1 Department of Human Genetics, University of California at Los Angeles, 695
Charles Young Drive South, Los Angeles, CA 90095, USA
2 UCLA MRRC, University of California at Los Angeles, 695 Charles Young Drive
South, Los Angeles, CA 90095, USA
3 Departments of Molecular and Medical Pharmacology, and Psychiatry and
Behavioral Sciences, University of California at Los Angeles, 695 Charles
Young Drive South, Los Angeles, CA 90095, USA
4 UCLA Neuropsychiatric Institute, University of California at Los Angeles, 695
Charles Young Drive South, Los Angeles, CA 90095, USA
5 Department of Biological Chemistry, University of California at Los Angeles,
695 Charles Young Drive South, Los Angeles, CA 90095, USA
6 Department of Medicine, University of California at Los Angeles, 695 Charles
Young Drive South, Los Angeles, CA 90095, USA
* Authors for correspondence (e-mail: gfan{at}mednet.ucla.edu and ysun{at}mednet.ucla.edu)
Accepted 20 May 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Dnmt1, CpG methylation, Neural differentiation, STAT1, Chromatin remodeling, MeCP2, Histone modification, Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mammalian CNS is established through a temporally and spatially
well-organized sequence of events during development. Starting as a single
layer of multipotent neural progenitor cells (NPCs), the developing CNS
sequentially produces neurons, astrocytes, and oligodendrocytes at specific
stages during development (Bayer,
1991; Qian et al.,
2000
; Sauvageot and Stiles,
2002
). The sequential differentiation of neurons and glia from
NPCs is not simply due to the sequential appearance of neuronal and glial
inducing cues, because early CNS progenitors are not capable of immediately
differentiating into glia, even when presented with strong glial-inducing
factors (Sauvageot and Stiles,
2002
; Takizawa et al.,
2001
). Therefore, during development, NPCs gradually acquire
competence for gliogenesis. It is intriguing that when cells become gliogenic,
they simultaneously lose their neurogenic potential, suggesting the existence
of a neurogenic to gliogenic switch mechanism during CNS development.
Both cell intrinsic factors and extracellular cues have been postulated to
influence the neuro- to gliogenic switch. For example, the presence of
neurogenic factors such as the proneural basic helix-loop-helix (bHLH) genes
has been shown to actively suppress the gliogenic state of NPCs during the
period of neurogenesis (Nieto et al.,
2001; Sun et al.,
2001
), contributing to the late onset of gliogenesis. In addition,
the transient rise of basic fibroblast growth factors (bFGFs) in the
ventricular zone of the mouse cortex around embryonic day 14.5 (E14.5) may
serve as a potential modulatory factor mediating the transition from
neurogenesis to gliogenesis. Low levels of bFGF have been shown to increase
neuronal differentiation, while high levels of bFGF expand the glial
progenitor pool (Dono et al.,
1998
; Qian et al.,
1997
). In addition, bFGF regulates the expression of the
epithelial growth factor receptor (EGFR), which is correlated with the
appearance of astrogliogenic progenitors
(Lillien and Raphael, 2000
).
The expression of EGFR in neural progenitors could also be influenced by other
factors, such as bone morphogenetic proteins (BMPs), WNT proteins and sonic
hedgehog (SHH) in vitro (Viti et al.,
2003b
). As oligodendrocytes are generated from the ventral part of
the forebrain, gliogenesis in the developing cortex is primarily confined to
astrocyte differentiation. Although it remains to be determined whether the
aforementioned glia-inducing factors are either necessary or sufficient to act
in vivo to regulate the neuro- to gliogenic switch, most of these factors have
been shown to influence the essential astrogliogenic JAK-STAT pathway
(Nakashima et al., 1999b
;
Song and Ghosh, 2004
;
Sun et al., 2001
;
He et al., 2005
).
The JAK-STAT pathway, which is activated by cytokine leukemia inhibitory
factor (LIF) through the heterodimeric receptor LIFRß and gp130 can
effectively induce astroglial differentiation
(Bonni et al., 1997;
Rajan and McKay, 1998
). BMPs
also use STATs to activate the expression of astrocyte marker genes via the
association between STATs and the transactivating complex composed of the
BMP-activated signaling factors Smad1 and P300/CBP
(Nakashima et al., 1999b
).
Gene knockouts of LIF (Bugga et al.,
1998
), LIFRß (Koblar et
al., 1998
), gp130 (Nakashima
et al., 1999a
) or STAT3 (He et
al., 2005
) all result in impaired astrocyte differentiation in
vivo, further indicating that JAK-STAT signaling contributes to
astrogliogenesis in the developing CNS.
Although LIF is a potent astroglial differentiation factor, it cannot
immediately trigger astrogliogenesis in early cortical NPCs. The lack of an
astrogliogenic response to LIF in early cortical NPCs has been primarily
attributed to methylation of astrocyte marker genes such as Gfap, as
it has been suggested that various components of the LIF-induced JAK-STAT
pathway are present in both early and late NPCs
(Molne et al., 2000;
Song and Ghosh, 2004
;
Takizawa et al., 2001
).
Furthermore, methylation of the STAT binding element within the Gfap
promoter was shown to inhibit the association of activated STATs with the
glial promoter, thereby repressing transcription of the Gfap gene
(Takizawa et al., 2001
).
However, we recently found that the overall activity of the JAK-STAT pathway
is strongly suppressed during the neurogenic period and becomes robustly
elevated during the gliogenic switch (He
et al., 2005
). In this report, we provide evidence that DNA
methylation is one of the key mechanisms inhibiting JAK-STAT signaling in
neurogenic NPCs. Global DNA hypomethylation in the developing CNS leads to
precocious astrogliogenesis. However, hypomethylation-induced precocious
astrogliogenesis is not simply due to demethylation of the STAT binding
element within the GFAP promoter (Takizawa
et al., 2001
), but primarily due to the elevation of overall
JAK-STAT signaling activity. Our findings suggest that DNA methylation
regulates the timing and magnitude of astrogliogenesis through both modulation
of JAK-STAT activity and its direct inhibition of glial marker genes via
inactive chromatin remodeling.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA methylation analysis
Bisulfite genomic sequencing analysis was performed essentially as
described Clark et al. (Clark et al.,
1994). Methylation-specific SNuPE assay was used to quantify the
percentage of methylation at a particular CpG site as described in detail by
Gonzalgo and Jones (Gonzalgo and Jones,
1997
). The quantification of radioactive signals was performed
with a phosphor-imager system (Molecular Dynamics).
Electrophoretic-mobility-shift assay (EMSA) and chromatin-immunoprecipitation (ChIP) analyses
Purification of nuclear extracts and EMSA assays were carried out as
described (Bonni et al., 1997).
For ChIP analysis, NPCs were left untreated or treated with LIF for 30 minutes
and crosslinked with 1% formaldehyde for 20 minutes at room temperature.
Experiments were performed as previously described
(Martinowich et al., 2003
).
Normal rabbit serum or anti-ß-gal antiserum was routinely used in ChIP
assays for negative controls (data not always shown). Antibodies used for pull
downs were anti-MeCP2 (Upstate), anti-H3 dmK9, anti-H3
d/tmK4 (Upstate), anti-STAT1 (generated by Ke Shuai's laboratory at
UCLA) (ten Hoeve et al., 2002
)
and anti-STAT3 (Santa Cruz). DNA samples obtained before (Input) and after
(ChIP) immunoprecipitation were subjected to PCR amplification. PCR primer
sequences for ChIP analyses are listed as follows. Gfap gene promoter
encompassing the 1.5 kb STAT binding element: forward 5' TAA GCT
GAA GAC CTG GCA GTG 3'; reverse, 5' GCT GAA TAG AGC CTT GTT CTC
3'. Stat1 gene promoter set I (1748 bp to 1478 bp
encompassing one of the STAT binding elements, used for anti-STAT3 ChIP):
forward, 5' AAG TGG TGC TGT TCA AGG 3'; reverse, 5' CAG AGG
TAA GCT GAT TCC 3'. Stat1 gene promoter set II (670 bp
to 449 bp encompassing the developmentally regulated CpG site, used for
anti-MeCP2 ChIP): forward, 5' GAC AGA GGG ATG TCC TGC 3'; reverse,
5' CTT CGG ACC TCC ACT GAC 3'. S100ß gene ChIP primers
(1142 bp to 817 bp encompassing both STAT binding and the
developmentally regulated CpG site): forward, 5' GGA ACA CGA GGG GCA AAG
3'; reserve, 5' CGC TCT TGC CCA GAA ATG 3'.
DNA constructs, cell transfection, and promoter activity luciferase-reporter assay
The construction of the 1.9 kb rat GFAP-luciferase and ß-gal reporter
plasmids has been described before (Bonni
et al., 1997). The mouse Dnmt1 expression plasmid was
kindly provided by Dr En Li (Chen et al.,
2003
). Plasmids were transfected into neural precursor cells using
the Fugene-6 reagent as instructed by the manufacturer (Roche). For the
promoter activity assay, fly-luciferase reporter plasmids were co-transfected
into cells using Fugene-6 (Roche) along with a TK-renilla luciferase plasmid
(Promega), which serves as a cell transfection control. Cultures were treated
with or without LIF (50 ng/ml) for
24 hours post-transfection and were
lysed and subjected to dual luciferase assays (Promega). Statistical analysis
of luciferase assay data and cell counting results was performed with StatView
5.0 software (SAS Institute)
TUNEL staining method
Cultured cells were fixed with 4% PFA/PBS and stained for TUNEL analysis
using the Apoptag Fluoroscein In Situ Apoptosis Detection Kit (Chemicon)
following the supplied manufacturer's protocol. TUNEL-positive cells were
counted at 40x magnification (20 fields/coverslip) and reported as a
ratio of total cell number as determined by DAPI staining.
Western and northern blot analysis
Proteins were extracted with NP40 lysis buffer and concentrations measured
using the Bradford method. After denaturing with SDS lysis buffer, protein
samples were fractionated on a SDS-PAGE gel (BioRad) and transferred to
nitrocellulose membrane for immunoblotting. To measure phosphorylation of
STAT1/3, cell cultures were harvested after a 20-minute stimulation with LIF
and subjected to western blot analysis using antibodies specific for the
phosphorylated form of STAT1/3 as described before
(Bonni et al., 1997;
Sun et al., 2001
). RNA was
extracted from brain samples with RNAzol and subjected to northern blot
analysis as reported previously (Sun et
al., 2001
).
Retroviral lineage tracing assay
E11.5 NPCs from control and Dnmt1/ CNS
were infected with a replication-deficient retrovirus containing a
ß-galactosidase gene at the time of plating as described before
(Bonni et al., 1997). At 4 DIV,
cells were triple-labeled with antibodies against MAP2, GFAP and ß-gal,
and the percentage of MAP2/ß-gal and GFAP/ß-gal double-positive
cells over the total number of ß-gal positive cells was scored.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To uncover the underlying mechanism by which hypomethylation leads to
precocious astrogliogenesis, we established an E11.5 mouse cortical NPC
culture system, in which the neuro- to gliogenic switch can be recapitulated
in vitro. Previous studies have demonstrated that cultured NPCs derived from
early (E11.5) mouse cortices differentiate mainly down the neurogenic pathway,
whereas NPCs derived from late (E14.5 and later) cortices have increased
potential for glial differentiation (Qian
et al., 2000). We found that when E11.5 mouse cortical NPCs were
cultured for an extended period, they switched to producing GFAP-positive
astrocytes after 4-5 days in vitro (DIV)
(Fig. 2A). The generation of
astrocytes in E11.5 cortical NPC cultures can be further promoted by treatment
with LIF (Fig. 2A). However,
even in the presence of LIF, no GFAP-positive astrocytes were observed in
short-term (fewer than 4-5 DIV) mouse E11.5 cortical cultures, suggesting that
there is an intrinsic mechanism blocking the initiation of astroglial
differentiation in early NPCs.
In contrast to the lack of GFAP positive astrocytes in control
(Dnmt1+/+) cultures, GFAP-positive cells with astrocytic
morphologies were observed in E11.5 Dnmt1/
cultures at 2 DIV with LIF treatment (Fig.
2B). The precocious induction of GFAP expression as well as
another astrocyte marker S100ß in
Dnmt1/ NPC cultures was also evident
(Fig. 2C,D). In these cultures,
virtually all of the cells from mutant mouse CNS lacked Dnmt1
(Fan et al., 2001). Using a
monoclonal antibody against 5'-methylcytosine (5'meC), we found
that the intensity of 5'meC staining was significantly weaker in
Dnmt1/ cells than that in control cells
after three days in culture, which is consistent with decreased DNA
methylation in Dnmt1/ cells
(Fig. 2E). However,
immunostaining with the 5'-methylcytosine antibody is not quantitative
enough to allow us to address whether newly generated GFAP-positive astroglial
cells are exclusively derived from those
Dnmt1/ NPCs with a more extensively
demethylated genome. Finally, we also carried out TUNEL staining and compared
cell death phenotypes between cultured control and Dnmt1 mutant NPCs after 5
DIV. There was no apparent difference in the percentage of TUNEL-positive
cells when mutant cultures were compared with control cultures [CON=2.7%
(75/2728); MUT=3.9% (92/2347)], indicating that the increase in GFAP and
S100ß positive cells in Dnmt1/ cultures
did not result from changes in cell survival in vitro.
|
Enhanced activation of the JAK/STAT astrogliogenic pathway due to DNA hypomethylation
Astroglial differentiation is regulated by LIF-induced activation of the
JAK-STAT pathway. The major STAT proteins expressed in the nervous system are
STAT1 and STAT3. Importantly, increased STAT1/3 expression and phosphorylation
in E11.5 NPCs is correlated with the neuro- to gliogenic switch in vitro
(Fig. 3A)
(He et al., 2005). In
addition, we noticed that genes encoding various components of the JAK-STAT
pathway including STAT1, STAT3, gp130 receptor, as well as JAK1 contain
STAT-binding elements in their promoters that can be activated by STATs,
suggesting that an autoregulatory/positive feedback loop of this pathway
exists. This positive-feedback loop may be important for the rapid and robust
activation of JAK-STAT signaling during the neuro- to gliogenic transition
(He et al., 2005
).
|
To examine whether the elevated STAT phosphorylation observed in
Dnmt1/ E11.5 CNS cultures had any functional
consequences in the activation of astrocyte marker genes, we transfected a 1.9
kb GFAP promoter-luciferase reporter construct
(Bonni et al., 1997) into 3 DIV
cultures from either control or mutant E11.5 mouse CNS. The cells were either
left untreated or treated with LIF (50 ng/ml) immediately following
transfection and analyzed for luciferase activity 24 hours later. LIF
treatment induced a stronger activation of the GFAP promoter in
Dnmt1/ cells than in
Dnmt1+/+ cells (Fig.
4A). To determine whether enhanced activation of the exogenous
GFAP promoter resulted from the elevated activity of STAT1/3 or other
transcription factors, we introduced a STAT-binding mutant of the GFAP
promoter-luciferase reporter and measured luciferase activities in both
control and methylation-deficient cells. When the canonical STAT1/3 binding
element was mutated (from TTCCGAGAA to CCAAGAGAA), the
LIF-induced GFAP promoter activation in both control and mutant cultures was
abolished (Fig. 4A). This
result indicated that the increase in GFAP promoter activity in
Dnmt1/ cells is caused by enhanced STAT
function (Fig. 4A). Consistent
with enhanced STAT1/3 activation in Dnmt1/
cells, both EMSA and chromatin immunoprecipitation (ChIP) assays further
indicated that Dnmt1/ CNS cells contain more
nuclear pSTAT1/3 and that more STAT1/3 were associated with the endogenous
GFAP promoter in Dnmt1/ CNS cells
(Fig. 4B,C). Taken together,
these data suggest that the enhanced activation of the JAK-STAT pathway in
methylation-deficient CNS cells leads to stronger activation of astrocyte
marker genes.
|
Dnmt1 deficiency accelerates the developmentally regulated demethylation of glial differentiation-related genes
Multiple CpG sites within the rat Gfap promoter are methylated
early on during CNS development and become less methylated during gliogenic
stages in vivo (Teter et al.,
1996). Takizawa et al. also reported that a single CpG site within
the STAT-binding element in the mouse Gfap promoter undergoes
demethylation during the neuro- to gliogenic switch in the developing CNS. To
further examine the relationship between changes in DNA methylation and
gliogenesis, we analyzed the methylation status of the mouse Gfap,
Stat1 and S100ß genes in neurogenic and gliogenic NPCs. Within the
Gfap promoter, we focused on a region from 1557 bp to
1280 bp, which is highly conserved across species of mouse, rat and
human. In fact, this region in the rat GFAP gene has been designated
as the neuroectoderm/astrocyte methylation domain
(Fig. 5A), because it undergoes
demethylation during the neurogenic to gliogenic phase transition
(Teter et al., 1996
).
Importantly, the relative position and sequence of the STAT-binding element
(5'TTCCGAGAA3') within this neuroectoderm methylation
region is 100% conserved among the three species. We performed bisulfite
genomic sequencing analyses on 8 CpG sites within this promoter region. Our
analysis showed that selective demethylation occurred at five out of the eight
CpG sites in E11.5 cortical NPCs over 4 days in culture, including the CpG
within the STAT-binding element (Fig.
5A). Similarly, selective CpG demethylation was also observed on
the Stat1 and S100ß promoters in E11.5 cortical culture during
the neuro- to gliogenic switch (Fig.
5B, see Fig. S1 in the supplementary material).
Loss of Dnmt1 activity would be predicted to cause accelerated
demethylation of the Gfap, Stat1 and S100ß promoters in the
developing CNS. To directly examine the methylation status of the STAT-binding
element within the Gfap promoter in both control and Dnmt1 mutant
cells, we performed bisulfite genomic sequencing analyses and the
methylation-site-specific single nucleotide primer extension (SNuPE) assays
(Gonzalgo and Jones, 1997).
The SNuPE assay showed a significant decrease in methylation of the
STAT-binding site in cultured E12.5 Dnmt1/
CNS cells compared with control cells (Fig.
5C). The CpG site within the Gfap STAT-binding site is
virtually completely unmethylated in E18.5
Dnmt1/ brains in vivo
(Fig. 5C), suggesting that
demethylation of this CpG site also occurs in the neuronal population.
However, neuronal Gfap expression was not detected, supporting the
notion that demethylation of the CpG within the STAT binding element is not
sufficient to induce GFAP expression. It is likely that additional mechanisms
exist in neurons to actively suppress the demethylated GFAP gene promoter in
Dnmt1/ neurons. Substantial demethylation of
the Stat1 and S100ß promoters was also observed in
Dnmt1/ brains during development, which
could contribute to the induction of STAT1 and S100ß in hypomethylated
NPCs and/or astroglia (Fig. 5D,
see Fig. S1 in the supplementary material).
|
It has previously been reported that methylation of the STAT cis-element
within the Gfap promoter blocks STAT3 association
(Takizawa et al., 2001). Using
EMSA, we confirmed that methylation of the STAT-binding element attenuates
phospho STAT1 (pSTAT1) and the STAT 1/3 complex from associating with the GFAP
promoter (see Fig. S2 in the supplementary material). However, in contrast to
the Gfap promoter, the STAT-binding elements of the Stat1
and S100ß promoters do not contain any CpG sites. Therefore, the
aforementioned mechanism, i.e. reduced binding affinity between STATs and the
methylated STAT cis-element, cannot contribute to DNA methylation mediated
suppression of Stat1 and S100ß genes. Therefore, we examined
whether DNA methylation inhibits expression of Stat1, S100ß and
Gfap through a general gene silencing mechanism, i.e. inactive
chromatin remodeling.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We hypothesize that during CNS development, the generation of neurons in
the absence of glia relies on the suppression of glial differentiation
programs during the neurogenic period. Many glial inducing factors, including
LIF, BMP, NOTCH and the oligodendrogliogenic bHLH factor, OLIG2, are unable to
induce glial differentiation during the neurogenic period
(Ge et al., 2002;
Sauvageot and Stiles, 2002
;
Sun et al., 2003
;
Viti et al., 2003a
). The
ability of these factors to regulate cell fate is thought to depend on the
intrinsic state of the precursor cell. Our study suggests that the JAK/STAT
pathway is a crucial control point for regulating the intrinsic responsiveness
of neural progenitors to astrogliogenic factors. We previously demonstrated
that the expression of a proneural bHLH factor, NGN1, which is exclusively
expressed in progenitor cells during the neurogenic period, inhibits STAT1/3
phosphorylation and prevents pSTATs from activating glial gene transcription
by sequestering the transcription co-activator, p300/CBP, away from glial
specific promoters (Sun et al.,
2001
). This present study indicates that DNA methylation is
another key mechanism inhibiting the expression of the various components of
the astrogliogenic JAK-STAT pathway. Thus, JAK-STAT activation in neural
precursor cells is under the tight control of several independent
mechanisms.
|
CpG methylation can directly inhibit gene transcription when methylation
blocks the association of transcription factor to the cis-element, as
demonstrated for the association of the GFAP promoter with STAT3
(Takizawa et al., 2001) and
STAT1 (see Fig. S2 in the supplementary material). However, the canonical
STAT-binding elements in many other genes involved in astroglial
differentiation such as STAT1, STAT3, gp130, JAK1 and S100ß do not
contain a CpG site (He et al.,
2005
), suggesting that the direct effect of CpG methylation on the
binding of STATs is not a general mechanism in the inhibition of JAK-STAT
signaling. Instead, our data (Fig.
6) demonstrate that DNA methylation suppresses the gliogenic
pathway genes via methyl-CpG-binding protein-mediated inactive chromatin
remodeling. Indeed, the association of methyl-CpG-binding proteins such as
MeCP2, which recruit histone modification enzymes associated with inactive
chromatin remodeling, could be involved in glial gene silencing
(Bird and Wolffe, 1999
;
Jones et al., 1998
;
Martinowich et al., 2003
). Our
ChIP study showed that MeCP2 was associated with methylated glial lineage
genes in neurogenic cells. However, we cannot rule out the possible
involvement of other methyl binding proteins (e.g. MBD1-3) in the nervous
system for their potential role in glial gene silencing during early CNS
development (Heinrich and Bird, 1998) (reviewed by
Fan and Hutnick, 2005
).
DNA methylation may be one of the many repressive mechanisms to prevent
superfluous transcription to achieve cell-specific gene expression during
differentiation. In this regard, methylation of the GFAP promoter is proposed
to be one of the key silencing mechanisms for inhibiting GFAP expression in
peripheral non-neural cell types such as fibroblasts
(Condorelli et al., 1997). In
the current study, we discovered that demethylation in the GFAP promoter in
E18 Dnmt1/ CNS neurons does not lead to
ectopic expression of GFAP in neuronal cells
(Fig. 1 and
Fig. 5B), arguing for the
existence of additional repression mechanism(s) in neurons that blocks ectopic
GFAP gene transcription.
In this study, we have demonstrated that nestin-cre-mediated Dnmt1
gene deletion in mitotic E11 NPCs leads to rapid demethylation in daughter
cells in culture (Fig. 2E),
indicating the essential role for Dnmt1 in maintaining DNA methylation in
embryonic CNS cells (Fan et al.,
2001). The observed phenotype of precocious activation and active
chromatin remodeling of gliogenic genes is consistent with the demethylation
phenotype in Dnmt1/ NPCs. However, it is
worth noting that Dnmt1 molecule has also been shown to directly inhibit gene
transcription with its N-terminal transcription repression domain, which
interacts with other transcription repressor components, including histone
deacetylases and histone lysine-methyltransferases for inactive chromatin
remodeling (Rountree et al.,
2000
; Fuks et al.,
2000
). To determine whether Dnmt1 molecule itself would repress
any astrogliogenic genes such as GFAP, STAT1 and S100ß in vivo, one
potential experiment is to generate a new conditional Dnmt1 mutant
allele that introduces mutations in the catalytic domain and examine
astrogliogenesis phenotype in E11 NPCs that would contain a full-length mutant
form of Dnmt1 without methylation activity. If we do not observe precocious
astroglial differentiation with this line of mutant mice, it will argue for
the direct role of Dnmt1 in repressing astrogliogenic genes. An alternative
result with no rescue of the precocious glial differentiation phenotype would
favor the idea that demethylation of gene promoters is the key mechanism that
mediates the effect of Dnmt1 mutant phenotype.
In summary, our study has provided evidence demonstrating that disruption of DNA methylation of GFAP and other glial differentiation-related genes (e.g. genes involved in the JAK-STAT pathway) alter the timing of glial cell lineage differentiation in vivo. We speculate that such a timing control mechanism may also regulate the differentiation of other cell type lineages. It is possible that the wave of de novo DNA methylation that occurs between the blastula and gastrula stages serves as a general mechanism to block differentiation of late cell lineages effectively such that a developmentally regulated gene-specific demethylation process controls sequential cell lineage differentiation.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/15/3345/DC1
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bayer, S. A. (1991). Neocortical Development. NY: Raven Press NY.
Bird, A. P. and Wolffe, A. P. (1999). Methylation-induced repression belts, braces, and chromatin. Cell 99,451 -454.[CrossRef][Medline]
Bonni, A., Sun, Y., Nadal-Vicens, M., Bhatt, A., Frank, D. A.,
Rozovsky, I., Stahl, N., Yancopoulos, G. D. and Greenberg, M. E.
(1997). Regulation of gliogenesis in the central nervous system
by the JAK-STAT signaling pathway. Science
278,477
-483.
Bugga, L., Gadient, R. A., Kwan, K., Stewart, C. L. and Patterson, P. H. (1998). Analysis of neuronal and glial phenotypes in brains of mice deficient in leukemia inhibitory factor. J. Neurobiol. 36,509 -524.[CrossRef][Medline]
Chen, T., Ueda, Y., Dodge, J. E., Wang, Z. and Li, E.
(2003). Establishment and maintenance of genomic methylation
patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol.
Cell Biol. 23,5594
-5605.
Condorelli, D. F., Dell'Albani, P., Conticello, S. G., Barresi, V., Nicoletti, V. G., Caruso, A., Kahn, M., Vacanti, M., Albanese, V., de Vellis, J. and Giuffrida, A. M. (1997). A neural-specific hypomethylated domain in the 5' flanking region of the glial fibrillary acidic protein gene. Dev. Neurosci. 19,446 -456.[Medline]
Clark, S. J., Harrison, J., Paul, C. L. and Frommer, M. (1994). High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22,2990 -2997.[Abstract]
Dono, R., Texido, G., Dussel, R., Ehmke, H. and Zeller, R.
(1998). Impaired cerebral cortex development and blood pressure
regulation in FGF-2-deficient mice. EMBO J.
17,4213
-4225.
Fan, G. and Hutnick, L. (2005). Methyl-CpG binding proteins in the nervous system. Cell Res. 15,255 -261.[Medline]
Fan, G., Beard, C., Chen, R. Z., Csankovszki, G., Sun, Y.,
Siniaia, M., Biniszkiewicz, D., Bates, B., Lee, P. P., Kuhn, R. et
al. (2001). DNA hypomethylation perturbs the function and
survival of CNS neurons in postnatal animals. J.
Neurosci. 21,788
-797.
Feng, J., Chang, H., Li, E. and Fan, G. (2005). Dynamic expression of De novo DNA methyltransferases Dnmt3a and Dnmt3b in the central nervous system. J. Neurosci. Res. 79,734 -746.[CrossRef][Medline]
Fuks, F., Burgers, W. A., Brehm, A., Hughes-Davies, L. and Kouzarides, T. (2000). DNA methyltransferase Dnmt1 associates with histone deacetylase activity. Nat. Genet. 24,88 -91.[CrossRef][Medline]
Ge, W., Martinowich, K., Wu, X., He, F., Miyamoto, A., Fan, G., Weinmaster, G. and Sun, Y. E. (2002). Notch signaling promotes astrogliogenesis via direct CSL-mediated glial gene activation. J. Neurosci. Res. 69,848 -860.[CrossRef][Medline]
Gonzalgo, M. L. and Jones, P. A. (1997). Rapid
quantitation of methylation differences at specific sites using
methylation-sensitive single nucleotide primer extension (Ms-SNuPE).
Nucleic Acids Res. 25,2529
-2531.
He, F., Ge, W., Zhu, W., Becker-Catania, S., Martinowich, K., Wu, H., Coskun, V., Fan, G., deVellis, J. and Sun, Y. (2005). A positive autoregulation loop of JAK-STAT signaling is part of the clock mechanism regulating astrogliogenesis. Nat. Neurosci. 8,616 -625.[CrossRef][Medline]
Hendrich, B. and Bird, A. 1998. Identification
and characterization of a family of mammalian methyl-CpG binding proteins.
Mol. Cell Biol. 18,6538
-6547.
Jaenisch, R. and Bird, A. (2003). Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33,245 -254.[CrossRef][Medline]
Jones, P. A. and Baylin, S. B. (2002). The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. 3,415 -428.[Medline]
Jones, P. L., Veenstra, G. J., Wade, P. A., Vermaak, D., Kass, S. U., Landsberger, N., Strouboulis, J. and Wolffe, A. P. (1998). Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat. Genet. 19,187 -191.[CrossRef][Medline]
Koblar, S. A. Turnley, A. M, Classon, B. J., Reid, K. L., Ware,
C. B., Cheema, S. S., Murphy, M. and Bartlett, P. F.
(1998). Neural precursor differentiation into astrocytes requires
signaling through the leukemia inhibitory factor receptor. Proc.
Natl. Acad. Sci. USA 95,3178
-3181.
Li, E., Bestor, T. H. and Jaenisch, R. (1992). Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69,915 -926.[CrossRef][Medline]
Lillien, L. and Raphael, H. (2000). BMP and FGF
regulate the development of EGF-responsive neural progenitor cells.
Development 127,4993
-5005.
Lunyak, V. V., Burgess, R., Prefontaine, G. G., Nelson, C., Sze,
S. H., Chenoweth, J., Schwartz, P., Pevzner, P. A., Glass, C., Mandel,
G. et al. (2002). Corepressor-dependent silencing of
chromosomal regions encoding neuronal genes. Science
298,1747
-1752.
Martinowich, K., Hattori, D., Wu, H., Fouse, S., He, F., Hu, Y.,
Fan, G. and Sun, Y. E. (2003). DNA methylation-related
chromatin remodeling in activity-dependent BDNF gene regulation.
Science 302,890
-893.
Molne, M., Studer, L., Tabar, V., Ting, Y. T., Eiden, M. V. and McKay, R. D. (2000). Early cortical precursors do not undergo LIF-mediated astrocytic differentiation. J. Neurosci. Res. 59,301 -311.[CrossRef][Medline]
Nakashima, K., Wiese, S., Yanagisawa, M., Arakawa, H., Kimura,
N., Hisatsune, T., Yoshida, K., Kishimoto, T., Sendtner, M. and Taga,
T. (1999a). Developmental requirement of gp130 signaling in
neuronal survival and astrocyte differentiation. J.
Neurosci. 19,5429
-5434.
Nakashima, K., Yanagisawa, M., Arakawa, H., Kimura, N.,
Hisatsune, T., Kawabata, M., Miyazono, K. and Taga, T.
(1999b). Synergistic signaling in fetal brain by STAT3-Smad1
complex bridged by p300. Science
284,479
-482.
Nieto, M., Schuurmans, C., Britz, O. and Guillemot, F. (2001). Neural bHLH genes control the neuronal versus glial fate decision in cortical progenitors. Neuron 29,401 -413.[CrossRef][Medline]
Okano, M., Bell, D. W., Haber, D. A. and Li, E. (1999). DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99,247 -257.[CrossRef][Medline]
Paulsen, M. and Ferguson-Smith, A. C. (2001). DNA methylation in genomic imprinting, development, and disease. J. Pathol. 195,97 -110.[CrossRef][Medline]
Qian, X., Davis, A. A., Goderie, S. K. and Temple, S. (1997). FGF2 concentration regulates the generation of neurons and glia from multipotent cortical stem cells. Neuron 18, 81-93.[CrossRef][Medline]
Qian, X., Shen, Q., Goderie, S. K., He, W., Capela, A., Davis, A. A. and Temple, S. (2000). Timing of CNS cell generation: a programmed sequence of neuron and glial cell production from isolated murine cortical stem cells. Neuron 28, 69-80.[CrossRef][Medline]
Rajan, P. and McKay, R. D. (1998). Multiple
routes to astrocytic differentiation in the CNS. J.
Neurosci. 18,3620
-3629.
Razin, A. and Cedar, H. (1991). DNA methylation and gene expression. Microbiol. Rev. 55,451 -458.[Medline]
Robertson, K. D. and Wolffe, A. P. (2000). DNA methylation in health and disease. Nat. Rev. Genet. 1, 11-19.[CrossRef][Medline]
Rountree, M. R., Bachman, K. E. and Baylin, S. B. (2000). DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. Nat. Genet. 25,269 -277.[CrossRef][Medline]
Sauvageot, C. M. and Stiles, C. D. (2002). Molecular mechanisms controlling cortical gliogenesis. Curr. Opin. Neurobiol. 12,244 -249.[CrossRef][Medline]
Song, M. R. and Ghosh, A. (2004). FGF2-induced chromatin remodeling regulates CNTF-mediated gene expression and astrocyte differentiation. Nat. Neurosci. 7, 229-235.[CrossRef][Medline]
Stancheva, I. and Meehan, R. R. (2000).
Transient depletion of xDnmt1 leads to premature gene activation in Xenopus
embryos. Genes Dev. 14,313
-327.
Stancheva, I., El-Maarri, O., Walter, J., Niveleau, A. and Meehan, R. R. (2002). DNA methylation at promoter regions regulates the timing of gene activation in Xenopus laevis embryos. Dev. Biol. 243,155 -165.[CrossRef][Medline]
Stancheva, I., Collins, A. L., Van den Veyver, I. B., Zoghbi, H. and Meehan, R. R. (2003). A mutant form of MeCP2 protein associated with human Rett syndrome cannot be displaced from methylated DNA by notch in Xenopus embryos. Mol. Cell 12,425 -435.[CrossRef][Medline]
Sun, Y., Nadal-Vicens, M., Misono, S., Lin, M. Z., Zubiaga, A., Hua, X., Fan, G. and Greenberg, M. E. (2001). Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms. Cell 104,365 -376.[CrossRef][Medline]
Sun, Y. E., Martinowich, K. and Ge, W. (2003). Making and repairing the mammalian brain signaling toward neurogenesis and gliogenesis. Semin. Cell Dev. Biol. 14,161 -168.[CrossRef][Medline]
Takizawa, T., Nakashima, K., Namihira, M., Ochiai, W., Uemura, A., Yanagisawa, M., Fujita, N., Nakao, M. and Taga, T. (2001). DNA methylation is a critical cell-intrinsic determinant of astrocyte differentiation in the fetal brain. Dev. Cell 1,749 -758.[CrossRef][Medline]
ten Hoeve, J., de Jesus Ibarra-Sanchez, M., Fu, Y., Zhu, W.,
Tremblay, M., David, M. and Shuai, K. (2002).
Identification of a nuclear Stat1 protein tyrosine phosphatase.
Mol. Cell Biol. 22,5662
-5668.
Teter, B., Rozovsky, I., Krohn, K., Anderson, C., Osterburg, H. and Finch, C. (1996). Methylation of the glial fibrillary acidic protein gene shows novel biphasic changes during brain development. Glia 17,195 -205.[CrossRef][Medline]
Viti, J., Feathers, A., Phillips, J. and Lillien, L.
(2003a). Epidermal growth factor receptors control competence to
interpret leukemia inhibitory factor as an astrocyte inducer in developing
cortex. J. Neurosci. 23,3385
-3393.
Viti, J., Gulacsi, A. and Lillien, L. (2003b).
Wnt regulation of progenitor maturation in the cortex depends on Shh or
fibroblast growth factor 2. J. Neurosci.
23,5919
-5927.