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First published online 19 December 2007
doi: 10.1242/dev.010090
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Department of Genetics and Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
* Author for correspondence (e-mail: terry_magnuson{at}med.unc.edu)
Accepted 1 November 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and β-globins, and subsequently undergo apoptosis. Additionally,
vascular remodeling of the extraembryonic yolk sac is abnormal in
Brg1fl/fl:Tie2-Cre+ embryos. Importantly,
Brm deficiency does not exacerbate the erythropoietic or vascular
abnormalities found in Brg1fl/fl:Tie2-Cre+
embryos, implying that Brg1-containing SWI/SNF-like complexes, rather
than Brm-containing complexes, play a crucial role in primitive
erythropoiesis and in early vascular development.
Key words: SWI/SNF, Brg1, Tie2-Cre, Erythropoiesis, β-globin, Vascular remodeling, Angiogenesis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Margueron et al., 2005).
The mammalian SWI/SNF-related chromatin-remodeling complexes comprise one
major family of ATP-dependent chromatin-modifying factors. These large,
multi-protein complexes use one of two different ATPases as their catalytic
subunit: brahma (BRM, also known as SMARCA2) and brahma-related gene 1 (BRG1,
also known as SMARCA4). The significance of SWI/SNF-related complexes in
mammalian development is particularly well demonstrated by the phenotypes
associated with mice carrying mutations in Brg1. Brg1-/-
embryos die at the peri-implantation stage of development, and conditional
alleles have been used to demonstrate the role of Brg1 in T-cell
development, limb morphogenesis, skin development, gliogenesis and zygotic
genome activation (Bultman et al.,
2000; Bultman et al.,
2006; Gebuhr et al.,
2003; Indra et al.,
2005; Matsumoto et al.,
2006). By contrast, Brm-/- mice develop
normally, although adult mutants are 15% heavier than their control
littermates, possibly because of increased cellular proliferation
(Reyes et al., 1998). As BRG1
is significantly upregulated in Brm-/- mice, it has been
hypothesized that BRG1 can functionally compensate for the loss of BRM during
development (Reyes et al.,
1998).
We previously isolated and characterized an
N-ethyl-N-nitrosourea (ENU)-induced point mutation in
Brg1 (Brg1ENU1) that changes a single amino-acid
residue (E1083G) in a highly conserved region of the catalytic ATPase domain
(Bultman et al., 2005). The
mutant protein is stable, assembles into SWI/SNF-related complexes, and
exhibits normal ATPase activity, but has diminished nucleosome-remodeling
capability. Brg1null/Brg1ENU1 embryos fail to
transcribe adult β-globin genes, thereby indicating that BRG1 plays an
important role in chromatin remodeling of the β-globin locus during
definitive erythropoiesis. However, because hypomorphic
Brg1null/Brg1ENU1 embryos express embryonic
β-globin genes and undergo normal primitive erythropoiesis, it has been
unclear whether BRG1 and SWI/SNF-related complexes are involved in this
earlier hematopoietic process. Using a conditional null allele, we now report
that BRG1, but not BRM, is indeed recruited to the β-globin locus control
region in primitive erythrocytes, and is required for the transcription of
embryonic β-globin genes and for erythroblast survival. We also
demonstrate for the first time that BRG1 is required for embryonic
-globin expression, although expression of adult -globins does
not depend upon BRG1-induced remodeling in primitive erythrocytes. Finally,
BRG1 appears to play an important role in vascular development that is
separable from its role in primitive erythropoiesis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), the Brm-/- mice
(Reyes et al., 1998), the
Tie2-Cre transgenic mice (Koni et
al., 2001) and the ROSA26R mice
(Soriano, 1999) have been
described. All mice were maintained on a mixed genetic background at the
University of North Carolina, Chapel Hill Animal Facility.
Brg1-floxed mice were genotyped by Southern blot analysis using a 449
bp probe against the 3' portion of intron 14 and genomic DNA digested
with BamHI. The PCR primers used to generate the Southern probe were
as follows: forward, 5'-TGGCATCTCATTTGTGTGGT-3'; and reverse,
5'-ACAGCCACTGGTTAGGGATG-3'. The Southern blot yields a 7.7 kb
floxed allele, a 6.0 kb wild-type allele and a 5.5 kb excised allele.
Brg1-floxed embryos were genotyped by PCR using the following
primers: forward, 5'-GTCATACTTATGTCATAGCC-3'; and reverse,
5'-GCCTTGTCTCAAACTGATAAG-3'. These primers flank the 3' LoxP
site, and yield a 387 bp floxed allele and a 230 bp wild-type allele. The PCR
was performed at an annealing temperature of 51°C.
Brm-/- mice and embryos were genotyped by PCR using the
following primers for the 197 bp wild-type allele: forward,
5'-ATATCTGGAGGAGGCCCAAC-3'; and reverse,
5'-TGCAGAGTTTCAGGGAGAGG-3'. The 600 bp targeted allele was
amplified using the same forward primer and the following reverse primer:
5'-CATCGCCTTCTATCGCCTTC-3'. The PCR was performed at an annealing
temperature of 55°C. Tie2-Cre transgenic mice and embryos were
genotyped using a gene-specific forward primer
(5'-GGGAAGTCGCAAAGTTGTGAGTT-3') and a Cre-specific reverse primer
(5'GTGAAACAGCATTGCTGTCACTT-3') that amplifies a 400 bp product.
Control primers amplifying a 324 bp product from the IL2 gene were
used as a template control: forward,
5'-CTAGGCCACAGAATTGAAAGATCT-3'; and reverse,
5'-GTAGGTGGAAATTCTAGCATCATCC-3'. The PCR was performed at an
annealing temperature of 51°C. ROSA26R mice were genotyped as described
(Soriano, 1999). All
histological sections were scraped from paraffin or OCT (Tissue-Tek) into
DEXPAT Reagent (TaKaRa) before genotyping.
Histology
Embryos and yolk sacs were dissected from maternal tissue, immersion-fixed
in 4% paraformaldehyde overnight, dehydrated, embedded in paraffin, sectioned
(10 µm), and stained with hematoxylin and eosin. For cryosections, embryos
were dissected, fixed, and passed through 10% sucrose (10 minutes), 15%
sucrose (10 minutes), 20% sucrose (1 hour) and 20% sucrose/OCT (overnight),
and then embedded in OCT on dry ice before sectioning (10 µm) and mounting
on Superfrost/Plus microscope slides (Fisher). Electron microscopy was
performed as described (Schwarz et al.,
2002) on embryonic day 10.5 (E10.5) yolk sacs from two mutant and
two control littermate embryos with visible heart beats.
Staining
Whole-mount immunostaining for platelet/endothelial cell adhesion molecule
1 (PECAM1) was performed as described using a rat anti-mouse PECAM1 monoclonal
antibody (BD PharMingen) (Schlaeger et
al., 1995). Whole-mount β-galactosidase staining was
performed as described (Schlaeger et al.,
1995), and stained embryos were subsequently cryosectioned and
counterstained with Nuclear Fast Red. Transferase-mediated deoxyuridine
triphosphate (dUTP) nick-end labeling (TUNEL) staining was performed on
paraffin-embedded tissues using the In Situ Cell Death Detection Kit,
Fluorescein (Roche), according to the manufacturer's instructions. Benzidine
staining was performed on cryosectioned embryos and yolk sacs. After a brief
submersion in PBS, slides were incubated in methanol (15 seconds), 1%
benzidine (Sigma-Aldrich) in methanol (5 minutes), 2.5% hydrogen peroxide in
70% ethanol (2.5 minutes), and washed in water (2.5 minutes).
In situ hybridization
Gene-specific antisense probes to the murine 
y,
βH1, 
and 1/2 globins have been described
(Kingsley et al., 2006). A 382
bp antisense probe for murine Band3 was amplified from E8.5 yolk sac
cDNA using the following primers: forward,
5'-AGAGACCTAACCATCCCTGTGA-3'; and reverse,
5'-TCTGATCCTCGTAGATGAAGCA-3'. A 425 bp antisense probe for murine
Alas2 was amplified from E8.5 yolk sac cDNA using the following
primers: forward, 5'-CCATGCTGTAGGACTGTATGGA-3'; and reverse,
5'-CATAGATGCTGTGCTTGGAGAG-3'. In vitro transcription was performed
to generate digoxigenin-labeled RNA probes. Cryosectioned embryos were
subjected to a 10-minute proteinase K (2.5 µg/ml) pre-treatment before in
situ hybridization. Prehybridization and hybridization incubations were
carried out at 60°C in a mixture of 50% formamide, 5xSSC, 2%
blocking reagent (Roche), 0.1% Triton-X100, 0.5% CHAPS, 50 µg/ml yeast
tRNA, 5 mM EDTA and 100 µg/ml heparin. After overnight hybridization,
slides were washed and incubated with anti-digoxigenin-AP Fab fragments
(Roche) overnight at 4°C. After further washing, slides were incubated
with NBT/BCIP at room temperature for several hours, or at 4°C for up to
three days.
Chromatin immunoprecipitation (ChIP)
To obtain primitive erythrocytes, approximately 40 wild-type E9.5-E10.5
embryos were separated from their placentae by severing the umbilical vessels.
The embryos with their attached yolk sacs were immediately placed in minimal
essential media containing 2% fetal bovine serum (JRH Biosciences) and were
rocked on a Nutator mixer for approximately 30 minutes at room temperature
while allowing the hearts to pump the majority of circulating blood out of the
embryos and yolk sacs. Embryos and yolk sacs were removed from the media, and
the remaining blood cells were counted on a hemocytometer. Typically
20x106 to 50x106 blood cells were collected.
The fresh cells were used in ChIP assays as previously described
(Bultman et al., 2005), with
some modifications: 7x106 cells were used for each ChIP or
mock reaction; 0.6% formaldehyde was used to cross-link the protein-protein
and protein-DNA interactions; and chromatin was sonicated with eleven
10-second pulses at 10% maximum power on a Branson Digital Sonifier. The
anti-BRG1 antibody J1 (a gift from G. Crabtree and W. Wang, Stanford
University) and the anti-acetyl-histone 3 antibody (Santa Cruz, 06-599) were
used for immunoprecipitations. The β-globin locus control region DNAseI
hypersensitive site 3 (HS3) primers that were used for amplification of the
ChIP products and controls were as follows: forward,
5'-AGGCCTCCTAGGGACTGAGA-3'; and reverse,
5'-AGACTCCACCCTGAGCTGAA-3'. The 158 bp product was amplified at an
annealing temperature of 55°C for 35 cycles. The amplicon spans
271bp of the promoter starting 535 bp upstream of the start site, and the PCR
primers were as follows: forward, 5'-TATGGAGGGCTAGCTGGAGA-3'; and
reverse, 5'-GGCCTTAGTCCCACACAGAA-3'. The product was amplified at
an annealing temperature of 55°C for 34 cycles. The 1/2
primers used for ChIP amplification were as follows: forward,
5'-GGGAGGAGACAGTGGACAAA-3'; and reverse,
5'-AGTGATGGCAGTTTGGGAAG-3'. The amplicon spans 257 bp of the
promoter starting 515 bp upstream of the 1 start site, and was
amplified at an annealing temperature of 55°C for 30 cycles. Cycle numbers
were determined for each amplicon based on the maximum amount of amplification
that could be achieved before background bands appeared in the mock (no
antibody) control lane.
Image acquisition
Brightfield histological images were obtained with a Leica DM IRB
microscope (Leica Microsystems) using 10x (NA 0.25) and 20x (NA
0.4) objectives with a 1.5x magnification changer and a SPOT RT-Slider
digital camera (Diagnostic Instruments). Images were acquired with SPOT RT
Software version 4.5 (Diagnostic Instruments) and were globally optimized for
brightness/contrast, if necessary, using Photoshop software (Adobe). Whole
yolk sacs were imaged on a Leica MZ FLIII microscope (Leica Microsystems)
using the camera and acquisition software described above. Fluorescent TUNEL
images were obtained with a Leica DM LB Microscope (Leica Microsystems) using
a 40x (NA 0.7) objective and a Retiga-2000R digital camera (QImaging).
Black and white images were acquired with QCapture Software version 3.0
(QImaging), and were pseudocolored and merged with SPOT RT Software.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We
recovered no live Brg1fl/fl:Tie2-Cre+/0
offspring from matings between Brg1fl/fl and
Brg1fl/+:Tie2-Cre+/0 mice
(Table 1), indicating that
expression of Brg1 on Tie2+ cells is critical for embryonic
development. These data also indicate that BRM does not compensate for loss of
BRG1 on Tie2+ cells, despite the observation that BRG1 and BRM can
play partially redundant roles in vitro
(Chiba et al., 1994;
Phelan et al., 1999;
Strobeck et al., 2002).
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; Orkin and Zon,
1997). We determined that
Brg1fl/fl:Tie2-Cre+/0 embryos die at
E10.5-E11.0, based on the absence of a visible heartbeat and the onset of
necrosis. By E9.5, mutant embryos are visibly paler than their control
littermates, and mutant yolk sacs lack the large, blood-filled vitelline
vessels that are readily visible in control yolk sacs. Importantly, no
exacerbation of the timing or severity of this gross mutant phenotype is
detected on a Brm-/- background (11
Brm-/-;Brg1fl/fl:Tie2-Cre+/0 embryos
were assessed at E9.5-E10.5), further demonstrating the importance of
Brg1 over Brm in hematopoietic and/or vascular
development. The extreme pallor of mutant embryos at E9.5 cannot be explained by hemorrhage. Upon gross dissection, no embryonic blood is visibly pooled in the extravascular exocoelomic, amniotic or pericardial cavities of mutant embryos. Likewise, histological analysis of control and mutant embryos from 19 litters (E8.5-E10.5), embedded intact within the maternal uterus so as not to disrupt any sites of pooled embryonic blood, failed to reveal any sign of hemorrhage.
The extraembryonic yolk sac is the initial site of hematopoiesis and
produces large, nucleated blood cells that begin to circulate between the yolk
sac and embryo at E8.25 (McGrath et al.,
2003). Such blood cells are abundant in histological sections of
E8.5 Brg1fl/fl:Tie2-Cre+/0 yolk sacs,
demonstrating that hematopoiesis is unimpaired
(Fig. 1B). However, by light
and electron microscopy, a dramatic scarcity of blood cells is found in mutant
embryos and yolk sacs at E9.5 and E10.5
(Fig. 1D). Furthermore, many of
those blood cells that remain are fragmented and show abnormal membrane or
nuclear morphology (Fig. 1F).
In order to determine whether embryonic blood cells undergo apoptosis between
E8.5 and E9.5, we performed TUNEL staining on sections of E9.5 mutant and
control littermate embryos and yolk sacs. Blood cells from mutant embryos show
more evidence of TUNEL staining than do those from control embryos
(13.6±0.75% versus 0.4±0.2%, mean±s.e.m.), indicating
that blood from Brg1fl/fl:Tie2-Cre+/0 embryos
is subject to aberrant apoptosis (Fig.
2).
To determine whether the blood cell death detected in mutant embryos is
cell autonomous, we crossed
Brg1fl/fl:Tie2-Cre+/0 embryos onto the ROSA26R
mouse line (Soriano, 1999).
ROSA26R mice express β-galactosidase upon Cre-mediated deletion of a
`STOP' signal; therefore, cells that express Tie2-Cre turn blue upon
X-gal staining in the presence of this reporter line. At E8.5, both control
and mutant embryos display blue endothelium and comparable amounts of blue
blood cells in their yolk sac vasculature
(Fig. 3A,B,E). These data
indicate that the majority of embryonic blood cells express Tie2-Cre
at E8.5 and that Brg1 is not required for production of these cells.
At E9.5, both control and mutant embryos continue to display blue endothelium,
but whereas control embryos reveal a preponderance of circulating blue blood
cells (86±3.9%), mutants (which contain fewer blood cells overall)
display a minority of blue blood cells (33.5±11.5%;
Fig. 3C-E). Additionally, many
of the blue blood cells in mutants are fragmented or display abnormal
morphology (Fig. 3D, inset),
whereas the non-blue blood cells retain normal morphology. This indicates that
blood cells undergoing Brg1 excision are selectively destroyed in a
cell-autonomous fashion while wild-type blood cells are spared from
destruction. Therefore, we conclude that
Brg1fl/fl:Tie2-Cre+/0 blood cells are initially
formed but subsequently undergo apoptosis between E8.5 and E10.5.
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; Wong et al.,
1986). Once formed, these `primitive' erythroblasts continue to
divide for several days before entering the blood stream, undergoing
maturation, and eventually enucleating
(Kingsley et al., 2004).
`Definitive' erythroblasts, which originate in the yolk sac, placenta and
aorta/gonad/mesonephros region, colonize the fetal liver where they expand and
differentiate before entering the blood stream as mature, enucleated
erythrocytes at E11.5-E12.5 (reviewed by
McGrath and Palis, 2005;
Mikkola et al., 2005).
Definitive erythrocytes are the predominant cell type in the embryonic
circulation after E12.5. As they mature, both primitive and definitive erythroblasts accumulate hemoglobin, the oxygen-carrying component of red blood cells. In order to assess the production of hemoglobin in Brg1fl/fl:Tie2-Cre+/0 embryonic blood cells, we stained histological sections of control and mutant E9.5 embryos with benzidine (see Fig. S1 in the supplementary material). Although blood cells in control embryos exhibit strong staining, many blood cells in mutant embryos show little or no benzidine staining. These data indicate that hemoglobin production and/or accumulation is defective in Brg1fl/fl:Tie2-Cre+/0 primitive erythrocytes.
Globin tetramers encoded by the - and β-globin gene loci are
critical components of hemoglobin. The globin loci are multi-gene clusters: in
mice there are three functional -globins (, 1 and
2) and four functional β-globins (y,
βH1, βmaj and
βmin). Whereas primitive erythroblasts express all of
the globin genes, definitive erythrocytes express only the adult globins
(1, 2, βmaj and
βmin) (Trimborn
et al., 1999). Therefore a transition occurs during development
(E10.5-E13.5) in which embryonic globin expression diminishes and adult globin
expression escalates.
Brg1 is recruited to the β-globin locus control region (LCR),
a long-distance upstream regulatory element, by erythroid-specific
transcription factors, where it mediates chromatin remodeling to allow
transcription of β-globin genes in vitro
(Kadam et al., 2000). We
hypothesized that the hemoglobin deficit observed in
Brg1fl/fl:Tie2-Cre+/0 embryos could result from
inadequate globin transcription. Because of the mixed population of wild-type
and mutant blood cells in Brg1fl/fl:Tie2-Cre+/0
embryos at E9.5 (see Fig. 3D),
we could not analyze the expression of -globin and β-globin genes
by RT-PCR. Instead, we performed in situ hybridization on sectioned embryos to
visualize globin expression on a cell-by-cell basis. Control embryos express
all of the predominant globins expected to be detected at E9.5 in the vast
majority of their blood cells (Fig.
4A,C,E,G,I). Likewise, the adult -globins
(1 and 2) are expressed in almost every blood
cell in mutant embryos at E9.5 (91.5±2.5%;
Fig. 4D,I). However, the
embryonic globins , y and βH1 are only
expressed in a subset of mutant blood cells at E9.5 (63.75±8.6%,
57.25±7.8% and 42.9±6%, respectively;
Fig. 4B,F,H,I).
Our laboratory previously showed that BRG1 is recruited to the
β-globin LCR in definitive erythrocytes in vivo, where it plays a crucial
role in mediating adult β-globin transcription
(Bultman et al., 2005). To
demonstrate that BRG1 is also recruited to the β-globin LCR in primitive
erythrocytes, we performed chromatin immunoprecipitation (ChIP) assays on
circulating blood cells collected from E9.5-E10.5 embryos. These cells are
predominantly primitive erythrocytes, as definitive erythrocytes first enter
the bloodstream between E11.5-E12.5
(Brotherton et al., 1979;
Kingsley et al., 2004). Using
an anti-BRG1 antibody, we were able to demonstrate BRG1 binding to the DNAse I
hypersensitive site 3 (HS3) of the β-globin LCR, where it is recruited in
definitive erythrocytes as well (Fig.
4J). These ChIP data indicate that the defects we detect in
embryonic β-globin expression by in situ hybridization directly result
from loss of BRG1-induced chromatin remodeling at the β-globin LCR in
mutant primitive erythrocytes. Furthermore, we were able to detect BRG1
binding at the promoter of the embryonic -globin , whereas BRG1
does not bind the promoter of the adult -globins 1/2
(Fig. 4J). These ChIP results
for the -globin genes are consistent with our in situ hybridization
results, which indicate BRG1 involvement in but not 1/2
expression at E9.5. Therefore, we provide the first evidence that BRG1 is
important for embryonic -globin transcription in primitive
erythrocytes. Overall, the reduction in blood cells expressing embryonic
globins, and the reduction in blood cells expressing Tie2-Cre by
reporter analysis in E9.5 mutant embryos (see
Fig. 3D,E), lead us to
hypothesize that primitive erythrocytes having undergone excision of
Brg1 are unable to support normal levels of embryonic globin
transcription, resulting in their destruction through apoptosis.
|
). By
E8.5, the time at which the primitive vascular plexus is formed,
Tie2-Cre-mediated excision occurs efficiently in yolk sac endothelium
(Fig. 3A,B). Yet
Brg1fl/fl:Tie2-Cre+/0 yolk sac vasculature is
indistinguishable from that seen in control yolk sacs at this time
(Fig. 5A,B), demonstrating that
the vascular plexus formation/maintenance stages of vasculogenesis proceed
normally in mutant yolk sacs. By E9.5, however,
Brg1fl/fl:Tie2-Cre+/0 yolk sacs demonstrate an
aborted vascular remodeling process (Fig.
5D,E). Whereas their littermate controls successfully undergo
angiogenesis, resulting in yolk sacs with interconnected large and small
vessels, mutant yolk sacs progress beyond the plexus stage of vascular
development but fail to establish a mature vascular tree. Instead, the mutant
yolk sac vasculature consists of small and medium-sized vessels that
occasionally fail to interconnect. Many dead-end or tapering vascular termini
are visible in the mutant yolk sacs, indicating that vascular sprouting or
pruning fails to progress normally (Fig.
5E). Dysregulated cardiac function and blood flow are unlikely
causes of these vascular abnormalities because heart beat rates are normal and
pericardial edema is rarely detected in
Brg1fl/fl:Tie2-Cre+/0 embryos at E9.5 when the
yolk sac vascular abnormalities are readily apparent. Interestingly, PECAM1
immunostaining of the vasculature within the mutant embryo proper appears
normal and comparable to that seen in control littermates or stage-matched
embryos at E9.5 (data not shown). Therefore, the vascular abnormalities we
observe on the Brg1fl/fl:Tie2-Cre+/0 background
are specific to the yolk sac. Finally, although BRM expression in the yolk sac
is restricted to vascular endothelium
(Dauvillier et al., 2001), we
did not see any exacerbation of the
Brg1fl/fl:Tie2-Cre+/0 vascular phenotype on a
Brm-/- background. Therefore BRM does not appear to play a
functionally redundant role with BRG1 during early vascular development. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Erythroblasts in Brg1null/ENU1 embryos contain a stable
BRG1 protein that assembles into SWI/SNF-like complexes and retains normal
ATPase activity. However, although the mutant BRG1ENU1 protein is
effectively recruited to the β-globin LCR, as demonstrated by ChIP
assays, the LCR does not establish DNAse I hypersensitive sites characteristic
of open chromatin. Because of this closed chromatin configuration at the LCR,
adult β-globin transcription is reduced in
Brg1null/ENU1 embryos, and definitive erythroblasts
undergo developmental arrest, leading to embryonic lethality by E14.5. By
contrast, Brg1fl/fl:Tie2-Cre+/0 erythroblasts
contain little or no BRG1 protein, and fail to transcribe embryonic
β-globins during primitive erythropoiesis, resulting in embryonic
lethality by E11.0. Together, these hypomorphic and conditional null mutations
demonstrate the requirement for Brg1-mediated chromatin remodeling
during both embryonic and adult β-globin transcription.
|
). However,
unlike Brg1fl/fl:Tie2-Cre+/0 mutants, which die
at E10.5-E11.0, Klf2-/- embryos persist until E12.5-E14.0,
when they succumb to heart failure (Lee et
al., 2006). Notably, Klf2 is dispensable for adult
β-globin gene expression in definitive erythrocytes
(Basu et al., 2005). Perhaps
Brg1fl/fl:Tie2-Cre+/0 mutants die at E10.5 from
anemia whereas Klf2-/- embryos do not become anemic,
because Brg1 is necessary for transcription of both embryonic and
adult β-globins whereas Klf2 is only critical for embryonic
β-globin transcription. We hypothesize that embryonic β-globins are
required for the survival of primitive erythrocytes, but that definitive
erythrocytes, which express adult β-globins, can compensate for loss of
primitive erythrocytes at the primitive/definitive transition. This hypothesis
is substantiated by the observation that mice depleted of both of the
embryonic β-globins survive development whereas embryos depleted of all
of the embryonic and adult β-globins, as a result of targeted deletion of
the β-globin locus control region, die early in embryogenesis
(Bender et al., 2000;
Hu et al., 2007).
|
-globin () transcription in
Brg1fl/fl:Tie2-Cre+/0 erythroblasts, although
adult -globin transcription occurs normally in these cells
(Fig. 4). Furthermore, we
demonstrate through ChIP assays that BRG1 binds the promoter of but not
the promoter of the adult -globins. These data indicate that BRG1
facilitates -globin expression at the individual gene promoters rather
than at the major regulatory element (MRE), which is a long-distance upstream
regulatory element comparable to the β-globin LCR. In this regard, the
regulation of -globin and β-globin expression by SWI/SNF-mediated
chromatin remodeling appear to be distinct in primitive erythrocytes.
β-Thalassemia occurs when a reduced synthesis of β-globin chains
leads to an excessive accumulation of insoluble -globin chains in red
blood cells. The resulting blood cells have morphological abnormalities
(reminiscent of those seen in Fig.
1F and Fig. 3D),
and are vulnerable to mechanical injury and death. Although embryonic
-globin expression is compromised in
Brg1fl/fl:Tie2-Cre+/0 erythroblasts, the adult
-globins are expressed normally and should be able to serve the
survival needs of an embryo when β-globin chains are available
(Leder et al., 1997). However,
because of defective β-globin transcription in our mutant blood cells,
the adult -globins have no binding partners and presumably accumulate
detrimentally. Therefore, we provide the first demonstration that loss of
Brg1 in primitive erythroblasts leads to severe β-thalassemia
and lethality.
Because SWI/SNF might be expected to mediate transcription of multiple
genes during erythropoiesis, we assessed expression of two other markers of
erythroid development: Alas2, an important enzyme in heme
biosynthesis; and Band3 (Slc4a1), an integral membrane
protein on the surface of erythrocytes that provides cellular mechanical
stability. By in situ hybridization, we found normal expression of
Alas2 but deficient expression of Band3 in
Brg1fl/fl:Tie2-Cre+/0 erythroblasts (see Fig.
S2 in the supplementary material), indicating that BRG1-mediated chromatin
remodeling may be important for Band3 transcription. Because
Band3-/- mice survive development
(Peters et al., 1996;
Southgate et al., 1996), we do
not believe that aberrant Band3 expression contributes to the
lethality we observe in Brg1fl/fl:Tie2-Cre+/0
embryos. Nevertheless, in combination with the globin expression data
presented here, these expression analyses provide interesting evidence for the
specificity of SWI/SNF activity. Altogether, we demonstrate changes in the
expression of embryonic globins and Band3, but no changes in the
expression of adult -globins or Alas2 in our mutant
erythroblasts. These data support a growing body of evidence that SWI/SNF
complexes control a limited number of targets and are not simply
indiscriminate transcriptional regulators (reviewed by
Kwon and Wagner, 2007). As
such, these and other chromatin remodeling complexes have the capacity to
regulate a wide variety of nuanced developmental processes.
Finally, we believe Brg1fl/fl:Tie2-Cre+/0
embryonic lethality is due to anemia resulting from failure of hemoglobin
synthesis and subsequent apoptosis of red blood cells, because other mutants
with defective primitive erythropoiesis, such as Gata1, Gata2 and
Rbtn2 mutants, all die from anemia at the same time in development as
Brg1fl/fl:Tie2-Cre+/0 embryos (E10-E11.5)
(Fujiwara et al., 1996;
Tsai et al., 1994;
Warren et al., 1994).
Importantly, these mutants with defects in primitive erythropoiesis do not
harbor secondary vascular defects. Therefore, we suspect that the vascular
phenotypes we observe in Brg1fl/fl:Tie2-Cre+/0
mutant yolk sacs are separable from the mutant blood phenotype. We plan to
verify this hypothesis in the future by rescuing expression of Brg1
in developing erythrocytes and determining whether the vascular abnormalities
in Brg1fl/fl:Tie2-Cre+/0 yolk sacs are still
apparent at E9.5.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/3/493/DC1
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ACKNOWLEDGMENTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Basu, P., Morris, P. E., Haar, J. L., Wani, M. A., Lingrel, J.
B., Gaensler, K. M. and Lloyd, J. A. (2005). KLF2 is
essential for primitive erythropoiesis and regulates the human and murine
embryonic beta-like globin genes in vivo. Blood
106,2566
-2571.
Bender, M. A., Bulger, M., Close, J. and Groudine, M. (2000). Beta-globin gene switching and DNase I sensitivity of the endogenous beta-globin locus in mice do not require the locus control region. Mol. Cell 5,387 -393.[CrossRef][Medline]
Brotherton, T. W., Chui, D. H., Gauldie, J. and Patterson,
M. (1979). Hemoglobin ontogeny during normal mouse fetal
development. Proc. Natl. Acad. Sci. USA
76,2853
-2857.
Bultman, S., Gebuhr, T., Yee, D., La Mantia, C., Nicholson, J., Gilliam, A., Randazzo, F., Metzger, D., Chambon, P., Crabtree, G. et al. (2000). A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes. Mol. Cell 6,1287 -1295.[CrossRef][Medline]
Bultman, S. J., Gebuhr, T. C. and Magnuson, T.
(2005). A Brg1 mutation that uncouples ATPase activity from
chromatin remodeling reveals an essential role for SWI/SNF-related complexes
in beta-globin expression and erythroid development. Genes
Dev. 19,2849
-2861.
Bultman, S. J., Gebuhr, T. C., Pan, H., Svoboda, P., Schultz, R.
M. and Magnuson, T. (2006). Maternal BRG1 regulates zygotic
genome activation in the mouse. Genes Dev.
20,1744
-1754.
Chiba, H., Muramatsu, M., Nomoto, A. and Kato, H.
(1994). Two human homologues of Saccharomyces cerevisiae
SWI2/SNF2 and Drosophila brahma are transcriptional coactivators cooperating
with the estrogen receptor and the retinoic acid receptor. Nucleic
Acids Res. 22,1815
-1820.
Coultas, L., Chawengsaksophak, K. and Rossant, J. (2005). Endothelial cells and VEGF in vascular development. Nature 438,937 -945.[CrossRef][Medline]
Dauvillier, S., Ott, M. O., Renard, J. P. and Legouy, E. (2001). BRM (SNF2alpha) expression is concomitant to the onset of vasculogenesis in early mouse postimplantation development. Mech. Dev. 101,221 -225.[CrossRef][Medline]
de la Serna, I. L., Ohkawa, Y. and Imbalzano, A. N. (2006). Chromatin remodelling in mammalian differentiation: lessons from ATP-dependent remodellers. Nat. Rev. Genet. 7,461 -473.[CrossRef][Medline]
Fujiwara, Y., Browne, C. P., Cunniff, K., Goff, S. C. and Orkin,
S. H. (1996). Arrested development of embryonic red cell
precursors in mouse embryos lacking transcription factor GATA-1.
Proc. Natl. Acad. Sci. USA
93,12355
-12358.
Gebuhr, T. C., Kovalev, G. I., Bultman, S., Godfrey, V., Su, L.
and Magnuson, T. (2003). The role of Brg1, a catalytic
subunit of mammalian chromatin-remodeling complexes, in T cell development.
J. Exp. Med. 198,1937
-1949.
Hu, X., Eszterhas, S., Pallazzi, N., Bouhassira, E. E., Fields,
J., Tanabe, O., Gerber, S. A., Bulger, M., Engel, J. D., Groudine, M. et
al. (2007). Transcriptional interference among the murine
beta-like globin genes. Blood
109,2210
-2216.
Indra, A. K., Dupe, V., Bornert, J. M., Messaddeq, N., Yaniv,
M., Mark, M., Chambon, P. and Metzger, D. (2005). Temporally
controlled targeted somatic mutagenesis in embryonic surface ectoderm and
fetal epidermal keratinocytes unveils two distinct developmental functions of
BRG1 in limb morphogenesis and skin barrier formation.
Development 132,4533
-4544.
Kadam, S., McAlpine, G. S., Phelan, M. L., Kingston, R. E.,
Jones, K. A. and Emerson, B. M. (2000). Functional
selectivity of recombinant mammalian SWI/SNF subunits. Genes
Dev. 14,2441
-2451.
Kingsley, P. D., Malik, J., Fantauzzo, K. A. and Palis, J.
(2004). Yolk sac-derived primitive erythroblasts enucleate during
mammalian embryogenesis. Blood
104, 19-25.
Kingsley, P. D., Malik, J., Emerson, R. L., Bushnell, T. P.,
McGrath, K. E., Bloedorn, L. A., Bulger, M. and Palis, J.
(2006). "Maturational" globin switching in primary
primitive erythroid cells. Blood
107,1665
-1672.
Koni, P. A., Joshi, S. K., Temann, U. A., Olson, D., Burkly, L.
and Flavell, R. A. (2001). Conditional vascular cell adhesion
molecule 1 deletion in mice: impaired lymphocyte migration to bone marrow.
J. Exp. Med. 193,741
-754.
Kwon, C. S. and Wagner, D. (2007). Unwinding chromatin for development and growth: a few genes at a time. Trends Genet. 23,403 -412.[CrossRef][Medline]
Leder, A., Daugherty, C., Whitney, B. and Leder, P.
(1997). Mouse zeta- and alpha-globin genes: embryonic survival,
alpha-thalassemia, and genetic background effects.
Blood 90,1275
-1282.
Lee, J. S., Yu, Q., Shin, J. T., Sebzda, E., Bertozzi, C., Chen, M., Mericko, P., Stadtfeld, M., Zhou, D., Cheng, L. et al. (2006). Klf2 is an essential regulator of vascular hemodynamic forces in vivo. Dev. Cell 11,845 -857.[CrossRef][Medline]
Margueron, R., Trojer, P. and Reinberg, D. (2005). The key to development: interpreting the histone code? Curr. Opin. Genet. Dev. 15,163 -176.[CrossRef][Medline]
Matsumoto, S., Banine, F., Struve, J., Xing, R., Adams, C., Liu, Y., Metzger, D., Chambon, P., Rao, M. S. and Sherman, L. S. (2006). Brg1 is required for murine neural stem cell maintenance and gliogenesis. Dev. Biol. 289,372 -383.[CrossRef][Medline]
McGrath, K. E. and Palis, J. (2005). Hematopoiesis in the yolk sac: more than meets the eye. Exp. Hematol. 33,1021 -1028.[CrossRef][Medline]
McGrath, K. E., Koniski, A. D., Malik, J. and Palis, J.
(2003). Circulation is established in a stepwise pattern in the
mammalian embryo. Blood
101,1669
-1676.
Mikkola, H. K., Gekas, C., Orkin, S. H. and Dieterlen-Lievre, F. (2005). Placenta as a site for hematopoietic stem cell development. Exp. Hematol. 33,1048 -1054.[CrossRef][Medline]
Orkin, S. H. and Zon, L. I. (1997). Genetics of erythropoiesis: induced mutations in mice and zebrafish. Annu. Rev. Genet. 31,33 -60.[CrossRef][Medline]
Palis, J., Robertson, S., Kennedy, M., Wall, C. and Keller, G. (1999). Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development 126,5073 -5084.[Abstract]
Peters, L. L., Shivdasani, R. A., Liu, S. C., Hanspal, M., John, K. M., Gonzalez, J. M., Brugnara, C., Gwynn, B., Mohandas, N., Alper, S. L. et al. (1996). Anion exchanger 1 (band 3) is required to prevent erythrocyte membrane surface loss but not to form the membrane skeleton. Cell 86,917 -927.[CrossRef][Medline]
Phelan, M. L., Sif, S., Narlikar, G. J. and Kingston, R. E. (1999). Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits. Mol. Cell 3, 247-253.[CrossRef][Medline]
Reyes, J. C., Barra, J., Muchardt, C., Camus, A., Babinet, C. and Yaniv, M. (1998). Altered control of cellular proliferation in the absence of mammalian brahma (SNF2alpha). EMBO J. 17,6979 -6991.[CrossRef][Medline]
Sato, T. N. and Loughna, S. (2002). Vasculogenesis and angiogenesis. In Mouse Development: Patterning, Morphogenesis, and Organogenesis (ed. J. Rossant and P. L. Tam), pp. 211-233. San Diego: Academic Press.
Schlaeger, T. M., Qin, Y., Fujiwara, Y., Magram, J. and Sato, T. N. (1995). Vascular endothelial cell lineage-specific promoter in transgenic mice. Development 121,1089 -1098.[Abstract]
Schwarz, D. G., Griffin, C. T., Schneider, E. A., Yee, D. and
Magnuson, T. (2002). Genetic analysis of sorting nexins 1 and
2 reveals a redundant and essential function in mice. Mol. Biol.
Cell 13,3588
-3600.
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Southgate, C. D., Chishti, A. H., Mitchell, B., Yi, S. J. and Palek, J. (1996). Targeted disruption of the murine erythroid band 3 gene results in spherocytosis and severe haemolytic anaemia despite a normal membrane skeleton. Nat. Genet. 14,227 -230.[CrossRef][Medline]
Strobeck, M. W., Reisman, D. N., Gunawardena, R. W., Betz, B.
L., Angus, S. P., Knudsen, K. E., Kowalik, T. F., Weissman, B. E. and Knudsen,
E. S. (2002). Compensation of BRG-1 function by Brm: insight
into the role of the core SWI-SNF subunits in retinoblastoma tumor suppressor
signaling. J. Biol. Chem.
277,4782
-4789.
Trimborn, T., Gribnau, J., Grosveld, F. and Fraser, P.
(1999). Mechanisms of developmental control of transcription in
the murine alpha- and beta-globin loci. Genes Dev.
13,112
-124.
Tsai, F. Y., Keller, G., Kuo, F. C., Weiss, M., Chen, J., Rosenblatt, M., Alt, F. W. and Orkin, S. H. (1994). An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 371,221 -226.[CrossRef][Medline]
Warren, A. J., Colledge, W. H., Carlton, M. B., Evans, M. J., Smith, A. J. and Rabbitts, T. H. (1994). The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell 78,45 -57.[CrossRef][Medline]
Wong, P. M., Chung, S. W., Reicheld, S. M. and Chui, D. H.
(1986). Hemoglobin switching during murine embryonic development:
evidence for two populations of embryonic erythropoietic progenitor cells.
Blood 67,716
-721.
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