SWI/SNF Chromatin Remodeling ATPase Brm Regulates the Differentiation of Early Retinal Stem Cells/Progenitors by Influencing Brn3b Expression and Notch Signaling

SWI/SNF Chromatin Remodeling ATPase Brm Regulates theDifferentiation of Early Retinal Stem Cells/Progenitors byInfluencing Brn3b Expression and Notch Signaling*

Received for publication, August 14, 2007 Published, JBC Papers in Press, September 11, 2007, DOI 10.1074/jbc.M706742200

Ani V. Das‡, Jackson James§, Sumitra Bhattacharya‡, Anthony N. Imbalzano¶, Marie Lue Antony‡, Ganapati Hegde‡,Xing Zhao‡, Kavita Mallya‡, Faraz Ahmad‡, Eric Knudsen�, and Iqbal Ahmad‡1

From the ‡Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198,the §Department of Neurobiology, Rajiv Gandhi Center for Biotechnology, Kerala 695014, India, the ¶Department of Cell Biology,University of Massachusetts Medical School, Worcester, Massachusetts 01655, and the �Department of Cell and Cancer Biology,University of Cincinnati, Cincinnati, Ohio 45267

Based on a variety of approaches, evidence suggests that dif-ferent cell types in the vertebrate retina are generated by multi-potential progenitors in response to interactions between cellintrinsic and cell extrinsic factors. The identity of some of thecellular determinants that mediate such interactions hasemerged, shedding light on mechanisms underlying cell differ-entiation. For example, we know now that Notch signalingmediates the influence of the microenvironment on states ofcommitment of the progenitors by activating transcriptionalrepressors. Cell intrinsic factors such as the proneural basichelix-loop-helix and homeodomain transcription factors regu-late a network of genes necessary for cell differentiation andmaturation. What is missing from this picture is the role ofdevelopmental chromatin remodeling in coordinating theexpression of disparate classes of genes for the differentiation ofretinal progenitors.Herewedescribe the role ofBrm, anATPasein the SWI/SNF chromatin remodeling complex, in the differ-entiation of retinal progenitors into retinal ganglion cells. Usingthe perturbation of expression and function analyses, we dem-onstrate that Brm promotes retinal ganglion cell differentiationby facilitating the expression and function of a key regulator ofretinal ganglion cells, Brn3b, and the inhibition of Notch signal-ing. In addition, we demonstrate that Brm promotes cell cycleexit during retinal ganglion cell differentiation. Together, ourresults suggest that Brm represents one of the nexus wherediverse information of cell differentiation is integrated duringcell differentiation.

Cell fate specification and subsequent cell differentiation inthe nervous system are orchestrated and finessed by interplaybetween cell intrinsic and cell extrinsic factors. This process isexemplified during the development of the retina, an excellent

model of the central nervous system. Recent evidence suggeststhat disparate transcription factors belonging to basic helix-loop-helix, homeodomain, and zinc finger classes cooperatetoward lineage specification and differentiation (1–6). Forexample, the basic helix-loop-helix transcription factor,Math5,and the homeodomain transcription factor, Pax6, have beenshown to cooperate during the specification of retinal progen-itors into retinal ganglion cells (RGCs)2 (2, 7–9). As progenitorsare committed along RGC lineage,Wt1, a zinc finger transcrip-tion factor, and Brn3b, a homeodomain transcription factor ofPOU class, are expressed and ultimately promote the differen-tiation and maturation of the specified progenitors into RGCs(10). The progress in the identification of intrinsic factors hasbeen paralleled by the characterization of extrinsic factors andintercellular signaling pathways, e.g.Notch pathway, thatmedi-ates the regulatory influence of the microenvironment on reti-nal progenitors’ maintenance and their differentiation intoRGCs (11–15).Although the identification of cell intrinsic and cell extrinsic

factors has helped our understanding of the mechanisms thatregulate the differentiation of retinal cell types, particularly thatof RGCs, it is not known as to how the remodeling of chroma-tin, which is necessary for eukaryotic gene expression, isrecruited toward the coordinated regulation of genes duringRGC differentiation. There are two major classes of chromatinremodeling complexes, that are associated with specificenzymes that remodel chromatin by changing nucleosomestructure or position (16, 17). The first class includes complexesconsisting of histone acetyltransferase or histone deacetylasethat covalently modify chromatin by adding and removingacetyl groups from the amino terminus of the four core his-tones, respectively. The second class includes SWI/SNF com-plexes that utilize the energy obtained from ATP hydrolysis todisrupt nucleosomal structure or position. The mammalianSWI/SNF complexes consist of 10–12 proteins, including thehomologous but mutually exclusive ATPases, Brahma (Brm)and Brm related gene 1 (Brg1) (16, 18).

* This work was supported by The Lincy Foundation, Pearsons Foundation,Nebraska Tobacco Fund for Biomedical Research, and Research to PreventBlindness. The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

1 To whom correspondence should be addressed: Dept. of Ophthalmologyand Visual Sciences, 4044 Durham Research Center, University of NebraskaMedical Center, Omaha, NE 68198-5840. Tel.: 402-559-4091; Fax: 402-559-3251; E-mail: [email protected].

2 The abbreviations used are: RGC, retinal ganglion cell; ChIP, chromatinimmunoprecipitation; Rb, retinoblastoma; BrdUrd, bromo-2-deoxyuri-dine; siRNA, short interfering RNA; E, embryonic day; RT, reverse transcrip-tion; Tunel, terminal dUTP nick-end labeling; NICD, Notch intracellulardomain; FACS, fluorescence-activated cell sorter; PN, postnatal; LM,ligation-mediated.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 48, pp. 35187–35201, November 30, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

NOVEMBER 30, 2007 • VOLUME 282 • NUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 35187

by guest on August 11, 2016

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Although both major classes of chromatin remodeling com-plexes are likely to contribute to developmental processes,including those in the central nervous system (19–21), evi-dence is emerging that the SWI/SNF chromatin remodelingcomplexes play an important role in the differentiation of spe-cific cell types (22). For example, these complexes have beenshown to facilitate the differentiation of a variety of cell typessuch as erythrocytes (7, 23), macrophages (24), myeloid cells(25), adipocytes (26), myoblasts (27), osteoblasts (28), neurons(29, 30), and glia (31). Here we demonstrate the role of Brm inthe differentiation of retinal progenitors into RGCs. Brm isexpressed in the developing rat retina, and its temporal andspatial patterns of expression correlate with retinal histogene-sis. Using perturbation of expression and function analyses, wedemonstrate that Brm influences the differentiation of retinalprogenitors into RGCs by facilitating Brn3b expression andfunction and inhibiting Notch signaling. In addition, we dem-onstrate that Brm may influence differentiation generically bypromoting cell cycle exit. Together, our results suggest thatchromatin remodeling by Brm may represent one of the nexuswhere cell intrinsic and cell extrinsic influence may be inte-grated toward the differentiation of retinal progenitors.

EXPERIMENTAL PROCEDURES

Progenitor Cell Culture—Timed-pregnant (E14) Sprague-Dawley rats were obtained from Sasco (Wilmington, MA). Thegestation daywas confirmed by themorphological examinationof embryos (32). Fertilized hen eggs were incubated in a humid-ified chamber at 38 °C, and embryos were staged according toHamburger and Hamilton (33). Embryos were harvested atappropriate gestation periods, and eyeswere enucleated. Retinawere dissected out and dissociated as described previously (34).Cells were cultured in RCM (Dulbecco’smodified Eagle’smedi-um/F-12, 1� N2 supplement (Invitrogen), 2 mM L-glutamine,100 units/ml penicillin, 100 �g/ml streptomycin) containingFGF2 (10 ng/ml), epidermal growth factor (20 ng/ml), and 0.1%fetal bovine serum for 5 days to generate clonal neurospheres.5-Bromo-2-deoxyuridine (BrdUrd) (10 �M) was added to theculture for the final 24 h. For co-culture, neurospheres werecollected, washed extensively to remove BrdUrd, and plated onpoly-D-lysine- and laminin-coated glass coverslips with E3chick/PN1 rat retinal cells in 1% fetal bovine serum. For RT-PCR analysis co-culture was performed across a 0.4-�mmem-brane (Millipore, Bedford, MA). Cells were either frozen forRNA extraction or fixed with 4% paraformaldehyde for 15 minat 4 °C for immunofluorescence analysis.ImmunofluorescenceAnalysis—Immunocytochemical analy-

sis for detection of cell-specific markers and BrdUrd wasperformed as described previously (35). Briefly, paraformalde-hyde-fixed cells/sections were incubated in 1� phosphate-buffered saline containing 5% Normal Goat Serum and 0.2–0.4% Triton X-100 followed by an overnight incubation inappropriate dilutions of antibodies against Brm (Santa CruzBiotechnology), Notch1 (Santa Cruz Biotechnology), Shh(Developmental Studies Hybridoma Bank), Brn3b (Covance),RPF1 (36), and BrdUrd (Accurate Chemical and Scientific Corp.)at 4 °C. Cells/sections were examined for epifluorescence afterincubation with IgG, conjugated to Cy3/fluorescein isothiocya-

nate. Images were captured using a CCD camera (PrincetonInstruments, Trenton, NJ) and Openlab software (Improvision,Lexington,MA).Cell Cycle Analysis—The DNA content of the retinal cells

was measured by flow cytometry using propidium iodide asdescribed (37). Cell cycle analysis was done using a FACStarflow cytometer (BD Biosciences).Semi-quantitative RT-PCR—Total RNA was isolated from

frozen cells or explants using Qiagen RNA isolation kit (Valen-cia, CA), and cDNA synthesis was performed as described pre-viously using�5�g of total RNA (38). Specific transcripts wereamplified with gene-specific forward and reverse primers usinga step-cycle program on a Robocycler (Stratagene, La Jolla, CA)for less than 30 cycles to keep the amplificationwithin the rangeof linearity. The gene-specific primers used for RT-PCR aredescribed in Table 1.Northern Analysis—Northern analysis was carried out to

detect rBrm transcripts as described previously (39). Briefly,�2�g of poly(A) RNA, isolated from adult retina, was electro-phoresed on a 1.2% formaldehyde gel and transferred toNytranPlus. Hybridization was carried out using a 32P-labeled rBrmcDNA probe overnight at 65 °C. Blot was washed sequentiallywith 2� SSC, 0.1% SDS at room temperature for 20 min, thentwice with 1� SSC, 0.1% SDS at 65 °C for 15 min, and finallywith 0.1� SSC, 0.1% SDS at 65 °C for 10 min, followed byautoradiography.siRNA Electroporation—Brm siRNA sequence was cloned

into pSuper vector (Oligogene) according to the vendor’s pro-tocol. Electroporation was carried out according to a modifiedprotocol of Matsuda and Cepko (40). Briefly, E14 retinas weredissected out and collected in Hanks’ balanced salt solutioncontaining pSuper-Brm and pEGFP-C3. The explants wereplaced in wells made in a 2% agarose gel. Electroporation inagarose gel reduced the shock, and thereby cell death. It alsoprevented aggregation of explants with one another. Electropo-ration was carried out by applying 5 pulses at 40 V for 50-msdurations with 950-ms intervals using gold-plated electrodesand an Electro Square Porator (BTX Inc.). The efficiency ofelectroporation was monitored by green fluorescent proteinepifluorescence, and the explants were cultured in RCM con-taining 5% fetal bovine serum for 4 days. The effect of siRNAwas ascertained by analyzing the Brm/�-actin protein andmRNA levels by Western and RT-PCR analyses, respectively.Recombinant Virus Preparation and Infection—Brm (pBabe-

Brm), dominant negative Brm (pBabe-dnBrm), and empty(pBabe) retrovirus were made in BOSC-23 cells using the cal-cium phosphate method, as described previously (27). Neuro-spheres/RGC-5 cells, at�60% confluency, were exposed to ret-rovirus containing medium for 24 h. After infection, themedium was replaced with fresh culture medium.Reporter Assay—RGC-5/293T cells, transduced with pBabe/

pBabe-Brm/pBabe-dnBrmwere transfectedwith pGL2-Brn3b-luciferase (RGC-5 cells) and pGV-B-Hes1/Hes5 luciferaseconstructs (293T cells) using Lipofectamine (Invitrogen).Transfection efficiency was examined by co-transfecting cellswith pGFP-C3 (Clontech). For luciferase assay, cells were lysedin 1� reporter lysis buffer (Promega), and 100 �l of lysate wasdiluted five times using assay reagent (Promega). Diluted sam-

Brm-mediated Differentiation of Retinal Stem Cells/Progenitors

35188 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 48 • NOVEMBER 30, 2007


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

ples (100�l) were analyzed for luciferase activities using a lumi-nometer (Pharmingen).Co-immunoprecipitation—Total protein was extracted from

RGC-5 cells using aM-PER protein extraction kit (Pierce). Fivehundredmicrograms of total protein in 1ml of RIPA buffer (50mMTris-HCl, pH 7.4, 15mMNaCl, 1%Triton X-100, 0.1% SDS,1mMEDTA, 1% sodiumdeoxycholate) was incubatedwith 5�gof antibody overnight at 4 °C. Protein-antibody complex wasprecipitated by incubating with protein A/G-Sepharose for 1 hat 4 °C. The protein-antibody-Sepharose complex was precipi-tated by centrifuging at 1000 rpm for 1 min, and precipitateswere washed three times with RIPA buffer, and the final pelletwas resuspended in appropriate volume of loading buffer. Themixturewas boiled to dissociate the complex and electrophore-sed in a 7–9% denaturing polyacrylamide gel. Negative controlsincluded reactions carried out without the antibody and withIgG.Western Blot Analysis—Western blot analysis was done as

described previously (39). Samples from co-immunoprecipita-tion or protein isolated from siRNA-transfected retinas wereelectroblotted onto polyvinylidene difluoride membranes, fol-lowing electrophoresis. Membranes were blocked for 1 h in 5%nonfat dry milk in TBST and incubated with anti-Brm/anti-Notch (SantaCruz Biotechnology), diluted 1:500 inTBSTover-night at 4 °C with shaking. After incubating with anti-mousehorseradish peroxidase, the blots were washed with TBST, andimmunoreactive bands were detected using ECLWestern blot-ting detection reagents (RPN 2108, Amersham Biosciences).The blots were then exposed to x-ray film to visualize immuno-reactive bands.Chromatin Immunoprecipitation—Chromatin immunopre-

cipitation (ChIP) assay was done using a modified procedurefrom Upstate Biotechnology, Inc. Briefly, transduced (pBabe/

pBabe-Brm) or transfected (pSuper-Brm siRNa/Wt1 (KTS�))RGC5 cells were grown until they reached confluency, and his-tones were cross-linked to DNA by adding formaldehydedirectly to culture medium to a final concentration of 1% andincubating for 10 min at room temperature on a rocking plat-form. Cells were washed three times with ice-cold phosphate-buffered saline containing protease inhibitors. Cell pellets wereresuspended in prewarmed SDS lysis buffer (1% SDS, 10 mM

EDTA, 50 mM Tris, pH 8.1). To reduce the nonspecific back-ground, the samples were pre-cleared using 80 �l of salmonsperm DNA/protein A-agarose slurry at 4 °C for 30 min. Sam-pleswere centrifuged at 100 rpm for 1min at 4 °C. Supernatantswere transferred to a new tube, and the immunoprecipitatingantibody was added, and incubation was carried out overnightat 4 °C on a rocking platform. For a negative control, we used noantibody or IgG. The histone-antibody complex was precipi-tated using 60 �l of salmon sperm DNA and protein A-Sepha-rose (Upstate) for 1 h at 4 °C. Precipitates were washed sequen-tially at room temperature for 5min, oncewith low salt immunecomplex wash buffer (0.1% SDS, 1%TritonX-100, 2mMEDTA,20 mM Tris-Cl, pH 8.1, 150 mM NaCl), high salt immune com-plex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20mM Tris-Cl, pH 8.1, 500 mM NaCl), and lithium salt immunecomplex wash buffer (2.25 M LiCl, 1% IGEPAL-CA630, 1%deoxycholic acid-sodiumsalt, 1mMEDTA, 10mMTris, pH8.1),and twice with TE buffer (10 mMTris-Cl, 1 mM EDTA, pH 8.0).After completely removing the TE buffer, the precipitate wasresuspended and extracted twice in 250 �l of freshly preparedelution buffer (1% SDS, 0.1MNaHCO3). To reverse the histone-DNA cross-linking, samples were heated at 65 °C for 4 h. 200�lof initial sonicated sample was reverse cross-linked and used asan input. After removing the antibodies by protease digestion of

TABLE 1List of primers and their respective sequences used for RT-PCR and ChIP analysis

Genes Primer sequences Productsize

GenBankTMaccession no. Temperature

bp °C�-actin Forward, 5�-GTGGGGCGCCCCAGGCACCA-3� 548 XM_037235 50

Reverse, 5�-CTCCTTAATGTCACGCACGATTTC-3�Hes1 Forward, 5�-GCTTTCCTCATCCCCAAAG-3� 224 NM_024360 56

Reverse, 5�-CGTATTTAGTGTCCGTCAGAAGAG-3�Ki67 Forward, 5�-GAGCAGTTACAGGGAACCGAAG3-� 262 X82786 58

Reverse, 5�-CCTACTTTGGGTGAAGAGGCTG3-�Brn3b Forward, 5�-GGCTGGAGGAAGCAGAGAAATC-3� 141 AF390076141 60

Reverse, 5�-TTGGCTGGATGGCGAAGTAG-3�RPF1 Forward, 5�-TTTCAGGGGATTCTGGTGTGC-3� 359 XM_344604 56

Reverse, 5�-CGCTTTTTGGATGGCTCACTC-3�Brm Forward, 5�-TGCCCTGTAATTCTCAGTTGGA-3� 180 XM_005383 52

Reverse, 5�-CTCCCAGGCTTCGGTACTTATG-3�Cyclin A Forward, 5�-TACACACACGAGGTAGTGACGCTG-3� 307 X60767 56

Reverse, 5�-CCAAGCCGTTTTCATCCAGG-3�Cyclin E Forward, 5�-TGACAAGACTGTGAAAAGCCAGG-3� 303 D14015 59

Reverse, 5�-AGAAGAACTGCTCTCATCCTCGC-3�Shh Forward, 5�-AGAGGCAGCACCCCAAAAAG-3� 464 NM_017221 56

Reverse, 5�-TTTCACAGAGCAGTGGATGCG-3�Hes5 Forward, 5�-TGGAGATGCTCAGTCCCAAG-3� 199 NM_024383 58

Reverse, 5�-GCTTTGCTGTGCTTCAGGTAG-3�Brn3b promoter Forward, 5�-CAGCCCCCGAGGGTGTGTGT-3� 256 NM_138944 58

Reverse, 5�-TCTGAACGGGTGCCGAGGTCT-3�Shh enhancer Forward, 5�-GGAGGCTTCAGGAACAACTTGG-3� 386 AF098925 60

Reverse, 5�-GGGTAGGATGGATAGGGTTTTGG-3�Hes1 promoter Forward, 5�-TCTCCTTGGTCCTGGAATAGTGC-3� 398 D16464 56

Reverse, 5�-ATCTGCCATTTCACCCCGAG-3�CD11b promoter Forward, 5�-GACCAGGCAGGGCTATGT-3� 122 M84477 52

Reverse, 5�-AAAGCAAAGAAGGGCAGAAA-3�




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

the samples, DNA was recovered and column-purified. PCRswere performed using gene-specific primers.Restriction Enzyme Accessibility Assay—Restriction enzyme

accessibility using LM-PCR assay was carried out as describedpreviously (41). Briefly, 5 � 106 nuclei from pBabe/pBabe-Brmtransduced RGC5 cells were subjected to BglII (75–100 units)restriction enzyme digest for 30 min at 37 °C. First strand syn-thesis of genomicDNA (100 ng) was carried out using PfuDNApolymerase (Stratagene) and gene-specific forward primer (5�-acccacgtctttctgcactag-3�). Following linker ligation, using T4DNA ligase overnight at 17 °C, PCR was carried out on theligated products using the gene-specific forward primer andlinker-specific reverse primer (5�-ccgggagatctgaattcgat-3�) for25 cycles at an annealing temperature at 65 °C. The PCR prod-ucts were resolved on 1.2% agarose gel followed by Southernanalysis using a PCR product corresponding to a sequencebetween the second TaqI site and the BgllII site in proximalBrn3b promoter as a probe (Fig. 6D).Statistics—Results were expressed as mean � S.E. of at least

three separate experiments. Statistical analyses were done

using Student’s t test to determine the significance of the dif-ferences between the various conditions.

RESULTS

Expression of Brm in the Developing Retina—Emerging evi-dence suggests that Brm plays an important role in cell differ-entiation (27, 42). To ascertainwhether or not Brmhas a similarrole in retinal development, we examined the temporal patternsof Brm expression during early (E12–E18) and late (E18-PN6)retinal histogenesis by RT-PCR analysis. Retinal cells are bornin an evolutionarily conserved temporal sequence; RGCs, conephotoreceptors, horizontal cells, and a majority of amacrinecells are born during early histogenesis, whereas rod photorecep-tors, bipolar cells, and Muller glia are generated during latehistogenesis (43, 44).Weobserved that levels of Brm transcriptsincreased at E14 stage, compared with those at the E12 stage(Fig. 1,A and B). The stages between E12 and E14 represent theperiod of active neurogenesis when the majority of RGCs, hor-izontal cells, and cone photoreceptors are born (45). Thedecrease in the expression of Brm at stage E16 is likely because

FIGURE 1. Temporal and spatial expression of Brm in rat retina. Temporal analysis of Brm expression in the developing and adult retina by RT-PCR analysisreveals that levels of Brm transcripts increase with the onset of differentiation in both early and late stages of retinal histogenesis (A and B). Northern analysis,carried out using rat Brm cDNA as a probe, on mRNA isolated from adult retina shows 5.8-kb transcripts corresponding to full-length Brm mRNA (C). Immuno-histochemical analysis, carried out on E14 (D–G) and E18 (H–K) retina, obtained from embryos in utero treated with BrdUrd, reveals Brm immunoreactivities(arrows) in E14 retina, distributed both toward scleral (s) and ventricular (v) sides. In contrast, in E18 retina, Brm immunoreactivities (arrows) are predominantlylocalized toward the scleral side in the inner retina (IR). As expected, BrdUrd immunoreactivities are distributed throughout the width of E14 retina, whereasthey are confined in the outer neuroblastic retina (OR) in E18 retina, where proliferating progenitors are distributed. Immunocytochemical analysis, carried outon cell dissociates from BrdUrd-exposed E14 (L–P) and E18 (P) retina, demonstrates that Brm immunoreactivities are associated with both BrdU� and BrdU�

cells (O). However, the proportion of BrdU� and Brm� cells is significantly (p � .001) higher, compared with that of BrdU� and Brm� cells, suggesting acorrelation of Brm expression with the process of retinal differentiation. Magnification, �200.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

of the fact that E16 represents a stage of relative quiescenceduring early histogenesis (12). Similarly, during late histogene-sis, there was a significant increase in levels of Brm transcriptsat PN1 and PN3, comparedwith those at E18when themajorityof rod photoreceptors, which constitute �80% of total cells inrodent retina, are born (45). The Brm expression persistedthrough postnatal stages and its levels were highest in adultretina, compared with all stages examined. In addition, thespecificity of Brm expression was determined by sequencingPCR products and Northern analysis of RNA from the adultretina, which revealed a 5.8-kb band corresponding to full-length Brm transcripts (46) (Fig. 1C). Together, these resultssuggested that Brm expression is associated with retinal histo-genesis. To obtain further insight into the involvement of Brmwith the process of cell differentiation in retina, we examinedthe cell-specific expression of Brm in the retina of E14 and E18embryos. We observed that Brm immunoreactivities were dis-tributed toward the scleral and ventricular sides in the develop-ing retina (Fig. 1,D–I). In both E14 and E18 retina, Brm immu-noreactivities were predominantly localized toward the scleralside where differentiating precursors are located. As reportedpreviously, Brm immunoreactivities hadnuclear aswell as cyto-plasmic localization (47). Next, to test whether or not Brmexpression is associated with differentiating precursor popula-tion, we examined the cellular distribution of Brm immu-noreativities in the retina of E14 and E18 embryos, pre-exposedto BrdUrd in utero to identify proliferating (BrdU�) cells (Fig.1J). We observed that Brm immunoreactivities were associatedwith both BrdU� and BrdU� cells. However, in the total cellpopulation at E14, the proportion of BrdU�Brm� cells (6.8 �0.23) was significantly higher than those of BrdU�Brm� cells(4.09 � 0.23), suggesting that Brm expression was predomi-nantly associated with post-mitotic cells (Fig. 1L). A compari-son of different classes of cells at the two different stagesrevealed that the proportion of BrdU�Brm� cells increased�2-fold in E18 retina relative to that in E14 retina (12.43� 0.75versus 6.8 � 0.23, p � 0.05), demonstrating a progressive asso-ciation of Brm with post-mitotic cells as development pro-gressed. Together, these observations suggested that theexpression of Brm correlatedwith the process of differentiationof retinal stem cells/progenitors.Next, we examined the involvement of Brm expression and

function in the differentiation of stem cells/progenitors along aspecific lineage. Although it is likely that Brm is involved in thedifferentiation of multiple retinal cell types, we studied Brm inthe context of RGC differentiation because these are the firstcells born (15), and their regulators and markers (10) are wellcharacterized, thus affording an unambiguous interpretation ofresults. To know whether or not there is a correlation betweenBrm expression and RGC differentiation, we examined the spa-tial and cellular distribution of Brm immunoreactivities in thedeveloping retina. Immunohistochemical analysis of E14 retinarevealed a spatial overlap of Brm immunoreactivities with thatof Brn3b (Fig. 2, A–D) and RPF1 (Fig. 2, E–H). Immunocyto-chemical analysis of freshly dissociated E14 retinal cellsrevealed that Brm immunoreactivities are co-localized withthat of regulators of RGCs, Brn3b, Wt1, RPF1, and Shh in asubset of cells (Fig. 2, I–L). Together these observations dem-

onstrated the association of Brm expressionwith nascent RGCsin E14 retina. To determine the temporal association of Brmexpression with RGC differentiation, we carried out immuno-histochemical analysis of retinal cell dissociates from embryosin different stages (E12, E14, E16, and E18 stages) of early his-togenesis. We observed that a subset of Brm� cells expressedBrn3b at all stages of early histogenesis, albeit at different pro-portions (Fig. 2M). For example, compared with those at E12stage, the proportion of Brm�Brn3b� cells increased by�2-fold at E14, the peak of RGC generation (23.9 � 3.0 versus46.0 � 2.6, p � 0.001). Subsequently, the number ofBrm�Brn3b� cells decreased with the time; compared withthose at E14, the proportion of such cells decreased at E18 stage,by which time themajority of RGCs is differentiated (46.0� 2.6versus 4.2� 0.89, p� 0.001). These results are indicated towardthe association of Brm with RGC differentiation in vivo; Brm isexpressed in differentiating RGCs, and the expression is pro-gressively extinguished from these cells as they complete theirdifferentiation. To know whether Brm had a specific role inRGC differentiation required information regarding thedynamic patterns of Brm expression during RGC differentia-tion. Does Brm expression correlate with the process of RGCdifferentiation? This issue was addressed using neurosphereassay, which serves as a robust model of RGC differentiation(48). Retinal stem cells/progenitors in neurospheres utilize nor-mal mechanism of RGC differentiation (38), and the resultingRGCs show target selectivity in terms of establishing contactswith cells in superior colliculus and not with those in inferiorcolliculus (49). Proliferating progenitors in E14 neurosphereswere tagged with BrdUrd, before shifting them to conditionsthat promote RGC differentiation. This was to ensure that weaccounted for the differentiation of only those cells that wereproliferating before the onset of differentiation, and not ofthose that had spontaneously differentiated by asymmetricaldivision. Samples of neurospheres were assayed at specifiedtimes (0, 6, 12, 24, and 48 h) to determine the temporal propor-tion of Brm� cells that were BrdUrd-tagged (BrdU�) orexpressed Brn3b immunoreactivities. We observed that moreBrm� cells were BrdUrd-tagged in differentiating conditions,and their proportions increased with the time, compared withthose in proliferating conditions (0 h), corroborating our earlierobservation that the expression of Brm is tightly correlatedwiththe process of differentiation (Fig. 2N). That the increase inBrm expression is specifically correlated with the differentia-tion of RGCs was demonstrated by a coincidental increasein the proportion of Brm� cells expressing Brn3b immunore-activities, comparedwith those in proliferating conditions (0 h).The difference in the proportion of Brm�Brn3b� andBrm�BrdU� cells is likely because of the presence of post-mi-totic precursors, which did not incorporate BrdUrd, and prolif-erating progenitors, which failed to incorporate BrdUrdbecause they were not in S phase of the cell cycle when exposedto BrdUrd.Functional Involvement of Brm in RGC Differentiation—The

correlation of Brm expression with RGC differentiation sug-gested that Brm influences the process of RGC differentiation.This premise was examined further by perturbing Brm expres-sion and function in retinal stem cells/progenitors and examin-




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

ing the effects on RGC differentiation. First, we used thesiRNA-mediated gene silencing approach to attenuate Brmexpression. pSuper vector (Oligogene) containing Brm siRNAwas co-electroporated with pEGFP-C3 plasmid in E14 rat reti-nal explants. Controls included explants, similarly electropo-rated with siRNA corresponding to scrambled Brm sequence.Because�90% of cells in E14 retina are mitotic (50), the major-

ity of cells electroporated with siRNA was likely to be prolifer-ating progenitors. The efficiency of siRNA electroporation wasdemonstrated by green fluorescent protein epifluorescence inexplant sections (Fig. 3A). The specificity of gene silencing wasdemonstrated by a decrease in levels of Brm protein and itscorresponding transcripts in Brm siRNA-treated explants,compared with controls (Fig. 3, B and H). As expected, the

FIGURE 2. Correlation of Brm expression with the process of RGC differentiation. Retina from E14 embryos were subjected to double immunohistochem-ical analysis to determine the spatial distribution of Brm immunoreactivities relative to that of Brn3b and RPF1. The distribution of Brm immunoreactivitiesoverlapped with Brn3b (A–D) and RPF1 (E–H) near the scleral side, where nascent RGCs are located. To demonstrate that Brm is expressed in nascent RGCs,double immunocytochemical analysis was carried out on freshly dissociated E14 retinal cells. Brm immunoreactivities are co-localized with multiple RGCregulators Wt1 (I–K), Brn3b (L–N), RPF1 (O–Q), and Shh (R–T). To determine the temporal association of Brm expression with RGC differentiation, retina from E12,E14, E16, and E18 embryos were dissociated and subjected to double immunohistochemical analyses to determine the proportion of Brm-positive cellsexpressing Brn3b (M). Compared with that at E12, the proportion of Brm� and Brn3b� cells increased significantly (p � 0.001) at E14, the peak of the generationof RGCs. The proportion of Brm� and Brn3b� cells decreased significantly (p � 0.001) at E18 stage, when the majority of RGCs have completed theirdifferentiation (U). To determine the association of Brm with the dynamics of RGC differentiation, proliferating E14 progenitors, tagged with BrdUrd in aneurosphere assay, were shifted to differentiating conditions, and double immunohistochemical analyses were carried out on an aliquot of cells at differenttime points to determine the temporal proportion of BrdU�Brm�/and Brm�Brn3b� cells (V). The proportion of BrdU�Brm� cells increased steadily indifferentiating conditions, compared with those in proliferating conditions (0 h), suggesting that as BrdUrd-tagged progenitors differentiate, they express Brm(N). The proportion of Brm�Brn3b� cells increased steadily in differentiating conditions, compared with those in proliferating conditions (0 h), suggesting that,with the time, Brm� cells differentiate into RGCs. Magnification, �200 (A–H), �400 (I–L); * � p � 0.05; ** � p � 0.001.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

expression levels of nontargeted �-actin protein and its corre-sponding transcripts were similar in both groups. The effectson RGC differentiation were examined by changes in the pro-portion of cells expressing immunoreactivities correspondingto the RGC regulators and markers, Brn3b and RPF1 (36).Immunoreactivities corresponding to both RPF1 (Fig. 3, C–F)and Brn3b (G–J) were decreased in siRNA-treated explants,compared with controls. The RPF1 immunoreactivities, dis-playing relatively discrete nuclear localization in explants thanBrn3b, were used for the quantitation of the number of nascentRGCs. We observed an �3-fold decrease in the proportion ofRPF1� cells in Brm siRNA-treated retinal explants, comparedwith controls (RPF1� cells, 70.0 � 12.41 versus 20 � 4.08, p �0.001), suggesting a decrease in RGC differentiation when Brmexpression is attenuated (Fig. 3K). RT-PCR analysis revealed adecrease in levels of regulators of RGCs, RPF1, Brn3b, and Shhtranscripts in Brm siRNA-treated explants, compared withcontrols, thus corroborating our immunohistochemical results.Together, these results demonstrated that the attenuation ofBrm expression adversely affected the number of cells express-ing RGC-specific markers. Second, we determined whether ornot Brm chromatin remodeling function was required for RGCdifferentiation. We overexpressed dominant negative Brm in

retinal progenitors through retrovi-rus transduction (pBabe-dnBrm) ofneurospheres culture. The domi-nant negative Brm contains a muta-tion in the ATPase domain, andwhen overexpressed it is expectedto participate in the formation of thenonfunctional ATPase chromatinremodeling complex, thus impair-ing activities of genes that requireBrm (46, 51). Controls includedneurospheres transduced with wildtype Brm (pBabe-Brm) and empty(pBabe) retrovirus. Following trans-duction, neurospheres were shiftedfrom proliferating to differentiatingconditions and scored for the pro-portion of cells expressing RGC-specific markers. Because retroviralinfection of retinal explants doesnot lead to a uniform transductionof retinal progenitors, which mightlead to ambiguous results, we usedE14 neurospheres instead. Weobserved a significant increase inthe proportion of both Brn3b� andRPF1� cells in neurospheresinfected with wild type Brm, com-pared with those infected withempty retrovirus (Brn3b� cells,68.3 � 2.3 versus 38.7 � 2.2, p �0.001; RPF1� cells, 56.0� 2.4 versus37.3� 4.2, p� 0.001) (Fig. 4,A–M).In contrast, there were significantlyfewer cells expressing RGCmarkers

in neurospheres infected with dominant negative Brm, com-pared with those infected with empty retrovirus (Brn3b� cells,38.7 � 2.2 versus 23.5 � 4.5, p � 0.05; RPF1� cells, 37.3 � 4.2versus 11.7 � 0.3, p � 0.005) (Fig. 4M) suggesting the involve-ment of Brm-mediated chromatin remodeling in RGC differ-entiation. RT-PCR analysis showed an increase and a decreasein levels of transcripts corresponding to Brn3b, RPF1, and Shhin neurospheres infected with wild type Brm retrovirus anddominant negative Brm retrovirus, respectively, comparedwithcontrols (Fig. 4N), corroborating the immunocytochemicalresults. There was no difference in Tunel-positive cells in con-trol and experimental groups demonstrating that the observeddifference in the number of RGCs was not because of Brm-induced survival of nascent RGCs (Fig. 4,O–T). Third, we wereinterested in knowing whether the lack of Brm could lead toRGC phenotype in vivo. We carried out immunohistochemicalanalysis of retinal sections obtained from adult Brm�/� andwild type mice. The retina of Brm�/� mice was comparablewith that of the wild type in terms of lamination and thick-ness. However, there were significantly fewer RPF1� andBrn3b� cells, observed more in the periphery than in thecenter, in Brm�/� retina, compared with wild controls(Fig. 5, A–E). Together, these observations suggested the

FIGURE 3. Effects of perturbation of Brm expression on RGC differentiation. To attenuate Brm expressionduring RGC differentiation, pSuper vector with Brm siRNA sequence was electroporated in E14 retinal explantswith pGFP-C3 plasmid (A). After 4 days in culture, retinal explants were subjected to Western blot, immunoflu-orescence, and RT-PCR analyses. Controls included explants electroporated with pSuper vector with scram-bled Brm siRNA sequence. The specificity of siRNA-mediated silencing of Brm expression was demonstrated bya decrease in the levels of Brm protein, compared with controls and no change in levels of �-actin protein (B).There was a significant decrease (p � 0.001) in the proportion of cells expressing RPF1 (C–F and K) and Brn3b(G–J) in Brm siRNA-treated explants, compared with controls. RT-PCR analysis showed a decrease in the levelsof Brm transcripts along with that of the regulators of RGCs (Brn3b, RPF1, and Shh) in Brm siRNA-treatedexplants (lane 2), compared with controls (lane 1), corroborating immunohistochemical results (L). Magnifica-tion, �200; * � p � 0.05; ** � p � 0.001.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

involvement of Brm expression and its function in RGCdifferentiation.Influence of Brm on the Regulation of Brn3b Expression and

Function—Next, we examined the mechanism of Brm-medi-ated RGCdifferentiation.Brn3b is a prominent RGC regulatorygene whose activation heralds phenotypic differentiation ofRGC precursors through subsequent activation of downstreamtarget genes such as Shh (10, 52). We were interested in know-ing whether Brm regulated RGC differentiation by facilitatingthe activation ofBrn3b and Shh. The decrease in levels ofBrn3band Shh transcripts in response to siRNA-mediated attenua-

tion of Brm expression (Fig. 3) andoverexpression of dominant nega-tive Brm (Fig. 4) suggested that Brmpositively regulated Brn3b and Shhexpression in retinal progenitors.This notion was further supportedby observations that the overex-pression of wild type Brm led to anincrease in levels of Brn3b and Shhtranscripts (Fig. 4). To know if Brmdirectly activated the Brn3b pro-moter, RGC-5 cells, a transformedRGC cell line (53, 54), transducedwith wild type Brm or empty retro-virus, were transiently transfectedwith Brn3b-Luc constructs (24). A4.5-fold increase in reporter activi-ties was observed in cells trans-duced with wild type Brm retrovi-rus, compared with those incontrols, suggesting a direct influ-ence of Brm on Brn3b promoteractivities (Fig. 6A). It has been dem-onstrated that ATPase chromatinremodeling complexes are recruitedto specific promoters by cell-spe-cific transcription factors where itremodels chromatin to facilitate theactivities of the promoters (55–57).To know if such a mechanism wasinvolved in Brm-mediated facilita-tion of Brn3b expression, we exam-ined whether or not Brm interactedwith one of the upstream regulatorsof Brn3b, Wt1 (4, 5). Co-immuno-precipitation analysis carried out onnuclear extracts from RGC5 cellsrevealed that Wt1 antibody precipi-tated protein complexes from thenuclear extract, which were immu-noreactive to Brm antibody (Fig.6B). This observation suggested thatBrm and Wt1 co-existed in proteincomplexes in RGC5 nuclei. Toknow whether Brmwas recruited toendogenous Brn3b promoter and ifthe recruitment was influenced by

Wt1, we carried out ChIP analysis on RGC5 cells. We observedthat DNA-protein complex precipitated by Brm antibody con-tained sequences corresponding to Brn3b promoter, confirm-ing the presence of BrmonBrn3b promoter (Fig. 6C). The spec-ificity of Brm interactions with the Brn3b promoter wasdemonstrated by an increase and decrease in the levels of PCRproducts in pBabe-Brm-transduced and Brm siRNA-treatedRGC5 cells, respectively, compared with those in respectivecontrols. To know whether or not Wt1 influenced interactionsof Brm with the Brn3b promoter, we carried out ChIP assay onRGC5 cells that were transfected with Wt1 expression con-

FIGURE 4. Effects of perturbation of Brm function on RGC differentiation. To perturb Brm function duringRGC differentiation, E14 neurospheres were transduced with dominant negative Brm (pBabe-dnBrm) and wildtype Brm (pBabe-Brm) retrovirus, followed by immunocytochemical analyses of cells expressing RGC markers,Brn3b and RPF1. Controls included neurospheres transduced with empty (pBabe) retrovirus. Expression of wildtype and dominant negative Brm led to a significant increase (p � 0.05) and decrease (p � 0.001) in theproportion of RPF1� (E, F, I, J, and M) and Brn3b� (G, H, K, L, and M) cells, respectively, compared with controls(A–D and M). RT-PCR analyses of transduced neurospheres revealed that expression of wild type Brm anddominant negative Brm led an increase and a decrease in levels of transcripts corresponding to Brn3b, RPF1,and Shh, respectively, compared with controls (N). Tunel analysis shows no significant difference in Tunel-positive cells in siRNA-treated and control explants (O–T). Magnification, �200. * � p � 0.05; ** � p � 0.001.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

structs, following transductionwith pBabe/pBabe-Brm retrovi-rus. We observed that the levels of amplified Brn3b promotersequence increasedwhen RGC5 cells overexpressedWt1, com-pared with controls. Together, these observations suggestedthat Brmwas recruited to the Brn3b promoter byWt1, and thisstepmay constitute amechanism to facilitateBrn3b expression.To know whether or not Brm expression is associated withnuclease accessibility at the endogenous Brn3b promoter, weexamined the restriction enzyme accessibility at the BglII site,�613 bp upstream of the first ATG site in Brn3b promoter inRGC5 cells transduced with pBabe/pBabe-Brm retrovirus. Inan LM-PCR assay, we observed a significant increase in theaccessibility to the BglII sites in RGC5 cells transduced withpBabe-Brm, compared with those transduced with empty ret-rovirus (pBabe), suggesting a Brm-mediated change in chroma-tin configuration at the Brn3b promoter (Fig. 6D).A similarmechanism could be invokedwhere the function of

Brn3b in activating Shh is facilitated byBrm.To test this notion,RGC5 cells, transducedwithwild type Brmor empty retrovirus,were transiently transfected with luciferase reporter constructsin which a highly conserved Shh regulatory sequence contain-ing a Brn3b-binding site was cloned upstream of a prolactin

minimal promoter (58). Controls included transfection oftransduced RGC5 cells with reporter constructs containingonly the minimal promoter. We observed an �6-fold increasein reporter activities in Brm-transduced cells, transfected withreporter constructs containing the Shh regulatory sequence,compared with activities in those that were transduced withempty retrovirus (Fig. 6E). Reporter activities were minimal intransduced cells, transfected with reporter constructs contain-ing the minimal prolactin promoter, demonstrating the speci-ficity of the Brn3b-binding site in Brm-mediated activation ofreporter. To know if Brm interacted with Brn3b, the upstreamregulator of Shh, we carried out co-immunoprecipitation anal-ysis on nuclear extracts from RGC5 cells. We observed Brn3bantibody-precipitated protein complexes that were immunore-active to Brm antibody, suggesting interactions between Brmand Brn3b (Fig. 6F). Next, to know whether Brm was recruitedto endogenous Shh promoter, we carried out ChIP analysis onRGC5 cells. We observed that the DNA-protein complex pre-cipitated by Brm antibody contained Shh regulatory sequenceswith Brn3b-binding site (Fig. 6G). The specificity of Brm inter-actions with Shh enhancer was demonstrated by increased anddecreased levels of PCR products in pBabe-Brm-transducedand Brm siRNA-treated RGC5 cells, respectively, comparedwith those in respective controls. Taken together, these resultsdemonstrated that Brm facilitated the expression and functionof Brn3b, a key regulator of RGC differentiation.Influence of Brm on Notch Signaling—One of the key regula-

tors of retinal progenitors during RGC differentiation is Notchsignaling. It is thought that attenuation of Notch signalingallows progenitors to commit along RGC lineage (11, 59).Because it has been shown previously that Brmmight interferewith Notch signaling by forming complexes with CSL (55), wewere interested in knowing if Brm influenced Notch signalingduring the differentiation of retinal progenitors into RGCs. ForBrm to regulateNotch signaling, it should be co-expressedwithNotch1 receptor in retinal progenitors. Immunocytochemicalanalysis of retinal progenitors in differentiating conditionsrevealed the co-localization of Brm and Notch1 immunoreac-tivities, suggesting their possible interactions (Fig. 7, A and B).To understand the nature of interactions between Brm onNotch signaling, we first wanted to know if Brm affected theexpression of Notch target genes,Hes1/Hes5, in retinal progen-itors. We observed that, in response to overexpression of dom-inant negative Brm, therewas an increase in levels of transcriptscorresponding to both Hes1 and Hes5, compared with those incontrol neurospheres, suggesting that Brmnegatively regulatedNotch signaling (Fig. 7C). Next, we wanted to know the mech-anism of Brm-mediated inhibition of Hes1 gene. Did Brmdirectly influence the activities of Hes1 promoter? To addressthis question, we transiently transfected Hes1-luc vectors inNotch intracellular domain (NICD) expressing 293T cells thatwere transduced with either wild type Brm retrovirus or emptyretrovirus. Reporter activities were easily detected in cellstransduced with Hes1-luc vectors and served as positive con-trols (Fig. 7D). We observed an �3-fold decrease in Hes1 pro-moter activities in cells transduced wild type Brm retrovirus,compared with controls, suggesting a direct negative effect ofBrm on Hes1 promoter activities (Fig. 7D). Next, we wanted to

FIGURE 5. RGCs in the retina of Brm�/� mice. To determine whether or notBrm�/� mice have an RGC phenotype, immunohistochemical analysis wascarried out on retina of 3-month-old Brm�/� and wild type mice (n � 3) (A–D).There was a significant decrease (p � 0.001) in the proportion of RPF1� (A–Dand I) and Brn3b� (E–I) cells in Brm�/� retina, compared with those in controls(E). Magnification, �200. * � p � 0.05; ** � p � 0.001.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

know whether or not these effectson promoter activities involve inter-actions between CSL/NICD andBrm. CSL is a transcriptionalrepressor, which is converted toa transcriptional activator whenNICD, cleaved upon the activationof Notch receptor, binds to it (60).To address this question, wecarried out a co-immunoprecipita-tion assay, performed on nuclearextracts from cells overexpressingBrm. We observed that proteincomplexes immunoprecipitated byCSL and NICD antibodies wereimmunoreactive to Brm, suggestinginteractions between Brm and CSL(Fig. 7E). To address the possibilitythat the faint the Brm immunoreac-tivity in the complex pulled down byNICD antibody was because of lowlevels Notch signaling, we carriedout co-immunoprecipitation on293T cells overexpressing NICD.There was an increase in Brmimmunoreactivity in a complex pre-cipitated by NICD antibody fromcells overexpressing NICD, com-pared with controls, confirming thespecificity of interactions betweenBrm and NICD. Next, to testwhether or not Brm is recruited tothe Hes1 promoter, we carried outChIP analysis in RGC5 cell lines.Weobserved that DNA-protein precip-itated by Brm contained Hes1 pro-moter sequences, suggesting thepresence of Brm as a part of Hes1transcriptional complex (Fig. 7F).The specificity of Brm interactionswith the Hes1 promoter was dem-onstrated by increase and decreasein the levels of PCR products inpBabe-Brm-transduced and BrmsiRNA-treated RGC5 cells, respec-tively, compared with those inrespective controls. Taken together,these observations suggested thatBrm negatively regulatedNotch sig-naling by facilitating the repressionof Hes1.Influence of Brm on Cell Cycle—

Recent observations have demon-strated that ATPase chromatinremodeling complexes are involvedin the facilitation of cell cycle exit(61–64). We were interested inknowing if Brm-mediated cell cycle

FIGURE 6. Influence of Brm on the regulation of Brn3b and Shh expression. To determine that Brm facilitates theactivation of Brn3b expression, RGC5 cells, transduced with wild type Brm (pBabe-Brm) (�)/empty (pBabe) (�)retrovirus, were transiently transfected with Brn3b-luc (A). There was a significant increase in luciferase activities incells overexpressing Brm, compared with controls, suggesting a direct effect of Brm on Brn3b promoter activities.Co-immunoprecipitation analyses were carried out to determine interactions between Brm and Wt1. Complexesimmunoprecipitated with Wt1 antibody from the nuclear extract of RGC5 cells were immunoreactive to Brm anti-body, suggesting a complex formation between Brm and Wt1 (B). DNA-protein complexes, immunoprecipitatedwith Brm antibody, in a ChIP assay on RGC5 cells, contained a sequence corresponding to Brn3b promoter asdemonstrated by the size- and sequence-specific PCR products, the levels of which were increased and decreasedwhen RGC5 overexpressed Brm (lane 4)/overexpressed Wt1 (lane 5)/overexpressed Brm�Wt1 (lane 6) and treatedwith Brm-siRNA (lane 8), respectively (C). Lane 3 (pBabe) and lane 7 (pSuper) represent controls for retrovirus-medi-ated gene transduction and siRNA-mediated attenuation of Brm expression, respectively. The analysis of input DNA(lane 1), ChIP assay with IgG (lane 2), and the amplification of CD11 promoter sequence constituted nonspecificcontrols. To examine the restriction enzyme accessibility at endogenous Brn3b promoter, we carried out LM-PCRassay on BglII-digested nuclei of RGC5 cells, transduced with Brm (lane 2) or empty retrovirus (lane 1) (D). The levelsof PCR products, amplified with gene-specific (forward arrow) and LM-PCR (reverse arrow) primers, were higher incells overexpressing Brm, compared with those transduced with empty retrovirus. LM-PCR assay was carried out onBglII-digested RGC5 cell genomic DNA-constituted controls (lane 3). To determine that Brm facilitates the activationof Shh, RGC5 cells, transduced with wild type Brm (pBabe-Brm) (�)/empty (pBabe) (�) retrovirus, were transientlytransfected with luciferase reporter vectors (E). The luciferase reporter vectors contained the rat minimal prolactinpromoter (A) or conserved 67-bp Shh sequence containing the Brn3b-binding site, upstream of the minimal prolac-tin promoter (B). There was a significant increase in luciferase activities in Brm-transduced cells, transfected withvector b compared with those transfected with vector a. Co-immunoprecipitation analyses were carried out todetermine the interactions between Brm and Brn3b. Complexes immunoprecipitated with Brn3b antibody from thenuclear extract of RGC5 cells were immunoreactive to Brm antibody, suggesting a complex formation between Brmand Brn3b (F). DNA-protein complexes, immunoprecipitated with Brm antibody, in a ChIP assay on RGC5 cells,contained sequence corresponding to the Shh enhancer as demonstrated by the size- and sequence-specific PCRproducts, the levels of which were increased and decreased when RGC5 overexpressed Brm (lane 4) and treated withBrm-siRNA (lane 6), respectively (G). Controls are same as described in C. * � p � 0.05; ** � p � 0.001.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

exit could constitute another mechanism by which Brm pro-motes the differentiation of E14 progenitors into RGCs. Weexamined this possibility from different angles. We first deter-mined if the proportion of proliferating cells changed inresponse to perturbations in Brm expression and function inE14 neurospheres. Because retroviral infection of retinalexplants does not lead to a uniform transduction of retinal pro-genitors, which might lead to ambiguous results, we used E14neurospheres, which are enriched for progenitors and trans-duced uniformly. E14 neurospheres were transduced with wildtype Brm or dominant negative Brm retrovirus in proliferatingconditions. Neurospheres were shifted to differentiating condi-tions and collected at 4-, 12-, 24-, and 48-h time points, after a4-h pulse with BrdUrd to gauge the temporal proportion ofproliferating cells.We observed that in comparison with that at

4 h, the proportion of BrdU� cellsdecreased with the time in neuro-spheres, transduced with wild typeBrm retrovirus, comparedwith con-trols (Fig. 8A). In contrast, the pro-portion of BrdU� cells increasedwith the time in neurospheres,transduced with dominant negativeBrm retrovirus, suggesting a role ofthe endogenous Brm and its chro-matin remodeling function in cellproliferation (Fig. 8B). Next, wewanted to know at which stage ofthe cell cycle Brm exerted its influ-ence, so that we could speculateabout themechanism bywhich Brmpromotes cell cycle exit. E14 neuro-spheres were transduced with wildtype Brm retrovirus andmaintainedin proliferating condition for 48 h.Cells were dissociated, stained withpropidium iodide, and subjected tocell cycle analysis by FACS. Weobserved that E14 neurospheres,transduced with the empty retrovi-rus, were enriched for cells thatwere in G2/M phase (Fig. 8C). Incontrast, the proportion of cells inG2/M phase had decreased, andmore cells were shifted to G1 phasein neurospheres transduced withwild type Brm retrovirus. Theseobservations suggested that Brminfluenced the G1-S transition. Oneof the mechanisms by which Brmmay influence G1-S transition is byinfluencing the expression of cyclinE, the G1 phase cyclin that regulatesG1 checkpoint (61). Therefore, weargued that the facilitation ofRB-E2F-mediated repression ofcyclin E, in particular, would ensurethat committed precursors do not

escape into S phase. We also examined the expression of cyclinA because, like cyclin E, it is also regulated via the RB-E2F com-plex and is therefore a likely target of Brm-mediated repression.To test the notion, E14 neurospheres were transduced withwild type Brm/dominant negative Brm/empty retrovirus asdescribed above, and levels of transcripts corresponding to cellcycle-related genes were examined by RT-PCR analysis. Weobserved that levels of transcripts corresponding to cyclin E,cyclin A, and Ki67, a cell cycle marker, were decreased in neu-rospheres transduced with wild type Brm retrovirus, comparedwith controls (Fig. 8D). In contrast, their levels increased inneurospheres transduced with dominant negative Brm retrovi-rus, compared with controls. Similarly, in a separate experi-ment that involved siRNA-mediated silencing of Brm in neuro-spheres, we observed an increase in levels of these transcripts in

FIGURE 7. Interactions between Brm and Notch signaling. Double immunocytochemical analysis carried outon E14 retinal cells shows the co-localization of Notch1 and Brm immunoreactivities, suggesting interactionsbetween the two (A and B). Examination of the expression of Hes1 and Hes5 in E14 neurospheres transducedwith dominant negative Brm (pBabe-dnBrm) and empty (pBabe) retrovirus revealed increase in levels of Hes1/Hes5 transcripts in the former, compared with latter, suggesting that Brm has a negative influence on Notchsignaling (C). To determine the mode of the regulation of Hes1 by Brm, NICD expressing 293T cells, transducedwith Brm (pBabe-Brm)/empty (pBabe) retrovirus, were transfected with luciferase reporter vectors, pGV-BHes1-Luc (positive control)/pGV-B-Luc (negative control) driven by Hes1 promoter (D). There was a significantdecrease in luciferase activities in Brm-transduced cells, compared with positive controls, suggesting a directeffect of Brm on Hes1 promoter activities (D). Complexes, immunoprecipitated with CSL and NICD antibodiesfrom the nuclear extracts of cells transduced with wild type Brm, were immunoreactive for Brm, suggestinginteractions between Brm and CSL/NICD (E1). There was an increase in Brm immunoreactivity in a compleximmunoprecipitated by NICD antibody from cells transfected with NICD expression constructs (lane 2), com-pared with those transfected with empty expression constructs (lane 1) (E2). DNA-protein complexes, immu-noprecipitated with Brm antibody, in a ChIP assay on RGC5 cells, contained sequence corresponding to Hes1promoter as demonstrated by the size- and sequence-specific PCR products, the levels of which were increasedand decreased when RGC5 overexpressed Brm (lane 4) and treated with Brm-siRNA (lane 6), respectively (F).Controls are same as described in Fig. 6C. Magnification, �200. * � p � 0.05; ** � p � 0.001.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

neurospheres treatedwith Brm siRNA, comparedwith controls(Fig. 8E). Together, these observations suggested that Brm neg-atively regulated cell cycle during RGC differentiation, and themechanism may involve inhibition of cyclin E and cyclin Aexpression.Because the expression of cyclin A is required for the entry

intoM phase, we surmise that a decrease in cyclin A expressionmay explain the persistence of cells in the S phase (61, 64).

DISCUSSION

The existence of eukaryotic DNA as chromatin renders con-straints on the accessibility of the regulatory sequence of genesto tissue-specific and basic transcription factors. The chroma-tin remodeling complexes, by relaxing (euchromatin) or com-pacting (heterochromatin) the chromatin organization, modu-late the accessibility of these sequences to transcription factorsand therefore facilitate gene activation or repression. ThatSWI/SNF chromatin remodeling complexes have roles to playin neurogenesis was apparent from long known expression ofBrg1 and Brm in the developing brain and retina (65, 66). Brmand Brg1 are highly homologous ATPases; however, severallines of evidence suggest that they have different functions andthey remodel chromatin in different cellular contexts (67).

First, the expression of Brg1 is con-stitutive and associated with prolif-erating cells since early embryonicstages, whereas that of Brm stronglycorrelates with cell differentiationin vivo (68, 69) and in vitro (70). Sec-ond, mice lacking Brg1 die at thepre-implantation stage (71), andthose without Brm survive with anovert phenotype of weight gain (72).Third, SWI/SNF complexes con-taining Brg1 or Brm have differentsubunit compositions, and Brm-containing complexes appear tohave lower chromatin remodelingactivities than those with Brg1 (67,73, 74). These distinct roles of Brg1and Brm have emerged from thestudy of higher vertebrates, particu-larly mammals. In nonamniotic ver-tebrates, like frogs and fish, Brg1 hasbeen observed to promote differen-tiation rather than the maintenanceof stem cells/progenitors (29, 30). Amore recent study, using the condi-tional knock-out strategy, has reaf-firmed the earlier observations thatBrg1 maintains stem cells, whereasBrm promotes their differentiationin mammals (31). Our study dem-onstrates a similar role for Brm inretinal progenitors.Our results suggest that Brm-me-

diated chromatin remodelingaffects three overlapping steps in

RGC differentiation. First, Brm may promote RGC differentia-tion by facilitating transcriptional activation and function ofBrn3b. Brn3b is a key RGC regulatory gene. UnlikeMath5, it isnot required for RGC specification, but it is essential for thenormal differentiation and survival of RGCs (75–77). There-fore, it occupies a lower position thanMath5 in the hierarchicalregulatory gene network of RGC differentiation and is thoughtto be under the regulation of Math5 (76, 78). Another impor-tant upstream regulator of Brn3b during RGC differentiation isWt1. A recent study has demonstrated that Brn3b is a directtarget of Wt1 because its proximal promoter contains a Wt1-binding site, WRE, and Wt1 can directly activate Brn3b pro-moter (5). Our results suggest that Brm interacts withWt1 andgets recruited to the Brn3b promoter, where it may relaxnucleosomal structure, facilitating transcriptional activities ofWt1. In addition, we have demonstrated that a similar mecha-nism may be involved in promoting the function of Brn3b inactivating Shh expression, thus facilitating a cascade of tran-scriptional activities needed for RGC differentiation. Ourobservations add to the evidence, emerging fromother systems,that the recruitment of Brm to specific promoters by cell-spe-cific transcription factors is a mechanism that provides cellspecificity to chromatin remodeling during differentiation (55).

FIGURE 8. Influence of Brm on cell proliferation during RGC differentiation. To determine the influence ofBrm on cell proliferation during RGC differentiation, temporal analysis of cell proliferation was carried out inresponse to perturbation of Brm expression and function. When E14 neurospheres were transduced with wildtype Brm (pBabe-Brm) retrovirus and shifted to RGC-differentiating conditions, the proportion of BrdU� cellsdecreased and increased with the time, compared with those transduced with empty retrovirus (A). In contrast,the proportion of BrdU� cells increased with the time in neurospheres transduced with the dominant negativeBrm (pBabe-dnBrm), compared with those transduced with the empty (pBabe) retrovirus (B). To know theinfluence of Brm on cell cycle regulation during RGC differentiation, E14 neurospheres, transduced with wildtype Brm (pBabe-Brm)/empty (pBabe) retrovirus, were dissociated, labeled with propidium iodide, and sub-jected to FACS analysis. The proportion of cells in G2/M phase decreased significantly (S phase, p � 0.001; G2/Mphase, p � 0.05) and those in G1/G0 phase increased (p � 0.05) in neurospheres transduced with wild type Brmretrovirus, compared with those in controls, suggesting the influence of Brm on G1-S check point (C). RT-PCRanalysis revealed a decrease in the levels of transcripts corresponding to cyclin A, cyclin E, and Ki67 in neuro-spheres transduced with wild type Brm, compared with controls (D). In contrast, levels of these transcripts wereincreased in neurospheres electroporated with Brm siRNA, compared with controls, suggesting a negativeinfluence of Brm on cell cycle regulators during RGC differentiation (E). * � p � 0.05; ** � p � 0.001.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

However, the observation that Brm interacts with Wt1 is atodds with a report that Brg1, and not Brm, interacts with zincfinger transcription factors (55). This discrepancy could be rec-onciled by the facts that SWI/SNF complexes consist of differ-ent subunits, which have tissue-specific isoforms and act in atissue-specific manner (72, 74, 79). Such a complex nature ofinteractions, with temporal and cellular contexts, may be thereason why interactions between Brg1 and �-catenin (80), andBrg1 and basic helix-loop-helix transcription factors (30)observed by others in different systems, were not detected byKadam and Emerson (55).Second, Brm may influence RGC differentiation by atten-

uating Notch signaling, thereby promoting cell commit-ment. This notion is supported by the observation that levelsof Hes1 and Hes5 increase when Brm expression and func-tion are compromised. We are proposing that Brm influ-ences Notch signaling by interacting with CSL (55). One ofthe consequences of such interactions could be the repres-sion of Hes1/Hes5. For example, Brm may prevent NICD-CSL interactions by binding CSL and gets recruited to theHes1/Hes5 promoter, where it could participate in CSL-me-diated suppression of promoter activities as CSL withoutNICD acts as a transcriptional repressor. Additionally oralternatively, Brmmay interact with NICD, accentuating therepressor function of CSL. In either case, Brm will inhibitNotch signaling by repressing Hes1/Hes5, thus promotingRGC differentiation. Repression of genes through Brm/Brg1-mediated chromatin remodeling complexes is not without prec-edence. For example, Brm/Brg1 forms a repressor complexwith Rb to inhibit the expression of E2F-mediated expres-sion of cyclin genes (77).Third, Brm facilitates differentiation by ensuring that com-

mitted precursors do not make the G1-S transition. The mam-malian somatic cell cycle alternates between the S phase and theMphase with gaps, G1 andG2, between them (81, 82). From theviewpoint of the maintenance of retinal progenitors, the G1phase has a specific significance. A point that comes late in theG1 phase, when crossed, progenitors irreversibly enter the Sphase (G1-S transition). That point is the G1 restriction/check-point. The G1 checkpoint is regulated by two types of cyclins,cyclin D and cyclin E, that regulate activities of their respectivecyclin-dependent kinases. D-type cyclins (D1, D2, and D3) aresensitive to growth factors (i.e. FGF2 and Wnts). The growthfactor-mediated activation and accumulation of cyclin D-de-pendent kinases phosphorylate Rb, rendering it incapable offorming repression complex with histone deacetylase and Brmcomplex. In the absence of the Rb repression complex, E2F isable to activate the cyclin E gene. The activation and accumu-lation of cyclin E-dependent kinases complete Rb phosphoryl-ation and, in addition, inactivate its own repressor, p27Kip1, amember of Cip/Kip family of peptides that inhibit cyclin E- andcyclin A-dependent kinases. These two processes ensure G1-Stransition, at which time cyclin E is degraded and replaced bycyclin A, the S phase cyclin, whose expression is also E2F-de-pendent. Retinal neurogenesis is intricately linked to the cellcycle (83, 84). Different cell cycle regulators, cyclin A, cyclin E,cyclin D1, cdk2, p27Kip1, and Rb, for example, are expressed inthe developing retina. In addition, levels of their expression are

different in stem cells, progenitors, and precursor populationssuggesting their modulation during retinal neurogenesis (83,84). Based on conditional and classical knock-out experimentsand perturbation of expression/function approaches, the rolessome of these regulators play in retinal neurogenesis havebegun to emerge. For example, cyclin D1 plays an essentialrole in progenitor populations because there is a remarkabledecrease in retinal thickness in cyclin D1 knock-out mice,because of compromised progenitor proliferation (85, 86).Both Rb and p27Kip1 play context-dependent roles in thedeveloping retina, i.e. depending on the cellular context theyregulate either proliferation or differentiation, the latter ofMuller cells and the former of rod photoreceptors (87, 88).We are proposing that Brm antagonizes G1-S transition byfacilitating the inhibition of cyclin E and cyclin A. Thisnotion is supported by our observations that both the posi-tion of cells in different phases of the cell cycle and theexpression levels of cyclin E and cyclin A change in experi-ments involving the perturbation of expression and functionof Brm. In one of the emerging mechanisms, based on avariety of approaches in transformed cell lines, Brm/Brg1constitute an integral part of the repressor complex consist-ing of the retinoblastoma protein and histone deacetylase.This complex inhibits the E2F-mediated expression of cyclinE and cyclin A, thus preventing G1-S transition (61–64). Asimilar mechanism may be involved Brm-mediated repres-sion of cyclin E and cyclin A during RGC differentiation. Insummary, our observations suggest that developmentalchromatin remodeling, mediated by Brm, may serve as thehub where diverse information for cell differentiation is inte-grated during neurogenesis. This mechanism is likely to berecruited reiteratively for temporal differentiation of retinalprogenitors into different retinal cell types.

Acknowledgments—We thank Dr. Mengquing Xing for the Brm pro-moter construct; Dr. William Klein for the Shh enhancer construct;Dr. Jonathan Licht for theWt1 expression construct; Dr. Neeraj Agar-wal for the RGC5 cell line; Dr. Angie Rizzino for the ChIP assay pro-tocol; andDr. Greg Bennett and Brittany Cody for a critical reading ofthe manuscript.

REFERENCES1. Bertrand, N., Castro, D. S., and Guillemot, F. (2002)Nat. Rev. Neurosci. 3,

517–5302. Hatakeyama, J., and Kageyama, R. (2004) Semin. Cell Biol. 15, 83–893. Jessell, T. M. (2000) Nat. Rev. Genet. 1, 20–294. Lee, S. K., and Pfaff, S. L. (2003) Neuron 38, 731–7455. Wagner, K. D., Wagner, N., Schley, G., Theres, H., and Scholz, H. (2003)

Gene (Amst.) 305, 217–2236. Wagner, K. D., Wagner, N., Vidal, V. P., Schley, G., Wilhelm, D., Schedl,

A., Englert, C., and Scholz, H. (2002) EMBO J. 21, 1398–14057. Brown, R. C., Pattison, S., van Ree, J., Coghill, E., Perkins, A., Jane, S. M.,

and Cunningham, J. M. (2002)Mol. Cell. Biol. 22, 161–1708. Kay, J. N., Finger-Baier, K. C., Roeser, T., Staub, W., and Baier, H. (2001)

Neuron 30, 725–7369. Marquardt, T., Ashery-Padan, R., Andrejewski, N., Scardigli, R., Guil-

lemot, F., and Gruss, P. (2001) Cell 105, 43–5510. Mu, X., and Klein, W. H. (2004) Semin. Cell Dev. Biol. 15, 115–12311. Ahmad, I., Dooley, C. M., and Polk, D. L. (1997) Dev. Biol. 185, 92–10312. Austin, C. P., Feldman, D. E., Ida, J. A., Jr., and Cepko, C. L. (1995) Devel-




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

opment (Camb.) 121, 3637–365013. James, J., Das, A. V., Rahnenfuhrer, J., and Ahmad, I. (2004) J. Neurobiol.

61, 359–37614. Perron, M., and Harris, W. A. (2000) Cell. Mol. Life Sci. 57, 215–22315. Rapaport, D. H., and Dorsky, R. I. (1998) Semin. Cell Dev. Biol. 9, 241–24716. Imbalzano, A. N., and Xiao, H. (2004) Adv. Protein. Chem. 67, 157–17917. Narlikar, G. J., Fan, H. Y., and Kingston, R. E. (2002) Cell 108, 475–48718. Martens, J. A., and Winston, F. (2003) Curr. Opin. Genet. Dev. 13,

136–14219. Hsieh, J., Nakashima, K., Kuwabara, T., Mejia, E., and Gage, F. H. (2004)

Proc. Natl. Acad. Sci. U. S. A. 101, 16659–1666420. Kondo, T., and Raff, M. (2004) Genes Dev. 18, 2963–297221. Song, M. R., and Ghosh, A. (2004) Nat. Neurosci. 7, 229–23522. Marenda, D. R., Zraly, C. B., and Dingwall, A. K. (2004) Dev. Biol. 267,

279–29323. Zhang, D., Johnson, M. M., Miller, C. P., Pircher, T. J., Geiger, J. N., and

Wojchowski, D. M. (2001) Exp. Hematol. 29, 1278–128824. Liu, R., Liu, H., Chen, X., Kirby, M., Brown, P. O., and Zhao, K. (2001)Cell

106, 309–31825. Kowenz-Leutz, E., and Leutz, A. (1999)Mol. Cell 4, 735–74326. Pedersen, T. A., Kowenz-Leutz, E., Leutz, A., and Nerlov, C. (2001)Genes

Dev. 15, 3208–321627. de la Serna, I. L., Carlson, K. A., and Imbalzano, A. N. (2001) Nat. Genet.

27, 187–19028. Young, D.W., Pratap, J., Javed, A.,Weiner, B., Ohkawa, Y., vanWijnen, A.,

Montecino, M., Stein, G. S., Stein, J. L., Imbalzano, A. N., and Lian, J. B.(2005) J. Cell. Biochem. 94, 720–730

29. Gregg, R. G., Willer, G. B., Fadool, J. M., Dowling, J. E., and Link, B. A.(2003) Proc. Natl. Acad. Sci. U. S. A. 100, 6535–6540

30. Seo, S., Richardson, G. A., and Kroll, K. L. (2005) Development (Camb.)132, 105–115

31. Matsumoto, S., Banine, F., Struve, J., Xing, R., Adams, C., Liu, Y., Metzger,D., Chambon, P., Rao, M. S., and Sherman, L. S. (2006) Dev. Biol. 289,372–383

32. Christie, G. A. (1964) J. Morphol. 114, 263–28333. Hamburger, V., and Hamilton, H. L. (1951) J. Morphol. 88, 49–9234. Ahmad, I., Dooley, C. M., Thoreson, W. B., Rogers, J. A., and Afiat, S.

(1999) Brain Res. 831, 1–1035. Das, A. V., Mallya, K. B., Zhao, X., Ahmad, F., Bhattacharya, S., Thoreson,

W. B., Hegde, G. V., and Ahmad, I. (2006) Dev. Biol. 299, 283–30236. Zhou, H., Yoshioka, T., and Nathans, J. (1996) J. Neurosci. 16, 2261–227437. Ringel, J., Jesnowski, R., Moniaux, N., Luttges, J., Choudhury, A., Batra,

S. K., Kloppel, G., and Lohr, M. (2006) Cancer Res. 66, 9045–905338. James, J., Das, A. V., Bhattacharya, S., Chacko, D. M., Zhao, X., and Ah-

mad, I. (2003) J. Neurosci. 23, 8193–820339. Dooley, C. M., James, J., JaneMcGlade, C., and Ahmad, I. (2003) J. Neuro-

biol. 54, 313–32540. Matsuda, T., and Cepko, C. L. (2004) Proc. Natl. Acad. Sci. U. S. A. 101,

16–2241. Hempel, W. M., and Ferrier, P. (2004)Methods Mol. Biol. 287, 53–6342. Roy, K., De La Serna, I. L., and Imbalzano, A. N. (2002) J. Biol. Chem. 277,

33818–3382443. Sidman, R. L. (1961) in Structure of the Eye (Smelser, G., ed) pp. 487–506,

Academic Press, New York44. Young, R. W. (1985) Anat. Rec. 212, 199–20545. Rapaport, D. H., Wong, L. L., Wood, E. D., Yasumura, D., and LaVail,

M. M. (2004) J. Comp. Neurol. 474, 304–32446. Muchardt, C., and Yaniv, M. (1993) EMBO J. 12, 4279–429047. Fukuoka, J., Fujii, T., Shih, J. H., Dracheva, T., Meerzaman, D., Player, A.,

Hong, K., Settnek, S., Gupta, A., Buetow, K., Hewitt, S., Travis, W. D., andJen, J. (2004) Clin. Cancer Res. 10, 4314–4324

48. Hegde, G. V., James, J., Das, A. V., Zhao, X., Bhattacharya, S., and Ahmad,I. (2007) Exp. Eye Res. 84, 577–590

49. Das, A. V., James, J., Edakkot, S., and Ahmad, I. (2005) in Stem Cells andCNSDevelopment (Rao,M., ed) 2nd Ed., pp. 235–247, Humana Press Inc.,Totowa, NJ

50. Alexiades, M. R., and Cepko, C. (1996) Dev. Dyn. 205, 293–30751. de La Serna, I. L., Carlson, K. A., Hill, D. A., Guidi, C. J., Stephenson, R. O.,

Sif, S., Kingston, R. E., and Imbalzano, A. N. (2000) Mol. Cell. Biol. 20,2839–2851

52. Mu, X., Fu, X., Sun, H., Beremand, P. D., Thomas, T. L., and Klein, W. H.(2005) Dev. Biol. 280, 467–481

53. Frassetto, L. J., Schlieve, C. R., Lieven, C. J., Utter, A. A., Jones, M. V.,Agarwal, N., and Levin, L. A. (2006) Investig. Ophthalmol. Vis. Sci. 47,427–438

54. Krishnamoorthy, R. R., Agarwal, P., Prasanna, G., Vopat, K., Lambert, W.,Sheedlo,H. J., Pang, I. H., Shade,D.,Wordinger, R. J., Yorio, T., Clark, A. F.,and Agarwal, N. (2001) Brain Res. Mol. Brain Res. 86, 1–12

55. Kadam, S., and Emerson, B. M. (2003)Mol. Cell 11, 377–38956. de la Serna, I. L., Ohkawa, Y., Berkes, C. A., Bergstrom, D. A., Dacwag,

C. S., Tapscott, S. J., and Imbalzano, A. N. (2005) Mol. Cell. Biol. 25,3997–4009

57. Ohkawa, Y., Marfella, C. G., and Imbalzano, A. N. (2006) EMBO J. 25,490–501

58. Mu, X., Beremand, P. D., Zhao, S., Pershad, R., Sun, H., Scarpa, A., Liang,S., Thomas, T. L., and Klein, W. H. (2004) Development (Camb.) 131,1197–1210

59. Austin, T. W., Solar, G. P., Ziegler, F. C., Liem, L., and Matthews, W.(1997) Blood 89, 3624–3635

60. Mumm, J. S., and Kopan, R. (2000) Dev. Biol. 228, 151–16561. Zhang, Z. K., Davies, K. P., Allen, J., Zhu, L., Pestell, R. G., Zagzag, D., and

Kalpana, G. V. (2002)Mol. Cell. Biol. 22, 5975–598862. Strobeck, M. W., Knudsen, K. E., Fribourg, A. F., DeCristofaro, M. F.,

Weissman, B. E., Imbalzano, A. N., and Knudsen, E. S. (2000) Proc. Natl.Acad. Sci. U. S. A. 97, 7748–7753

63. Coisy, M., Roure, V., Ribot, M., Philips, A., Muchardt, C., Blanchard, J. M.,and Dantonel, J. C. (2004)Mol. Cell 15, 43–56

64. Zraly, C. B., Marenda, D. R., and Dingwall, A. K. (2004) Genetics 168,199–214

65. Randazzo, F. M., Khavari, P., Crabtree, G., Tamkun, J., and Rossant, J.(1994) Dev. Biol. 161, 229–242

66. Schofield, J., Isaac, A., Golovleva, I., Crawley, A., Goodwin, G., Tickle, C.,and Brickell, P. (1999)Mech. Dev. 80, 115–118

67. Wang, W. (2003) Curr. Top. Microbiol. Immunol. 274, 143–16968. LeGouy, E., Thompson, E. M., Muchardt, C., and Renard, J. P. (1998)Dev.

Dyn. 212, 38–4869. Reisman, D. N., Sciarrotta, J., Bouldin, T. W., Weissman, B. E., and Funk-

houser,W. K. (2005)Appl. Immunohistochem.Mol. Morphol. 13, 66–7470. Machida, Y., Murai, K., Miyake, K., and Iijima, S. (2001) J. Biochem. (To-

kyo) 129, 43–4971. Bultman, S., Gebuhr, T., Yee, D., La Mantia, C., Nicholson, J., Gilliam, A.,

Randazzo, F., Metzger, D., Chambon, P., Crabtree, G., and Magnuson, T.(2000)Mol. Cell 6, 1287–1295

72. Reyes, J. C., Barra, J., Muchardt, C., Camus, A., Babinet, C., and Yaniv, M.(1998) EMBO J. 17, 6979–6991

73. Sif, S., Saurin, A. J., Imbalzano, A.N., andKingston, R. E. (2001)GenesDev.15, 603–618

74. Wang, W., Cote, J., Xue, Y., Zhou, S., Khavari, P. A., Biggar, S. R., Mu-chardt, C., Kalpana, G. V., Goff, S. P., Yaniv, M., Workman, J. L., andCrabtree, G. R. (1996) EMBO J. 15, 5370–5382

75. Gan, L., Xiang, M., Zhou, L., Wagner, D. S., Klein, W. H., and Nathans, J.(1996) Proc. Natl. Acad. Sci. U. S. A. 93, 3920–3925

76. Wang, S. W., Kim, B. S., Ding, K., Wang, H., Sun, D., Johnson, R. L., Klein,W. H., and Gan, L. (2001) Genes Dev. 15, 24–29

77. Wang, S. W., Mu, X., Bowers, W. J., Kim, D. S., Plas, D. J., Crair, M. C.,Federoff, H. J., Gan, L., and Klein,W.H. (2002)Development (Camb.) 129,467–477

78. Brown, N. L., Patel, S., Brzezinski, J., and Glaser, T. (2001) Development(Camb.) 128, 2497–2508

79. Olave, I., Wang, W., Xue, Y., Kuo, A., and Crabtree, G. R. (2002) GenesDev. 16, 2509–2517

80. Barker, N., Hurlstone, A., Musisi, H., Miles, A., Bienz, M., and Clevers, H.(2001) EMBO J. 20, 4935–4943

81. Baek, S. H., Kioussi, C., Briata, P., Wang, D., Nguyen, H. D., Ohgi, K. A.,Glass, C. K.,Wynshaw-Boris, A., Rose, D.W., and Rosenfeld, M. G. (2003)Proc. Natl. Acad. Sci. U. S. A. 100, 3245–3250




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

82. Sherr, C. J. (2000) Cancer Res. 60, 3689–369583. Ohnuma, S., and Harris, W. A. (2003) Neuron 40, 199–20884. Ohnuma, S., Hopper, S.,Wang, K.C., Philpott, A., andHarris,W.A. (2002)

Development (Camb.) 129, 2435–244685. Fantl, V., Stamp, G., Andrews, A., Rosewell, I., and Dickson, C. (1995)

Genes Dev. 9, 2364–2372

86. Sicinski, P., Donaher, J. L., Parker, S. B., Li, T., Fazeli, A., Gardner, H.,Haslam, S. Z., Bronson, R. T., Elledge, S. J., andWeinberg, R. A. (1995)Cell82, 621–630

87. Dyer, M. A., and Cepko, C. L. (2001) J. Neurosci. 21, 4259–427188. Levine, E. M., Close, J., Fero, M., Ostrovsky, A., and Reh, T. A. (2000)Dev.

Biol. 219, 299–314




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Iqbal AhmadAntony, Ganapati Hegde, Xing Zhao, Kavita Mallya, Faraz Ahmad, Eric Knudsen and Ani V. Das, Jackson James, Sumitra Bhattacharya, Anthony N. Imbalzano, Marie Lue

SignalingEarly Retinal Stem Cells/Progenitors by Influencing Brn3b Expression and Notch SWI/SNF Chromatin Remodeling ATPase Brm Regulates the Differentiation of

doi: 10.1074/jbc.M706742200 originally published online September 11, 20072007, 282:35187-35201.J. Biol. Chem.

10.1074/jbc.M706742200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/282/48/35187.full.html#ref-list-1

This article cites 86 references, 34 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M706742200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;282/48/35187&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/282/48/35187

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=282/48/35187&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/282/48/35187

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/282/48/35187.full.html#ref-list-1

http://www.jbc.org/

SWI/SNF Chromatin Remodeling ATPase Brm Regulates the Differentiation of Early Retinal Stem Cells/Progenitors by Influencing Brn3b Expression and Notch Signaling

Documents