Chromatin remodeling enzyme Brg1 is required for mouse lens fiber cell terminal differentiation and its denucleation.

BACKGROUND: Brahma-related gene 1 (Brg1, also known as Smarca4 and Snf2ß) 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. RESULTS: 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 IIß, 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 IIß, for lens fiber cell denucleation and indirectly for retinal development. CONCLUSIONS: These studies demonstrate a cell-autonomous role for Brg1 in lens fiber cell terminal differentiation and identified DNase IIß 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.

Regulation of alphaA-crystallin via Pax6, c-Maf, CREB and a broad domain of lens-specific chromatin.

Pax6 and c-Maf regulate multiple stages of mammalian lens development. Here, we identified novel distal control regions (DCRs) of the alphaA-crystallin gene, a marker of lens fiber cell differentiation induced by FGF-signaling. DCR1 stimulated reporter gene expression in primary lens explants treated with FGF2 linking FGF-signaling with alphaA-crystallin synthesis. A DCR1/alphaA-crystallin promoter (including DCR2) coupled with EGFP virtually recapitulated the expression pattern of alphaA-crystallin in lens epithelium and fibers. In contrast, the DCR3/alphaA/EGFP reporter was expressed only in 'late' lens fibers. Chromatin immunoprecipitations showed binding of Pax6 to DCR1 and the alphaA-crystallin promoter in lens chromatin and demonstrated that high levels of alphaA-crystallin expression correlate with increased binding of c-Maf and CREB to the promoter and of CREB to DCR3, a broad domain of histone H3K9-hyperacetylation extending from DCR1 to DCR3, and increased abundance of chromatin remodeling enzymes Brg1 and Snf2h at the alphaA-crystallin locus. Our data demonstrate a novel mechanism of Pax6, c-Maf and CREB function, through regulation of chromatin-remodeling enzymes, and suggest a multistage model for the activation of alphaA-crystallin during lens differentiation.

Regulation of gene expression by Pax6 in ocular cells: a case of tissue-preferred expression of crystallins in lens.

Lens development is an excellent model for genetic and biochemical studies of embryonic induction, cell cycle regulation, cellular differentiation and signal transduction. Differentiation of lens is characterized by lens-preferred expression and accumulation of water-soluble proteins, crystallins. Crystallins are required for light transparency, refraction and maintenance of lens integrity. Here, we review mechanisms of lens-preferred expression of crystallin genes by employing synergism between developmentally regulated DNA-binding transcription factors: Pax6, c-Maf, MafA/L-Maf, MafB, NRL, Sox2, Sox1, RARbeta/RXRbeta, RORalpha, Prox1, Six3, gammaFBP-B and HSF2. These factors are differentially expressed in lens precursor cells, lens epithelium and primary and secondary lens fibers. They exert their function in combination with ubiquitously expressed factors (e.g. AP-1, CREB, pRb, TFIID and USF) and co-activators/chromatin remodeling proteins (e.g. ASC-2 and CBP/p300). A special function belongs to Pax6, a paired domain and homeodomain-containing protein, which is essential for lens formation. Pax6 is expressed in lens progenitor cells before the onset of crystallin expression and it serves as an important regulatory factor required for expression of c-Maf, MafA/L-Maf, Six3, Prox1 and retinoic acid signaling both in lens precursor cells and the developing lens. The roles of these factors are illustrated by promoter studies of mouse alphaA-, alphaB-, gammaF- and guinea pig zeta-crystallins. Pax6 forms functional complexes with a number of transcription factors including the retinoblastoma protein, pRb, MafA, Mitf and Sox2. We present novel data showing that pRb antagonizes Pax6-mediated activation of the alphaA-crystallin promoter likely by inhibiting binding of Pax6 to DNA.

Transcriptional regulation of mouse alphaB- and gammaF-crystallin genes in lens: opposite promoter-specific interactions between Pax6 and large Maf transcription factors.

Mammalian alphaB-crystallin is highly expressed both in lens epithelium and lens fibers. In contrast, gammaF-crystallin is highly expressed in the lens fiber cells. Crystallin gene expression in lens is regulated at the level of transcription by a sparse number of specific DNA-binding transcription factors. Here, we report studies on transcriptional regulation of mouse alphaB- and gammaF-crystallin promoters by specific combinations of Pax6/Pax6(5a), large Mafs (MafA, MafB, c-Maf, and NRL), Sox1, Sox2, Six3, and RARbeta/RXRbeta. Two sets of these factors, co-expressed both in lens epithelium and in lens fibers, were tested in co-transfection assays using cultured lens and non-lens cells. Regulation of alphaB-crystallin was studied in the presence of lens epithelial-factors Pax6, MafB, and RARbeta/RXRbeta, and lens fiber-factors Pax6, MafA, c-Maf, and NRL. Pax6 proteins activated the alphaB-crystallin promoter (-162 to +45) with any combination of Mafs. Addition of RARbeta/RXRbeta further increased its promoter activity. Gel shift assays using lens nuclear extracts demonstrated interactions of Pax6, Maf, and retinoic acid nuclear receptor proteins with two lens-specific regions, the distal LSR1 (-147/-118) and proximal LSR2 (-78/-40), of the alphaB-crystallin promoter. In contrast, Pax6 proteins acted as repressors of gammaF-crystallin promoter activity elicited by a combination of large Mafs, Sox, and RARbeta/RXRbeta proteins in transiently transfected lens and non-lens cells. The results show that Pax6 conversely regulates these two lens crystallin promoters. We propose that the opposite roles of Pax6 in crystallin gene regulation are results of different promoter architectures of the alphaB- and gammaF-crystallin genes, developmentally regulated association of transcription factors with the corresponding cis-regulatory sites, and specific recruitment of transcriptional co-activators and co-repressors by Pax6.

Functional interactions between alternatively spliced forms of Pax6 in crystallin gene regulation and in haploinsufficiency.

Pax6 is essential for development of the eye, olfactory system, brain and pancreas. Haploinsufficiency of Pax6 causes abnormal eye development. Two forms of Pax6 protein, PAX6 and PAX6(5a), differ in a 14 amino acid insertion encoded by an alternatively spliced exon 5a in the N-terminal DNA-binding paired domain (PD), and they are simultaneously expressed. Here, we show that PAX6 and PAX6(5a) together synergistically activate transcription from promoters recognized by Pax6 PD and PD5a, but not by their homeodomain. This synergism promotes activation of transcription by c-Maf and MafA on the alphaB-crystallin promoter, and is required for transcriptional co-activation by RARbeta/RXRbeta and PAX6/PAX6(5a) on the gammaF-crystallin promoter. To determine the role of this synergism in haploinsufficiency, we tested four human missense (G18W, R26G, G64V and R128C) and one nonsense (R317X) mutants, with reporters driven by Pax6 PD consensus binding sites and the alphaB-crystallin promoter. The simultaneous activity of Pax6 proteins [PAX6, mutated PAX6, PAX6(5a) and mutated PAX6(5a)] modeling haploinsufficiency yielded results not predicted by properties of individual PAX6 or PAX6(5a). Taken together, these results indicate that complex ocular phenotypes due to Pax6 haploinsufficiency originate, at least partially, from functional interactions between alternatively spliced PAX6 and PAX6(5a) variants and other factors, e.g. MafA/c-Maf.

Functional properties of natural human PAX6 and PAX6(5a) mutants.

PURPOSE: Pax6 is essential for development of the eye, brain, and pancreas. Two major products of PAX6 are specific DNA-binding proteins, PAX6 and PAX6(5a). PAX6(5a) contains a short insertion influencing its DNA-binding activity. Heterozygous mutations in PAX6 result in abnormal eye development implicating haploinsufficiency. Deletions of one PAX6 allele result in aniridia characterized by severe ocular phenotypes. Approximately 10% of PAX6 mutations encode missense mutations. These mutations usually cause less severe abnormalities than does aniridia. The moderate phenotypes raise the possibility that different ocular tissues are differently sensitive to specific mutations. To test this hypothesis, we probed functional properties of individual mutated Pax6 proteins in a variety of conditions. METHODS: Mutations in PAX6 and PAX6(5a) were introduced by site-directed mutagenesis and tested by transfections in four cell lines using reporters containing three different Pax6 binding sites. Pax6 binding to DNA was studied by electrophoretic mobility shift assays. RESULTS: Functional studies of PAX6 and PAX6(5a) and their eight natural missense (G18W, R26G, A33P, S43P, G64V, I87R, V126D and R128C) and two nonsense (R317X and S353X) disease-causing mutants revealed unexpected pleiotropic effects in gene regulation, not predicted by the PAX6-DNA crystal structure. Transactivation by PAX6 and PAX6(5a) was dependent on the location of mutation, type of DNA-binding site, and cellular environment. CONCLUSIONS: This work provides evidence that activation by PAX6 and PAX6(5a) is modulated by specific cellular environments. It is likely that moderate phenotypes associated with PAX6 missense mutations originate from abnormal protein function in a restricted number of ocular cell types.

DACH1 inhibits transforming growth factor-beta signaling through binding Smad4.

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.

Identification of differentially expressed genes in mouse Pax6 heterozygous lenses.

PURPOSE: Pax6 is a critical regulator of the developing lens, other ocular tissues, central nervous system, and pancreas. Downstream targets of Pax6 are largely unknown. The present study was designed to identify differentially expressed genes in Pax6 heterozygous and normal mouse lenses. METHODS: RNAs from 8-week-old normal and Pax6 heterozygous mouse lenses were analyzed by both RT-PCR differential display and a candidate-gene approach. The expression levels of identified genes were confirmed by semiquantitative RT-PCR. RESULTS: Eight transcripts encoding phosphatase inhibitor Pip-1 protein, heat shock protein Hsp40, Purkinje cell protein Pcp4, an expressed sequence tag (EST; AA331381) originally reported in a brain-specific library, Pitx3, and CBP, were confirmed to be downregulated in the Pax6 heterozygous mouse lenses. alphaB- and betaA3/A1-crystallin transcripts exhibited decreased expression in Pax6 heterozygous lenses, whereas the expression levels of other crystallins were virtually unchanged. CONCLUSIONS: The present data identify eight genes with expression levels that are decreased in Pax6 heterozygous lenses and provide evidence that four functional categories of transcripts-namely, small hsps (alphaB-crystallin and Hsp40), crystallins (alphaB- and betaA3/A1-crystallin), transcription factors (Pitx-3 and CBP), and components of signal transduction cascades (Pip-1) are under direct or indirect transcriptional control by Pax6.