Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA
* Author for correspondence (e-mail: kkroll{at}molecool.wustl.edu)
Accepted 28 October 2004
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SUMMARY |
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Key words: Brg1, Chromatin remodeling, Differentiation, NeuroD, Neurogenesis, Neurogenin, P19, SWI/SNF, Xenopus
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Introduction |
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Non-amniotic vertebrates such as Xenopus laevis generate a simple
pattern of primary neurons regulating early larval behavior, which represents
an attractive experimental system for analyzing molecular aspects of
neurogenesis. Primary neurons are observed in three longitudinal domains on
either side of the dorsal midline. Cells differentiating in these domains
correspond to the three classes of primary neurons - motoneurons, interneurons
and sensory neurons - in a medialto-lateral order. Neurogenesis within these
domains is regulated by bHLH proneural genes. The earliest proneural gene
expression is that of Neurogenin-related-1 (Ngnr-1), which
induces later-acting bHLH factors including NeuroD. Ngn also activates the
Notch ligand Delta-1 and the Zn-finger transcription factor MyT1 in neuronal
precursor cells. Activation of Delta-1 expression stimulates lateral
inhibition, a negative feedback loop mediated by the Notch pathway, in
neighboring cells, whereas MyT1 renders neuronal precursors resistant to Notch
signaling (Bellefroid et al.,
1996). Therefore, sequential activities of Ngn, MyT1 and NeuroD
promote neuronal differentiation of some competent neural precursors, while
activated Notch signaling maintains neighboring cells in an undifferentiated
state (Bellefroid et al., 1996
;
Chitnis et al., 1995
;
Lee et al., 1995
;
Ma et al., 1996
;
Wettstein et al., 1997
).
Factors modulating chromatin structure, such as chromatin-remodeling
complexes, histone acetyltransferases (HATs) and deacetylases (HDACs) play
crucial roles in transcriptional regulation and participate in diverse
processes, including cell proliferation, differentiation, embryonic patterning
and tumorigenesis. The SWI/SNF complex was the first chromatin-remodeling
complex identified and its biochemical properties have been actively
characterized (Kadonaga, 1998;
Martens and Winston, 2003
).
The SWI/SNF complex consists of 7-13 subunits with a total molecular mass of
2 MDa and uses energy provided by ATP hydrolysis to locally disrupt
histone-DNA associations and relocate nucleosomes to alternate positions
(Kingston and Narlikar, 1999
;
Whitehouse et al., 1999
).
Various sequence-specific transcription factors, HATs and HDACs are known to
interact with the SWI/SNF complex to activate or repress target genes
(Kadam and Emerson, 2003
;
Peterson and Logie, 2000
).
SWI/SNF complexes in mammalian cells have either one of the two catalytic
subunits, Brahma (Brm) or Brahma-related gene 1 (Brg1), but not both
(Martens and Winston, 2003
).
Brm- and Brg1-containing complexes share most other subunits and have similar
in vitro biochemical activities but appear to have some target gene
specificity in vivo (Kadam and Emerson,
2003
).
Prior evidence has suggested that Brg1 (and by inference the SWI/SNF
complex) may be involved in neural development. First, although Brg1
is ubiquitously expressed in early stage mouse embryos, its expression becomes
enriched in neural tissue during embryogenesis
(Randazzo et al., 1994). For
example, at stage E15, Brg1 is abundantly expressed within the brain,
spinal cord and retina. Within the spinal cord, Brg1 is more abundant
in the mantle zone, where differentiating neurons exist, compared with the
ventricular zone. Second, Brg1-null mice die at peri-implantation
stages, but 15-30% of heterozygotes display exencephaly, a neural tube
abnormality (Bultman et al.,
2000
). In addition, heterozygotes of Brg1 or other
essential SWI/SNF complex components are predisposed to tumors of neural
origin (Bultman et al., 2000
;
Kim et al., 2001
;
Klochendler-Yeivin et al.,
2000
). Finally, Brg1 mutant zebrafish have defects in
terminal differentiation of retinal cells
(Gregg et al., 2003
;
Link et al., 2000
). These
observations suggest Brg1 is involved in neural development, but the precise
role of Brg1 in neural development had not previously been defined.
Here, we describe cloning and characterization of a Xenopus Brg1 homolog, and determination of Brg1 requirements for vertebrate neuronal differentiation. Brg1 is ubiquitously expressed in Xenopus embryos until the neurula stage but its expression is gradually restricted to neural tissues at later stages. Roles of Brg1 in Xenopus neural development were studied by either reducing its expression by antisense morpholino oligonucleotide (MO) injection or by introducing a well-characterized dominant-negative form. Upon reduction of Brg1 activity, Sox2-positive neuroectoderm was properly induced, but neuronal differentiation was blocked. Neural precursors appeared to remain as proliferating progenitors. Ectopic neurogenesis driven by Ngn and NeuroD was also blocked by loss of Brg1 function. We further demonstrated that Brg1 physically interacts with the proneural bHLH proteins, Ngn and NeuroD, and mediates their transcriptional activities for neurogenesis. Our results define Brg1 as an essential regulator of neuronal differentiation during vertebrate nervous system formation.
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Materials and methods |
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Embryos and RNA/morpholino oligonucleotide injections
Embryos were obtained by in vitro fertilization and raised as described
previously (Kroll et al.,
1998). When indicated, hydroxyurea was added to media at a final
concentration of 30 mM at stage 12.5 onwards until embryos were fixed. Embryos
were staged after Nieuwkoop and Faber
(Nieuwkoop and Faber, 1967
).
For generating capped mRNAs, vectors encoding xNgnr1
(Ma et al., 1996
), xNeuroD
(Lee et al., 1995
), xMyT1
(Bellefroid et al., 1996
),
xBrg1, DN-xBrg1 and ß-galactosidase were transcribed in vitro (mMessage
mMachine kit, Ambion). RNAs for xNgnr1 (30 pg), xNeuroD (50 pg), xMyT1 (50
pg), DN-xBrg1 (1 ng) or antisense morpholino oligonucleotides (20 ng of
xBrg1MO; 5'-tcactgctaacctgtccccgaatcc-3') (Gene Tools LLC) were
co-injected with 30 pg of ß-galactosidase mRNA in a volume of 10 nl into
one blastomere of stage 2 embryos. For rescue experiments, wild-type
xBrg1 mRNA (900 ng) was co-injected with xBrg1MO. In parallel with
xBrg1MO injections, embryos were injected with 20 ng of standard control MO
5'-cctcttacctcagttacaatttata-3' from Gene Tools LLC; injection of
up to 40 ng of this MO did not cause any apparent embryonic defects.
Xenopus whole-mount in situ hybridization, TUNEL assay, and phospho-histone H3 immunostaining
Embryos were raised until indicated stages, fixed in MEMFA for 1 hour,
X-gal (5-bromo-4-chloro-3-indolyl ß-galactopyranoside) stained and
analyzed by whole-mount in situ hybridization
(Harland, 1991). Probes for
Sox2 (Mizuseki et al.,
1998
) and N-tubulin
(Oschwald et al., 1991
) were
generated by in vitro transcription with digoxigenin-11-UTP (Roche) and
detected using alkaline phosphatase (AP)-conjugated anti-digoxigenin
antibodies (Roche) with Nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP; Roche).
Whole-mount TUNEL assay was performed after
(Hensey and Gautier, 1998
).
Labeled cells were visualized as described for whole-mount in situ
hybridization. Phosphorylated histone H3 (PH3) immunostaining was carried out
essentially as previously described (Saka
and Smith, 2001
) using a 1:1000 dilution of primary antibody
(Upstate). Secondary antibody was AP-conjugated goat anti-rabbit IgG (Jackson
ImmunoResearch) diluted to 1:1000. Labeled cells were visualized as described
for TUNEL. After staining, some specimens were embedded in 4% low-melting
agarose and vibratome sectioned (50 µm). TUNEL or PH3-positive cells were
counted within regions of equal surface area on injected and uninjected
bilateral halves of each embryo; fold changes were then determined for each
embryo as the ratio of injected/uninjected values. Fold change values shown
represent averaged results from at least five embryos.
P19 cell culture and immunohistochemistry
Maintenance of P19 cells and transfection of bHLH plasmids was performed
according to Farah et al. (Farah et al.,
2000). For immunohistochemistry, cells were grown on
poly-L-lysine-coated coverslips and transfected with mouse NeuroD2
(800 ng), mouse E12 (300 ng), eGFP (700 ng) and
DN-hBrg1 (1200 ng) using FuGENE6 (Roche). When applicable, pCS2+MT
was included in transfection to maintain constant levels of transfected DNA.
Three days post-transfection cells were fixed for 6 minutes in 4%
formaldehyde/PBS, permeabilized for 6 minutes in 0.2% TritonX-100/PBS, washed
and then blocked in 5% BSA/PBS. TuJ1 (Covance) and anti-GFP antibody (BD
Biosciences) were used at a dilution of 1:600 and 1:1500, respectively.
Secondary antibodies used were Alexa Fluor 488 anti-rabbit IgG and Alexa Fluor
568 anti-mouse IgG (Molecular Probes) diluted to 1:1500 and 1:500,
respectively. Digital images were captured using a Zeiss Axioskop and
Axiovision software and overlaid in Adobe Photoshop.
Luciferase assays
The E1X3-TATA reporter contains three copies of the E1 E-box from the
neuroD1/ß2 promoter in a luciferase reporter vector
(Huang et al., 2000).
Transfections were performed in six-well plates using FuGENE6 (Roche) as
described previously. Plasmid amounts were: 400 ng pCS2+Ngn3 or pCS2+NeuroD2,
1200 ng pCS2+DN-hBrg1, and 1 µg E1X3-TATA. pSV40 ß-gal (Promega) (300
ng) was cotransfected to normalize transfection efficiency. When applicable,
pCS2+MT was included to bring total DNA content up to 3 µg. Cells were
harvested 60 hours post-transfection and analyzed using the Luciferase assay
system (Promega) and the ß-galactosidase enzyme assay system (Promega).
Samples were assayed in triplicate and experiments were repeated multiple
times with similar results. Representative experiments are shown in
Fig. 8.
|
HeLa cells were grown in DMEM supplemented with 10% FBS, transfected using PolyFect transfection reagent (Qiagen) and harvested 30-48 hours after transfection. Cells were disrupted in lysis buffer [20 mM HEPES (pH 7.2), 150 mM NaCl, 0.3% Triton X-100, 3 mM EDTA] supplemented with Protease inhibitor cocktail (Roche) on ice. After clarification by centrifugation, lysates were applied to co-IP as described above.
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Results |
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To determine the expression profile of xBrg1 during embryonic
development, we performed whole-mount in situ hybridization
(Fig. 1C-I) using a probe
corresponding to a region of Domain I (Fig.
1A), which is relatively less conserved among various Brg1 and Brm
homologs. Xenopus Brg1 was expressed maternally and its mRNA was
detected throughout the animal hemisphere
(Fig. 1C). During gastrulation,
xBrg1 transcripts were still detected throughout the entire embryo
except the yolk plug (Fig. 1D).
However, as RNA in situ signals can be quenched in the yolk-rich vegetal
hemisphere, lack of signals in the vegetal hemisphere and yolk plug does not
exclude a ubiquitous distribution of maternal transcripts. No dorsoventral
bias was observed until late gastrulation (stage 13; data not shown). At stage
14, for example, xBrg1 was expressed broadly in dorsal tissues by
comparison with more restricted expression of N-tubulin
(Fig. 1I,J). From the late
gastrula stage, however, xBrg1 expression began to be enriched in the
neural plate and this bias became obvious by stage 16
(Fig. 1E). At stage 20-22,
xBrg1 expression was maintained at high levels throughout the neural
tube (Fig. 1F). Cranial and
trunk neural crest cells also expressed xBrg1, whereas expression in
epidermal cells decreased dramatically by these stages
(Fig. 1F). At tailbud and
tadpole stages, xBrg1 mRNA was detected throughout the CNS, including
the eye, brain and spinal cord, and additionally in the branchial arches and
otic vesicle (Fig. 1G,H). A
similar expression pattern was also observed at stage 33/34, using alternate,
non-overlapping probes corresponding to Domain II or 3' coding sequences
(Fig. 1A; data not shown). The
xBrg1 expression pattern is in accordance with expression of mouse
and Drosophila homologs, showing ubiquitous expression at early
stages but later neural-enriched expression
(Elfring et al., 1998;
Randazzo et al., 1994
). This
expression pattern suggests that Brg1 may play a general role in early
development but could have a more specific function in neural development at
post-gastrula stages.
Loss-of-function approaches to characterize Brg1 function during neural development
To study requirements for Brg1 in Xenopus embryogenesis, we used
two independent and complementary approaches. First, we used a
well-characterized dominant-negative form of Brg1 (DN-xBrg1). The ATPase
domain of Brg1 is highly conserved in all Brg1 homologs, and point mutations
in the ATP-binding pocket are known to disrupt ATPase activity
(de la Serna et al., 2001a;
Khavari et al., 1993
). This
mutant protein can still associate with the other proteins of the SWI/SNF
complex, but the mutant Brg1-containing complex is enzymatically inactive.
Thus, mutant Brg1 behaves in a dominant-negative manner. Here, we used this
mutant to perturb function of wild-type xBrg1. At tadpole stages, injection of
DN-xBrg1 consistently caused a series of related morphological defects ranging
from apparent truncation of anterior structures to reduction of the eye
(Fig. 2A). As a second,
independent method for interfering with Brg1 function, we designed morpholino
oligonucleotides (MOs) complementary to the 5' untranslated region of
xBrg1 mRNA (xBrg1MO). After titration experiments, using doses from 1
ng to 33 ng, we found dose-dependent morphological defects in a range between
15 ng and 25 ng, and used 20 ng for experiments. Morphological defects
observed in xBrg1MO-injected embryos (Fig.
2B) were similar to those observed in DN-xBrg1-injected
embryos (Fig. 2A). These
defects were not obtained following control injections of standard MO
(Fig. 2C) or lineage tracer
mRNA (data not shown) performed in parallel. The similarity of the defects
produced by either xBrg1MO or DN-xBrg1 injection suggests that these
effects are specific to reduction of xBrg1 activity.
|
To determine specific Brg1 requirements during neural development, embryos were injected with either DN-xBrg1 or xBrg1MO and neural marker expression was examined. At stage 13, both DN-xBrg1 and xBrg1MO-injected embryos showed normal expression of Sox2, an early neural marker expressed in proliferating neural progenitors (Fig. 3A,D). These results imply that early aspects of neural induction and neural cell fate determination occurred normally. However, at stage 15-16, the Sox2-positive domain was expanded on the DN-xBrg1 (67%, n=43) or xBrg1MO (80%, n=51) injected side of the embryo (Fig. 3B,E). By contrast, expression of type II neuron-specific tubulin (N-tubulin), which marks terminally differentiated neurons, was severely reduced or abolished in DN-xBrg1-injected embryos (Fig. 3C; Table 1). N-tubulin expression was also severely reduced or abolished in xBrg1MO-injected embryos (Fig. 3F; Table 1). However, injection of xBrg1MO along with wild-type xBrg1 restored normal or near normal N-tubulin expression (Fig. 3K; Table 1). These data indicate that loss of N-tubulin expression is specific to reduction of Brg1 activity. Likewise, co-injection of wild-type xBrg1 with xBrg1MO suppressed the Sox2 expansion previously observed (no expansion in 78%, n=60) (Fig. 3J), indicating that this defect was also specific to loss of Brg1 activity. Injection of standard MO, a negative control, at doses up to 30 ng did not change either Sox2 or N-tubulin expression (Fig. 3G-I). These results suggest that initial specification of the neural territory occurs normally but that neuronal differentiation is blocked by reduction of Brg1 activity.
|
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We next examined whether loss of N-tubulin-positive cells could have resulted from apoptosis by performing TUNEL assays. Although the standard MO did not cause significant changes in apoptosis (Fig. 4I), injection of xBrg1MO resulted in a twofold increase in the number of apoptotic cells (Fig. 4J). However, the actual number of apoptotic cells that appeared following xBrg1MO injection was much fewer than the number of primary neurons normally produced. Furthermore, most apoptotic cells were observed in the anterior neural plate rather than the posterior neural plate, where the primary neurons arise. Therefore, quantitatively and qualitatively, increased apoptosis was not sufficient to explain the almost complete loss of N-tubulin positive neurons obtained in xBrg1MO-injected embryos. The lack of differentiated neurons appeared to be due to a failure of differentiation rather than selective cell death.
Brg1 is required for neurogenesis by proneural bHLH transcription factors
To further analyze requirements for and the position of Brg1 within the
proneural pathway, we examined whether loss of Brg1 function affected
proneural activities of Ngnr1, NeuroD and MyT1. RNAs encoding Ngnr1, NeuroD or
MyT1 were injected alone or together with xBrg1MO. In agreement with previous
studies (Lee et al., 1995;
Ma et al., 1996
), Ngnr-1 and
NeuroD induced strong ectopic expression of N-tubulin
(Fig. 5A,B). However, ectopic
expression of N-tubulin induced by Ngnr1 and NeuroD was greatly
reduced by co-injection of xBrg1MO (Fig.
5D,E). MyT1 injection caused an increase of
N-tubulin-positive cells within the primary neuronal stripes
(Bellefroid et al., 1996
)
(Fig. 5C), and co-injection of
Brg1MO also blocked N-tubulin induction by MyT1
(Fig. 5F). These results
suggest that the proneural activities of Ngn1, NeuroD and MyT1 are
Brg1-dependent, and that Brg1 acts in concert with and/or downstream of
NeuroD.
|
To test the requirement of Brg1 for neuronal differentiation in mammalian
cells, P19 cells were transiently transfected with plasmids expressing mouse
NeuroD2 and mouse E12 together with or without
DN-hBrg1. To identify transfected cells, a green fluorescent protein
(GFP) expression vector was cotransfected. Three days after
transfection, cells were fixed and examined for the expression of
neuron-specific class III ß-tubulin protein, detected by the antibody
TuJ1. Consistent with previous reports
(Farah et al., 2000),
NeuroD2 transfection efficiently induced formation of TuJ1-positive
neurons (75.1±4.5% of transfected cells: average data from three
experiments; Fig. 6A-C,J). By
contrast, co-transfection of DN-hBrg1 decreased the generation of
TuJ1-positive cells by NeuroD2 to 35.0±12.1%
(Fig. 6D-F,J). Overexpression
of GFP or E12 alone did not induce TuJ1-positive cells
(Fig. 6G-I). These results
indicate that normal Brg1 function is essential for neuronal differentiation
driven byNeuroD2 and that the role of Brg1 in neurogenesis is likely to be
evolutionarily conserved in vertebrates.
|
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Brg1 mediates transactivation of proneural bHLH proteins
As Ngnr1 and NeuroD were found to interact with Brg1, we next investigated
whether Brg1 is required for transcriptional activation by proneural bHLH
proteins. Previously, it has been shown that a multimerized E-box derived from
the mouse NeuroD1 promoter stimulated reporter expression in response
to Ngn3 and NeuroD2 in transfected P19 cells
(Farah et al., 2000). We
tested whether this transcriptional activation was sensitive to interference
with Brg1 activity. P19 cells were transfected with plasmids for Ngn3,
NeuroD2 and E-box reporter (E1X3-TATA-luc) together with or without
DN-hBrg1 and promoter activities were measured by luciferase assay.
Transient expression of proneural bHLH proteins, Ngn3 or NeuroD2, robustly
increased the transcription of the reporter gene
(Fig. 8). However, the presence
of DN-hBrg1 decreased the transcriptional activity of Ngn3 by 55.6±7.9%
on average and that of mNeuroD2 by 59.9±2.4%
(Fig. 8). Taken together with
our data regarding the association of Brg1 with proneural bHLHs, these results
suggest that Brg1 might act by mediating the transactivation of proneural bHLH
proteins.
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Discussion |
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Brg1 requirements for cell cycle withdrawal during neurogenesis
The process of cell differentiation is tightly linked to cell cycle
withdrawal. Expansion of the Sox2-positive territory at stage 15 but
not at stage 13 combined with loss of N-tubulin expression is
interesting because it suggests that the neural progenitor cells which failed
to differentiate in the absence of Brg1 remain as progenitor cells. However,
as Sox2 expansion was observed in not only posterior but also
anterior neural plate where no N-tubulin-positive cells arise at this
stage, expansion of neural progenitors cannot be explained solely by a failure
of differentiation. Further study is required to understand this
phenomenon.
Proneural genes not only determine neuronal fate but also promote cell
cycle withdrawal, at least partly by inducing cyclin-dependent kinase (Cdk)
inhibitors, and are therefore involved in coupling these two processes
(Ohnuma et al., 2001). For
example, Ngn2 overexpression in the chick neural tube resulted both
in premature neuronal differentiation of neuroepithelial cells and premature
cell cycle exit (Mizuguchi et al.,
2001
; Novitch et al.,
2001
). Similarly, NeuroD associates with the promoter of
p21Cip1, a Cdk inhibitor, and can induce its expression
upon RA treatment in neuroblastoma cells
(Liu et al., 2004
). Brg1 also
appeared to associate with promoter regions of the Cip/Kip family of Cdk
inhibitors and was required for their induction
(Hendricks et al., 2004
). The
implication of both Brg1 and proneural bHLHs in cell cycle withdrawal and the
physical and functional interaction of Brg1 with proneural bHLHs (our study)
suggest Brg1 and bHLHs may cooperate on the same target genes to coordinate
cell cycle withdrawal during neurogenesis. We have examined whether expression
of p27Xic1, which is the only currently available Cdk
inhibitor in Xenopus, is reduced in the absence of Brg1 activity
(Vernon et al., 2003
).
However, we did not observe a significant change in
p27Xic1 transcript levels in the neural plate (data not
shown). It is unclear whether other Cdk inhibitors are involved or whether
p27Xic1 activity is post-transcriptionally regulated in
Xenopus.
Brg1 mediates the transcriptional activities of Neurogenin and NeuroD
How does Brg1 mediate the activity of bHLH transcription factors during
neurogenesis? The physical interaction we observed between proneural bHLHs and
Brg1 raises the possibility that Brg1 (and the SWI/SNF complex) is recruited
to Ngn and NeuroD target loci and remodels the chromatin structure to activate
transcription of these target genes. Alternatively, chromatin remodeling might
be a prerequisite for binding of proneural bHLHs to their target loci. In this
case, the SWI/SNF complex binds target loci before bHLH factors and exposes
bHLH target sites, permitting recruitment of bHLH proteins and transcriptional
activation of target genes. Currently, only a few genes are known to be direct
targets of Ngn and NeuroD, and chromatin remodeling at these loci during
neurogenesis has not yet been analyzed. It will therefore be of interest to
investigate the recruitment of the SWI/SNF complex to Ngn target genes such as
NeuroD1 (Huang et al.,
2000) and to NeuroD target genes such as
p21Cip1 (Liu et al.,
2004
) and the recruitment order of SWI/SNF complex and proneural
bHLHs to target promoters. In addition to interacting with Brg1, both Ngn and
NeuroD are also known to interact with p300/CBP HATs and HAT activity has been
shown to be necessary for neurogenesis
(Koyano-Nakagawa et al., 1999
;
Mutoh et al., 1998
). Thus,
neurogenesis might be a process that requires extensive chromatin
remodeling.
Parallels between neurogenesis and myogenesis
In many respects, molecular regulation of neurogenesis parallels that of
myogenesis, with a cascade of myogenic bHLH transcription factors regulating
myoblast cell fate determination, cell cycle withdrawal and upregulation of
muscle-specific gene expression (McKinsey
et al., 2001; Pownall et al.,
2002
). During myogenesis, myogenic regulatory factors (MRFs), such
as MyoD, induce muscle-specific gene expression, and a subset of MRF target
genes shows Brg1 dependent-expression. Brg1 mediates MyoD transactivation of
some targets through recruitment of SWI/SNF chromatin remodeling activity, and
Brg1 has also recently been shown to interact with MyoD and Mef2C during
myogenesis (de la Serna et al.,
2001a
; Roy et al.,
2002
; Simone et al.,
2004
). Thus, our finding of an interaction between Brg1 and
proneural bHLH proteins suggests that myogenic and proneural bHLH proteins may
use similar mechanisms to activate their targets and induce differentiation.
Intriguingly, recruitment of Brg1 and chromatin remodeling activity to MyoD
targets has also recently been shown to be under additional regulatory
controls. For example, activated p38 kinase recruits Brg1 to a subset of MyoD
target genes (Simone et al.,
2004
). Transcriptional activation of an overlapping set of MyoD
targets depends on cooperative interactions between the Pbx homeodomain
protein and the C/H and helix III domains of MyoD that have been previously
shown to recruit chromatin remodeling activity to MyoD target genes
(Berkes et al., 2004
;
Gerber et al., 1997
).
Conversely, transactivation of other MyoD targets (notably those involved in
cell cycle withdrawal) is independent of Brg1 activity
(de la Serna et al., 2001b
;
Roy et al., 2002
), and some
MyoD target gene transactivation is not regulated by p38 kinase or
Pbx-dependent mechanisms (Bergstrom et al.,
2002
; Berkes et al.,
2004
). Therefore, distinct subsets of MyoD targets have different
requirements both for Brg1-dependent chromatin remodeling and for the
additional regulatory controls that may impinge on that remodeling.
By comparison with myogenesis, we still know very little about how
proneural bHLH proteins activate transcription of their targets in a chromatin
context during neurogenesis. It remains to be seen whether, as for myogenesis,
subsets of proneural bHLH targets show Brg1-dependent versus-independent
transcriptional activation during neurogenesis. Additionally, although
proneural bHLH proteins act as general regulators of neuronal cell fate,
specification of distinct neuronal subtypes involves cooperative functioning
of proneural bHLHs and homeodomain proteins at particular loci
(Lee and Pfaff, 2003). It will
therefore be of great interest to determine whether target gene subsets
regulated by particular proneural bHLH and homeodomain protein combinations
show differential Brg1 requirements in a manner potentially analogous to the
MyoDPbx cooperativity, which was hypothesized to recruit chromatin remodeling
activity to some loci during myogenesis
(Berkes et al., 2004
).
In summary, our results show that Brg1 is required for cell cycle arrest and for neuronal differentiation and can bind to and mediate transcriptional activities of Ngn and NeuroD. Although future work will enable a more complete understanding of the role of chromatin remodeling in regulating target gene activation by proneural bHLH factors, our findings here have defined Brg1 (and the SWI/SNF complex) as essential for neurogenesis.
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ACKNOWLEDGMENTS |
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