FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2022551903> ?p ?o ?g. }

• W2022551903 endingPage "35201" @default.
• W2022551903 startingPage "35187" @default.
• W2022551903 abstract "Based on a variety of approaches, evidence suggests that different cell types in the vertebrate retina are generated by multipotential progenitors in response to interactions between cell intrinsic and cell extrinsic factors. The identity of some of the cellular determinants that mediate such interactions has emerged, shedding light on mechanisms underlying cell differentiation. For example, we know now that Notch signaling mediates the influence of the microenvironment on states of commitment of the progenitors by activating transcriptional repressors. Cell intrinsic factors such as the proneural basic helix-loop-helix and homeodomain transcription factors regulate a network of genes necessary for cell differentiation and maturation. What is missing from this picture is the role of developmental chromatin remodeling in coordinating the expression of disparate classes of genes for the differentiation of retinal progenitors. Here we describe the role of Brm, an ATPase in the SWI/SNF chromatin remodeling complex, in the differentiation of retinal progenitors into retinal ganglion cells. Using the perturbation of expression and function analyses, we demonstrate that Brm promotes retinal ganglion cell differentiation by facilitating the expression and function of a key regulator of retinal ganglion cells, Brn3b, and the inhibition of Notch signaling. In addition, we demonstrate that Brm promotes cell cycle exit during retinal ganglion cell differentiation. Together, our results suggest that Brm represents one of the nexus where diverse information of cell differentiation is integrated during cell differentiation. Based on a variety of approaches, evidence suggests that different cell types in the vertebrate retina are generated by multipotential progenitors in response to interactions between cell intrinsic and cell extrinsic factors. The identity of some of the cellular determinants that mediate such interactions has emerged, shedding light on mechanisms underlying cell differentiation. For example, we know now that Notch signaling mediates the influence of the microenvironment on states of commitment of the progenitors by activating transcriptional repressors. Cell intrinsic factors such as the proneural basic helix-loop-helix and homeodomain transcription factors regulate a network of genes necessary for cell differentiation and maturation. What is missing from this picture is the role of developmental chromatin remodeling in coordinating the expression of disparate classes of genes for the differentiation of retinal progenitors. Here we describe the role of Brm, an ATPase in the SWI/SNF chromatin remodeling complex, in the differentiation of retinal progenitors into retinal ganglion cells. Using the perturbation of expression and function analyses, we demonstrate that Brm promotes retinal ganglion cell differentiation by facilitating the expression and function of a key regulator of retinal ganglion cells, Brn3b, and the inhibition of Notch signaling. In addition, we demonstrate that Brm promotes cell cycle exit during retinal ganglion cell differentiation. Together, our results suggest that Brm represents one of the nexus where diverse information of cell differentiation is integrated during cell differentiation. Cell fate specification and subsequent cell differentiation in the nervous system are orchestrated and finessed by interplay between cell intrinsic and cell extrinsic factors. This process is exemplified during the development of the retina, an excellent model of the central nervous system. Recent evidence suggests that disparate transcription factors belonging to basic helixloop-helix, homeodomain, and zinc finger classes cooperate toward lineage specification and differentiation (1Bertrand N. Castro D.S. Guillemot F. Nat. Rev. Neurosci. 2002; 3: 517-530Crossref PubMed Scopus (1141) Google Scholar, 2Hatakeyama J. Kageyama R. Semin. Cell Biol. 2004; 15: 83-89Crossref Scopus (219) Google Scholar, 3Jessell T.M. Nat. Rev. Genet. 2000; 1: 20-29Crossref PubMed Scopus (1618) Google Scholar, 4Lee S.K. Pfaff S.L. Neuron. 2003; 38: 731-745Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 5Wagner K.D. Wagner N. Schley G. Theres H. Scholz H. Gene (Amst.). 2003; 305: 217-223Crossref PubMed Scopus (35) Google Scholar, 6Wagner K.D. Wagner N. Vidal V.P. Schley G. Wilhelm D. Schedl A. Englert C. Scholz H. EMBO J. 2002; 21: 1398-1405Crossref PubMed Scopus (122) Google Scholar). For example, the basic helix-loop-helix transcription factor, Math5, and the homeodomain transcription factor, Pax6, have been shown to cooperate during the specification of retinal progenitors into retinal ganglion cells (RGCs) 2The abbreviations used are: RGC, retinal ganglion cell; ChIP, chromatin immunoprecipitation; Rb, retinoblastoma; BrdUrd, bromo-2-deoxyuridine; siRNA, short interfering RNA; E, embryonic day; RT, reverse transcription; Tunel, terminal dUTP nick-end labeling; NICD, Notch intracellular domain; FACS, fluorescence-activated cell sorter; PN, postnatal; LM, ligation-mediated. 2The abbreviations used are: RGC, retinal ganglion cell; ChIP, chromatin immunoprecipitation; Rb, retinoblastoma; BrdUrd, bromo-2-deoxyuridine; siRNA, short interfering RNA; E, embryonic day; RT, reverse transcription; Tunel, terminal dUTP nick-end labeling; NICD, Notch intracellular domain; FACS, fluorescence-activated cell sorter; PN, postnatal; LM, ligation-mediated. (2Hatakeyama J. Kageyama R. Semin. Cell Biol. 2004; 15: 83-89Crossref Scopus (219) Google Scholar, 7Brown R.C. Pattison S. van Ree J. Coghill E. Perkins A. Jane S.M. Cunningham J.M. Mol. Cell. Biol. 2002; 22: 161-170Crossref PubMed Scopus (49) Google Scholar, 8Kay J.N. Finger-Baier K.C. Roeser T. Staub W. Baier H. Neuron. 2001; 30: 725-736Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 9Marquardt T. Ashery-Padan R. Andrejewski N. Scardigli R. Guillemot F. Gruss P. Cell. 2001; 105: 43-55Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar). As progenitors are committed along RGC lineage, Wt1, a zinc finger transcription factor, and Brn3b, a homeodomain transcription factor of POU class, are expressed and ultimately promote the differentiation and maturation of the specified progenitors into RGCs (10Mu X. Klein W.H. Semin. Cell Dev. Biol. 2004; 15: 115-123Crossref PubMed Scopus (92) Google Scholar). The progress in the identification of intrinsic factors has been paralleled by the characterization of extrinsic factors and intercellular signaling pathways, e.g. Notch pathway, that mediates the regulatory influence of the microenvironment on retinal progenitors' maintenance and their differentiation into RGCs (11Ahmad I. Dooley C.M. Polk D.L. Dev. Biol. 1997; 185: 92-103Crossref PubMed Scopus (71) Google Scholar, 12Austin C.P. Feldman D.E. Ida Jr., J.A. Cepko C.L. Development (Camb.). 1995; 121: 3637-3650PubMed Google Scholar, 13James J. Das A.V. Rahnenfuhrer J. Ahmad I. J. Neurobiol. 2004; 61: 359-376Crossref PubMed Scopus (57) Google Scholar, 14Perron M. Harris W.A. Cell. Mol. Life Sci. 2000; 57: 215-223Crossref PubMed Scopus (97) Google Scholar, 15Rapaport D.H. Dorsky R.I. Semin. Cell Dev. Biol. 1998; 9: 241-247Crossref PubMed Scopus (27) Google Scholar).Although the identification of cell intrinsic and cell extrinsic factors has helped our understanding of the mechanisms that regulate the differentiation of retinal cell types, particularly that of RGCs, it is not known as to how the remodeling of chromatin, which is necessary for eukaryotic gene expression, is recruited toward the coordinated regulation of genes during RGC differentiation. There are two major classes of chromatin remodeling complexes, that are associated with specific enzymes that remodel chromatin by changing nucleosome structure or position (16Imbalzano A.N. Xiao H. Adv. Protein. Chem. 2004; 67: 157-179Crossref PubMed Scopus (17) Google Scholar, 17Narlikar G.J. Fan H.Y. Kingston R.E. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1240) Google Scholar). The first class includes complexes consisting of histone acetyltransferase or histone deacetylase that covalently modify chromatin by adding and removing acetyl groups from the amino terminus of the four core histones, respectively. The second class includes SWI/SNF complexes that utilize the energy obtained from ATP hydrolysis to disrupt nucleosomal structure or position. The mammalian SWI/SNF complexes consist of 10-12 proteins, including the homologous but mutually exclusive ATPases, Brahma (Brm) and Brm related gene 1 (Brg1) (16Imbalzano A.N. Xiao H. Adv. Protein. Chem. 2004; 67: 157-179Crossref PubMed Scopus (17) Google Scholar, 18Martens J.A. Winston F. Curr. Opin. Genet. Dev. 2003; 13: 136-142Crossref PubMed Scopus (292) Google Scholar).Although both major classes of chromatin remodeling complexes are likely to contribute to developmental processes, including those in the central nervous system (19Hsieh J. Nakashima K. Kuwabara T. Mejia E. Gage F.H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16659-16664Crossref PubMed Scopus (599) Google Scholar, 20Kondo T. Raff M. Genes Dev. 2004; 18: 2963-2972Crossref PubMed Scopus (187) Google Scholar, 21Song M.R. Ghosh A. Nat. Neurosci. 2004; 7: 229-235Crossref PubMed Scopus (209) Google Scholar), evidence is emerging that the SWI/SNF chromatin remodeling complexes play an important role in the differentiation of specific cell types (22Marenda D.R. Zraly C.B. Dingwall A.K. Dev. Biol. 2004; 267: 279-293Crossref PubMed Scopus (53) Google Scholar). For example, these complexes have been shown to facilitate the differentiation of a variety of cell types such as erythrocytes (7Brown R.C. Pattison S. van Ree J. Coghill E. Perkins A. Jane S.M. Cunningham J.M. Mol. Cell. Biol. 2002; 22: 161-170Crossref PubMed Scopus (49) Google Scholar, 23Zhang D. Johnson M.M. Miller C.P. Pircher T.J. Geiger J.N. Wojchowski D.M. Exp. Hematol. 2001; 29: 1278-1288Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), macrophages (24Liu R. Liu H. Chen X. Kirby M. Brown P.O. Zhao K. Cell. 2001; 106: 309-318Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar), myeloid cells (25Kowenz-Leutz E. Leutz A. Mol. Cell. 1999; 4: 735-743Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar), adipocytes (26Pedersen T.A. Kowenz-Leutz E. Leutz A. Nerlov C. Genes Dev. 2001; 15: 3208-3216Crossref PubMed Scopus (158) Google Scholar), myoblasts (27de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (269) Google Scholar), osteoblasts (28Young D.W. Pratap J. Javed A. Weiner B. Ohkawa Y. van Wijnen A. Montecino M. Stein G.S. Stein J.L. Imbalzano A.N. Lian J.B. J. Cell. Biochem. 2005; 94: 720-730Crossref PubMed Scopus (83) Google Scholar), neurons (29Gregg R.G. Willer G.B. Fadool J.M. Dowling J.E. Link B.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6535-6540Crossref PubMed Scopus (81) Google Scholar, 30Seo S. Richardson G.A. Kroll K.L. Development (Camb.). 2005; 132: 105-115Crossref PubMed Scopus (156) Google Scholar), and glia (31Matsumoto S. Banine F. Struve J. Xing R. Adams C. Liu Y. Metzger D. Chambon P. Rao M.S. Sherman L.S. Dev. Biol. 2006; 289: 372-383Crossref PubMed Scopus (112) Google Scholar). Here we demonstrate the role of Brm in the differentiation of retinal progenitors into RGCs. Brm is expressed in the developing rat retina, and its temporal and spatial patterns of expression correlate with retinal histogenesis. Using perturbation of expression and function analyses, we demonstrate that Brm influences the differentiation of retinal progenitors into RGCs by facilitating Brn3b expression and function and inhibiting Notch signaling. In addition, we demonstrate that Brm may influence differentiation generically by promoting cell cycle exit. Together, our results suggest that chromatin remodeling by Brm may represent one of the nexus where cell intrinsic and cell extrinsic influence may be integrated toward the differentiation of retinal progenitors.EXPERIMENTAL PROCEDURESProgenitor Cell Culture—Timed-pregnant (E14) Sprague-Dawley rats were obtained from Sasco (Wilmington, MA). The gestation day was confirmed by the morphological examination of embryos (32Christie G.A. J. Morphol. 1964; 114: 263-283Crossref PubMed Scopus (112) Google Scholar). Fertilized hen eggs were incubated in a humidified chamber at 38 °C, and embryos were staged according to Hamburger and Hamilton (33Hamburger V. Hamilton H.L. J. Morphol. 1951; 88: 49-92Crossref PubMed Scopus (9958) Google Scholar). Embryos were harvested at appropriate gestation periods, and eyes were enucleated. Retina were dissected out and dissociated as described previously (34Ahmad I. Dooley C.M. Thoreson W.B. Rogers J.A. Afiat S. Brain Res. 1999; 831: 1-10Crossref PubMed Scopus (109) Google Scholar). Cells were cultured in RCM (Dulbecco's modified Eagle's medium/F-12, 1× N2 supplement (Invitrogen), 2 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin) containing FGF2 (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 μm) was added to the culture for the final 24 h. For co-culture, neurospheres were collected, washed extensively to remove BrdUrd, and plated on poly-d-lysine- and laminin-coated glass coverslips with E3 chick/PN1 rat retinal cells in 1% fetal bovine serum. For RT-PCR analysis co-culture was performed across a 0.4-μm membrane (Millipore, Bedford, MA). Cells were either frozen for RNA extraction or fixed with 4% paraformaldehyde for 15 min at 4 °C for immunofluorescence analysis.Immunofluorescence Analysis—Immunocytochemical analysis for detection of cell-specific markers and BrdUrd was performed as described previously (35Das A.V. Mallya K.B. Zhao X. Ahmad F. Bhattacharya S. Thoreson W.B. Hegde G.V. Ahmad I. Dev. Biol. 2006; 299: 283-302Crossref PubMed Scopus (262) Google Scholar). Briefly, paraformaldehyde-fixed cells/sections were incubated in 1× phosphatebuffered saline containing 5% Normal Goat Serum and 0.2-0.4% Triton X-100 followed by an overnight incubation in appropriate dilutions of antibodies against Brm (Santa Cruz Biotechnology), Notch1 (Santa Cruz Biotechnology), Shh (Developmental Studies Hybridoma Bank), Brn3b (Covance), RPF1 (36Zhou H. Yoshioka T. Nathans J. J. Neurosci. 1996; 16: 2261-2274Crossref PubMed Google Scholar), and BrdUrd (Accurate Chemical and Scientific Corp.) at 4 °C. Cells/sections were examined for epifluorescence after incubation with IgG, conjugated to Cy3/fluorescein isothiocyanate. Images were captured using a CCD camera (Princeton Instruments, 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 as described (37Ringel J. Jesnowski R. Moniaux N. Luttges J. Choudhury A. Batra S.K. Kloppel G. Lohr M. Cancer Res. 2006; 66: 9045-9053Crossref PubMed Scopus (57) Google Scholar). Cell cycle analysis was done using a FACStar flow cytometer (BD Biosciences).Semi-quantitative RT-PCR—Total RNA was isolated from frozen cells or explants using Qiagen RNA isolation kit (Valencia, CA), and cDNA synthesis was performed as described previously using ∼5 μg of total RNA (38James J. Das A.V. Bhattacharya S. Chacko D.M. Zhao X. Ahmad I. J. Neurosci. 2003; 23: 8193-8203Crossref PubMed Google Scholar). Specific transcripts were amplified with gene-specific forward and reverse primers using a step-cycle program on a Robocycler (Stratagene, La Jolla, CA) for less than 30 cycles to keep the amplification within the range of linearity. The gene-specific primers used for RT-PCR are described in Table 1.TABLE 1List of primers and their respective sequences used for RT-PCR and ChIP analysisGenesPrimer sequencesProduct sizeGenBank™ accession no.Temperaturebp°Cβ-actinForward, 5′-GTGGGGCGCCCCAGGCACCA-3′548XM_03723550Reverse, 5′-CTCCTTAATGTCACGCACGATTTC-3′Hes1Forward, 5′-GCTTTCCTCATCCCCAAAG-3′224NM_02436056Reverse, 5′-CGTATTTAGTGTCCGTCAGAAGAG-3′Ki67Forward, 5′-GAGCAGTTACAGGGAACCGAAG3-′262X8278658Reverse, 5′-CCTACTTTGGGTGAAGAGGCTG3-′Brn3bForward, 5′-GGCTGGAGGAAGCAGAGAAATC-3′141AF39007614160Reverse, 5′-TTGGCTGGATGGCGAAGTAG-3′RPF1Forward, 5′-TTTCAGGGGATTCTGGTGTGC-3′359XM_34460456Reverse, 5′-CGCTTTTTGGATGGCTCACTC-3′BrmForward, 5′-TGCCCTGTAATTCTCAGTTGGA-3′180XM_00538352Reverse, 5′-CTCCCAGGCTTCGGTACTTATG-3′Cyclin AForward, 5′-TACACACACGAGGTAGTGACGCTG-3′307X6076756Reverse, 5′-CCAAGCCGTTTTCATCCAGG-3′Cyclin EForward, 5′-TGACAAGACTGTGAAAAGCCAGG-3′303D1401559Reverse, 5′-AGAAGAACTGCTCTCATCCTCGC-3′ShhForward, 5′-AGAGGCAGCACCCCAAAAAG-3′464NM_01722156Reverse, 5′-TTTCACAGAGCAGTGGATGCG-3′Hes5Forward, 5′-TGGAGATGCTCAGTCCCAAG-3′199NM_02438358Reverse, 5′-GCTTTGCTGTGCTTCAGGTAG-3′Brn3b promoterForward, 5′-CAGCCCCCGAGGGTGTGTGT-3′256NM_13894458Reverse, 5′-TCTGAACGGGTGCCGAGGTCT-3′Shh enhancerForward, 5′-GGAGGCTTCAGGAACAACTTGG-3′386AF09892560Reverse, 5′-GGGTAGGATGGATAGGGTTTTGG-3′Hes1 promoterForward, 5′-TCTCCTTGGTCCTGGAATAGTGC-3′398D1646456Reverse, 5′-ATCTGCCATTTCACCCCGAG-3′CD11b promoterForward, 5′-GACCAGGCAGGGCTATGT-3′122M8447752Reverse, 5′-AAAGCAAAGAAGGGCAGAAA-3′ Open table in a new tab Northern Analysis—Northern analysis was carried out to detect rBrm transcripts as described previously (39Dooley C.M. James J. Jane McGlade C. Ahmad I. J. Neurobiol. 2003; 54: 313-325Crossref PubMed Scopus (35) Google Scholar). Briefly, ∼2 μg of poly(A) RNA, isolated from adult retina, was electrophoresed on a 1.2% formaldehyde gel and transferred to Nytran Plus. Hybridization was carried out using a 32P-labeled rBrm cDNA probe overnight at 65 °C. Blot was washed sequentially with 2× SSC, 0.1% SDS at room temperature for 20 min, then twice with 1× SSC, 0.1% SDS at 65 °C for 15 min, and finally with 0.1× SSC, 0.1% SDS at 65 °C for 10 min, followed by autoradiography.siRNA Electroporation—Brm siRNA sequence was cloned into pSuper vector (Oligogene) according to the vendor's protocol. Electroporation was carried out according to a modified protocol of Matsuda and Cepko (40Matsuda T. Cepko C.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16-22Crossref PubMed Scopus (738) Google Scholar). Briefly, E14 retinas were dissected out and collected in Hanks' balanced salt solution containing pSuper-Brm and pEGFP-C3. The explants were placed in wells made in a 2% agarose gel. Electroporation in agarose gel reduced the shock, and thereby cell death. It also prevented aggregation of explants with one another. Electroporation was carried out by applying 5 pulses at 40 V for 50-ms durations with 950-ms intervals using gold-plated electrodes and an Electro Square Porator (BTX Inc.). The efficiency of electroporation was monitored by green fluorescent protein epifluorescence, and the explants were cultured in RCM containing 5% fetal bovine serum for 4 days. The effect of siRNA was ascertained by analyzing the Brm/β-actin protein and mRNA 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 calcium phosphate method, as described previously (27de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (269) Google Scholar). Neurospheres/RGC-5 cells, at ∼60% confluency, were exposed to retrovirus containing medium for 24 h. After infection, the medium was replaced with fresh culture medium.Reporter Assay—RGC-5/293T cells, transduced with pBabe/pBabe-Brm/pBabe-dnBrm were transfected with pGL2-Brn3b-luciferase (RGC-5 cells) and pGV-B-Hes1/Hes5 luciferase constructs (293T cells) using Lipofectamine (Invitrogen). Transfection efficiency was examined by co-transfecting cells with pGFP-C3 (Clontech). For luciferase assay, cells were lysed in 1× reporter lysis buffer (Promega), and 100 μl of lysate was diluted five times using assay reagent (Promega). Diluted samples (100 μl) were analyzed for luciferase activities using a luminometer (Pharmingen).Co-immunoprecipitation—Total protein was extracted from RGC-5 cells using a M-PER protein extraction kit (Pierce). Five hundred micrograms of total protein in 1 ml of RIPA buffer (50 mm Tris-HCl, pH 7.4, 15 mm NaCl, 1% Triton X-100, 0.1% SDS, 1mm EDTA, 1% sodium deoxycholate) was incubated with 5 μg of antibody overnight at 4 °C. Protein-antibody complex was precipitated by incubating with protein A/G-Sepharose for 1 h at 4 °C. The protein-antibody-Sepharose complex was precipitated by centrifuging at 1000 rpm for 1 min, and precipitates were washed three times with RIPA buffer, and the final pellet was resuspended in appropriate volume of loading buffer. The mixture was boiled to dissociate the complex and electrophoresed in a 7-9% denaturing polyacrylamide gel. Negative controls included reactions carried out without the antibody and with IgG.Western Blot Analysis—Western blot analysis was done as described previously (39Dooley C.M. James J. Jane McGlade C. Ahmad I. J. Neurobiol. 2003; 54: 313-325Crossref PubMed Scopus (35) Google Scholar). Samples from co-immunoprecipitation or protein isolated from siRNA-transfected retinas were electroblotted onto polyvinylidene difluoride membranes, following electrophoresis. Membranes were blocked for 1 h in 5% nonfat dry milk in TBST and incubated with anti-Brm/antiNotch (Santa Cruz Biotechnology), diluted 1:500 in TBST overnight at 4 °C with shaking. After incubating with anti-mouse horseradish peroxidase, the blots were washed with TBST, and immunoreactive bands were detected using ECL Western blotting detection reagents (RPN 2108, Amersham Biosciences). The blots were then exposed to x-ray film to visualize immunoreactive bands.Chromatin Immunoprecipitation—Chromatin immunoprecipitation (ChIP) assay was done using a modified procedure from Upstate Biotechnology, Inc. Briefly, transduced (pBabe/pBabe-Brm) or transfected (pSuper-Brm siRNa/Wt1 (KTS−)) RGC5 cells were grown until they reached confluency, and histones were cross-linked to DNA by adding formaldehyde directly to culture medium to a final concentration of 1% and incubating for 10 min at room temperature on a rocking platform. Cells were washed three times with ice-cold phosphatebuffered saline containing protease inhibitors. Cell pellets were resuspended in prewarmed SDS lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris, pH 8.1). To reduce the nonspecific background, the samples were pre-cleared using 80 μl of salmon sperm DNA/protein A-agarose slurry at 4 °C for 30 min. Samples were centrifuged at 100 rpm for 1 min at 4 °C. Supernatants were transferred to a new tube, and the immunoprecipitating antibody was added, and incubation was carried out overnight at 4 °C on a rocking platform. For a negative control, we used no antibody or IgG. The histone-antibody complex was precipitated using 60 μl of salmon sperm DNA and protein A-Sepharose (Upstate) for 1 h at 4°C. Precipitates were washed sequentially at room temperature for 5 min, once with low salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-Cl, pH 8.1, 150 mm NaCl), high salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-Cl, pH 8.1, 500 mm NaCl), and lithium salt immune complex wash buffer (2.25 m LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid-sodium salt, 1 mm EDTA, 10 mm Tris, pH 8.1), and twice with TE buffer (10 mm Tris-Cl, 1 mm EDTA, pH 8.0). After completely removing the TE buffer, the precipitate was resuspended and extracted twice in 250 μl of freshly prepared elution buffer (1% SDS, 0.1 m NaHCO3). To reverse the histone-DNA cross-linking, samples were heated at 65 °C for 4 h. 200 μl of initial sonicated sample was reverse cross-linked and used as an input. After removing the antibodies by protease digestion of the samples, DNA was recovered and column-purified. PCRs were performed using gene-specific primers.Restriction Enzyme Accessibility Assay—Restriction enzyme accessibility using LM-PCR assay was carried out as described previously (41Hempel W.M. Ferrier P. Methods Mol. Biol. 2004; 287: 53-63PubMed Google Scholar). Briefly, 5 × 106 nuclei from pBabe/pBabe-Brm transduced RGC5 cells were subjected to BglII (75-100 units) restriction enzyme digest for 30 min at 37 °C. First strand synthesis of genomic DNA (100 ng) was carried out using Pfu DNA polymerase (Stratagene) and gene-specific forward primer (5′-acccacgtctttctgcactag-3′). Following linker ligation, using T4 DNA ligase overnight at 17 °C, PCR was carried out on the ligated products using the gene-specific forward primer and linker-specific reverse primer (5′-ccgggagatctgaattcgat-3′) for 25 cycles at an annealing temperature at 65 °C. The PCR products were resolved on 1.2% agarose gel followed by Southern analysis using a PCR product corresponding to a sequence between the second TaqI site and the BgllII site in proximal Brn3b 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 differences between the various conditions.RESULTSExpression of Brm in the Developing Retina—Emerging evidence suggests that Brm plays an important role in cell differentiation (27de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (269) Google Scholar, 42Roy K. De La Serna I.L. Imbalzano A.N. J. Biol. Chem. 2002; 277: 33818-33824Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). To ascertain whether or not Brm has a similar role in retinal development, we examined the temporal patterns of Brm expression during early (E12-E18) and late (E18-PN6) retinal histogenesis by RT-PCR analysis. Retinal cells are born in an evolutionarily conserved temporal sequence; RGCs, cone photoreceptors, horizontal cells, and a majority of amacrine cells are born during early histogenesis, whereas rod photoreceptors, bipolar cells, and Müller glia are generated during late histogenesis (43Sidman R.L. Smelser G. Structure of the Eye. Academic Press, New York1961: 487-506Google Scholar, 44Young R.W. Anat. Rec. 1985; 212: 199-205Crossref PubMed Scopus (875) Google Scholar). We observed that levels of Brm transcripts increased at E14 stage, compared with those at the E12 stage (Fig. 1, A and B). The stages between E12 and E14 represent the period of active neurogenesis when the majority of RGCs, horizontal cells, and cone photoreceptors are born (45Rapaport D.H. Wong L.L. Wood E.D. Yasumura D. LaVail M.M. J. Comp. Neurol. 2004; 474: 304-324Crossref PubMed Scopus (294) Google Scholar). The decrease in the expression of Brm at stage E16 is likely because of the fact that E16 represents a stage of relative quiescence during early histogenesis (12Austin C.P. Feldman D.E. Ida Jr., J.A. Cepko C.L. Development (Camb.). 1995; 121: 3637-3650PubMed Google Scholar). Similarly, during late histogenesis, there was a significant increase in levels of Brm transcripts at PN1 and PN3, compared with those at E18 when the majority of rod photoreceptors, which constitute ∼80% of total cells in rodent retina, are born (45Rapaport D.H. Wong L.L. Wood E.D. Yasumura D. LaVail M.M. J. Comp. Neurol. 2004; 474: 304-324Crossref PubMed Scopus (294) Google Scholar). The Brm expression persisted through postnatal stages and its levels were highest in adult retina, compared with all stages examined. In addition, the specificity of Brm expression was determined by sequencing PCR products and Northern analysis of RNA from the adult retina, which revealed a 5.8-kb band corresponding to full-length Brm transcripts (46Muchardt C. Yaniv M. EMBO J. 1993; 12: 4279-4290Crossref PubMed Scopus (522) Google Scholar) (Fig. 1C). Together, these results suggested that Brm expression is associated with retinal histogenesis. To obtain further insight into the involvement of Brm with the process of cell differentiation in retina, we examined the cell-specific expression of Brm in the retina of E14 and E18 embryos. We observed that Brm immunoreactivities were distributed toward the scleral and ventricular sides in the developing retina (Fig. 1, D-I). In both E14 and E18 retina, Brm immunoreactivities were predominantly localized toward the scleral side where differentiating precursors are located. As reported previously, Brm immunoreactivities had nuclear as well as cytoplasmic localization (47Fukuoka 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. Jen J. Clin. Cancer Res. 2004; 10: 4314-4324Crossref PubMed Scopus (174) Google Scholar). Next, to test whether or not Brm expression is associated with differentiating precursor population, we examined the cellular distribution of Brm immunoreativities in the retina of E14 and E18 embryos, pre-exposed to BrdUrd in utero to identify proliferating (BrdU+) cells (Fig. 1J). We observed that Brm immunoreactivities were associated with both BrdU+ and BrdU− cells. However, in the total cell population 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 predominantly associated with post-mitotic cells (Fig. 1L). A comparison of different classes of cells at the two different stages revealed that the proportion of BrdU−Brm+ cells increased ∼2-fold in E18 retina relative to that in E14 retina (12.43 ± 0.75 versus 6.8 ± 0.23, p < 0.05), demonstrating a progressive association of Brm with post-mitotic cells as development progressed. Together, these observations suggested that the expression of Brm correlated with the process of differentiation of retinal stem cells/progenitors.FIG" @default.
• W2022551903 created "2016-06-24" @default.
• W2022551903 creator A5005591732 @default.
• W2022551903 creator A5032067317 @default.
• W2022551903 creator A5037386161 @default.
• W2022551903 creator A5042534461 @default.
• W2022551903 creator A5049456907 @default.
• W2022551903 creator A5050216645 @default.
• W2022551903 creator A5051464513 @default.
• W2022551903 creator A5053641440 @default.
• W2022551903 creator A5066312889 @default.
• W2022551903 creator A5073140225 @default.
• W2022551903 creator A5074549939 @default.
• W2022551903 date "2007-11-01" @default.
• W2022551903 modified "2023-10-03" @default.
• W2022551903 title "SWI/SNF Chromatin Remodeling ATPase Brm Regulates the Differentiation of Early Retinal Stem Cells/Progenitors by Influencing Brn3b Expression and Notch Signaling" @default.
• W2022551903 cites W129269179 @default.
• W2022551903 cites W1515408155 @default.
• W2022551903 cites W1533487943 @default.
• W2022551903 cites W1547610032 @default.
• W2022551903 cites W1604048158 @default.
• W2022551903 cites W1636569427 @default.
• W2022551903 cites W1835721351 @default.
• W2022551903 cites W1863364305 @default.
• W2022551903 cites W1963788105 @default.
• W2022551903 cites W1964318436 @default.
• W2022551903 cites W1965772224 @default.
• W2022551903 cites W1967047395 @default.
• W2022551903 cites W1968800607 @default.
• W2022551903 cites W1970709734 @default.
• W2022551903 cites W1970794606 @default.
• W2022551903 cites W1981429455 @default.
• W2022551903 cites W1982021610 @default.
• W2022551903 cites W1988939522 @default.
• W2022551903 cites W1989312217 @default.
• W2022551903 cites W1989312919 @default.
• W2022551903 cites W1997272979 @default.
• W2022551903 cites W1997987643 @default.
• W2022551903 cites W2004391147 @default.
• W2022551903 cites W2008638145 @default.
• W2022551903 cites W2010544272 @default.
• W2022551903 cites W2011204532 @default.
• W2022551903 cites W2012179207 @default.
• W2022551903 cites W2013851169 @default.
• W2022551903 cites W2014046637 @default.
• W2022551903 cites W2019855082 @default.
• W2022551903 cites W2022179732 @default.
• W2022551903 cites W2029763858 @default.
• W2022551903 cites W2030802556 @default.
• W2022551903 cites W2031330495 @default.
• W2022551903 cites W2034310924 @default.
• W2022551903 cites W2036684921 @default.
• W2022551903 cites W2038439057 @default.
• W2022551903 cites W2043655216 @default.
• W2022551903 cites W2046741131 @default.
• W2022551903 cites W2047341780 @default.
• W2022551903 cites W2049279317 @default.
• W2022551903 cites W2054244366 @default.
• W2022551903 cites W2055689986 @default.
• W2022551903 cites W2055946685 @default.
• W2022551903 cites W2057924308 @default.
• W2022551903 cites W2058705034 @default.
• W2022551903 cites W2058902552 @default.
• W2022551903 cites W2059128085 @default.
• W2022551903 cites W2059679976 @default.
• W2022551903 cites W2060997280 @default.
• W2022551903 cites W2061304668 @default.
• W2022551903 cites W2074974586 @default.
• W2022551903 cites W2079229603 @default.
• W2022551903 cites W2079462555 @default.
• W2022551903 cites W2080937698 @default.
• W2022551903 cites W2081235375 @default.
• W2022551903 cites W2082401415 @default.
• W2022551903 cites W2093001237 @default.
• W2022551903 cites W2095666604 @default.
• W2022551903 cites W2100189018 @default.
• W2022551903 cites W2101728847 @default.
• W2022551903 cites W2107766440 @default.
• W2022551903 cites W2109652663 @default.
• W2022551903 cites W2110050626 @default.
• W2022551903 cites W2114967857 @default.
• W2022551903 cites W2119855916 @default.
• W2022551903 cites W2131214833 @default.
• W2022551903 cites W2133162368 @default.
• W2022551903 cites W2136582666 @default.
• W2022551903 cites W2136904483 @default.
• W2022551903 cites W2140034535 @default.
• W2022551903 cites W2146977555 @default.
• W2022551903 cites W2148768745 @default.
• W2022551903 cites W2152907543 @default.
• W2022551903 cites W2157156910 @default.
• W2022551903 cites W2157410753 @default.
• W2022551903 cites W2161225294 @default.
• W2022551903 cites W2161852106 @default.
• W2022551903 cites W2162008350 @default.
• W2022551903 cites W2163517157 @default.
• W2022551903 cites W2166844117 @default.
• W2022551903 cites W2167940414 @default.

• next