1 Department of Pediatrics and Molecular Medicine Program, University of
California, Parnassus Avenue, San Francisco, CA 94143, USA
2 Department of Cell and Tissue Biology, University of California, Parnassus
Avenue, San Francisco, CA 94143, USA
3 Obstetrics and Gynecology, Cedars-Sinai Medical Center, 8700 Beverly
Boulevard, West Hollywood, CA 90048, USA
4 Howard Hughes Medical Institute, Abramson Family Cancer Research Institute,
University of Pennsylvania, Philadelphia, PA 19104, USA
5 Department of Cell and Developmental Biology, University of Pennsylvania,
Philadelphia, PA 19104, USA
6 Abramson Cancer Research Institute, University of Pennsylvania, Philadelphia,
PA 19104, USA
7 Obstetrics, Gynecology and Reproductive Sciences, University of California,
Parnassus Avenue. San Francisco, CA 94143, USA
8 Department of Pharmaceutical Chemistry, University of California, Parnassus
Avenue, San Francisco, CA 94143, USA
9 Department of Anatomy, University of California, Parnassus Avenue, San
Francisco, CA 94143, USA
* Author for correspondence (e-mail: sfisher{at}cgl.ucsf.edu)
Accepted 31 May 2005
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SUMMARY |
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Key words: HIF, ARNT, HDAC, Stem cell, Syncytiotrophoblast, Placenta, Mouse
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Introduction |
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Murine and human trophoblasts exhibit many unique characteristics. For
example, spongiotrophoblasts, the murine equivalent of human cytotrophoblast
progenitors, proliferate in response to hypoxia a characteristic they
share with hematopoietic precursors
(Adelman et al., 1999). This
unique feature is likely to be one important component of the mechanisms that
contribute to the explosive growth of the placenta as compared with the early
embryo (Genbacev et al., 1997
;
Adelman et al., 2000
). Murine
trophoblast giant cells are among the few mammalian cells to undergo
endoreduplication repeated DNA replication without intervening mitoses
that produces a polyploid state (Cross et
al., 1994
; Zybina and Zybina,
2000
). Likewise, differentiation of human invasive extravillous
cytotrophoblasts produces aneuploid cells
(Weier et al., 2005
). In
addition, trophoblast giant cells and invasive cytotrophoblasts carry out
endovascular invasion, a process that includes transdifferentiation into
endothelial-like cells that line the maternal vasculature
(Cross et al., 1994
;
Zhou et al., 1997
). The
multinucleated syncytiotrophoblasts, which surround fetal blood vessels and
maternal blood sinuses in mice, and form the surface of vascularized chorionic
villi in humans, are responsible for transport. These cells arise from the
underlying chorionic plate in mice or villous cytotrophoblasts in humans,
where they are also largely responsible for the endocrine functions of the
placenta. Thus, the mature placenta is a complex organ with multiple
specialized cell types produced through an equally complex series of
differentiation processes.
Hypoxia-inducible factor 1 (HIF1) is an important regulator of the
responses of a cell to oxygen tension. HIF1 is a widely expressed basic
helix-loop-helix (bHLH)-PAS transcription factor composed of two subunits:
HIF1 and ARNT/HIF1ß (Wang et
al., 1995
). This family is responsible for mediating the response
of cells and organisms to various environmental stimuli, including xenobiotic
exposure, hypoxia and light (Gu et al.,
2000
). We have previously demonstrated a requirement for ARNT in
murine development (Maltepe et al.,
1997
; Adelman et al.,
1999
). As the heterodimerization partner for HIF1
and the
related HIF2
in most tissues, ARNT mediates transcriptional responses
to oxygen deprivation (Wang et al.,
1995
; Ema et al.,
1997
; Tian et al.,
1997
). In the mature organism, this pathway is often activated in
response to pathology. By contrast, mammalian development normally transpires
in a hypoxic environment (Maltepe and
Simon, 1998
). We initially focused on the role of ARNT in
embryonic vascularization and hematopoiesis
(Maltepe et al., 1997
;
Adelman et al., 1999
). It later
became apparent that the primary cause of lethality of Arnt-null
embryos was placental failure caused by a loss of the spongiotrophoblast
population (Kozak et al.,
1997
; Adelman et al.,
2000
). As a result, the placental labyrinth, which is crucial for
maternal-fetal gas and nutrient exchange, fails to form.
In vitro models of differentiation are important adjuncts to in vivo
analyses. The recent derivation of TS cells from mouse blastocysts
(Tanaka et al., 1998) created
a reliable system to study trophoblast giant cell and spongiotrophoblast
differentiation in vitro. Using the TS model, we now report an unexpected role
for ARNT in placental development. Specifically, differentiation of
Arnt-null TS cells in vitro produced syncytiotrophoblasts and
chorionic trophoblasts. As to the mechanisms involved, our data suggest that
the interplay between HIFs and histone deacetylases (HDACs) is crucial for TS
cell differentiation, linking HIF function with epigenetic effectors. These
results are evidence that the HIFs can coordinate diverse epigenetic
mechanisms with profound effects on intrauterine development.
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Materials and methods |
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Labeling of apoptotic cells
Differentiated wild-type or Arnt-null cells grown on glass
coverslips were fixed in freshly prepared 4% paraformaldehyde in PBS, pH 7.4,
for 1 hour at room temperature. DNA breaks were labeled with the In Situ Cell
Death Detection kit (Roche Applied Science, Penzberg, Germany) and apoptotic
cells were visualized by fluorescence microscopy.
Time-lapse Video Microscopy
Arnt-null TS cells at near confluence were allowed to
differentiate in an environmental chamber mounted on a motorized microscope
stage (Carl Zeiss MicroImaging). Time-lapse images were collected every 15
minutes beginning 96 hours after the initiation of differentiation using a
SPOT-RT CCD camera (Molecular Dynamics).
RNA FISH
TS cells were cultured on gelatin-coated glass coverslips, fixed in 4%
formaldehyde/PBS for 15 minutes, permeabilized on ice in PBS containing 0.5%
Triton X-100 for 4 minutes, washed with PBS and then rinsed twice in 2 x
SSC. RNA FISH and washes were performed as described
(Heard et al., 1999) using DNA
probes labeled with Spectrum Red or Green dUTP (Vysis, Downer Grove, IL). DNA
was visualized by staining for 2 minutes with DAPI.
Northern blot analysis
Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA)
according to the manufacturer's instructions. The Errß2, placental
lactogen I and 4311 probes (Adelman et al.,
2000) as well as VEGF, PGK1 and GLUT1 probes have been described
previously (Maltepe et al.,
1997
). The Gcm1 probe was the kind gift of Dr James Cross (U
Calgary). A Tfeb probe corresponding to the terminal 300 bp of the coding
region along with 300 bp of the 3'UTR was generated by RT-PCR. The
product was cloned into the PCR 2.1 vector (Invitrogen, Carlsbad, CA).
Northern blot hybridization was performed using standard methodologies.
Briefly, 20 µg of total RNA was loaded per lane and resolved on 1.5%
agarose gels in a MOPS buffer containing formaldehyde. Then the samples were
transferred to Hybond N+ membranes (Amersham Biosciences, Piscataway, NJ) and
hybridized in QuickHyb hybridization solution (Stratagene, La Jolla, CA) to
32P-labeled DNA probes generated with High Prime reagent (Roche
Diagnostics GMBH, Mannheim, Germany). Binding of labeled probes was detected
using BIOMAX MS film (Kodak, Rochester, NY).
RNase protection assay
Total RNA, isolated by using Trizol reagent as described above, was
analyzed using the RiboQuant Multi-Probe RNase protection assay kit with the
hCR-8 Multi-Probe Template purchased from Pharmingen (San Diego, CA) according
to the manufacturer's instructions. Briefly, a probe set containing the C-X-C
chemokine receptor 4 was labeled with 32P-CTP and hybridized to 10
µg total RNA isolated from either undifferentiated or differentiated
wild-type or mutant TS cells. After RNase digestion, the protected products
were resolved on sequencing gels and identified by size.
Glyceraldehyde-3-phosphate dehydrogenase transcripts were used to assess
sample loading.
|
Histone deacetylase activity
Nuclear extracts of undifferentiated or differentiated wild-type and
Arnt-null TS cells (obtained as described above) were analyzed for
HDAC activity via the HDAC Fluorometric Assay kit (Upstate, Lake Placid, NY)
according to the manufacturer's instructions.
Chemicals and reagents
Trichostatin A (TSA), geldanamycin A (GA) and sodium butyrate (NaB) were
obtained from Sigma-Aldrich (St Louis, MO). The following antibodies were used
for immunoblotting: HIF1 NB 100-105 (Novus Biologicals, Littleton, CO),
anti human/mouse HIF1
(R&D Systems, Minneapolis, MN), HIF2
NB 100-122 (Novus Biologicals, Littleton, CO), ARNT 2B10 (Abcam, Cambridge,
MA), E-cadherin (BD Transduction Pharmingen, San Diego, CA), Hsp90ß Ab-1
(Neomarkers, Fremont, CA), HDAC1 (Upstate, Lake Placid, NY), HDAC2 (Zymed,
South San Francisco, CA), HDACs 3-7 (Cell Signaling Technology, Beverly, MA),
HDAC9 (Biovision Research Products, Mountain View, CA), Ac-H4 (Serotec,
Raleigh, NC), PARP clone C-2-10 (Biomol, Plymouth Meeting, PA) and Granzyme B
(Labvision, Fremont, CA).
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Results |
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To confirm these morphological observations, we exploited two unique
features of trophoblast cells. First, TS cell differentiation along the giant
cell lineage is accompanied by endoreduplication in which the normal
chromosome complement increases by severalfold
(Tanaka et al., 1998). Second,
allocation of the trophoblast lineage entails X inactivation
(Mak et al., 2004
): Xist RNA
expression spreads in cis from the Xic locus, which inactivates the paternal X
chromosome (Brockdorff, 1998
).
To determine if differentiated Arnt-null TS cells have a normal karyotype, we
subjected them to fluorescence in situ hybridization (FISH) analysis for Xist
RNA. As shown in Fig. 1H, 90%
of the cells, which are female (data not shown), contained a single inactive X
chromosome. This was consistent with the conclusion that differentiating
Arnt-null TS cells, like syncytiotrophoblasts, do not undergo
endoreduplication, the hallmark of trophoblast giant cell formation.
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Arnt-null TS cells undergo granzyme B-mediated apoptosis during differentiation
In humans, syncytial turnover mediated by a region of the apoptotic cascade
has been proposed as an important component of placental development in vivo
(Huppertz and Kingdom, 2004).
Thus, we investigated whether differentiation of Arnt-null TS cells is
associated with apoptosis. At a morphological level, DAPI-staining of
Arnt-null TS cell nuclei 48 hours after initiation of differentiation revealed
many pyknotic nuclei (frequently a sign of apoptosis) that were not commonly
observed in cultures of wild-type TS cells
(Fig. 2A). This observation was
confirmed by TUNEL staining (Fig.
2A, inset). Quantification of these results revealed an
approximate sevenfold increase (14% versus 2%) in the number of apoptotic
cells in Arnt-null versus wild-type cultures. To better understand the
consequences, we used timelapse video microscopy to analyze this process. As
shown in Fig. 2B, in
approximately half of the differentiating Arnt-null cells, syncytialization
was followed at 6 days by large-scale membrane breakdown and cytoplasmic
vacuolization that commonly resulted in cell sloughing (not shown). The
remaining cells had normal morphological characteristics.
Next, we used poly (ADP-ribose) polymerase (PARP) cleavage into specific
fragments as a readout of the proapoptotic pathways involved. In addition to
classic caspase family members, granzyme B which after birth is
largely restricted to cytotoxic T-lymphocytes and natural killer (NK) cells
is highly expressed in human villous trophoblast
(Bladergroen et al., 2001;
Hirst et al., 2001
).
Consistent with this observation, immunoblot analyses showed that TS cells
expressed granzyme B protein (Fig.
2C). In wild-type cells, expression of this molecule, which was
predominantly cytoplasmic, was markedly induced during differentiation. By
comparison, mutant cells expressed constitutively elevated levels that did not
change upon withdrawal of FGF4 and heparin. In addition, undifferentiated
wild-type and Arnt-null TS cells expressed PARP protein, which was largely
confined to the nucleus (Fig.
2C). Upon differentiation, nuclear PARP levels were dramatically
decreased in both cell types, with a 64 kDa fragment that is indicative of
Granzyme B-mediated proteolysis apparent in both the nuclear and cytoplasmic
fractions of Arnt-null cells (Fig.
2C, arrow). This was in contrast to an 89 kDa fragment that is
generated by caspase activity (Soldani and
Scovassi, 2002
). TS cells also expressed the PARP-related enzyme
tankyrase 2 (TANK2). Overexpression of this molecule has been shown to lead to
cell death (Kaminker et al.,
2001
). Differentiated Arnt-null cells expressed much higher
cytoplasmic and nuclear levels of TANK2 than were observed in wild-type cells.
Taken together, these results indicated that, in some cases, differentiating
Arnt-null TS cells underwent programmed cell death via mechanisms that
included the actions of granzyme B and TANK2.
Oxygen-independent modulation of HIF expression during TS cell differentiation
To more clearly define the function of HIF family members in TS cells, we
analyzed their expression in wild-type and Arnt-null cells during
differentiation. As expected, only wild-type cells expressed Arnt mRNA
(Fig. 3A). HIF1, but not
HIF2
, mRNA was expressed in undifferentiated TS cells. Interestingly,
levels of both transcripts increased dramatically with differentiation
(Fig. 3A).
HIF activity is coupled to oxygen tension by mechanisms that operate at the
post-translational level. Specifically, the constitutive hydroxylation of
critical proline residues within the oxygen-dependent degradation domain of
HIF by the PHD family of prolyl hydroxylases enables recognition and
subsequent ubiquitination by the Von Hippel-Lindau (pVHL) tumor suppressor
protein (Epstein et al., 2001
;
Ivan et al., 2001
). Under
standard tissue culture conditions of 20% O2, ubiquitinated
HIF
is degraded in the proteasome. This process, which is inhibited by
hypoxia, enables accumulation of the active protein under conditions of
reduced oxygen tension.
|
To determine the functional significance of these observations, we analyzed
mRNA abundance of HIF1 target genes in undifferentiated TS cells by Northern
blot hybridization. As expected, exposure to 2% O2 increased
expression of vascular endothelial growth factor (VEGF) and glucose
transporter 1 (GLUT1) only in wild-type TS cells
(Fig. 3D). Unexpectedly, there
was a dramatic difference in phosphoglycerate kinase 1 (PGK1) expression
between wild-type and Arnt-null TS cells. In wild-type cells, PGK1
expression was regulated similarly to the other HIF1 targets we analyzed. In
sharp contrast, the abundance of mRNA encoding this molecule was significantly
diminished in mutant cells maintained in 20% O2, and transcript
abundance did not change when the cells were cultured in hypoxic conditions
(Fig. 3D). This observation is
consistent with the fact that PGK1 mRNA expression is crucially dependent on
HIF1 during embryonic development in the mouse
(Iyer et al., 1998
).
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Impaired HDAC activity and nuclear localization in Arnt-null TS cells
Multiple proteins have been shown to interact with HIF1. Among
these, the HDACs are of particular interest. HIF1
interacts with HDAC1,
HDAC2 and HDAC3 via pVHL (Mahon et al.,
2001
), as well as HDAC4 and HDAC7
(Kato et al., 2004
). The HDAC
inhibitor trichostatin A (TSA) inhibits hypoxia-induced gene expression.
Conversely, hypoxia induces HDAC activity
(Kim et al., 2001
;
Mie Lee et al., 2003
). To
determine whether the latter activity was altered in the absence of functional
HIF, nuclear extracts isolated from either wild-type or Arnt-null TS
cells were assayed using a fluorometric approach. The results showed that
undifferentiated wild-type TS cells contained significantly more nuclear HDAC
activity than their Arnt-null counterparts (64,520±1893 RFU
versus 58,664±3483 RFU; n=5, P<0.05). Based on
these results, we estimated histone acetylation as a function of
differentiation. Consistent with the activity assays, undifferentiated
Arnt-null TS cells had higher levels of the acetylated form of
histone H4 (Ac-H4) than did wild-type cells. In both cases, acetylation
increased during differentiation, but the pattern of higher levels in
Arnt-null cells was maintained
(Fig. 5A).
Targeted disruption of individual HDAC family members in mouse embryonic
stem cells reveals compensatory increases in the expression of other family
members (Lagger et al., 2002).
To determine whether a similar phenomenon occurs in Arnt-null TS
cells, which have diminished HDAC activity, the relative levels of multiple
HDACs were assayed using an immunoblot approach. As shown in
Fig. 5B, whole-cell lysates
derived from undifferentiated cells showed a pattern of increased HDAC levels
(HDAC1, HDAC2, HDAC3, HDAC4, HDAC5 and HDAC9) in the absence of ARNT. Because
the nuclear localization of individual HDACs largely determines their
biological functions, nuclear extracts from wild-type and Arnt-null
TS cells were also assayed (Fig.
5C). In addition to the class I HDAC HDAC3, the class II HDACs
(HDAC4, HDAC5, HDAC7 and HDAC9), the activities of which are implicated in
differentiation, showed the highest degree of misregulated nuclear expression
in Arnt-null cells.
|
Finally, we tested the effects of TSA and GA on TS cell differentiation. As seen in Fig. 6B, TSA treatment of differentiating wild-type cells abolished expression of placental lactogen I (Pl-I), indicative of giant cell formation, and greatly reduced expression of the spongiotrophoblast marker 4311. Instead, as with Arnt-null TS cells, Tfeb mRNA expression, indicative of chorionic trophoblast formation, was upregulated. TSA treatment of differentiating wild-type TS cells prevented the formation of trophoblast giant cells and spongiotrophoblasts and instead promoted syncytialization (Fig. 6C, arrows) as well as cytoplasmic vacuolization and membrane breakdown (Fig. 6C, arrowheads) akin to that observed with Arnt-null cells. Similar results were obtained in multiple independently derived wild-type TS cell lines, as well as with another HDAC inhibitor, sodium butyrate (data not shown). Like TSA, GA treatment inhibited trophoblast giant cell differentiation and augmented Tfeb-positive chorionic trophoblast expansion in wild-type cells (Fig. 6B). However, HSP90 inhibition with GA preserved formation of 4311-positive spongiotrophoblast during wild-type TS cell differentiation (Fig. 6B). Thus, while both HDAC and HSP90 activity are required for HIF1 induction of target gene expression, only HDAC inhibition phenocopies the effects of ARNT deletion on TS cell differentiation.
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Discussion |
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Together, our data provide evidence for a model of trophoblast stem cell differentiation in which the progenitors either proliferate in an undifferentiated state or enter one of two differentiation pathways (Fig. 7). In vitro, TS cell differentiation is skewed down the pathway that gives rise to spongiotrophoblasts and trophoblast giant cells. Our previous work showed that ARNT deficiency in vivo results in impaired maintenance of the spongiotrophoblast population, and a concomitant increase in the number of trophoblast giant cells, suggesting that spongiotrophoblasts act as precursors to trophoblast giant cells. We show that when wild-type TS cells are subjected to a cycle of hypoxia (2% O2) and reoxygenation (20% O2), conditions that mimic the stage when blood flow to the placenta begins in vivo, the spongiotrophoblast population also expands. Thus, oxygen plays a crucial role in regulating TS cell differentiation both in vivo and in vitro.
Trophoblast differentiation in vivo also gives rise to chorionic
trophoblasts and syncytiotrophoblasts. TS cell differentiation in vitro fails
to recapitulate this process as only a small fraction (5%) of the cells
adopt this fate, suggesting that the culture conditions do not support
differentiation along this pathway. The reasons may involve culture-induced
differences in the epigenetic mechanisms involved in this process. As evidence
for this theory, we found that global HDAC inhibition in wild-type TS cells
promoted differentiation to chorionic trophoblasts and syncytiotrophoblasts in
vitro. In addition, ARNT-deficiency resulted in altered HDAC nuclear
localization and activity, along with global alterations in histone
acetylation patterns, and redirected TS cell differentiation. To our knowledge
this is the first time that TS cell differentiation into chorionic trophoblast
and syncytiotrophoblast has been induced at the expense of spongiotrophoblast
and trophoblast giant cell formation in vitro.
The lack of robust models of trophoblast differentiation into syncytium has
made it difficult to study this process, which is crucial to placental
function. The chorionic plate of the murine placenta begins to produce
Gcm1-expressing syncytiotrophoblasts on embryonic day E7.5
(Basyuk et al., 1999).
Formation of the mature labyrinthine layer is initiated when invading
embryonic endothelial cells from the allantois are surrounded by a bilayer of
multinucleated syncytiotrophoblasts. As an important interface between mother
and embryo/fetus, the syncytium plays crucial barrier and transport roles. The
derivation of syncytiotrophoblasts from trophoblast stem cells provides an in
vitro system for studying these functions. In addition, the syncytium, which
is bathed in maternal blood, must play important immunomodulatory roles. For
example, human syncytiotrophoblasts lack MHC class I and II expression
(Faulk et al., 1977
). In
addition, these cells express other molecules that have immune functions such
as CXCR4 (Douglas et al.,
2001
), which our data show is also expressed by murine
syncytiotrophoblasts in vitro. As this molecule is an important HIV
co-receptor (Coakley et al.,
2005
), Arnt-null TS cells could be a model for studying
viral transmission during pregnancy.
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Differentiating TS cells also exhibited other unique phenomena. Whereas
ARNT protein levels remained relatively constant, differentiation of wild-type
TS cells cultured in 20% O2 induced both HIF1 and
HIF2
mRNA and protein expression. The mechanism, as yet unknown,
represents a novel mode of regulating the abundance of these transcription
factors, which in most tissues are responsible for modulating gene expression
in response to hypoxia. Whether or not the response pathways include IGF
receptor signaling, which has been implicated in the oxygen-independent
activation of HIF1
(Feldser et al.,
1999
), or estrogen- and progesterone-mediated HIF
induction, as described in the peri-implantation mouse embryo, remains to be
determined (Daikoku et al.,
2003
). Interestingly, the expression of pVHL, the E3 ubiquitin
ligase responsible for the degradation of HIF
, was also induced with
differentiation, suggesting novel regulatory mechanisms. In human
cytotrophoblasts, differentiation is also associated with oxygen-independent
upregulation of HIF
and pVHL expression
(Genbacev et al., 2001
),
another example of important parallels between human and murine
placentation.
HSP90 also plays a role in regulating HIF stabilization
(Isaacs et al., 2002;
Katschinski et al., 2002
). In
addition, disruption of HSP90ß in mice leads to midgestational embryonic
lethality because of impaired placentation
(Voss et al., 2000
).
Interestingly, the HSP90 inhibitor GA, which selectively targets HIF1
over HIF2
(Park et al.,
2003
), is a useful reagent for dissecting the individual roles of
these transcription factors. Here, we show that this inhibitor impairs HIF1
dependent gene expression, which in turn inhibits trophoblast giant cell
differentiation in vitro.
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Because histone deacetylase activity can be induced by hypoxia
(Kim et al., 2001), we
investigated possible links between HIF functions in the placenta and
epigenetic phenomena. This association could have important ramifications
because histone acetylation is a crucial regulator of chromatin structure and
hence gene expression. Many transcription factors interact with both histone
acetyltransferases (HAT) and HDACs, which are components of large
multimolecular complexes (Legube and
Trouche, 2003
). In general, histone acetylation relaxes chromatin
structure, whereas deacetylation has the opposite effect. Consequently, the
balance of HAT and HDAC activities helps regulate gene expression.
HDACs belong to one of three classes that were first described in yeast
(Kurdistani and Grunstein,
2003). Class I HDACs (HDAC1, HDAC2, HDAC3 and HDAC8) are
ubiquitous regulators of transcription. Interestingly, HIF1
interacts
with the HAT p300/CBP and several HDACs. Class II HDACs play important roles
in regulating tissue-specific gene expression and hence differentiation
(Verdin et al., 2003
). For
example, the class II HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) mediate the
myoblast-to-myocyte transition by modifying MEF2C transcriptional activity
(McKinsey et al., 2000
;
Dressel et al., 2001
;
Miska et al., 2001
).
Interestingly, subcellular localization is an important determinant of class
II HDAC activity. For example, HDAC5 phosphorylation unmasks docking sites for
molecular chaperones of the 14-3-3 family, which results in nuclear export and
subsequent inhibition of HDAC activity. Class III HDACs, which bear homology
to the NAD-dependent SIR2 family in yeasts, do not appear to be involved in
hypoxia responses. Thus, our results with regard to ARNT effects on HDAC
expression and localization are consistent with the known effects of these
molecules during differentiation and in response to physiological stimuli.
Our findings are also consistent with the molecular details of HIF and HDAC
interactions. For example, we have shown that ARNT is involved in the nuclear
translocation of HIF1 and HDACs, one possible mechanism whereby hypoxia
induces HDAC activity. Conversely, HDAC inhibition represses hypoxia-induced
gene expression and subsequent angiogenesis in murine tumor models
(Kim et al., 2001
). Here, we
have shown that both undifferentiated and differentiated Arnt-null TS
cells express less HDAC activity than their wild-type counterparts. These
cells, similar to HDAC1-deficient ES cells, exhibited compensatory
upregulation of many other family members. Additionally, the absence of ARNT
dramatically impaired the nuclear localization of multiple HDACs, which was
consistent with the reduction we observed in the cells' HDAC activity. The
fact that in wild-type TS cells HDAC inhibition recapitulated the
Arnt-null phenotype provides a functional link between impaired HDAC
localization/activity and changes in TS cell fate. Additionally,
differentiating TS cells upregulated both HIF
and pVHL expression. As
physical interactions between HIF1
and multiple HDACs occur in a
complex that also includes pVHL (Mahon et
al., 2001
), parallel regulation of these molecules would be
required for changes in gene expression. Thus, ARNT interaction with HDACs
regulated by both oxygen-dependent and -independent mechanisms is crucial to
placental development.
In conclusion, the results of this study significantly broaden our understanding of the functions of ARNT. In accordance with its known actions, ARNT mediates oxygen-dependent gene expression and expansion of the spongiotrophoblast lineage in vivo and in vitro. However, in TS cells, ARNT also influences HDAC expression, localization and activity in an oxygen-independent fashion. Thus, during differentiation this transcription factor integrates multiple epigenetic inputs, such as oxygen tension and histone acetylation, with classic transcriptional regulatory mechanisms. The study of ARNT-deficient TS cells will allow the further dissection of this complex regulatory network into its component parts. Whether or not similar networks function during the development of other organs remains to be determined. If these principles apply to embryonic development, they could shed light on the mechanisms involved in fetal programming of adult disease, a process by which the physiological environment, perhaps working through epigenetic pathways, eventually alters gene expression.
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ACKNOWLEDGMENTS |
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