The Chromatin-Remodeling Enzymes BRG1 and CHD4Antagonistically Regulate Vascular Wnt Signaling

Carol D. Curtisa and Courtney T. Griffina,b

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA,a and Department of Cell Biology, University ofOklahoma Health Sciences Center, Oklahoma City, Oklahoma, USAb

Canonical Wnt signaling plays an important role in embryonic and postnatal blood vessel development. We previously reportedthat the chromatin-remodeling enzyme BRG1 promotes vascular Wnt signaling. Vascular deletion of Brg1 results in aberrantyolk sac blood vessel morphology, which is rescued by pharmacological stimulation of Wnt signaling with lithium chloride(LiCl). We have now generated embryos lacking the chromatin-remodeling enzyme Chd4 in vascular endothelial cells. UnlikeBrg1 mutants, Chd4 mutant embryos had normal yolk sac vascular morphology. However, concomitant deletion of Chd4 andBrg1 rescued vascular abnormalities seen in Brg1 mutant yolk sacs to the same extent as LiCl treatment. We hypothesized thatWnt signaling was upregulated in Chd4 mutant yolk sac vasculature. Indeed, we found that Chd4 deletion resulted in upregula-tion of the Wnt-responsive transcription factor Tcf7 and an increase in Wnt target gene expression in endothelial cells. Further-more, we identified one Wnt target gene, Pitx2, that was downregulated in Brg1 mutant endothelial cells but was rescued follow-ing LiCl treatment and in Brg1 Chd4 double mutant vasculature, suggesting that PITX2 helps to mediate the restoration of yolksac vascular remodeling under both conditions. We conclude that BRG1 and CHD4 antagonistically modulate Wnt signaling indeveloping yolk sac vessels to mediate normal vascular remodeling.

Chromatin, which consists of DNA tightly wrapped around ascaffold of histone proteins, provides a means for the eu-

karyotic cell to fit a large amount of genetic information intothe nucleus. It also controls gene regulation, since large tran-scriptional machinery cannot readily access gene promotersthat are embedded in tightly compacted chromatin. Enzymesthat modify chromatin structure therefore play importantroles in modulating gene transcription. For example, ATP-de-pendent chromatin-remodeling complexes transiently disruptbonds between histones and DNA to allow transcription fac-tors, coregulatory proteins, and large transcriptional machin-ery to access gene promoters (14). Importantly, these com-plexes exercise striking specificity in selecting their genomictargets and can positively or negatively regulate transcriptionto modulate a number of developmental processes (15, 20).

Chromatin-remodeling complexes contain various numbersof proteins that contribute to their specific temporal and spatialeffects on gene transcription (37). Importantly, these complexescontain catalytic ATPase subunits that are required for their func-tion. Mammalian switch/sucrose-nonfermentable (SWI/SNF)-like complexes utilize the ATPases brahma (BRM, also known asSMARCA2) and brahma-related gene 1 (BRG1, also known asSMARCA4). While mice with global Brm deletion are viable andfertile (31), Brg1�/� embryos die at implantation (1). Neverthe-less, tissue-specific mutations have revealed a role for BRG1 in avariety of later developmental processes (2, 9, 11, 13, 16, 32, 33, 38,40). For example, deletion of Brg1 from developing endothelialcells with a transgenic Tie2-Cre line (Brg1fl/fl:Tie2-Cre�) results inyolk sac vascular abnormalities due in part to misregulated Wntsignaling (12).

Canonical Wnt signaling occurs when soluble extracellularWnt ligands interact with a cell surface receptor complex contain-ing a seven-transmembrane-domain frizzled (Fzd) family protein(5, 25). Interactions between Wnt ligands and their receptors sta-bilize the intracellular signaling molecule �-catenin by inactivat-

ing a cytoplasmic destruction complex that would otherwise tar-get �-catenin for degradation. Stabilized �-catenin translocates tothe nucleus, where it coregulates transcription of Wnt target genesby interacting with transcription factors in the T-cell factor(TCF)/lymphoid enhancer factor (LEF) family. BRG1 impacts theWnt signaling pathway at two levels: it directly coregulates tran-scription of multiple Fzd receptors and a subset of Wnt targetgenes in yolk sac endothelial cells (YSECs) (12). As a result, overallWnt signaling is downregulated in Brg1fl/fl:Tie2-Cre� yolk sac vas-culature. In vivo pharmacological stimulation of Wnt signalingsignificantly restores Wnt target gene transcription in yolk sacblood vessels and rescues vascular morphology in Brg1fl/fl:Tie2-Cre� yolk sacs.

BRG1-containing SWI/SNF complexes can coordinate withother ATP-dependent chromatin-remodeling complexes, such asthe nucleosome remodeling and deacetylase (NuRD) complexes,to modulate target gene transcription (8, 30). NuRD complexescombine multiple aspects of epigenetic regulation to modulatetranscription of target genes. NuRD complexes contain, in addi-tion to ATPase chromatin-remodeling enzymes, subunits thatmediate histone deacetylation and demethylation and proteinsthat bind methylated DNA (29). Together, these subunits typicallymediate transcriptional repression of NuRD target genes, al-though NuRD can promote transcription of certain target genes invivo (36). The ATPase chromatin-remodeling enzymes associated

Received 1 September 2011 Returned for modification 28 October 2011Accepted 13 January 2012

Published ahead of print 30 January 2012

Address correspondence to Courtney T. Griffin, Courtney-Griffin@omrf.org.

Supplemental material for this article may be found at http://mcb.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/MCB.06222-11

1312 mcb.asm.org 0270-7306/12/$12.00 Molecular and Cellular Biology p. 1312–1320

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 13

Jan

uary

202

2 by

1.4

.174

.198

.

with mammalian NuRD complexes are the chromodomain heli-case DNA-binding (CHD) proteins CHD3 (Mi-2�) and CHD4(Mi-2�). CHD3 and CHD4 have been studied less extensivelythan the SWI/SNF ATPases BRG1 and BRM in vivo. No tar-geted mutation of Chd3 has yet been described, but a condi-tional allele has been used to delete Chd4 from thymocytes andkeratinocytes (18, 36). CHD4 is an autoantigen in a subset ofpatients with dermatomyositis, which is characterized by in-flammation of muscles and skin (27). Despite the fact that mul-tiple vascular pathologies, such as capillary fragmentation, in-flammation, and misregulation of vascular markers, areassociated with dermatomyositis (28), no role has yet beenassigned to Chd4 or NuRD remodeling complexes in vasculardevelopment or homeostasis.

We have now deleted Chd4 from developing endothelial cellswith a Tie2-Cre transgene and report that a number of Wnt targetgenes were upregulated in Chd4-floxed (Chd4fl/fl):Tie2-Cre� yolksac vasculature. This study demonstrates that CHD4 modulatesWnt signaling in endothelial cells during vascular development bydirectly regulating expression of both the Wnt-responsive tran-scription factor Tcf7 and a subset of Wnt-responsive target genes.Moreover, concurrent deletion of Chd4 and Brg1 from vascularendothelium strikingly rescued vascular abnormalities seen inBrg1fl/fl:Tie2-Cre� yolk sacs. We conclude that Brg1 and Chd4 an-tagonistically modulate vascular Wnt signaling to mediate yolk sacangiogenesis.

MATERIALS AND METHODSMice. A Tie2-Cre� transgene was used to delete floxed Brg1 and Chd4alleles from embryonic endothelial cells in vivo. Brg1-floxed mice(Brg1fl/fl) (9), Chd4-floxed mice (Chd4fl/fl) (36), and Tie2-Cre� trans-genic mice (19) were maintained on a mixed genetic background at theOklahoma Medical Research Foundation animal facility. All animal useprotocols were approved by the Institutional Animal Care and Use Com-mittee.

Genotyping. PCR genotyping of Brg1-floxed and Tie2-Cre� trans-genic embryos and mice was performed as described previously (12).Chd4-floxed mice and embryos were genotyped by PCR using the follow-ing primers: 5=-TCCAGAAGAAGACGGCAGAT-3= (forward) and 5=-CTGGTCATAGGGCAGGTCTC-3= (reverse). These primers flank the 5=LoxP site and yield a 400-bp floxed allele and a 278-bp wild-type allele.The PCR was performed at an annealing temperature of 56°C. All histo-logical sections were scraped from paraffin or OCT (Tissue-Tek) intoDEXPAT reagent (TaKaRa) before genotyping.

LiCl injections. Lithium chloride (LiCl) (400 mg/kg of body weight,dissolved in water) was injected intraperitoneally into pregnant femalemice at embryonic day 8.5 (E8.5) and E9.5. Embryos were harvested forendothelial cell isolation at E10.5.

Yolk sac staining. Whole-mount yolk sac immunostaining was per-formed as described previously (12). Hematoxylin and eosin staining,terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end la-beling (TUNEL) staining, and benzidine staining were performed on his-tological sections of yolk sacs as described previously (11).

Immunofluorescence. Brg1fl/fl:Tie2-Cre�, Brg1fl/fl; Chd4fl/fl:Tie2-Cre�, and littermate control embryos were cryoembedded and sectioned(8 �m). Double immunostaining for BRG1 and PECAM-1 was per-formed as follows: cryosections were thawed and blocked in 3% normaldonkey serum (Jackson ImmunoResearch)–3% bovine serum albumin(BSA; Rockland Immunochemicals)– 0.3% Triton X-100 –phosphate-buffered saline (PBS) for 1 h at room temperature. Anti-PECAM-1 (1:500;BD Biosciences catalog number 553370) was diluted in 1% normal don-key serum–1% BSA– 0.1% Triton X-100 –PBS and applied to sections for1 h at 37°C. Sections were washed three times (2 min each) in 0.1% Triton

X-100 –PBS. Cy3– donkey anti-rat IgG (1:500; Jackson ImmunoResearch)and Hoechst stain (20 �g/ml) were diluted as described above and appliedto sections for 1 h at room temperature. Sections were washed as describedabove and then blocked and stained with anti-BRG1 (1:100; Santa CruzBiotechnology catalog number sc-17796) using the M.O.M. kit (VectorLaboratories). Alexa 488-streptavidin (1:500; Invitrogen) was used to de-tect the biotinylated mouse IgG supplied in the M.O.M. kit. Sections werewashed three times (3 min each) in PBS, and a coverslip was applied with2.5% 1,4-diazabicyclo[2.2.2]octane (DABCO)–90% glycerol–PBS, pH8.6. Double immunostaining for CHD4 and PECAM-1 was performedsimilarly with the following exception: cryosections were blocked in 3%normal donkey serum–3% normal goat serum (Jackson Immuno-Research)–3% BSA– 0.3% Triton X-100 –PBS for 1 h at room tempera-ture. Anti-PECAM-1 (1:500) and anti-CHD4 (1:1,000; Active Motif cat-alog number 39289) were diluted in 1% normal donkey serum–1%normal goat serum–1% BSA– 0.1% Triton X-100 –PBS and applied tosections for 1 h at room temperature. Cy3– donkey anti-rat IgG, Alexa488 – goat anti-rabbit IgG (1:500, Invitrogen), and Hoechst dye were di-luted and applied to sections for 1 h at room temperature.

Microscopy. Fluorescent and light microscopic images were acquiredas previously described (12).

Primary endothelial cell isolation. Yolk sac endothelial cells (YSECs)were isolated with anti-PECAM-1-conjugated magnetic beads from E10.5embryos as described previously (12).

Custom array. Total RNA from primary YSECs was isolated from 4 to6 independent experiments as described previously (12), and cDNA wasprepared using the RT2 first-strand kit (SABiosciences). Custom-de-signed Wnt signaling target gene RT2 Profiler PCR arrays (SA Biosciences)were used as described previously (12). Data analysis and statistical deter-minations were performed using the Web-based PCR array data analysistool available through the SABiosciences website.

qPCR. To analyze transcript levels, total RNA from primary YSECswas isolated using TRIzol (Invitrogen) according to the manufacturer’sinstructions. The DNA-free kit (Ambion) was used to digest any contam-inating DNA. cDNA was prepared using the iScript cDNA synthesis kit(Bio-Rad), and real-time quantitative PCR (qPCR) was performed usingRT2 Fast SYBR green qPCR master mix (SABiosciences) and the CFX96detection system (Bio-Rad) with gene-specific primers.

qPCR primers. The following qPCR primers were used: for �-actin,5=-TGTTACCAACTGGGACGACA-3= and 5=-GGGGTGTTGAAGGTCTCAAA-3=; for glyceraldehyde-3-phosphate dehydrogenase (GAPDH),5=-TCAACGGCACAGTCAAGG-3= and 5=-ACTCCACGACATACTCAGC-3=; for Pitx2, 5=-CGTTGAATGTCTCTTCTCCA-3= and 5=-CTGGCCCTTATCTTTCTCAT-3=; for Chd4, 5=-GCTATGCCCGGTGGCAGGAC-3= and 5=-CGGGTGAGAGGGGTCCTCGG-3=; for �-catenin, 5=-TGGCAGCAGCAGTCTTAC-3= and 5=-GAGGTGTCAACATCTTCTTCC-3=; for Tcf3, 5=-CAGATGGTGGCCTGGATACT-3= and 5=-CATCCCTGCTGTAGCTGTCA-3=; for Tcf4, 5=-GTCCTCGCTGGTCAATGAAT-3=and 5=-CCCTTAAAGAGCCCTCCATC-3=; for Tcf7, 5=-GCCAGAAGCAAGGAGTTCAC-3= and 5=-ACAGGGGGTAGAGAGGAGGA-3=; and forLef1, 5=-TATGAACAGCGACCCGTACA-3= and 5=-TCGTCGCTGTAGGTGATGAG-3=.

qPCR analysis. The relative fold change in transcription was deter-mined using the comparative threshold cycle (CT) method and the �-actinand GAPDH housekeeping genes as internal controls. Data from six inde-pendent experiments were combined and are presented as means � stan-dard errors of the means (SEM). Statistical differences were detected usinga two-tailed Student t test.

Cell culture and transfections. C166 yolk sac endothelial cells (ATCCaccession number CRL-2581) were maintained and transfected with 100nM CHD4 siGENOME SMARTpool or nontargeting control small inter-fering RNA (siRNA) oligonucleotides (Dharmacon catalog numberM-052142-01 or D-001210-01, respectively) as described previously (12).

Western blotting. Total protein harvested from siRNA-transfectedC166 endothelial cells was fractionated on a 9% SDS-polyacrylamide gel

BRG1 and CHD4 Regulate Wnt Signaling

April 2012 Volume 32 Number 7 mcb.asm.org 1313

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 13

Jan

uary

202

2 by

1.4

.174

.198

.

and transferred to a polyvinylidene difluoride (PVDF) membrane forWestern blotting with antibodies to CHD4 (Abcam; catalog number72418), �-catenin (BD Biosciences; catalog number 610153), andGAPDH (Sigma; catalog number G9545). Relative band intensity wasdetermined using ImageJ software (National Institutes of Health).

ChIP. Chromatin immunoprecipitation (ChIP) assays were per-formed as described previously (12) with modifications. Chromatin wasimmunoprecipitated using a CHD4-specific antibody (Abcam; catalognumber 70469). Mouse IgG (Invitrogen; catalog number 100005291) orthe polyhistidine epitope tag (His) antibody (Rockland; catalog number600-401-382) was used as a negative control. For total histone H3 ChIP,chromatin was harvested as described previously (12) from cells trans-fected with nonspecific or CHD4-specific siRNAs and immunoprecipi-tated using the histone H3-specific and negative-control antibodiessupplied in the ChIPAb� histone H3 kit (Millipore; catalog number 17-10046). Real-time quantitative PCR was performed using RT2 Fast SYBRgreen qPCR master mix (SABiosciences) and the CFX96 detection system(Bio-Rad) with primer sets within the promoter region of Tcf7 (5=-TGTTCACACCAAGGTTCCAA-3= and 5=-GGCCCACTGGGAATAATCTT-3=), Lef1 (5=-ATTTTCGCTAGGGTGGTGTG-3= and 5=-TTCAGCAAAGGCAAACAGAA-3=), Pitx2 (5=-CGGTTTTCCTGGAGACTGAA-3= and5=-CAGGCAAACAAACTTGCTCA-3=), Myc (5=-AGGGATCCTGAGTCGCAGT-3= and 5=-CGCTCACTCCCTCTGTCTCT-3=), Ccnd1 gene (5=-CACACGGACTACAGGGGAGT-3= and 5=-CGCGGAGTCTGTAGCTCTCT-3=), plaur (5=-ACTGAGCCGCTCTGAGTGAT-3= and 5=-CCAGGGGAAAAACAAGTTGA-3=), or a negative-control region �5 kb upstreamof the Fzd5 promoter designated Fzd5UP (5=-GGTGACTTAGGGCAAAACCA-3= and 5=-AGGCCACCATACCAGGTTCT-3=). Data from threeindependent experiment were combined and are presented as n-fold lev-els of enrichment over the level of expression with the negative-controlantibody �SEM. Statistical differences were detected using a two-tailedStudent t test.

RESULTSVascular deletion of Chd4 rescues vessel morphology in Brg1fl/fl:Tie2-Cre� yolk sacs. Brg1fl/fl:Tie2-Cre� (here referred to as Brg1fl/fl:Cre�) yolk sac vasculature is thin and disconnected by E10.5 (Fig.1B and F) (12). These abnormalities result in part from misregu-

lated vascular Wnt signaling, since pharmacological stimulationof the Wnt signaling pathway with LiCl treatment, which stabilizesthe intracellular signaling molecule �-catenin, significantly res-cues vascular morphology in vivo (12).

In order to assess the role of the NuRD complex in vasculardevelopment, we deleted the NuRD chromatin-remodeling en-zyme Chd4 from embryonic endothelial cells using the Tie2-Cretransgenic line. In contrast to Brg1fl/fl:Cre� yolk sacs, Chd4fl/fl:Cre� yolk sacs displayed normal vascular development and re-modeling at E10.5 (Fig. 1C and G). Furthermore, vascular luminalspaces, which were flattened in Brg1fl/fl:Cre� yolk sacs (Fig. 1J)(12), presumably due to plasma leakage through discontinuities inthe endothelial cell lining (12), were normally inflated in Chd4fl/fl:Cre� yolk sacs (Fig. 1K).

We predicted that deletion of both Brg1 and Chd4 from yolksac vasculature would result in more severe morphological defectsthan those seen in Brg1fl/fl:Cre� yolk sacs if SWI/SNF and NuRDplayed redundant roles in yolk sac vascular development. To oursurprise, the vascular thinning and misconnections observed inBrg1fl/fl:Cre� yolk sac microvasculature were significantly rescuedin Brg1fl/fl; Chd4fl/fl:Cre� yolk sacs (Fig. 1D and H). Likewise,Brg1fl/fl; Chd4fl/fl:Cre� yolk sac vascular luminal spaces were in-flated comparably to those of control and Chd4fl/fl:Cre� vessels(Fig. 1L, I, and K). These data indicate that vascular deletion ofChd4 rescues Brg1fl/fl:Cre� yolk sac vascular thinning, disconnect-edness, and luminal flattening.

Brg1 and Chd4 are efficiently excised in Brg1fl/fl; Chd4fl/fl:Cre� endothelial cells. In order to generate a Brg1fl/fl; Chd4fl/fl:Cre� mutant, the Tie2-Cre transgene must effectively delete 4floxed alleles from embryonic endothelial cells in vivo. Thus, weimmunostained E10.5 embryos for BRG1 to confirm that themorphological rescue observed was attributable to genetic inter-action between Brg1 and Chd4 and not an artifact due to dimin-ished Cre efficiency and mosaic Brg1 excision. BRG1 was normallyexpressed in the nuclei of endothelial cells and surrounding tissues

FIG 1 Chd4 deletion rescues Brg1fl/fl:Cre� yolk sac vascular morphology. (A to H) Anti-PECAM1 staining of E10.5 yolk sacs for blood vessel visualization. Vesselsin Brg1fl/fl:Cre� yolk sacs (B and F) are thin and disconnected (arrowheads) compared to those in control (A and E) and Chd4fl/fl:Cre� (C and G) yolk sacs. (Dand H) Brg1fl/fl; Chd4fl/fl:Cre� yolk sacs have a substantial restoration of vessel size and interconnectedness. (I to L) Hematoxylin and eosin (H&E)-stainedparaffin sections of E10.5 yolk sac vessels. Brg1fl/fl:Cre� yolk sac vessel lumens (J) are flat compared to those in control (I) and Chd4fl/fl:Cre� (K) yolk sacs. (L)Luminal space is rescued in Brg1fl/fl; Chd4fl/fl:Cre� yolk sac vessels. (I to L) Arrows indicate embryonic blood cells within yolk sac vascular luminal spaces. Scalebars, 100 �m (A to D) and 50 �m (E to L).

Curtis and Griffin

1314 mcb.asm.org Molecular and Cellular Biology

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 13

Jan

uary

202

2 by

1.4

.174

.198

.

of the developing embryo at E10.5 (Fig. 2A and D). In contrast,BRG1 was undetectable in the endothelium of Brg1fl/fl:Cre� (Fig.2B and E) and Brg1fl/fl; Chd4fl/fl:Cre� embryos (Fig. 2C and F).Likewise, CHD4 was detectable in endothelial cells from control em-bryos (Fig. 2G and J) and was efficiently excised in both Chd4fl/fl:Cre�

(Fig. 2H and K) and Brg1fl/fl; Chd4fl/fl:Cre� (Fig. 2I and L) endo-thelial cells. These data indicate that the phenotypic rescue docu-mented in Brg1fl/fl; Chd4fl/fl:Cre� yolk sac vasculature (Fig. 1) re-sults from a genetic interaction between Brg1 and Chd4.

Deletion of Chd4 from primitive erythrocytes does not res-cue Brg1fl/fl:Cre� anemia. The Tie2-Cre transgene that we used toexcise Brg1 and Chd4 from vascular endothelium is also expressedin a subset of hematopoietic cells during embryonic development(19). Brg1fl/fl:Cre� embryos die from anemia at midgestation due

to excision of Brg1 from primitive erythrocytes (11). BRG1 is re-quired for expression of embryonic globin genes, and Brg1-defi-cient primitive erythrocytes undergo apoptosis due to insufficienthemoglobin production (11).

Despite the role that CHD4 plays in erythrocyte and mega-karyocyte maturation and in T cell development in vivo (10, 36),we saw no evidence of hematopoietic abnormalities in Chd4fl/fl:Cre� embryos by E10.5. Unlike Brg1fl/fl:Cre� embryos, Chd4fl/fl:Cre� embryos were not grossly anemic (Fig. 3B and C). Likewise,primitive Chd4fl/fl:Cre� erythrocytes did not undergo apoptosis(Fig. 3G) or display deficient hemoglobin accumulation (Fig. 3K).

Since blood flow biomechanics can contribute to yolk sac vas-cular remodeling (24), we sought to determine whether the res-cued vascular morphology observed in Brg1fl/fl; Chd4fl/fl:Cre� yolksacs could result from rescued primitive erythrocyte survival.Gross assessment of Brg1fl/fl; Chd4fl/fl:Cre� embryos revealed thatthey were anemic, like Brg1fl/fl:Cre� embryos at E10.5 (Fig. 3D andB). Likewise, both Brg1fl/fl; Chd4fl/fl:Cre� embryos and Brg1fl/fl:Cre� embryos had apoptotic primitive erythrocytes (Fig. 3H andF) that failed to accumulate normal levels of hemoglobin (Fig. 3Land J). Therefore, Chd4 deletion did not rescue erythroblast he-moglobin production and apoptosis or pallor in Brg1fl/fl:Cre� em-bryos, although it did rescue yolk sac vascular patterning (Fig.1D). These data indicate that BRG1 and CHD4 have separabletissue-specific roles during development. They also provide strongevidence that the vascular anomalies seen in Brg1fl/fl:Cre� yolk sacsare caused primarily by genetic rather than biomechanical factors.

Wnt signaling is upregulated in Chd4fl/fl:Cre� yolk sac endo-thelial cells. The extent to which vascular morphology was res-cued in Brg1fl/fl; Chd4fl/fl:Cre� yolk sacs (Fig. 1D) is highly remi-niscent of that seen when we treat Brg1fl/fl:Cre� embryos with LiClin vivo (12). Because LiCl stimulates Wnt signaling by stabilizingthe intracellular signaling molecule �-catenin (25), we questionedwhether Chd4 deletion similarly rescued Brg1fl/fl:Cre� yolk sacvascular morphology by stimulating Wnt signaling. Using a cus-tom-designed qPCR array, we assessed the relative transcript lev-els of 28 direct Wnt target genes in primary YSECs isolated fromE10.5 yolk sacs (see Table S1 in the supplemental material). Wefound that 64% of these target genes were downregulated inBrg1fl/fl:Cre� YSECs (Fig. 4A), which is consistent with our previ-ous finding that BRG1 promotes vascular Wnt signaling (12). Incontrast, 79% of the 28 Wnt target genes were upregulated inChd4fl/fl:Cre� YSECs (Fig. 4B). In addition, several of the genesthat were downregulated in Brg1fl/fl:Cre� YSECs were upregulatedin Chd4fl/fl:Cre� YSECs (Fig. 4C). These data indicate that BRG1and CHD4 modulate Wnt signaling in opposite directions duringyolk sac vascular development.

In order to determine how CHD4 inhibits Wnt signaling, weanalyzed �-catenin protein levels following siRNA-mediatedknockdown of CHD4 in the C166 yolk sac endothelial cell line(35). Western blots revealed that �-catenin levels were normalfollowing CHD4 depletion (Fig. 4D). This differs from the signif-icant �-catenin degradation seen in BRG1-depleted endothelialcells, which results from reduced Fzd receptor transcription (12).Since we detected normal �-catenin expression in CHD4-de-pleted endothelial cells, we suspected that CHD4 impacted theWnt signaling pathway downstream of �-catenin.

�-Catenin mediates Wnt target gene transcription by interact-ing with and activating TCF/LEF transcription factors (25). Toaddress the possibility that numerous Wnt target genes were

FIG 2 Tie2-Cre efficiently excises BRG1 and CHD4 in Brg1fl/fl; Chd4fl/fl:Cre�

endothelial cells. E10.5 embryos were cryosectioned and immunostained withan anti-BRG1 antibody (green) (A to F) or an anti-CHD4 antibody (green) (Gto L) and an anti-PECAM antibody (red) to mark endothelial cells. Nucleiwere stained with Hoechst dye (blue). Arrows designate individual endothelialcells. (A to F) Brg1fl/fl:Cre� (B and E) and Brg1fl/fl; Chd4fl/fl:Cre� (C and F)endothelial cells display significantly reduced expression of BRG1 compared tocontrol cells (A and D). Likewise, Chd4fl/fl:Cre� (H and K) and Brg1fl/fl; Chd4fl/

fl:Cre� (I and L) endothelial cells have considerably reduced expression ofCHD4 compared to controls (G and J). (A to L) Scale bars, 50 �m.

BRG1 and CHD4 Regulate Wnt Signaling

April 2012 Volume 32 Number 7 mcb.asm.org 1315

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 13

Jan

uary

202

2 by

1.4

.174

.198

.

upregulated in Chd4fl/fl:Cre� YSECs due to misregulated TCF/LEF factors, we assessed the expression of all known mamma-lian TCF/LEF genes. We detected significant upregulation ofTcf7 and Lef1 upon siRNA-mediated depletion of CHD4 inC166 cells (Fig. 4E). We also performed qPCR on primaryYSECs isolated from Chd4fl/fl:Cre� embryos and confirmedthat the transcript level of Tcf7 was significantly upregulated inmutant cells (not shown).

Because transcript levels of Tcf7 and Lef1 were increased withCHD4 depletion, we were interested in determining whether thesegenes are direct targets of CHD4. Chromatin immunoprecipita-tion (ChIP) assays indicated that CHD4 associates with the pro-moter region of the Tcf7 gene, but not the Lef1 promoter, in C166endothelial cells (Fig. 5A). To determine the functional conse-quence of CHD4 association with the Tcf7 promoter, we per-formed ChIP following siRNA-mediated knockdown of CHD4 inthe C166 yolk sac endothelial cell line with an antibody that rec-ognizes total, unmarked histone H3. This assay allowed us to mea-sure nucleosome density at the Tcf7 promoter in the presence orabsence of CHD4. We found greater H3 enrichment in CHD4knockdown cells than in cells that had been transfected with non-specific siRNA (Fig. 5B), indicating that CHD4 mediates chroma-tin decondensation at the Tcf7 promoter along with transcrip-tional repression. This is consistent with the hypothesis thatNuRD complexes mediate transcriptional repression by first usingCHD4 to remodel chromatin in order to make it accessible toNuRD-associated histone deacetylases (34, 39). Our data indicatethat CHD4 epigenetically modulates vascular Wnt signalingdownstream of �-catenin through chromatin remodeling andsubsequent transcriptional repression at the Tcf7 promoter.

CHD4 associates with the promoter region of a subset ofWnt-responsive target genes. Since BRG1 impacts the Wnt sig-naling pathway through transcriptional regulation of multiple Fzdreceptors as well as a subset of Wnt target genes in yolk sac endo-thelial cells (12), we examined the possibility that CHD4 mightalso be directly involved in Wnt-responsive target gene transcrip-tion. Although several Wnt-responsive genes were upregulated inChd4fl/fl:Tie2-Cre� yolk sac vasculature (see Table S1 in the sup-plemental material), we randomly selected four genes, Pitx2, Myc,Ccnd1, and Plaur, to test by ChIP assay in C166 endothelial cells.We found that CHD4 associates with the promoter region of theMyc, cyclin D1, and uPAR genes but not the Pitx2 promoter (Fig.5C), indicating that CHD4 impacts the Wnt signaling pathwaythrough regulation of the Wnt-responsive transcription factorTcf7 but also directly coregulates transcription of a subset of Wnttarget genes.

Vascular deletion of Chd4 rescues Wnt signaling in Brg1fl/fl:Cre� yolk sacs. Since BRG1 and CHD4 influence Wnt target genetranscription in opposing manners in primary YSECs (Fig. 4A andB), we sought to determine how Wnt target genes were affectedupon concomitant deletion of both Brg1 and Chd4 from the vas-culature. Thus, we analyzed Wnt target gene qPCR arrays withYSECs isolated from Brg1fl/fl; Chd4fl/fl:Cre� mutants. We foundthat 50% of Wnt target gene transcripts were upregulated, 18%were downregulated, and 32% were unchanged in Brg1fl/fl; Chd4fl/fl:Cre� YSECs compared to YSECs from littermate controls (Fig.6A). Furthermore, a subset of genes that were downregulated inBrg1fl/fl:Cre� YSECs and/or upregulated in Chd4fl/fl:Cre� YSECswere normalized upon deletion of both Brg1 and Chd4 from thevasculature (Fig. 6C). We propose that this subset of genes may

FIG 3 Chd4 deletion does not rescue Brg1fl/fl:Cre� anemia. (A to D) Gross photos of E10.5 control and mutant embryos with attached yolk sacs andplacentae. Vessels in control (A) and Chd4fl/fl:Cre� (C) yolk sacs contain visible red blood. Vessels in Brg1fl/fl:Cre� (B) and Brg1fl/fl; Chd4fl/fl:Cre� (D) yolksacs are pale. Arrowheads designate blood vessels. (E to H) Cryosections from E10.5 control and mutant yolk sacs were stained by TUNEL (green) toidentify apoptotic cells. Primitive erythrocytes in control (E) and Chd4fl/fl:Cre� (G) yolk sac vessels are TUNEL negative. A subset of primitive erythrocytesin Brg1fl/fl:Cre� (F) and Brg1fl/fl; Chd4fl/fl:Cre� (H) yolk sac vessels are TUNEL positive (arrows). Nuclei were stained with Hoechst dye (blue). Yolk sacvessels are outlined (yellow). (I to L) Cryosections from E10.5 control and mutant embryos were stained with benzidine (yellow) for detection ofhemoglobin in developing blood cells. The majority of primitive erythrocytes in control (I) and Chd4fl/fl:Cre� (K) vessels stained with benzidine. Incontrast, the majority of primitive erythrocytes in Brg1fl/fl:Cre� (J) and Brg1fl/fl; Chd4fl/fl:Cre� (L) vessels failed to stain with benzidine (arrows). Scale bars,1 mm (A to D), 50 �m (E to H), and 100 �m (I to L).

Curtis and Griffin

1316 mcb.asm.org Molecular and Cellular Biology

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 13

Jan

uary

202

2 by

1.4

.174

.198

.

contribute to the vascular morphological rescue observed inBrg1fl/fl; Chd4fl/fl:Cre� yolk sacs.

Pharmacological stabilization of �-catenin rescues tran-scription of select Wnt target genes. We previously showed thatpharmacological stimulation of Wnt signaling with LiCl treat-ment rescues select Wnt target genes in Brg1fl/fl:Cre� YSECs (12).In order to expand upon these results, we examined 28 Wnt targetgenes in YSECs isolated from LiCl-treated Brg1fl/fl:Cre� yolk sacsand nontreated Brg1fl/fl:Cre� yolk sacs using our customizedqPCR array (compare Fig. 6B and 4A). We found fewer genesdownregulated in Brg1fl/fl:Cre� YSECs following LiCl treatment(39%) than following no treatment (64%), implying that LiCltreatment rescued the transcription of specific Wnt target genes.Since a subset of Wnt target genes require BRG1 for coregulationof their transcription during yolk sac vascular development (12),we hypothesize the genes that were not rescued in LiCl-treatedBrg1fl/fl:Cre� YSECs are direct targets of BRG1. In support of thishypothesis, BRG1 directly associates with the promoter of Wisp1(12), a Wnt target gene that was downregulated in Brg1fl/fl:Cre�

YSECs and was not rescued by LiCl treatment (Fig. 6D). Further-more, we propose that genes that were rescued by LiCl treatmentrepresent a subset of genes that may be involved in the morpho-

logical rescue of the Brg1fl/fl:Cre� yolk sac vasculature observedupon treatment of Brg1fl/fl:Cre� embryos with LiCl (12).

Identification of genes coordinately regulated by BRG1,CHD4, and Wnt signaling. Because of the comparable rescues ofvascular morphology in Brg1fl/fl; Chd4fl/fl:Cre� and LiCl-treatedBrg1fl/fl:Cre� yolk sacs, we hypothesized that similar gene targetswere rescued under both conditions. Using our custom Wnt tar-get gene arrays, we found a small subset of genes that were down-regulated in Brg1fl/fl:Cre� YSECs but were normalized in eitherBrg1fl/fl; Chd4fl/fl:Cre� or LiCl-treated Brg1fl/fl:Cre� YSECs (Fig. 6Cand D). Only one of these genes, Pitx2, was downregulated inBrg1fl/fl:Cre� YSECs and unchanged in both Brg1fl/fl; Chd4fl/fl:Cre�

and LiCl-treated Brg1fl/fl:Cre� YSECs. We verified these array databy direct qPCR in YSECs and confirmed that Pitx2 was downregu-lated in Brg1fl/fl:Cre� YSECs, upregulated in Chd4fl/fl:Cre� YSECs,and normalized in both Brg1fl/fl; Chd4fl/fl:Cre� and LiCl-treatedBrg1fl/fl:Cre� YSECs (Fig. 6E). Our results indicate that BRG1 andCHD4 act in opposing manners to regulate Pitx2 in the yolk sacvasculature during development and furthermore suggest thatPitx2 may contribute to the rescue phenotype observed in bothBrg1fl/fl; Chd4fl/fl:Cre� and LiCl-treated Brg1fl/fl:Cre� yolk sac vas-culatures.

FIG 4 CHD4 acts downstream of �-catenin to modulate vascular Wnt signaling. (A to C) Endothelial cells from littermate control and mutant yolk sacswere isolated, RNA was purified, and cDNA was synthesized. qPCR was performed using a custom-designed array containing primer sets for 28 Wnt targetgenes. Data from at least four independent experiments were combined and analyzed using SABiosciences Excel-based software. Pie charts display thepercentage of genes in each group that were downregulated, upregulated, or unchanged in Brg1fl/fl:Cre� (A) or Chd4fl/fl:Cre� (B) YSECs compared tolittermate control YSECs. (C) A Venn diagram shows the transcripts that were downregulated in Brg1fl/fl:Cre� YSECs compared to control YSECs andupregulated in Chd4fl/fl:Cre� YSECs compared to control YSECs. (D to E) C166 yolk sac endothelial cells were transfected with nonspecific (NS) orCHD4-specific siRNA oligonucleotides for 48 h. (D) Western blot analysis was performed using antibodies that recognize CHD4, �-catenin, or GAPDH.�-Catenin band intensity was determined and normalized to the intensity of GAPDH. The results from five independent experiments were combined, anddata are presented as the means � SEM. (E) RNA was isolated, cDNA was synthesized, and qPCR was performed using gene-specific primers (Chd4,�-catenin, Tcf3, Tcf4, Tcf7, and Lef1). Relative fold change was calculated for transcripts in CHD4 knockdown cells and normalized to nonspecificsiRNA-transfected samples (dotted line). Error bars represent �SEM of results from four independent experiments. Significant differences werecalculated using a two-tailed Student t test (*, P � 0.05).

BRG1 and CHD4 Regulate Wnt Signaling

April 2012 Volume 32 Number 7 mcb.asm.org 1317

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 13

Jan

uary

202

2 by

1.4

.174

.198

.

DISCUSSION

In order to understand how different ATP-dependent chromatin-remodeling complexes influence vascular development, we gener-ated embryos lacking the SWI/SNF catalytic subunit BRG1 or theNuRD catalytic subunit CHD4 in vascular endothelium. We dem-onstrated that these two chromatin-remodeling enzymes work inopposition to regulate vascular Wnt signaling. While BRG1 pri-marily promoted Wnt target gene transcription in yolk sac endo-thelial cells, CHD4 repressed expression of most Wnt target genesthat we tested. Furthermore, vascular deletion of Chd4 rescuedBrg1 mutant yolk sac phenotypes and restored a subset of Wnttarget genes to basal transcription levels. These findings indicatethat BRG1 and CHD4 antagonistically modulate Wnt signaling indeveloping yolk sac vessels.

BRG1 impacts Wnt signaling at two different levels in vascularendothelium: (i) through transcriptional activation of multipleWnt receptor genes within the Fzd family and (ii) through coregu-lation of a subset of Wnt/�-catenin target genes (Fig. 7) (12).

Therefore, BRG1 interfaces with the Wnt signaling pathway bothupstream and downstream of �-catenin to promote Wnt targetgene transcription. Our new data support a model in which CHD4modulates Wnt signaling downstream of �-catenin (Fig. 7). Ge-netic evidence that CHD4 does not impact Wnt signaling up-stream of �-catenin comes from our observation that Chd4fl/fl:Cre� yolk sacs do not phenocopy �-catenin gain-of-functionmutants (4), which have dramatic yolk sac vascular patterningdefects presumably due to upregulation of all vascular Wnt targetgenes. Like BRG1, CHD4 impacts Wnt signaling at two differentlevels in vascular endothelium: through transcriptional repressionof the Wnt-responsive transcription factor Tcf7 and also throughdirect coregulation and transcriptional repression of a subset ofWnt/�-catenin target genes (Fig. 7). Thus, upregulated Wnt targetgenes in Chd4fl/fl; Cre� endothelial cells may be a primary or sec-ondary consequence of CHD4 depletion. More extensive ChIPexperiments will be required to distinguish the genes that are di-rect targets of CHD4 and those that are secondarily affected bymisregulation of Tcf7.

We are particularly encouraged that our Wnt target gene arraysprovided a novel candidate for mediating yolk sac vascular devel-opment. The Wnt target gene Pitx2 was downregulated in Brg1fl/fl:Cre� yolk sac endothelium but normalized in both Brg1fl/fl; Chd4fl/fl:Cre� and LiCl-treated Brg1fl/fl:Cre� endothelium. Thus, wepredict that Pitx2 may contribute to the rescue of vascular thin-ning and disconnectedness observed in each of these mutants.Although Pitx2 is involved in several developmental processes,including heart development, it has not yet been directly impli-cated in vascular development (6, 7, 17, 21). Nevertheless, PITX2has been shown to repress Bmp4 and enhance Fgf8 in the regula-tion of cell motility during craniofacial development (22, 23).Since roles for BMP and FGF signaling in vascular developmenthave been previously described (3, 26), we speculate that misregu-lation of these pathways may contribute to the aberrant vasculardevelopment observed in Brg1fl/fl:Cre� mutant yolk sacs. There-fore, further studies are warranted to elucidate the unanticipatedrole that PITX2 may play in yolk sac vascular remodeling.

Our finding that BRG1 and CHD4 have opposing effects onvascular Wnt signaling in vivo complements previous in vitro ev-idence that these enzymes antagonistically regulate target genesinvolved in B cell specification and inflammatory responses (8,30). Notably, previous studies demonstrate that BRG1 and CHD4can bind and act antagonistically on the same gene promoters inplasmacytoma and macrophage cell lines. We now report thatBRG1 and CHD4 can act on separate targets within the same path-way to regulate Wnt signaling in endothelial cells. While BRG1modulates transcription of Fzd receptors upstream of �-catenin,CHD4 inhibits transcription of Tcf7, a transcription factor thatacts downstream of �-catenin to regulate Wnt target gene tran-scription. Furthermore, our current data demonstrate that bothBRG1 and CHD4 directly modulate Wnt target genes. However,further ChIP experiments will be necessary to determine whetherBRG1 and CHD4 bind simultaneously to a subset of Wnt targetgenes.

We predict that there is more coordination between chroma-tin-remodeling complexes than has been previously appreciated.Tandem deletion of Brg1 and Chd4 in additional cell types willhelp to elucidate more developmental processes that utilize SWI/SNF and NuRD to regulate and titrate transcription. In addition,deletion of Brg1 and Chd4 from blood vessels at later embryonic

FIG 5 CHD4 modulates vascular Wnt signaling at two levels. (A) Chromatinimmunoprecipitation (ChIP) assays were performed using antibodies againstCHD4 or a polyhistidine epitope tag as a negative control. DNA was isolatedand amplified by qPCR to determine whether CHD4 bound the promoterregion of Tcf7 or Lef1. (B) Chromatin harvested from nonspecific (NS) orCHD4 siRNA-transfected C166 yolk sac endothelial cells was immunoprecipi-tated with an antibody against histone H3 or a negative-control antibody.DNA was isolated and amplified by qPCR to examine the relative nucleosomedensity at the Tcf7 promoter. (C) Chromatin immunoprecipitation assayswere carried out using antibodies against CHD4 or IgG (negative control) todetermine whether CHD4 was associated with the promoter region of the Wnttarget genes Pitx2, Myc, Ccnd1, and Plaur. (A to C) A region greater than 5 kbupstream of the Fzd5 transcription start site (Fzd5UP) was used as a negativecontrol. Data from three independent experiments were combined and arepresented as fold enrichment over the level with the negative-control antibody.Significant differences were calculated using a two-tailed Student t test (*, P �0.05).

Curtis and Griffin

1318 mcb.asm.org Molecular and Cellular Biology

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 13

Jan

uary

202

2 by

1.4

.174

.198

.

and postnatal time points will reveal whether these chromatin-remodeling enzymes antagonistically modulate Wnt signaling un-der a variety of angiogenic conditions.

ACKNOWLEDGMENTS

We thank Katia Georgopoulos (Harvard Medical School) for Chd4-floxedmice, Vijay Muthukumar and James Riddle for their assistance withmouse husbandry and genotyping, and Rodger McEver and members ofthe Griffin lab for helpful discussions and critical reading of the manu-script.

This work was supported by National Institutes of Health grants toC.T.G. (R00HL087621 and P20RR018758).

REFERENCES1. Bultman S, et al. 2000. A Brg1 null mutation in the mouse reveals func-

tional differences among mammalian SWI/SNF complexes. Mol. Cell6:1287–1295.

2. Bultman SJ, Gebuhr TC, Magnuson T. 2005. A Brg1 mutation thatuncouples ATPase activity from chromatin remodeling reveals an essen-tial role for SWI/SNF-related complexes in beta-globin expression anderythroid development. Genes Dev. 19:2849 –2861.

3. Chappell JC, Bautch VL. 2010. Vascular development: genetic mecha-nisms and links to vascular disease. Curr. Top. Dev. Biol. 90:43–72.

FIG 6 A subset of Wnt target genes are rescued in Brg1fl/fl; Chd4fl/fl:Cre� YSECs and LiCl-treated Brg1fl/fl:Cre� YSECs. Endothelial cells from littermate controland mutant yolk sacs were isolated, RNA was purified, and cDNA was synthesized. (A to D) qPCR was performed using a custom-designed array containingprimer sets for 28 Wnt target genes. Data from at least four independent experiments were combined and analyzed using SABiosciences Excel-based software. (Ato B) Pie charts display the percentage of genes in each group that were downregulated, upregulated, or unchanged in Brg1fl/fl; Chd4fl/fl:Cre� (A) or LiCl-treatedBrg1fl/fl:Cre� (B) YSECs compared to littermate control YSECs. (C to D) Venn diagrams summarize the transcripts that were downregulated in Brg1fl/fl:Cre�

YSECs compared to control YSECs, upregulated in Chd4fl/fl:Cre� YSECs compared to control YSECs, and unchanged (i.e., rescued) in Brg1fl/fl; Chd4fl/fl:Cre�

YSECs compared to control YSECs (C) or downregulated in Brg1fl/fl:Cre� YSECs compared to control YSECs and unchanged (i.e., rescued) in LiCl-treatedBrg1fl/fl:Cre� YSECs compared to LiCl-treated control YSECs (D). (E) qPCR was performed using gene-specific primers for Pitx2. Relative fold change wascalculated and normalized to littermate control samples (dotted line). Error bars represent �SEM of results from six independent experiments. Significantdifferences between littermate control and mutant samples were calculated using a two-tailed Student t test (*, P � 0.05).

FIG 7 Model for how BRG1 and CHD4 antagonistically influence Wnt sig-naling during yolk sac vascular development. BRG1 activates the Wnt signal-ing pathway in two ways: through transcriptional regulation of multiple Fzdreceptor genes upstream of �-catenin and through coregulation of Wnt targetgenes downstream of �-catenin (12). CHD4 represses the Wnt signaling path-way downstream of �-catenin through transcriptional regulation of the Wnt-responsive transcription factor Tcf7 and select Wnt target genes.

BRG1 and CHD4 Regulate Wnt Signaling

April 2012 Volume 32 Number 7 mcb.asm.org 1319

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 13

Jan

uary

202

2 by

1.4

.174

.198

.

4. Corada M, et al. 2010. The Wnt/beta-catenin pathway modulates vascu-lar remodeling and specification by upregulating Dll4/Notch signaling.Dev. Cell 18:938 –949.

5. Dejana E. 2010. The role of wnt signaling in physiological and patholog-ical angiogenesis. Circ. Res. 107:943–952.

6. Eferl R, et al. 1999. Functions of c-Jun in liver and heart development. J.Cell Biol. 145:1049 –1061.

7. Gage PJ, Suh H, Camper SA. 1999. Dosage requirement of Pitx2 fordevelopment of multiple organs. Development 126:4643– 4651.

8. Gao H, et al. 2009. Opposing effects of SWI/SNF and Mi-2/NuRD chro-matin remodeling complexes on epigenetic reprogramming by EBF andPax5. Proc. Natl. Acad. Sci. U. S. A. 106:11258 –11263.

9. Gebuhr TC, et al. 2003. The role of Brg1, a catalytic subunit of mamma-lian chromatin-remodeling complexes, in T cell development. J. Exp.Med. 198:1937–1949.

10. Gregory GD, et al. 2010. FOG1 requires NuRD to promote hematopoiesisand maintain lineage fidelity within the megakaryocytic-erythroid com-partment. Blood 115:2156 –2166.

11. Griffin CT, Brennan J, Magnuson T. 2008. The chromatin-remodelingenzyme BRG1 plays an essential role in primitive erythropoiesis and vas-cular development. Development 135:493–500.

12. Griffin CT, Curtis CD, Davis RB, Muthukumar V, Magnuson T. 2011.The chromatin-remodeling enzyme BRG1 modulates vascular Wnt sig-naling at two levels. Proc. Natl. Acad. Sci. U. S. A. 108:2282–2287.

13. Hang CT, et al. 2010. Chromatin regulation by Brg1 underlies heartmuscle development and disease. Nature 466:62– 67.

14. Hargreaves DC, Crabtree GR. 2011. ATP-dependent chromatin remod-eling: genetics, genomics and mechanisms. Cell Res. 21:396 – 420.

15. Ho L, Crabtree GR. 2010. Chromatin remodelling during development.Nature 463:474 – 484.

16. Indra AK, et al. 2005. Temporally controlled targeted somatic mutagen-esis in embryonic surface ectoderm and fetal epidermal keratinocytes un-veils two distinct developmental functions of BRG1 in limb morphogen-esis and skin barrier formation. Development 132:4533– 4544.

17. Jochum W, Passegue E, Wagner EF. 2001. AP-1 in mouse developmentand tumorigenesis. Oncogene 20:2401–2412.

18. Kashiwagi M, Morgan BA, Georgopoulos K. 2007. The chromatin re-modeler Mi-2beta is required for establishment of the basal epidermis andnormal differentiation of its progeny. Development 134:1571–1582.

19. Kisanuki YY, et al. 2001. Tie2-Cre transgenic mice: a new model forendothelial cell-lineage analysis in vivo. Dev. Biol. 230:230 –242.

20. Kwon CS, Wagner D. 2007. Unwinding chromatin for development andgrowth: a few genes at a time. Trends Genet. 23:403– 412.

21. Lin CR, et al. 1999. Pitx2 regulates lung asymmetry, cardiac positioningand pituitary and tooth morphogenesis. Nature 401:279 –282.

22. Liu W, Selever J, Lu MF, Martin JF. 2003. Genetic dissection of Pitx2 incraniofacial development uncovers new functions in branchial arch mor-phogenesis, late aspects of tooth morphogenesis and cell migration. De-velopment 130:6375– 6385.

23. Lu MF, Pressman C, Dyer R, Johnson RL, Martin JF. 1999. Function ofRieger syndrome gene in left-right asymmetry and craniofacial develop-ment. Nature 401:276 –278.

24. Lucitti JL, et al. 2007. Vascular remodeling of the mouse yolk sac requireshemodynamic force. Development 134:3317–3326.

25. MacDonald BT, Tamai K, He X. 2009. Wnt/beta-catenin signaling:components, mechanisms, and diseases. Dev. Cell 17:9 –26.

26. Murakami M, et al. 2008. The FGF system has a key role in regulatingvascular integrity. J. Clin. Invest. 118:3355–3366.

27. Nilasena DS, Trieu EP, Targoff IN. 1995. Analysis of the Mi-2 autoan-tigen of dermatomyositis. Arthritis Rheum. 38:123–128.

28. Pestronk A, Schmidt RE, Choksi R. 2010. Vascular pathology indermatomyositis and anatomic relations to myopathology. MuscleNerve 42:53– 61.

29. Ramirez J, Hagman J. 2009. The Mi-2/NuRD complex: a critical epige-netic regulator of hematopoietic development, differentiation and cancer.Epigenetics 4:532–536.

30. Ramirez-Carrozzi VR, et al. 2006. Selective and antagonistic functions ofSWI/SNF and Mi-2beta nucleosome remodeling complexes during an in-flammatory response. Genes Dev. 20:282–296.

31. Reyes JC, et al. 1998. Altered control of cellular proliferation in theabsence of mammalian brahma (SNF2alpha). EMBO J. 17:6979 – 6991.

32. Stankunas K, et al. 2008. Endocardial Brg1 represses ADAMTS1 to main-tain the microenvironment for myocardial morphogenesis. Dev. Cell 14:298 –311.

33. Takeuchi JK, et al. 2011. Chromatin remodelling complex dosage mod-ulates transcription factor function in heart development. Nat. Commun.2:187.

34. Tong JK, Hassig CA, Schnitzler GR, Kingston RE, Schreiber SL. 1998.Chromatin deacetylation by an ATP-dependent nucleosome remodellingcomplex. Nature 395:917–921.

35. Wang SJ, Greer P, Auerbach R. 1996. Isolation and propagation ofyolk-sac-derived endothelial cells from a hypervascular transgenic mouseexpressing a gain-of-function fps/fes proto-oncogene. In Vitro Cell. Dev.Biol. Anim. 32:292–299.

36. Williams CJ, et al. 2004. The chromatin remodeler Mi-2beta is requiredfor CD4 expression and T cell development. Immunity 20:719 –733.

37. Wu JI, Lessard J, Crabtree GR. 2009. Understanding the words of chro-matin regulation. Cell 136:200 –206.

38. Wu JI, et al. 2007. Regulation of dendritic development by neuron-specific chromatin remodeling complexes. Neuron 56:94 –108.

39. Zhang J, et al. 2012. Harnessing of the nucleosome-remodeling-deacetylase complex controls lymphocyte development and prevents leu-kemogenesis. Nat. Immunol. 13:86 –94.

40. Zhang M, et al. 2011. SWI/SNF complexes containing Brahma orBrahma-related gene 1 play distinct roles in smooth muscle development.Mol. Cell. Biol. 31:2618 –2631.

Curtis and Griffin

1320 mcb.asm.org Molecular and Cellular Biology

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 13

Jan

uary

202

2 by

1.4

.174

.198

.