_g1). The sequences of the specific pri-
mers and probes for PR-B and total PR amplifications are
depicted in Table 1. Negative controls without cDNA and
with non-retrotranscribed RNA were included in all the
experiments. All PCR products were always studied and
analyzed together throughout the experiments. In order to
obtain the relative quantification by the DDCt method,
TaqMan
Ò
probes (Life Technologies
Ò
, California, USA)
were used as the detection system. Master Mix and
TaqMan
Ò
assays were used at 1X. Cycling conditions were
followed as specified by the distributor, with the exception
of PR-B (50 °C for 2 min; 95 °C for 10 min; 50 cycles of
95 °C for 30 s and 64 °C for 3 min). PR-A expression
levels were calculated by subtracting total PR minus PR-B
expression levels. All assays produced a single PCR pro-
duct at the molecular weight expected, as confirmed using
2 % agarose gels stained with GelRed
Ò
(Biotium
Ò
, USA).
Western blot
Cells were homogenized in RIPA buffer (50 mM Tris–HCl
pH 7.4, 150 mM NaCl, 1 % v/v NP40, 0.25 % w/v sodium
deoxycholate) containing protease and phosphatase inhi-
bitor cocktails (Roche, Basel, Switzerland). Proteins were
obtained by centrifugation at 12,500 rpm at 4 °C for
30 min and quantified by the Bradford method (BioRad,
California, USA). Proteins (40 lg) were separated by
denaturing electrophoresis in a 7.5 % polyacrylamide gel at
95 V. Gels were transferred to a polyvinylidene fluoride
membrane (Millipore, Massachusetts, USA) by semidry
transfer (BioRad, California, USA). Membranes were
blocked with 10 % w/v nonfat dry milk and 0.1 % v/v
Tween-20 at room temperature for 30 min. Membranes
were incubated with primary antibodies against PR, S118
phosphorylation of ERaand SP1 (Santa Cruz, Texas, USA)
at 4 °C overnight. The respective secondary antibodies
conjugated to horseradish peroxidase were incubated at
room temperature for 1 h. Membranes were stripped with
glycine (25 mM, pH 2.5; 1 % w/v SDS) at room temper-
ature for 30 min and reprobed with the ERaand GAPDH
primary antibodies (Santa Cruz, Texas, USA) as previously
mentioned. Proteins were detected by chemiluminescence
with the ECL kit (Amersham Piscataway, USA) and ana-
lyzed by densitometry using the Vision Works LS Software
(UVP
Ò
, USA).
Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation was carried out as previ-
ously described [26], with minor modifications. Briefly,
treated cells were incubated with 1 % w/v of formaldehyde
for 10 min and with 125 mM of glycine for 5 min. Cells
were harvested and homogenized in a lysis buffer (1 % w/v
SDS, 10 mM EDTA, 50 mM Tris pH 8) containing a
protease inhibitor cocktail (Roche, Basel, Switzerland).
Chromatin shearing was performed in a Vibra Cell
Ò
son-
icator (Sonics, Connecticut, USA) by 6 pulses of 10 s ON,
60 s OFF at 75 % amplitude, to obtain DNA fragments
with a modal size of 1000 bp, which was confirmed by a
2 % w/v agarose gel stained with GelRed
Ò
(Biotium
Ò
,
USA). 25 lg of chromatin were diluted 1:10 with buffer
containing protease inhibitor cocktail. Precleared chro-
matin was incubated at 4 °C overnight with 2–5 lgof
antibodies against H3K9ac, H3K4me3, H3K9me2, and
RNA Pol II (Abcam
Ò
, Cambridge, UK), ERa, and SP1
(Santa Cruz, TX, USA). The immune complexes were
incubated with protein A/G agarose (Santa Cruz, TX, USA)
in agitation at 4 °C for 1 h. The immunoprecipitated
products were washed with low salt wash buffer, high
Table 1 Primers and probes
used for real-time PCR assays Target Sequence (50–30) Detection Application
PR-B Forward-CAGCACCGGCCACACCAGTT TaqMan
Ò
Gene expression
Reverse-CCAAGCGTGCAAGCAAGGGG
Probe-ACACGTCTGGCGCTTCGCCCTCCCC
Total PR Forward-ATTCTACTCGCTGTGCCTTACC TaqMan
Ò
Gene expression
Reverse-CATGGGTCACCTGGAGTTTGA
Probe-ATGTGGCAAATCCC
PR-B promoter Forward-GGTGGGGCTGGCATGCTTCT TaqMan
Ò
ChIP
Reverse-ACTCCCGCTATCTCCGGACTTCT
Probe-TGGGCGGGCCTTCCTAGAGCGCCA
PR-A promoter Forward-CAGCACCGGCCACACCAGTT TaqMan
Ò
ChIP
Reverse-CCAAGCGTGCAAGCAAGGGG
Probe-ACACGTCTGGCGCTTCGCCCTCCCC
Endocrine
123

salt wash buffer, lithium chloride wash buffer, and
TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0). The
complexes were eluted twice with elution buffer (1 % w/v
SDS, 100 mM NaHCO
3
) for 15 min at room temperature.
Samples obtained were incubated with RNase A solution
(Promega, Wisconsin, USA) for 30 min at 37 °C. Reverse
crosslinking was performed by incubating with Proteinase
K (Roche, Basel, Switzerland) at 55 °C overnight and then
with NaCl (5 M) for 6 h at 65 °C. The enriched genomic
DNA was purified with the Wizard
Ò
SV Gel and PCR
Clean-Up System (Promega, Wisconsin, USA). The puri-
fied products were amplified using the 7500 real-time PCR
system (Life Technologies
Ò
, California, USA) to obtain the
fold enrichment of each immunoprecipitated sample.
TaqMan
Ò
probes (Life Technologies
Ò
, California, USA)
were used for detection. TaqMan
Ò
assays were designed
using Primer3 software in order to amplify the promoter
regions of both PR isoforms according to previous studies
and databases [2,17,27,28] (Fig. 1). Cycling conditions
were the following: 50 °C for 2 min; 95 °C for 10 min; 50
cycles of 95 °C for 30 s and 64 °C for 3 min. Normaliza-
tion was carried out using the input sample (1 % v/v of
diluted chromatin). Primers and probe used for PR isoform
promoters are shown in Table 1. Both assays produced a
single PCR product at the molecular weight expected, as
confirmed using a 2 % w/v agarose gel stained with
GelRed
Ò
(Biotium
Ò
, USA).
Methylated and hydroxymethylated DNA
immunoprecipitation
Immunoprecipitation of methylated and hydroxymethy-
lated DNA was performed as previously reported [29,30]
with minor modifications. Briefly, genomic DNA was
extracted from mHypoE-N42 cells using the Wizard
Ò
Genomic DNA purification kit (Promega, Wisconsin,
USA). 15 lg of genomic DNA were fragmented in a
Vibra Cell
Ò
sonicator by 6 pulses of 10 s ON, 60 s OFF
at 40 % amplitude, to obtain DNA fragments with a
modal size of 1000 bp, confirmed by a 1.5 % w/v agarose
gel stained with GelRed
Ò
(Biotium
Ò
, USA). 2.5 lgof
sonicated DNA was diluted in TE buffer and denatured
for 10 min at 100 °C, followed by snap-chilling samples
on wet ice. 10 % v/v of sample was transferred on a clean
tube for the input. Diluted DNA was incubated with
immunoprecipitation buffer (10 mM Na-phosphate, 140
mM NaCl, 0.05 % v/v Triton X-100) and 1 lg of anti-
methylcytosine or anti-hydroxymethylcytosine antibodies
(Zymo
Ò
, California, USA) at 4 °C overnight on a rotating
wheel. Collection and washing of the immune complexes
were performed as described for ChIP. To release the
DNA from the beads, samples were incubated with
digestion buffer (50 mM Tris, 10 mM EDTA, 0.5 % w/v
SDS) and Proteinase K overnight at 55 °C in agitation.
DNA purification and real-time PCR were performed as
described for ChIP.
Statistical analysis
Statistical analysis was performed using the Graph Pad
Prism
Ò
6 software (Graph Pad software, USA). Experi-
mental data are presented as mean with standard deviation
from three or more independent experiments. One way
ANOVA tests were performed in all data followed by
Tukey post hoc test. Statistical differences were considered
when P\0.05.
Results
Regulation of PR isoforms expression in mHypoE-
N42 cells
A series of experiments was performed to assess the effects
of estradiol in both PR-A and PR-B expression in mHy-
poE-N42 cells. An estradiol dose–response curve was
performed after 6 h of treatment, and a significant induc-
tion of PR-B protein content with 100 nM of estradiol was
observed by Western blot (Fig. 2a, b). PR-A was identified
as the predominant isoform (Fig. 2a). The regulation of PR
isoforms expression by estradiol at mRNA level was
assessed by RT-qPCR. A significant induction of PR-B
mRNA expression after 6 h of treatment was followed by a
marked decrease after 12 h (Fig. 2c). No significant
changes in PR-A mRNA expression after estradiol
TSS PR-B (+1) TSS PR-A (+646)
Rat PPR-B (-150 - +65) Rat PPR-A (+471 - +686)
Human PPR-B (-940 - +32) Human PPR-A (+367 - +1013)
Sp1 sites ERE or half ERE sites
Primers delimited region for PPR-B amplification(-184 - +247)
Primers delimited region for PPR-A amplification (+300 - +712)
+1 +300 +600 +900 +1200-300 +1500-600
Fig. 1 Schematic representation of PR gene promoters of Mus
musculus. Promoter regions and primers delimited regions for PCR
amplification are represented. TSS transcription start site, PPR-B PR-
B isoform promoter region, PPR-A PR-A isoform promoter region,
ERE estrogen responsive element. SP1 sites are represented by
triangles, ERE or half ERE sites are represented by circles
Endocrine
123

treatments were detected as compared to vehicle, although
a decrease in PR-A mRNA expression after 12 h of
estradiol treatment as compared with hormone treatment
after 6 h was observed.
Estradiol regulates the expression
and phosphorylation of ERaand SP1 transcription
factors
The expression and phosphorylation of ERaand SP1, two
main transcription factors that regulate PR gene expression
[17,19,31], were assessed by Western blot (Fig. 3a). An
increase in ERaexpression was observed after 12 h of
estradiol treatment, while SP1 expression was increased at
6 h. In order to determine the functional status of these
transcription factors, ERa(Ser118) and SP1 (total) phos-
phorylation was analyzed. Higher levels of Ser118 phos-
phorylation were found after 6 h of estradiol treatment as
compared to vehicle and 12 h of estradiol treatment
(Fig. 3a, b). An increase in the total phosphorylation levels
of SP1 was found after 12 h of estradiol treatment (Fig. 3a,
c).
As expected, treatment with the highly selective ERa
antagonist (MPP) blocked the estradiol-dependent induc-
tion of PR-B protein content, without affecting PR-A
protein content (Fig. 3d, e).
Estradiol induces a differential occupancy
of estrogen-related transcription factors and RNA
pol II on PR isoform promoters
The local estradiol effects on both PR isoform promoters
were assessed by ChIP. The recruitment of specific tran-
scription factors of estrogen signaling (ERaand SP1) and
RNA pol II was assessed after estradiol treatments (Fig. 4).
RNA pol II was differentially recruited at PR-B promoter
after 6 and 12 h of estradiol treatments (Fig. 4a). However,
a significant difference between the 6 h of estradiol and
vehicle treatments was not observed, but a significant
decrease occurred at 12 h (Fig. 4a). In contrast, RNA pol II
did not show a differential occupancy on PR-A promoter at
any of the studied conditions (Fig. 4b).
SP1 occupancy was detected on PR-B promoter after
vehicle and 6 h of estradiol treatment, but it was not
detected after 12 h of estradiol treatment (Fig. 4a). On the
other hand, SP1 and ERaoccupancy on PR-A promoter
was very low at all the studied conditions, and it was not
statistically different from the negative control of ChIP
assay (Fig. 4b and data not shown). Interestingly, ERawas
only recruited on PR-B promoter after 6 h of estradiol
treatment (Fig. 4a).
Estradiol induces a specific enrichment of active
and repressive histone marks on PR isoform
promoters
In order to determine the epigenetic status of both PR
isoform promoters after estradiol treatments, the analysis of
a
PR-B -
PR-A -
GAPDH -
E2 (nM) Veh. 1 10 100 1000
b
*
*
+
Estradiol (nM)
Protein expression (% Vehicle)
Veh.
1
10
100
1000
Veh.
1
10
100
1000
0
50
100
150
200 PR-B
PR-A
*
Veh.
6h
12 h
Veh.
6h
12 h
0.00
0.01
0.02
0.03
1
2
3
4
Estradiol 100 nM (h)
Relative mRNA expression
cPR-B
PR-A
#
(GAPDH normalized)
Fig. 2 Estradiol differentially regulates PR isoforms expression in
the mHypoE-N42 cell line. Cells were treated as specified in the
‘‘Materials and methods’’ section and then total protein or RNA were
extracted. PR isoforms expression was normalized using GAPDH.
Transcripts relative expression of PR-B and PR-A was calculated by
the DDCt method, and data were normalized using GAPDH gene as
endogenous control. aand bWestern blot analysis of PR isoforms
content after 6 h of estradiol treatment. cPR isoforms relative mRNA
expression after 6 and 12 h of estradiol (100 nM) and vehicle (ethanol
0.02 %) treatments. Significant differences between vehicle treat-
ments at 6 and 12 h were not observed (data not shown). Data are
expressed as mean ±SD of four independent replicates. Veh vehicle,
E2 estradiol. *P\0.05 versus Veh.;
#
P\0.01 versus 6 h;
?
P\0.05 versus 6 h
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123

histone marks related with active gene expression
(H3K9Ac and H3K4me3) and gene repression (H3K9me2)
was performed by ChIP (Fig. 5). The H3K9Ac histone
mark was not altered at any of the studied conditions
(Fig. 5a, b), although a higher enrichment of this histone
mark on PR-B promoter was observed, as compared with
PR-A promoter. A differential enrichment of the H3K4me3
histone mark on PR promoters was observed (Fig. 5). A
higher enrichment of the H3K4me3 mark on PR-B pro-
moter as compared to PR-A promoter was found after
vehicle and 6 h of estradiol treatment. Interestingly,
H3K4me3 enrichment on PR-B promoter was not detected
a
Time (h) 0 6 12
ERα -
P-SP1 -
GAPDH -
P-ERα -
SP1 -
PR-B -
PR-A -
GAPDH -
Antagonist Veh. - MPP
(5 nM)
E2 (100 nM)
b
c
d
#
+
*
e
Estradiol 100 nM (h)
Protein expression (% Vehicle)
Veh.
6h
12 h
Veh.
6h
12 h
0
100
200
300
400 ERα
P-ERα
Estradiol 100 nM (h)
Protein expression (% Vehicle)
Veh.
6h
12 h
Veh.
6h
12 h
0
100
200
300
400
500
SP1
P-SP1
Treatment
Protein expression (% Vehicle)
V
E2
MPP
V
E2
MPP
0
50
100
150
200 PR-B
PR-A
#
*
Fig. 3 Regulation of estrogen-related transcription factors expression
by estradiol in the mHypoE-N42 cell line. Cells were treated with
estradiol (100 nM) or vehicle (ethanol 0.02 %) for 6 and 12 h.
Proteins obtained were analyzed by Western blot. Data were
normalized using GAPDH as loading control. Significant differences
between vehicle treatments at 6 and 12 h were not observed in any of
the studied proteins (data not shown). aRepresentative Western blots
of ERa, phosphorylated ERa(Ser 118), SP1 and phosphorylated SP1
(total), and GAPDH at the studied conditions. Densitometric analysis
of bERaand phosphorylated ERaand cSP1 and phosphorylated
SP1. Levels are expressed as percent of the vehicle (mean ±SD of
three to six independent replicates). Phosphorylated ERaand SP1
expression levels were normalized using the total levels of each
protein. dand eBlocking of the estradiol-dependent induction of PR-
B by the ERaselective antagonist, MPP, expressed as percent of the
vehicle (mean ±SD of three to six independent replicates). Veh
vehicle, E2 estradiol, P-ERaphosphorylated ERaat Ser118; P-SP1
total phosphorylated SP1. *P\0.05 versus Veh. and 6 h;
#
P\0.05
versus Veh. and 12 h;
?
P\0.05 versus Veh. and MPP
Endocrine
123

after 12 h of estradiol treatment (Fig. 5a). In contrast, we
did not find any estradiol-dependent change in the
H3K4me3 histone mark recruitment at PR-A promoter
(Fig. 5b). Unexpectedly, estradiol-dependent transient enrich-
ment of H3K9me2 after 6 h was found at both PR isoform
promoters (Fig. 5a, b).
DNA methylation and hydroxymethylation marks
at PR promoter are not affected by estradiol
treatment
Another key component in epigenetic regulation is DNA
methylation. In addition, the 5-hydroxymethylcytosine has
a
b
*
*
#
+
Veh.
6h
12 h
Veh.
6h
12 h
Veh.
6h
12 h
0
20
40
60
200
400
600
800
Estradiol 100 nM (h)
Fold enrichment
RNA Pol II
SP1
ERα
PPR-B
Veh.
6h
12h
Veh.
6h
12h
Veh.
6h
12 h
0
20
40
60
Estradiol 100 nM (h)
Fold enrichment
PPR-A
RNA Pol II
SP1
ERα
Fig. 4 Transcription factors
and RNA pol II occupancy on
PR isoform promoters
regulation by estradiol.
mHypoE-N42 cells were treated
with estradiol (100 nM) and
vehicle (ethanol 0.02 %) for 6
and 12 h. RNA pol II, SP1, and
ERaoccupancy on aPR-B and
bPR-A promoters was analyzed
by ChIP coupled to real-time
PCR. Significant differences
between vehicle treatments at 6
and 12 h were not observed in
any of the immunoprecipitated
studied proteins (data not
shown). Data are expressed as
fold enrichment (mean ±SD of
three to six independent
replicates). Veh vehicle, PPR-B
PR-B promoter, PPR-A PR-A
promoter. *P\0.01 versus
12 h;
#
P\0.05 versus Veh.;
?
P\0.05 versus Veh. and 12 h
Endocrine
123

recently emerged as an intermediary of active DNA
demethylation [32]. In the present study, estradiol-depen-
dent changes in 5-methylcytosine and 5-hydroxymethylcy-
tosine content at either PR isoform promoters were not
observed (Fig. 6). Interestingly, the 5-methylcytosine and
5-hydroxymethylcytosine levels were higher on PR-A pro-
moter than on PR-B promoter. An increase in both hydroxy
and methyl cytosine levels on PR-A promoter was observed
after 12 h of both vehicle and estradiol treatments (Fig. 6b).
Discussion
In the present study, we demonstrate that PR isoforms are
differentially regulated by estradiol at the transcriptional
level through changes in promoter occupancy by transcrip-
tion factors as well as by epigenetic changes in PR-A and
PR-B promoters in a mouse embryonic hypothalamic cell
line. The transient ERa-dependent induction of PR-B
mRNA expression observed after 6 h of estradiol treatment
a
b
Estradiol 100 nM (h)
Fold enrichment
PPR-B
Estradiol 100 nM (h)
Fold enrichment
PPR-A
Veh.
6h
12h
Veh.
6h
12h
Veh.
6h
12 h
0
20
40
60
80
100
2000
4000
6000
8000 H3K9Ac
H3K4me3
H3K9me2
*
Veh.
6h
12 h
Veh.
6h
12 h
Veh.
6h
12h
0
20
40
60
200
400
600
800 H3K9Ac
H3K4me3
H3K9me2
*
#
Fig. 5 Active and repressive
histone marks enrichment on PR
isoform promoters is
differentially regulated by
estradiol. Cells were treated
with estradiol (100 nM) and
vehicle (ethanol 0.02 %) for 6
and 12 h. H3K9Ac, H3K4me3,
and H3K9me2 enrichment on
PR-B (a) and PR-A
(b) promoters was analyzed by
ChIP coupled to real-time PCR.
Significant differences between
vehicle treatments at 6 and 12 h
were not observed in any of the
immunoprecipitated studied
proteins (data not shown). Data
are expressed as fold
enrichment (mean ±SD of
three to six independent
replicates). Veh vehicle, PPR-B
PR-B promoter, PPR-A PR-A
promoter. *P\0.01 versus
Veh. and 12 h;
#
P\0.001
versus Veh
Endocrine
123

was associated with an increase in ERaphosphorylation at
Ser118, as well as its recruitment on the PR-B promoter. In
line with these data, a down regulation of PR-B mRNA
expression after 12 h of estradiol treatment was associated
with a decrease of SP1, H3K4me3, and RNA pol II
occupancy on its promoter. In contrast, estradiol modified
neither PR-A mRNA expression nor the occupancy of
transcription factors and epigenetic markers on its promoter.
We first demonstrated that estradiol (100 nM) produced
a transient induction of PR-B mRNA expression in the
a
b
Fold enrichment
PPR-B
Fold enrichment
PPR-A
Veh. 6 h
E2100 nM 6 h
Veh. 12 h
E2100 nM 12 h
Veh. 6h
E2100n
M6h
Veh.12h
E2100nM12h
0
10
20
30
40
50
50
100
150
200
250
Treatment
5-methylcytosine
5-hydroxymethylcytosine
Veh.6h
E2100 nM6h
Veh. 12h
E2100nM12h
Veh. 6 h
E2100 nM 6 h
Veh. 12 h
E2100 nM12h
0
50
100
150
200
500
1000
1500
2000
2500
Treatment
*
5-methylcytosine
5-hydroxymethylcytosine
#
Fig. 6 DNA methylation and
hydroxymethylation levels in
PR isoform promoters are not
affected by estradiol treatments.
mHypoE-N42 cells were treated
with estradiol (100 nM) and
vehicle (ethanol 0.02 %) for 6
and 12 h. 5-methylcytosine and
5-hydroxymethylcytosine
detection in PR-B (a) and PR-A
(b) promoters was analyzed by
immunoprecipitation coupled to
real-time PCR. Data are
expressed as fold enrichment
(mean ±SD of three to six
independent replicates). Veh
vehicle, E2 estradiol, PPR-B
PR-B promoter, PPR-A PR-A
promoter. *P\0.0001 versus
6h;
#
P\0.05 versus 6 h
Endocrine
123

mHypoE-N42 cell line (Fig. 2c). Although this estradiol
dose is considered higher than those reported in other
studies [33], the functional relevance of this estradiol
concentration has also been demonstrated in previous
reports using this cell line, in terms of aromatase gene
expression regulation in an ERa-dependent manner [23].
This differential response to estradiol doses between
hypothalamic cultures could be due to the differences in
cell type and developmental stage, since it has been pro-
posed that PR expression within the ventromedial nucleus
of the hypothalamus becomes more responsive to estradiol
as development advances, which supports the idea that
higher doses of estradiol are required to induce PR
expression during early stages of development than those
required in highly estradiol responsive adult cells [34].
Interestingly, the transient induction of PR-B and the
absence of changes in PR-A mRNA expression after
estradiol treatments observed in the present study were
similar to that observed in the hypothalamus of proestrus
rats and during development in several hypothalamic
nuclei [10,35]. In contrast to our results, the induction of
PR mRNA expression is still observed after 48 h of
estradiol administration in the hypothalamus of adult
ovariectomized female rats [7,12]. This suggests that time
differences in the regulation of PR mRNA expression by
estradiol depend on the developmental stage of the animal.
In fact, PR protein content is transiently upregulated in the
ventromedial nucleus of rat hypothalamus during embry-
onic development [35]. On the other hand, the 6 h transient
induction of PR expression by estradiol observed in the
present study has also been reported in the hypothalamus of
adult ovariectomized female mice [24], which suggests that
this short-term regulation of PR expression is species
dependent. The transient induction of PR-B was also
observed at protein level in the present study (data not
shown), suggesting that the effects of estradiol on the
transcription factors and epigenetic marks studied in the
present work are also related with PR isoforms protein
content, which in turn indicates that this regulation could
have an impact on the functional role of PR in this cell line.
Furthermore, PR-A was identified as the predominant iso-
form in the present study, as previously reported in
embryonic rat hypothalamic cells [13].
ERais the main transcription factor that regulates PR
expression in the brain [7,8,36]. We therefore, assessed
the phosphorylation status of ERaat Ser118, since it has
been reported that phosphorylation at this residue is
related with the transcriptional activity of ERain its
target genes [37,38]. Accordingly, higher levels of
phosphorylated ERa(Ser118) were found after 6 h of
estradiol treatment (Fig. 2a). This finding correlates with
the transient induction of PR-B mRNA expression
observed after 6 h of estradiol treatment, suggesting that
there is a correlation between the transcriptional activity
of ERaand PR-B mRNA expression in rodent embryonic
hypothalamic cells.
The participation of ERaon PR expression was
demonstrated using a selective ERaantagonist (Fig. 2d).
Moreover, our ChIP data show that ERais recruited on PR-
B promoter after 6 h of estradiol treatment (Fig. 4a), which
is in line with the induction of PR-B mRNA expression. On
the other hand, and according to several studies, ERa
occupancy on PR-A promoter was not detected at any of
the studied conditions (Fig. 4b) [20,24,39] despite the
presence of a half ERE site in this promoter [27]. The
absence of ERaon PR-B promoter after 12 h of estradiol
treatment could be due to the lack of SP1 presence on PR-B
promoter region at this period of time, since it has been
reported that SP1 is required for ERarecruitment [19].
Furthermore, we have recently reported that the
dynamic DNA methylation pattern found in PR-B promoter
in rat hypothalamus during proestrus transition involves a
consensus and highly conserved SP1 site [10]. Our ChIP
data suggest that SP1 is present on PR-B promoter after 6 h
of estradiol and vehicle treatments, as previously reported
[40,41]. Interestingly, a downregulation of SP1 together
with an increase in its phosphorylation after 12 h of
estradiol treatment was observed (Fig. 3a), when PR-B
mRNA expression was markedly decreased (Fig. 2c). In
line with these results, hyperphosphorylation of SP1 has
been associated with a decrease of its occupancy on target
genes [42,43]. On the other hand, we did not find SP1
occupancy on PR-A promoter. However, further studies are
needed to investigate the role of other SP proteins such as
SP3 or SP4 in PR isoforms regulation [44].
Regarding RNA pol II, our results demonstrate that a
decrease of its recruitment on PR-B promoter at 12 h of
estradiol treatment correlates with a marked decrease on
PR-B mRNA expression (Fig. 2c, 4a), suggesting that SP1
is required for the recruitment of RNA pol II to maintain
the transcriptional basal levels of PR-B. Interestingly, the
transient occupancy of RNA pol II in the promoters of
other estrogen-regulated genes has been previously repor-
ted in MCF7 human breast cancer cells [45]. In contrast, no
differential occupancy of RNA pol II on PR-A promoter at
the studied conditions was found (Fig. 4b), correlating with
the absence of changes in PR-A mRNA expression after
6 h of estradiol treatments. Interestingly, no significant
differences of RNA pol II occupancy on PR-A promoter
between the negative control of ChIP assay and the 12 h of
estradiol treatment were observed (data not shown), which
correlates with the significant decrease on PR-A mRNA
expression at this time point as compared with 6 h of
estradiol treatment. Further studies are required in order to
describe the recruitment of specific phosphorylated forms
of RNA pol II that actively participate in ERa-dependent
Endocrine
123

PR transcription, as we did not find changes in total RNA
pol II occupancy after 6 h of estradiol treatment.
Histone acetylation is one of the characteristic features
of transcriptionally active chromatin [46]. A higher
enrichment of the H3K9Ac histone mark on PR-B pro-
moter as compared to PR-A promoter was observed
(Fig. 5). This finding could explain the differential levels
of RNA pol II occupancy on the corresponding PR isoform
promoters (Fig. 3), which correlate with previous reports
[41,47].
The H3K4me3 histone mark is another of the classical
hallmarks of transcriptionally active chromatin. As in the
case of H3K9Ac histone mark, a higher enrichment of the
H3K4me3 mark on PR-B promoter was found as compared
with PR-A promoter, suggesting that PR-B promoter is
transcriptionally more permissive than PR-A promoter
(Fig. 5). In addition, the increase of the H3K4me3 histone
mark after 6 h of estradiol treatment, as well as the marked
decrease at 12 h, is in line with the transient induction of
PR-B mRNA expression (Fig. 2c) and the corresponding
decrease of RNA pol II and estrogen-related transcription
factors occupancy on this promoter (Fig. 4a). In contrast,
no changes in the H3K4me3 enrichment on PR-A promoter
were observed at any of the studied conditions (Fig. 5b).
The H3K9me2 histone mark is a typical hallmark of
transcriptional silencing on promoter regions [48]. The
enrichment of both transcriptional active (H3K4me3) and
repressive (H3K9me2) histone marks was increased after
6 h of estradiol treatment on PR-B promoter, which could
be the result of a fine-tune regulation mechanism to avoid
ligand-independent expression as previously reported on
ERa-regulated genes in MCF7 breast cancer cells [49].
According to our results, it has been reported that both
transcriptional active and repressive histone marks are also
present on PR isoform promoters in the human term
myometrium, in which the upregulated isoform (PR-A)
was more extensively marked for transcriptional activation
than the non-upregulated one (PR-B) [47]. On the other
hand, the enrichment of the H3K9me2 histone mark after
6 h of estradiol treatment was the only estradiol-dependent
change on PR-A promoter observed in the present study;
however, this modification was not associated with estra-
diol-dependent changes on PR-A mRNA expression.
The participation of DNA methylation in PR gene
expression regulation has been extensively described in
pathological and physiological models [10,50–54]. In the
present study, we did not find any estradiol-dependent
change in DNA methylation or hydroxymethylation of PR
isoform promoters (Fig. 6). It has been reported that DNA
methylation on PR-B promoter is observed as a late event
after disrupting ERasignaling in MCF7 cells [55]. The
higher enrichment of methylated DNA on PR-A promoter
as compared with PR-B promoter would confirm the
transcription factor accessibility on the later one (Fig. 6).
Interestingly, the increase of both 5-methylcytosine and
5-hydroxymethylcytosine marks on PR-A promoter observed
at 12 h was independent of estradiol exposure (Fig. 6b),
which could be related with a particular intrinsic regulation
mechanism of PR-A mRNA expression.
The fact that PR-B was regulated by estradiol in the
present study suggests that the mHypoE-N42 cell line has a
male origin, as previous studies have reported that estradiol
induces PR expression in the neonatal ventromedial
nucleus of hypothalamus in male but not in female rats
[16]. However, it has been demonstrated that prenatal
exposure to a synthetic estrogen (diethylstilbestrol) induces
PR expression in other reproductive relevant brain areas of
female rats such as the medial preoptic nucleus and the
anteroventral periventricular nucleus [56]. Further studies
are required to the complete understanding of molecular
mechanisms involved in the differential regulation of PR
isoforms mRNA expression by estradiol in embryonic
hypothalamic cells, which in turn could be associated with
estradiol mediated prenatal programming processes and
sexual dimorphism in the rodent brain [15,16].
The overall results of this study show that estradiol
induces a differential regulation of PR isoforms expression
in an embryonic hypothalamic cell line, which is associated
with specific promoter occupancy of transcription factors
and histone marks on PR-A and PR-B promoters.
Acknowledgments We acknowledge the technical support of Lucı
´a
Flores Peredo (Departamento de Bioquı
´mica, Facultad de Medicina,
Universidad Nacional Auto
´noma de Me
´xico, Mexico) regarding ChIP
technique and Patricia Mendoza-Lorenzo (Divisio
´n Acade
´mica de
Ciencias Ba
´sicas, Unidad Chontalpa, Universidad Jua
´rez Auto
´noma
de Tabasco, Mexico) for her assistance in real-time PCR experiments.
This work was supported by the Programa de Apoyo a Proyectos de
Investigacio
´n e Innovacio
´n Tecnolo
´gica (PAPIIT) No. IN210412 and
IA202814, and Programa de Apoyo a la Investigacio
´n para Estudi-
antes de Posgrado (PAIP) No. 5000-9108 and 5000-9141, from the
Universidad Nacional Auto
´noma de Me
´xico (UNAM, Me
´xico).
E.R.V-M. is recipient of a Ph.D. scholarship from CONACYT (CVU
288806).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict
of interest.
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