Chromatin remodeling enzyme Brg1 is required for mouse lens fiber cell terminal differentiation and its denucleation
© He et al; licensee BioMed Central Ltd. 2010
Received: 7 July 2010
Accepted: 30 November 2010
Published: 30 November 2010
Brahma-related gene 1 (Brg1, also known as Smarca4 and Snf2β) encodes an adenosine-5'-triphosphate (ATP)-dependent catalytical subunit of the (switch/sucrose nonfermentable) (SWI/SNF) chromatin remodeling complexes. SWI/SNF complexes are recruited to chromatin through multiple mechanisms, including specific DNA-binding factors (for example, heat shock transcription factor 4 (Hsf4) and paired box gene 6 (Pax6)), chromatin structural proteins (for example, high-mobility group A1 (HMGA1)) and/or acetylated core histones. Previous studies have shown that a single amino acid substitution (K798R) in the Brg1 ATPase domain acts via a dominant-negative (dn) mechanism. Genetic studies have demonstrated that Brg1 is an essential gene for early (that is, prior implantation) mouse embryonic development. Brg1 also controls neural stem cell maintenance, terminal differentiation of multiple cell lineages and organs including the T-cells, glial cells and limbs.
To examine the roles of Brg1 in mouse lens development, a dnBrg1 transgenic construct was expressed using the lens-specific αA-crystallin promoter in postmitotic lens fiber cells. Morphological studies revealed abnormal lens fiber cell differentiation in transgenic lenses resulting in cataract. Electron microscopic studies showed abnormal lens suture formation and incomplete karyolysis (that is, denucleation) of lens fiber cells. To identify genes regulated by Brg1, RNA expression profiling was performed in embryonic day 15.5 (E15.5) wild-type and dnBrg1 transgenic lenses. In addition, comparisons between differentially expressed genes in dnBrg1 transgenic, Pax6 heterozygous and Hsf4 homozygous lenses identified multiple genes coregulated by Brg1, Hsf4 and Pax6. DNase IIβ, a key enzyme required for lens fiber cell denucleation, was found to be downregulated in each of the Pax6, Brg1 and Hsf4 model systems. Lens-specific deletion of Brg1 using conditional gene targeting demonstrated that Brg1 was required for lens fiber cell differentiation, for expression of DNase IIβ, for lens fiber cell denucleation and indirectly for retinal development.
These studies demonstrate a cell-autonomous role for Brg1 in lens fiber cell terminal differentiation and identified DNase IIβ as a potential direct target of SWI/SNF complexes. Brg1 is directly or indirectly involved in processes that degrade lens fiber cell chromatin. The presence of nuclei and other organelles generates scattered light incompatible with the optical requirements for the lens.
Eukaryotic DNA is organized as chromatin in the nucleus. Chromatin is a copolymer of DNA, histone and nonhistone proteins and small noncoding RNA. During embryonic development, specific regions of the genome alter their chromatin organization . Gene expression is regulated at the level of the chromatin structure of individual genes and/or loci in the context of the three-dimensional organization of chromatin inside the cell nucleus. Local chromatin structure affects multiple stages of transcription, including the accessibility of sequence-specific DNA-binding transcription factors to promoters, enhancers and other genomic regulatory regions. Two major modifications of local chromatin structure (that is, chromatin remodeling) include posttranslational modifications of histones and adenosine-5'-triphosphate (ATP)-dependent alteration of nucleosomes .
ATP-dependent chromatin remodeling refers to dynamic processes in which multiprotein switch/sucrose nonfermentable (SWI/SNF), ISWI (Imitation Switch) and nucleosome remodeling and deacetylase (NuRD) complexes use nucleosomes as substrates and change positions of individual histone octamers and/or change the topology of DNA that is wrapped around the individual nucleosome particles . Mammalian SWI/SNF complexes, SWI/SNF-A and SWI/SNF-B/polybromo-associated Brg1-associated factor (PBAF), are composed of a catalytical and several additional regulatory subunits, Brg1-associated factors (BAFs). Brg1 (Smarca4/Snf2β) and Brahma (Brm; Smarca2/Snf2α) are structurally similar chromatin remodeling ATP-dependent helicases that play distinct roles during embryonic development . Brahma-related gene 1 (Brg1, also known as Smarca4 and Snf2β) is essential for early mammalian development as mutated embryos die during the preimplanation phase . In contrast, loss of function of Brm leads to increased cellular proliferation in adult mouse tissues . To study Brg1 function during organogenesis, conditional gene targeting of Brg1 was performed in T-cells , embryonic ectoderm/keratinocytes , hematopoietic/endothelial cells  and neural stem cells . These studies found a wide range of cell autonomous defects, including the control of T-cell proliferation and survival , terminal differentiation of keratinocytes , differentiation and apoptosis of primitive erythrocytes  and neural stem maintenance and gliogenesis . Mammalian SWI/SNF complexes participate in DNA double-strand break repair as they bind to the phosphorylated H2A histone family, member X (H2AX), histone variant, and promote its phosphorylation . Recent studies have also established specific roles of Brg1 in DNA replication . Additional insights into the role of Brg1 in muscle [13, 14], mammary epithelium , smooth muscle [16, 17] and myeloid  differentiation have been generated through the studies of a specific point mutation (K798R) in the ATP-binding domain of Brg1 that act via a dominant-negative (dn) mechanism . In the eye, studies using zebrafish showed that Brg1 plays specific roles in lens and retinal development [20–22]. Eye differentiation defects found in zebrafish mutation young (yng) were linked to the presence of an Y390X mutation in the Brg1/Smarca4 gene on chromosome 3 [20, 21]. Nevertheless, the existence of two Brg1-homologous genes, located on chromosomes 3 and 6 of the duplicated zebrafish genome, requires additional experimentation to clarify the roles of Brg1 enzymes in vertebrate eye development.
Central to understanding chromatin remodeling in embryonic development is to identify those genes that are regulated by specific chromatin remodeling systems and to elucidate the molecular mechanisms that recruit the remodelers to specific regions of chromatin. The molecular mechanisms of chromatin remodeling mediated by SWI/SNF complexes were probed using a combination of biochemical and genetic experiments. These experiments mostly examined the function of Brg1 as this enzyme alone can remodel nucleosomes [23, 24]. Genes regulated by SWI/SNF complexes in vertebrate systems were identified using candidate gene approaches [15, 25, 26] and RNA expression profiling [14, 22, 27]. The SWI/SNF complexes are recruited to DNA by at least four different mechanisms. Several lineage-specific DNA-binding transcription factors, including cAMP response element-binding factor (CREB), Hsf4, microphthalmia-associated transcription factor (Mitf), Pax6 and T-box transcription factor 2 (Tbx2), were shown to associate with Brg1 using various in vitro protein interaction and whole cell extract coimmunoprecipitation assays [28–30]. Other transcription factors associate with Brg1-associated factor (BAF) subunits, that is, BAF60c interacts with retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers . Brg1 contains a 110-amino-acid-long bromodomain that recognizes acetylated lysines in core histones . Brg1 also interacts with chromosomal architectural proteins such as high mobility group A1 (HMGA1) . Thus, CREB transcription factor, Hsf4 and Pax6 (see above) can potentially regulate lens development via recruitment of Brg1-containing SWI/SNF complexes [34, 35].
Embryonic lens development is an excellent system to study both individual cell lineage formation and terminal differentiation. Lens lineage originates from the preplacodal region that is established around the anterior neural plate of the vertebrate embryo [36, 37]. The lens placode, a thickened surface ectoderm, is the first morphologically distinct structure composed of lens progenitor cells. Invagination of the lens placode generates the lens vesicle, a polarized structure composed of lens precursor cells. The posterior cells of the lens vesicle exit the cell cycle and undergo terminal differentiation to generate primary lens fibers. The primary lens fibers are highly elongated cells filling the bulk of space of the original lens vesicle. The anterior cells of the lens vesicle subsequently differentiate into the anterior lens epithelium . The lens grows through the entire lifespan as a result of epithelial cell division and migration toward the lens equator. When the epithelial cells reach the equator, they undergo terminal differentiation as secondary lens fibers. The hallmark of lens fiber cell differentiation is the expression of lens-preferred genes, the crystallins , and synchronized degradation of all subcellular organelles . Lens fiber cell denucleation (karyolysis) is a final stage of this process that destroys the lens chromatin and/or epigenome. Aside from the active role of the acid DNase IIβ in this process, very little is known about molecular pathways that regulate lens fiber cell denucleation .
To investigate the role of Brg1 in lens fiber cell differentiation, we expressed a dn mutant of Brg1 using the lens-specific αA-crystallin promoter in postmitotic lens fibers. We examined lens growth and differentiation, focusing on the potential defects in the lens fiber cell denucleation process. Next, we identified differentially expressed genes in this system and compared these genes with genes regulated by Hsf4 and Pax6, two lineage-specific DNA-binding transcription factors shown to associate with SWI/SNF complexes through the Brg1 subunits [29, 30]. The role of Brg1 during embryonic lens development was examined by conditional Brg1 gene inactivation in mouse embryos.
Expression of Brg1 in mouse embryonic eye
Expression of dominant-negative Brg1 (dnBrg1) in transgenic lens disrupts lens fiber cell differentiation and induces cataract formation
Analysis of lens fiber cell terminal differentiation in dnBrg1 transgenic mouse
To evaluate the expression of two key markers of lens fiber cell differentiation in wild-type and transgenic lenses, we used antibodies specific to αA-crystallin and major intrinsic protein of lens fibers (main intrinsic polypeptide (MIP), also known as aquaporin O and MIP26) to perform immunochemical staining. We found reduced expression of both lens structural proteins in the transgenic mice (Additional file 1). Next, to formally exclude the possibility that transgenic lens fibers reentered cell cycle, we performed bromodeoxyuridine (BrdU) incorporation assays and detected proliferating cells only in the lens epithelium (Additional file 2). From these morphological, microscopic and immunohistochemical studies, we concluded that lens-specific expression of dnBrg1 disrupted proper fiber cell organization and suture formation, which impaired the optical quality of transgenic lenses and contributed to progressive cataract formation.
Identification of differentially expressed genes between dnBrg1 transgenic and wild-type lenses
Several genes implicated in lens differentiation were found among the 6,828 differentially expressed transcripts. We selected 16 of these genes for secondary validation using qRT-PCR (Additional files 3 and 4). The analysis was carried out with cDNA prepared from independent pools of day E15.5 wild-type and dnBrg1 total lens RNA preparations as described in Methods. Internal controls (B2m, Hprt and Sdha) were included for data normalization . These results confirmed upregulation of 12 genes (Bfsp1, Cdkn1b/p27, Dnmt3a, Fgfr1, Gsn, Hif1a, Hod/Hop, Mab21l1, Prox1, Smarcd1, Smarce1 and Vim) and downregulation of 3 genes (Dnase2b, Jag1 and Pitpnm2) in the dnBrg1 lenses (Additional file 3). Expression of Six3 showed no change on the arrays and minor downregulation by qRT-PCR (Additional file 3). In addition, we examined expression of Brm (Smarca2) in this system to address potential increase or decrease of expression of this ATPase . We found a moderate upregulation of Smarca2 transcripts (1.92-fold for array data and 1.50-fold for qPCR data). Upregulation of Smarcd1 and Smarce1, two genes encoding noncatalytic subunits of SWI/SNF complexes, was also validated (Additional file 3). From these data, we concluded that approximately one third of the lens transcriptome was directly or indirectly affected by the transgenic overexpression of dnBrg1 with comparable numbers of up- and downregulated transcripts.
Using the Database for Annotation, Visualization and Integrated Discovery (DAVID), we further classified those 178 genes that were commonly deregulated in both Pax6 heterozygous and dnBrg1 transgenic lenses (Figure 10A). A total of 130 genes (73%) were classified using the DAVID tool. Among these, there were 63 individual genes with partially redundant presence in multiple Gene Ontology (GO) categories. For example, the Dnase2b gene was found in GO Biological Process category "DNA metabolic process," GO Molecular Function category "nuclease activity," GO Cellular Compartment "vacuole" and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway "lysozome" (Additional file 6). Among the top-ranking GO categories we identified were GO Molecular Function "nucleotide binding" (n = 20, P = 0.005), "DNA metabolic process" (n = 6, P = 0.043) and "DNA replication" (n = 4, P = 0.032). These results are in agreement with Brg1's serving as a global regulator of chromatin and chromatin-associated processes including DNA repair  and DNA replication .
Targeted deletion of Brg1 causes multiple eye developmental abnormalities
A summary of gross morphology of the lens-specific Brg1 conditional knockout mice
Total number of embryos
No obvious phenotype
Abnormal eye development
Brg1flox/+ ; Le-Cre
Using a combination of two complementary genetic approaches, the present studies demonstrate that Brg1 is required for mouse lens fiber cell differentiation. Lens-specific expression of the dnBrg1 perturbed lens fiber cell differentiation process at multiple levels and resulted in cataract formation. In these abnormal lens fibers, nuclei were not degraded, suggesting that Brg1 participates in normal lens fiber cell karyolysis. The advantage of this system is that function of Brg1 was disrupted only in postmitotic lens fiber cells; however, this system is unlikely to produce complete inactivation of Brg1 biological activity . To address this problem, conditional inactivation of Brg1 using a MLR39 Cre line, active only in differentiating lens fibers, would be required . In parallel, conditional inactivation of Brg1 in the surface ectoderm resulted in a range of lens and/or eye developmental abnormalities, including the retention of nuclei in lens fiber cells. Incomplete deletion of floxed Brg1 alleles by Le-cre followed by clonal selection of viable cells and/or prolonged stability of Brg1 proteins in lens cells precludes any definitive conclusions about the potential role of Brg1 in lens lineage formation and lens placode invagination. In the majority of mutants, lens vesicles were formed and differentiation of primary lens fibers was compromised. Absence of lens in the mutated eye was accompanied by an aberrant infolding of the retina. Similar defects were found in Pax6 embryos conditionally inactivated in the surface ectoderm . Collectively, the present studies reveal an essential, novel role of Brg1 in lens fiber differentiation and denucleation. In addition, secondary defects in retinal formation suggest that Brg1 can play cell nonautonomous roles in retinal development that originate from aberrant lens morphogenesis as described elsewhere [47, 52, 55–58].
Although the use of two loss-of-function approaches to study the Brg1 function in lens development generated comparable results at the morphological and cellular levels, molecular studies using RNA expression profiling identified only a small number of commonly regulated genes (Figure 14). There are at least three factors that could contribute to these findings. First, loss of function of Brg1 from day E9.0, that is, prior to the morphological formation of lens pit and/or vesicle, should impair lens development more severely compared to the transgenic dnBrg1 system with later onset expression (from day ~ E11.5) in postmitotic lens fibers. Second, we could not isolate mutated lenses from comparable, that is, E15.5-day-old, embryos because of their structural fragility. Instead, we had to dissect eyeballs including mutated lenses and other affected tissues, and this tissue heterogeneity was reflected in the eyeball transcriptome. Third, the variability of the Brg1 cKO phenotypes (Table 1 and Figure 12) makes it difficult to microdissect lenses, even under ideal conditions, with abnormalities comparable to the dnBrg1 transgenic lenses.
Previous studies of Brg1 function in other cells and tissues established Brg1 as a specific regulator of cell proliferation, differentiation and survival [7–9, 11, 12]. The present studies in lens suggest that Brg1 plays a major role in lens fiber cell differentiation. Though Brg1 is highly expressed in the surface ectoderm that gives rise to the lens placode (Figures 1A and 1B), Brg1's role in the formation of lens lineage remains to be determined through detailed analysis of early stage (days E9-E10) embryos. Expression of Brg1 is reduced in differentiating primary and secondary lens fibers. Brg1's role in lens fiber cell differentiation is supported by ectopic expression of the dnBrg1 transgene in lens and by conditional inactivation of Brg1 in the presumptive lens ectoderm. Three transgenic mouse lines were established and generated similar lens-specific differentiation defects. Although the lens-specific knockout resulted in variable eye defects, in the majority of embryos, we detected rudimentary lens formation. This variability could originate from incomplete deletion of both Brg1 alleles, compensation via Brm/Smarca2 and/or via other mechanisms such as prolonged stability of the Brg1 proteins. Upregulation of Brm/Smarca2 was indeed found in the dnBrg1-transgenic model. A large number of transgenic lens studies utilizing the αA-crystallin promoter induced cell cycle reentry and/or apoptosis in the lens fiber cell compartment [41, 42, 59–62]. In the present study, no evidence for apoptosis (data not shown) and cell cycle reentry (see Additional file 2) in postmitotic dnBrg1 transgenic lens fibers was found.
Earlier studies identified αA- and αB-crystallins (among ~ 80 other genes) upregulated in the human adrenal carcinoma cell line SW-13, deficient in both Brg1 and Brm expression, in which Brg1 was reintroduced [27, 33]. Here we show reduced expression and accumulation of αA-crystallin in dnBrg1-transgenic and Brg1-cKO lenses that is consistent with our earlier findings that abundant quantities of Brg1 are present within a 16-kb region of lens-specific chromatin of the mouse Cryaa locus . On the basis of the data shown here and in our earlier studies , we conclude that αA-crystallin gene and/or locus is regulated directly by at least three DNA-binding transcription factors, Pax6, c-Maf and CREB, and by chromatin remodeling enzyme Brg1. We propose that Brg1, and, by inference, SWI/SNF complexes, are recruited to the Cryaa locus by Pax6 and possibly by other mechanisms, including the recognition of acetylated lysine residues by Brg1 bromodomain.
To identify genes downstream of Brg1 in lens, we performed RNA expression profiling studies in day E15.5 dnBrg1 transgenic lenses followed by comparative analyses of differentially expressed genes in Pax6 heterozygous and Hsf4 homozygous lenses. We reasoned that if lens lineage-specific DNA-binding transcription factors Pax6 and Hsf4 serve in vivo as recruiters of SWI/SNF complexes to specific regions of chromatin, we could find commonly regulated genes in these three model systems. Among those 6,828 transcripts regulated in dnBrg1 transgenic lenses, 715 (~ 10.5%) transcripts were also regulated in Pax6 +/- and Hsf4 -/- mutated lenses. Within the 559 differentially expressed genes in Pax6 +/- lenses, 178 (~ 32%) transcripts were shared between these two systems. Similarly, within the 1,428 differentially expressed genes in Hsf4 -/- mutated lenses, 559 (~ 39%) transcripts were commonly deregulated. Finally, 22 genes, including lens-preferred acid DNase IIβ endonuclease, were found to be dysregulated in all three mutated lenses. These results suggest that Brg1, Hsf4 and Pax6 exert their function through commonly regulated genes. In addition, the use of the DAVID and GSEA analysis tools for interpretation of genomewide expression profiles identified several functionally related groups of genes suggesting the presence of specific Brg1-dependent coregulated biological processes. Additional molecular studies using chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) and related methods are required to demonstrate co-localization of Brg1, Hsf4 and Pax6 proteins in lens chromatin at their target genes.
Our results demonstrate that lens fiber cell terminal differentiation, including their denucleation (karyolysis), requires the ATP-dependent chromatin remodeling enzyme Brg1. Our data suggest that Brg1, together with two lens lineage transcription factors, Pax6 and Hsf4, is required for the transcriptional regulation of DNase IIβ, the key enzyme for lens fiber cell denucleation. In addition, the present data are consistent with our earlier findings suggesting that Brg1 regulates directly the expression of the αA-crystallin gene, the key structural protein of the mammalian lens. These results provide new molecular insights into the process of lens fiber terminal differentiation and open new research avenues to probe chromatin structure prior to and during lens fiber cell denucleation.
The primary antibodies used were Brg1, αA-crystallin, γ-crystallin, Pax6, MIP/MIP26/aquaporin 0 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Flag M2 (Sigma-Aldrich, St. Louis, MO, USA). The secondary antibodies used were Alexa 568 goat anti-rabbit (Molecular Probes, Eugene, OR, USA) and biotin-conjugated secondary anti-rabbit (EO466; Dako, Carpintera, CA, USA).
Generation of transgenic dnBrg1 mice
The plasmid pACP3  was modified to insert Eco RV and Mlu I restriction sites into both 5'- and 3'-Not I sites that flank the αA-crystallin promoter/polylinker/simian virus 40 (SV40)/polyA sequences. A Brg1 cDNA containing the K798R mutation was cloned as a 5.2-kb Cla I-Spe I fragment obtained from pBluescript KS(+)-FLBrg1Mut  to generate pCryaa/dnBrg1 (see Figure 2A). A 7.2-kb Eco RV fragment was released from this plasmid and used to generate transgenic mice by injection into FVB/N fertilized oocytes. Transgene integration was confirmed by genomic PCR using tail DNA with the following primers: 5'-ATGGCTCCAGGGGAAGG-3' and 5'-CATTCCTTTCATCTGGTTG-3'. The cycling parameters were 94°C for 30 s, 55°C for 45 s and 72°C for 70 s for 35 cycles.
Conditional inactivation of Brg1 in the presumptive lens ectoderm
Brg1 flox/flox mice  were mated with Le-Cre-transgenic mice , and the progeny were crossed to generate litters containing homozygous floxed alleles and heterozygous for the Cre transgene. Mice genotyping was performed as described previously in the literature [7, 47]. Animal husbandry and experiments were conducted in accordance with the approved protocol of the Albert Einstein College of Medicine Animal Institute Committee and the Association for Research in Vision and Ophthalmology Statement for the use of animals in ophthalmic and vision research. Noon of the day the vaginal plug was observed was considered to be day E0.5 of embryogenesis.
Animals were killed by CO2, and either the embryos were dissected from pregnant females or whole eyeballs were removed from postnatal animals. Tissues were then fixed in 10% neutral buffered paraformaldehyde overnight at 4°C, processed and embedded in paraffin. Serial sections were cut in 5-μm thickness through the midsection of the lens and stained with hematoxylin and eosin or used for subsequent experiments. Immunohistochemistry was performed as described previously  with the 3,3'-diaminobenzidine (DAB) kit (Vector Laboratories, Burlingame, CA, USA). Indirect immunohistological staining was conducted following standard procedures. Briefly, sections were washed twice in Tris-buffered saline (TBS) and blocked for 30 min with Image-iT™ FX signal enhancer (Molecular Probes). Then sections were washed twice in TBS before undergoing primary antibody incubation in 1% Bovine Albumin Fraction Solution (Invitrogen, Grand Island, NY, USA) and 0.1% (vol/vol) Triton X in TBS overnight at 4°C in a humidified chamber. After being washed twice in PBS, sections were incubated with the secondary antibody for 1 h at room temperature. Sections were then washed three times with PBS and mounted with fluorescence preserve mounting medium (Vector Laboratories). Primary antibodies were diluted (vol/vol) as follows: Brg1 (1:200), αA-crystallin (1:1,000), γ-crystallin (1:1,000), Flag (1:150), MIP26 (1:200) and Pax6 (1:400). Secondary antibodies were diluted as follows: Alexa 568 goat antirabbit (1:500) and biotin-conjugated secondary antirabbit (1:400). For immunofluorescence staining, primary antibodies were diluted in blocking solution and nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Images were obtained with a Zeiss Axioskop II light microscope or a Leica MZ FLIII fluorescence stereomicroscope (Heerbrugg, Switzerland).
Scanning and transmission electron microscopy
Three-month-old mouse lenses from wild-type and dnBrg1 animals were fixed overnight at room temperature in 0.08 M sodium cacodylate, 1.25% glutaraldehyde and 1% paraformaldehyde (pH 7.4). After fixation, the lens capsule and several of the outermost layers of the fiber cells were peeled off to show the fiber pattern. Then the lens samples were dehydrated through a graded series of ethanol and processed by critical point dry (CPD) using liquid carbon dioxide in a Tousimis Samdri 795 Critical Point Drier (Rockville, MD, USA). Subsequently, lens samples were transferred to a filter paper, placed in vacuum desiccators, mounted on a stub and sputter-coated for 2 min with gold-palladium in Denton Vacuum Desk-2 Sputter Coater (Cherry Hill, NJ, USA). Specimens were examined using a JEOL JSM6400 Scanning Electron Microscope (Peabody, MA, USA) with an accelerating voltage of 10 kV.
For transmission electron microscopy, eyes from 3-month-old mice were fixed for several hours in a 2% paraformaldehyde/2.5% glutaraldehyde solution. While in fixative, the posterior hemispheres of eyeballs were pierced with a fine needle. After being rinsed in 0.1 M cacodylate buffer, eyeballs were postfixed in a mixture of 1% OsO4 and 0.8% potassium ferrocyanide in 0.1 M cacodylate buffer for 2 h at 4°C. Specimens were then dehydrated in a graded series of ethanol and embedded in Epon (Serva, Heidelberg, Germany). Semithin sections (1 μm) were collected on uncoated glass slides and stained with methylene blue/azure II  for light microscopy. Ultrathin sections were mounted on uncoated copper grids, stained with uranyl acetate and lead citrate and examined on a Zeiss Libra electron microscope.
Oligonucleotide microarrays and mRNA expression profiling
Lenses were isolated from day E15.5 transgenic and wild-type embryos and stored in RNA Later (Ambion, Woodlands, TX, USA). Newborn wild-type and Hsf4 -/- lenses were described previously . Whole eyeballs were isolated from newborn wild-type and conditional Brg1 knockouts as microdissected lenses were difficult to obtain because of their mechanical fragility. RNA isolations were performed using the RNeasy MiniElute Kit and RNase-Free DNase set (Qiagen, Valencia, CA, USA). RNA quality was assessed using the Agilent 2100 Bioanalyzer with the Nano LabChip Kit (Agilent Technologies, Palo Alto, CA, USA) following the manufacturer's instructions. Replicate sets of RNA from distinct day E15.5 dnBrg1 embryonic lenses (n = 4) and individual newborn eyeballs (n = 3) and corresponding numbers of stage-matched wild-type littermates were prepared for microarray analyses. cDNA synthesis and amplifications were performed with the Ovation™ RNA Amplification System V2 (Nugen, San Carlos, CA, USA) using 50 ng of total RNA per sample. Amplified cDNA were cleaned and purified with the DNA clean and Concentrator™-25 kit (Zymo Research, Orange, CA, USA). Fragmentation and labeling were performed using the FL Ovation™ cDNA Biotin Module V2 (Nugen) according to the manufacturer's instructions. The samples were subsequently hybridized on Mouse Genome 430A 2.0 arrays (Affymetrix, Santa Clara, CA, USA) following the manufacturer's specification.
Bioinformatic tools and statistical filtering of RNA microarray results
Differentially regulated genes and/or mRNA between dnBrg1 and wild-type lenses were identified using biological quadruplicate sets (n = 4) of robust multichip average (RMA)-normalized Affymetrix CEL files  by a conjunction of Student's t-test (P < 0.05) and significance analysis of microarrays (SAM; false discovery rate FDR set to < 1%), using the TIGR Multiexperiment Viewer of the TM4 suite (Dana-Farber Cancer Institute, Boston, MA, USA) . A similar strategy was used to identify differentially regulated genes and/or mRNA between Hsf4-null and wild-type lens (biological triplicates, RMA normalization, conjunction of t-test; P < 0.05 and SAM FDR < 5%). Primary data from this study were deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus database under accession numbers GSE22322 (the dnBrg1 part), GSE22362 (the Hsf4 part) and GSE25168 (the Brg1 cKO part). The R-based extension to GeneSpring GS7 (Agilent Technologies, Santa Clara, CA, USA) was used to create a boxplot representation of 6,828 Brg1 target genes in Figure 8 to generate a five-number summary including the smallest observation, lower quartile, median, upper quartile, largest observation and indications of outlier observations. The GO and KEGG pathway functional annotations were performed using the National Institutes of Health web-based tool DAVID (Database for Annotation, Visualization and Integrated Discovery) . GSEA (http://www.broad.mit.edu/gsea/) was additionally used to identify significantly enriched pathways disrupted in dnBrg1-transgenic lenses .
Relative expression levels of 16 genes were verified by qRT-PCR. Total RNA from biological triplicates of transgenic and wild-type littermate lenses were isolated using RNeasy MiniElute Kit and RNase-Free DNase set (Qiagen, Valencia, CA, USA). Subsequently, cDNA was synthesized with Random Haxamer primers (Invitrogen) and Superscript TM III Reverse Transcriptase (Invitrogen) following the manufacturers' instruction. cDNA was diluted 10 times, and qRT-PCR was performed using the Applied Biosystems (ABI, Foster City, CA, USA) 7900HT fast Real-Time PCR system with Power SYBR Green PCR master mix (ABI). Specific primers for qRT-PCR are listed in Additional file 12. Primers were designed across neighboring introns using NCBI Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi). Transcripts encoding β2 microglobulin (B2M), succinate dehydrogenase complex subunit A (SDHA), and hypoxanthine-guanine phosphoribosyltransferase (HPRT) genes were used for normalization of expression levels of both transgenic and wild-type results as described previously [45, 70].
A list of abbreviations used
- ATP :
- BAF :
Brg1 Associated Factor
- Brg1 :
Brahma-Related Gene 1
- CREB :
cAMP Response Element Binding Factor
- DAVID :
Database for Annotation, Visualization and Integrated Discovery
- dn :
- GO :
- GSEA :
Gene Set Enrichment Analysis
- HMGA 1:
High Mobility Group A 1
- Hsf4 :
Heat Shock Transcription Factor 4
- INL :
Inner Nuclear Layer
- ISWI :
- KEGG :
Kyoto Encyclopedia of Genes and Genomes
- MIP :
Main Intrinsic Polypeptide
- Mitf :
Microphthalmia-associated Transcription Factor
- NES :
Normalized Enrichment Score
- NuRD :
Nucleosome Remodeling and Deacetylase
- OFZ :
Organelle Free Zone
- Pax6 :
Paired Box Gene 6
- RAR :
Retinoic Acid Receptor
- RXR :
Retinoid X Receptor
scanning electron microscopy
- SWI/SNF :
- Tbx2 :
T-box Transcription Factor 2
transmission electron microscopy.
We thank Dr Wei Liu for critical comments on the manuscript. We thank Dr Said Sif for providing the Brg1 (K798R)-containing plasmid. Margit Schimmel provided excellent technical help for transmission electron microscopy. Core facilities were the AECOM Genomics and Transgenic Mouse Facility and the New York University Genome Technology Core. Data in this paper are from a thesis to be submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in the Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University. Grant support was provided by NIH grants R01 EY012200 and EY014237 (AC). AC is a recipient of the Irma T. Hirschl Career Scientist Award.
- Ho L, Crabtree G: Chromatin remodelling during development. Nature. 2010, 463: 474-484. 10.1038/nature08911.PubMed CentralView ArticlePubMedGoogle Scholar
- Allis C, Jenuwein T, Reinberg D: Overview and concepts. Epigenetics. Edited by: Allis C, Jenuwein T, Reinberg D. 2007, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 23-61.Google Scholar
- Saha A, Wittmeyer J, Cairns BR: Chromatin remodelling: the industrial revolution of DNA around histones. Nat Rev Mol Cell Biol. 2006, 7: 437-447. 10.1038/nrm1945.View ArticlePubMedGoogle Scholar
- Yoo A, Crabtree G: ATP-dependent chromatin remodeling in neural development. Curr Opin Neurobiol. 2009, 19: 120-126. 10.1016/j.conb.2009.04.006.PubMed CentralView ArticlePubMedGoogle Scholar
- Bultman S, Gebuhr T, Yee D, La Mantia C, Nicholson J, Gilliam A, Randazzo F, Metzger D, Chambon P, Crabtree G: A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes. Mol Cell. 2000, 6: 1287-1295. 10.1016/S1097-2765(00)00127-1.View ArticlePubMedGoogle Scholar
- Reyes JC, Barra J, Muchardt C, Camus A, Babinet C, Yaniv M: Altered control of cellular proliferation in the absence of mammalian brahma (SNF2α). EMBO J. 1998, 17: 6979-6991. 10.1093/emboj/17.23.6979.PubMed CentralView ArticlePubMedGoogle Scholar
- Gebuhr TC, Kovalev GI, Bultman S, Godfrey V, Su L, Magnuson T: The role of Brg1, a catalytic subunit of mammalian chromatin-remodeling complexes, in T cell development. J Exp Med. 2003, 198: 1937-1949. 10.1084/jem.20030714.PubMed CentralView ArticlePubMedGoogle Scholar
- Indra AK, Dupe V, Bornert JM, Messaddeq N, Yaniv M, Mark M, Chambon P, Metzger D: Temporally controlled targeted somatic mutagenesis in embryonic surface ectoderm and fetal epidermal keratinocytes unveils two distinct developmental functions of BRG1 in limb morphogenesis and skin barrier formation. Development. 2005, 132: 4533-4544. 10.1242/dev.02019.View ArticlePubMedGoogle Scholar
- Griffin CT, Brennan J, Magnuson T: The chromatin-remodeling enzyme BRG1 plays an essential role in primitive erythropoiesis and vascular development. Development. 2008, 135: 493-500. 10.1242/dev.010090.PubMed CentralView ArticlePubMedGoogle Scholar
- Matsumoto S, Banine F, Struve J, Xing R, Adams C, Liu Y, Metzger D, Chambon P, Rao M, Sherman L: Brg1 is required for murine neural stem cell maintenance and gliogenesis. Dev Biol. 2006, 289: 372-383. 10.1016/j.ydbio.2005.10.044.View ArticlePubMedGoogle Scholar
- Park J, Park E, Lee H, Kim S, Hur S, Imbalzano A, Kwon J: Mammalian SWI/SNF complexes facilitate DNA double-strand break repair by promoting -H2AX induction. EMBO J. 2006, 25: 3986-3997. 10.1038/sj.emboj.7601291.PubMed CentralView ArticlePubMedGoogle Scholar
- Cohen SM, Chastain PD, Rosson GB, Groh BS, Weissman BE, Kaufman DG, Bultman SJ: BRG1 co-localizes with DNA replication factors and is required for efficient replication fork progression. Nucleic Acids Res. 2010, 38: 6906-6919. 10.1093/nar/gkq559. 2PubMed CentralView ArticlePubMedGoogle Scholar
- de la Serna IL, Roy K, Carlson KA, Imbalzano AN: MyoD can induce cell cycle arrest but not muscle differentiation in the presence of dominant negative SWI/SNF chromatin remodeling enzymes. J Biol Chem. 2001, 276: 41486-41491. 10.1074/jbc.M107281200.View ArticlePubMedGoogle Scholar
- de la Serna IL, Ohkawa Y, Berkes CA, Bergstrom DA, Dacwag CS, Tapscott SJ, Imbalzano AN: MyoD targets chromatin remodeling complexes to the myogenin locus prior to forming a stable DNA-bound complex. Mol Cell Biol. 2005, 25: 3997-4009. 10.1128/MCB.25.10.3997-4009.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Xu R, Spencer VA, Bissell MJ: Extracellular matrix-regulated gene expression requires cooperation of SWI/SNF and transcription factors. J Biol Chem. 2007, 282: 14992-14999. 10.1074/jbc.M610316200.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang M, Fang H, Zhou J, Herring BP: A novel role of Brg1 in the regulation of SRF/MRTFA-dependent smooth muscle-specific gene expression. J Biol Chem. 2007, 282: 25708-25716. 10.1074/jbc.M701925200.View ArticlePubMedGoogle Scholar
- Zhou J, Zhang M, Fang H, El-Mounayri O, Rodenberg JM, Imbalzano AN, Herring BP: The SWI/SNF chromatin remodeling complex regulates myocardin-induced smooth muscle-specific gene expression. Arterioscler Thromb Vasc Biol. 2009, 29: 921-928. 10.1161/ATVBAHA.109.187229.PubMed CentralView ArticlePubMedGoogle Scholar
- Vradii D, Wagner S, Doan DN, Nickerson JA, Montecino M, Lian JB, Stein JL, van Wijnen AJ, Imbalzano AN, Stein GS: Brg1, the ATPase subunit of the SWI/SNF chromatin remodeling complex, is required for myeloid differentiation to granulocytes. J Cell Physiol. 2006, 206: 112-118. 10.1002/jcp.20432.View ArticlePubMedGoogle Scholar
- Khavari PA, Peterson CL, Tamkun JW, Mendel DB, Crabtree GR: BRG 1 contains a conserved domain of the SWI2/SNF2 family necessary for normal mitotic growth and transcription. Nature. 1993, 366: 170-174. 10.1038/366170a0.View ArticlePubMedGoogle Scholar
- Gregg RG, Willer GB, Fadool JM, Dowling JE, Link BA: Positional cloning of the young mutation identifies an essential role for the Brahma chromatin remodeling complex in mediating retinal cell differentiation. Proc Natl Acad Sci USA. 2003, 100: 6535-6540. 10.1073/pnas.0631813100.PubMed CentralView ArticlePubMedGoogle Scholar
- Kurita R, Sagara H, Aoki Y, Link BA, Arai K, Watanabe S: Suppression of lens growth by αA-crystallin promoter-driven expression of diphtheria toxin results in disruption of retinal cell organization in zebrafish. Dev Biol. 2003, 255: 113-127. 10.1016/S0012-1606(02)00079-9.View ArticlePubMedGoogle Scholar
- Leung YF, Ma P, Link BA, Dowling JE: Factorial microarray analysis of zebrafish retinal development. Proc Natl Acad Sci USA. 2008, 105: 12909-12914. 10.1073/pnas.0806038105.PubMed CentralView ArticlePubMedGoogle Scholar
- Sif S, Saurin AJ, Imbalzano AN, Kingston RE: Purification and characterization of mSin3A-containing Brg1 and hBrm chromatin remodeling complexes. Genes Dev. 2001, 15: 603-618. 10.1101/gad.872801.PubMed CentralView ArticlePubMedGoogle Scholar
- Fan HY, Trotter KW, Archer TK, Kingston RE: Swapping function of two chromatin remodeling complexes. Mol Cell. 2005, 17: 805-815. 10.1016/j.molcel.2005.02.024.View ArticlePubMedGoogle Scholar
- Wurster AL, Pazin MJ: BRG1-mediated chromatin remodeling regulates differentiation and gene expression of T helper cells. Mol Cell Biol. 2008, 28: 7274-7285. 10.1128/MCB.00835-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim SI, Bultman SJ, Kiefer CM, Dean A, Bresnick EH: BRG1 requirement for long-range interaction of a locus control region with a downstream promoter. Proc Natl Acad Sci USA. 2009, 106: 2259-2264. 10.1073/pnas.0806420106.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu R, Liu H, Chen X, Kirby M, Brown PO, Zhao K: Regulation of CSF1 promoter by the SWI/SNF-like BAF complex. Cell. 2001, 106: 309-318. 10.1016/S0092-8674(01)00446-9.View ArticlePubMedGoogle Scholar
- Trotter KW, Archer TK: The BRG1 transcriptional coregulator. Nucl Recept Signal. 2008, 6: e004.PubMed CentralPubMedGoogle Scholar
- Tu N, Hu Y, Mivechi NF: Heat shock transcription factor (Hsf) 4b recruits Brg1 during the G1 phase of the cell cycle and regulates the expression of heat shock proteins. J Cell Biochem. 2006, 98: 1528-1542. 10.1002/jcb.20865.View ArticlePubMedGoogle Scholar
- Yang Y, Stopka T, Golestaneh N, Wang Y, Wu K, Li A, Chauhan BK, Gao CY, Cveklova K, Duncan MK, et al: Regulation of αA-crystallin via Pax6, c-Maf, CREB and a broad domain of lens-specific chromatin. EMBO J. 2006, 25: 2107-2118. 10.1038/sj.emboj.7601114.PubMed CentralView ArticlePubMedGoogle Scholar
- Flajollet S, Lefebvre B, Cudejko C, Staels B, Lefebvre P: The core component of the mammalian SWI/SNF complex SMARCD3/BAF60c is a coactivator for the nuclear retinoic acid receptor. Mol Cell Endocrinol. 2007, 270: 23-32. 10.1016/j.mce.2007.02.004.View ArticlePubMedGoogle Scholar
- Zeng L, Zhou MM: Bromodomain: an acetyl-lysine binding domain. FEBS Lett. 2002, 513: 124-128. 10.1016/S0014-5793(01)03309-9.View ArticlePubMedGoogle Scholar
- Duncan B, Zhao K: HMGA1 mediates the activation of the CRYAB promoter by BRG1. DNA Cell Biol. 2007, 26: 745-752. 10.1089/dna.2007.0629.View ArticlePubMedGoogle Scholar
- Cvekl A, Duncan MK: Genetic and epigenetic mechanisms of gene regulation during lens development. Prog Retin Eye Res. 2007, 26: 555-597. 10.1016/j.preteyeres.2007.07.002.PubMed CentralView ArticlePubMedGoogle Scholar
- Cvekl A, Mitton K: Epigenetic regulatory mechanisms in vertebrate eye development and disease. Heredity. 2010, 105: 135-151. 10.1038/hdy.2010.16.PubMed CentralView ArticlePubMedGoogle Scholar
- Bhattacharyya S, Bailey AP, Bronner-Fraser M, Streit A: Segregation of lens and olfactory precursors from a common territory: cell sorting and reciprocity of Dlx5 and Pax6 expression. Dev Biol. 2004, 271: 403-414. 10.1016/j.ydbio.2004.04.010.View ArticlePubMedGoogle Scholar
- Streit A: The preplacodal region: an ectodermal domain with multipotential progenitors that contribute to sense organs and cranial sensory ganglia. Int J Dev Biol. 2007, 51: 447-461. 10.1387/ijdb.072327as.View ArticlePubMedGoogle Scholar
- Lovicu FJ, McAvoy JW: Growth factor regulation of lens development. Dev Biol. 2005, 280: 1-14. 10.1016/j.ydbio.2005.01.020.View ArticlePubMedGoogle Scholar
- Bassnett S: On the mechanism of organelle degradation in the vertebrate lens. Exp Eye Res. 2009, 88: 133-139. 10.1016/j.exer.2008.08.017.PubMed CentralView ArticlePubMedGoogle Scholar
- Randazzo FM, Khavari P, Crabtree G, Tamkun J, Rossant J: brg 1: a putative murine homologue of the Drosophila brahma gene, a homeotic gene regulator. Dev Biol. 1994, 161: 229-242. 10.1006/dbio.1994.1023.View ArticlePubMedGoogle Scholar
- Wang WL, Li Q, Xu J, Cvekl A: Lens fiber cell differentiation and denucleation are disrupted through expression of the N-terminal nuclear receptor box of Ncoa6 and result in p53-dependent and p53-independent apoptosis. Mol Biol Cell. 2010, 21: 2453-2468. 10.1091/mbc.E09-12-1031.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen Q, Liang D, Yang T, Leone G, Overbeek PA: Distinct capacities of individual E2Fs to induce cell cycle re-entry in postmitotic lens fiber cells of transgenic mice. Dev Neurosci. 2004, 26: 435-445. 10.1159/000082285.View ArticlePubMedGoogle Scholar
- de la Serna IL, Carlson KA, Imbalzano AN: Mammalian SWI/SNF complexes promote MyoD-mediated muscle differentiation. Nat Genet. 2001, 27: 187-190. 10.1038/84826.View ArticlePubMedGoogle Scholar
- Kuszak JR, Zoltoski RK, Sivertson C: Fibre cell organization in crystalline lenses. Exp Eye Res. 2004, 78: 673-687. 10.1016/j.exer.2003.09.016.View ArticlePubMedGoogle Scholar
- Wolf LV, Yang Y, Wang J, Xie Q, Braunger B, Tamm ER, Zavadil J, Cvekl A: Identification of Pax6-dependent gene regulatory networks in the mouse lens. PLoS ONE. 2009, 4: e4159-10.1371/journal.pone.0004159.PubMed CentralView ArticlePubMedGoogle Scholar
- Subramanian A, Tamayo P, Mootha V, Mukherjee S, Ebert B, Gillette M, Paulovich A, Pomeroy S, Golub T, Lander E: Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005, 102: 15545-15550. 10.1073/pnas.0506580102.PubMed CentralView ArticlePubMedGoogle Scholar
- Ashery-Padan R, Marquardt T, Zhou X, Gruss P: Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye. Genes Dev. 2000, 14: 2701-2711. 10.1101/gad.184000.PubMed CentralView ArticlePubMedGoogle Scholar
- Cvekl A, Yang Y, Chauhan BK, Cveklova K: Regulation of gene expression by Pax6 in ocular cells: a case of tissue-preferred expression of crystallins in lens. Int J Dev Biol. 2004, 48: 829-844. 10.1387/ijdb.041866ac.PubMed CentralView ArticlePubMedGoogle Scholar
- Fujimoto M, Izu H, Seki K, Fukuda K, Nishida T, Yamada S, Kato K, Yonemura S, Inouye S, Nakai A: HSF4 is required for normal cell growth and differentiation during mouse lens development. EMBO J. 2004, 23: 4297-4306. 10.1038/sj.emboj.7600435.PubMed CentralView ArticlePubMedGoogle Scholar
- Nishimoto S, Kawane K, Watanabe-Fukunaga R, Fukuyama H, Ohsawa Y, Uchiyama Y, Hashida N, Ohguro N, Tano Y, Morimoto T: Nuclear cataract caused by a lack of DNA degradation in the mouse eye lens. Nature. 2003, 424: 1071-1074. 10.1038/nature01895.View ArticlePubMedGoogle Scholar
- Pontoriero GF, Deschamps P, Ashery-Padan R, Wong R, Yang Y, Zavadil J, Cvekl A, Sullivan S, Williams T, West-Mays JA: Cell autonomous roles for AP-2α in lens vesicle separation and maintenance of the lens epithelial cell phenotype. Dev Dyn. 2008, 237: 602-617. 10.1002/dvdy.21445.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu W, Lagutin OV, Mende M, Streit A, Oliver G: Six3 activation of Pax6 expression is essential for mammalian lens induction and specification. EMBO J. 2006, 25: 5383-5395. 10.1038/sj.emboj.7601398.PubMed CentralView ArticlePubMedGoogle Scholar
- Kammandel B, Chowdhury K, Stoykova A, Aparicio S, Brenner S, Gruss P: Distinct cis-essential modules direct the time-space pattern of the Pax6 gene activity. Dev Biol. 1999, 205: 79-97. 10.1006/dbio.1998.9128.View ArticlePubMedGoogle Scholar
- Zhao H, Yang Y, Rizo C, Overbeek P, Robinson M: Insertion of a Pax6 consensus binding site into the αA-crystallin promoter acts as a lens epithelial cell enhancer in transgenic mice. Invest Ophthalmol Vis Sci. 2004, 45: 1930-1939. 10.1167/iovs.03-0856.View ArticlePubMedGoogle Scholar
- Coulombre A, Coulombre J: Corneal development*: III. The role of the thyroid in dehydration and the development of transparency. Exp Eye Res. 1964, 3: 105-114. 10.1016/S0014-4835(64)80024-5.View ArticlePubMedGoogle Scholar
- Vihtelic T, Yamamoto Y, Springer S, Jeffery W, Hyde D: Lens opacity and photoreceptor degeneration in the zebrafish lens opaque mutant. Dev Dyn. 2005, 233: 52-65. 10.1002/dvdy.20294.View ArticlePubMedGoogle Scholar
- Strickler A, Byerly M, Jeffery W: Lens gene expression analysis reveals downregulation of the anti-apoptotic chaperone αA-crystallin during cavefish eye degeneration. Dev Genes Evol. 2007, 217: 771-782. 10.1007/s00427-007-0190-z.View ArticlePubMedGoogle Scholar
- Liu Y, Kawai K, Khashabi S, Deng C, Yiu S: Inactivation of Smad4 leads to impaired ocular development and cataract formation. Biochem Biophys Res Commun. 2010, 400: 476-482. 10.1016/j.bbrc.2010.08.065.View ArticlePubMedGoogle Scholar
- Duncan MK, Cvekl A, Li X, Piatigorsky J: Truncated forms of Pax-6 disrupt lens morphology in transgenic mice. Invest Ophthalmol Vis Sci. 2000, 41: 464-473.PubMedGoogle Scholar
- Pan H, Griep A: Temporally distinct patterns of p53-dependent and p53-independent apoptosis during mouse lens development. Genes Dev. 1995, 9: 2157-2196. 10.1101/gad.9.17.2157.View ArticlePubMedGoogle Scholar
- Chen Q, Dowhan DH, Liang D, Moore DD, Overbeek PA: CREB-binding protein/p300 co-activation of crystallin gene expression. J Biol Chem. 2002, 277: 24081-20489. 10.1074/jbc.M201821200.View ArticlePubMedGoogle Scholar
- Xie L, Overbeek PA, Reneker LW: Ras signaling is essential for lens cell proliferation and lens growth during development. Dev Biol. 2006, 298: 403-414. 10.1016/j.ydbio.2006.06.045.View ArticlePubMedGoogle Scholar
- De Maria A, Bassnett S: DNase II distribution and activity in the mouse lens. Invest Ophthalmol Vis Sci. 2007, 48: 5638-5646. 10.1167/iovs.07-0782.View ArticlePubMedGoogle Scholar
- Caceres A, Shang F, Wawrousek E, Liu Q, Avidan O, Cvekl A, Yang Y, Haririnia A, Storaska A, Fushman D, et al: Perturbing the ubiquitin pathway reveals how mitosis is hijacked to denucleate and regulate cell proliferation and differentiation in vivo. PLoS One. 2010, 5: e13331-10.1371/journal.pone.0013331.PubMed CentralView ArticlePubMedGoogle Scholar
- Pirity MK, Wang WL, Wolf LV, Tamm ER, Schreiber-Agus N, Cvekl A: Rybp, a polycomb complex-associated protein, is required for mouse eye development. BMC Dev Biol. 2007, 7: 39-10.1186/1471-213X-7-39.PubMed CentralView ArticlePubMedGoogle Scholar
- Richardson K, Jarett L, Finke E: Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Biotech Histochem. 1960, 35: 313-323. 10.3109/10520296009114754.View ArticleGoogle Scholar
- Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP: Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 2003, 31: e15-10.1093/nar/gng015.PubMed CentralView ArticlePubMedGoogle Scholar
- Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M: TM4: a free, open-source system for microarray data management and analysis. Biotechniques. 2003, 34: 374-378.PubMedGoogle Scholar
- Huang DW, Sherman BT, Lempicki RA: Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2008, 37: 1-13. 10.1093/nar/gkn923.PubMed CentralView ArticleGoogle Scholar
- Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002, 3: RESEARCH0034-10.1186/gb-2002-3-7-research0034.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang Y, Frappart P, Frappart L, Wang Z, Tong W: A novel function of DNA repair molecule Nbs1 in terminal differentiation of the lens fibre cells and cataractogenesis. DNA Repair. 2006, 5: 885-893. 10.1016/j.dnarep.2006.05.004.View ArticlePubMedGoogle Scholar
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