3T3 Cell Lines Stably Expressing Pax6 or Pax6(5a) – A New Tool Used for Identification of Common and Isoform Specific Target Genes

Kiselev, Yury; Eriksen, Tonje Engevik; Forsdahl, Siri; Nguyen, Lan Huong; Mikkola, Ingvild

doi:10.1371/journal.pone.0031915

Cited by 32 publications

(31 citation statements)

References 81 publications

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“…Both Dkk3 and Sfrp2 function as negative regulators of Wnt/β-catenin signaling [102]. Expression of Dkk3 is reduced in E12 Pax6 mutated cortex [45] and directly regulated by Pax6 [103]. The regulation of three canonical Wnt signaling modulators, Dkk1, Sfrp1 and Sfrp2, through Pax6 has already been shown in lens and during neurogenesis [54].…”

Section: Discussionmentioning

confidence: 98%

Pax6 Interactions with Chromatin and Identification of Its Novel Direct Target Genes in Lens and Forebrain

et al. 2013

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Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of α-, β- and δ-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip) were performed using three distinct sources of chromatin (lens, forebrain and β-cells). ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133) of these promoter regions were shared between at least two (three) distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6+/− lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6−/− lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6 interacts preferentially with promoter regions in a tissue-specific fashion. Nevertheless, nearly 20% of the regions identified are accessible to Pax6 in multiple tissues.

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Section: Discussionmentioning

confidence: 98%

Pax6 Interactions with Chromatin and Identification of Its Novel Direct Target Genes in Lens and Forebrain

et al. 2013

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“…Various isoforms of PAX6 protein may be translated due to multiple alternative promoters and splice sites (Kim and Lauderdale, 2006;Bandah et al, 2007;Lakowski et al, 2007;Bandah et al, 2008). The isoforms were expressed together in various tissues and seem to functionally interact to regulate transcription of target genes (Chauhan et al, 2002a,b;Kiselev et al, 2012). Some of these transcripts have a unique expression pattern, which is highly conserved during evolution, but the transcripts have not yet been studied in humans (Lakowski et al, 2007;Bandah et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

A novel mutation of PAX6 identified in a Chinese twin family with congenital aniridia complicated with nystagmus

et al. 2014

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ABSTRACT. Genetic variations within the paired box gene 6 (PAX6) gene are associated with congenital aniridia. To detect the genetic defects in a Chinese twin family with congenital aniridia and nystagmus, exons of PAX6 were amplified by polymerase chain reaction (PCR), sequenced and compared with a reference database. Six members from the family of three generations were included in the study. The twins' father presented with congenital aniridia, nystagmus and cataract at birth, while the twins presented with congenital aniridia and nystagmus. A novel mutation c.888 insA in exon 10 of PAX6 was identified in all affected individuals. This study suggests that the novel mutation c.888 insA is likely responsible for the pathogenesis of the congenital aniridia and nystagmus in this pedigree. To the best of our knowledge, this is the first report of this mutation in PAX6 gene in pedigree with aniridia. Furthermore, no PAX6 gene defect was reported in twins with congenital aniridia.

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“…Although many differentially expressed isoforms have been found and are able to regulate the transcription or protein activation of the ‘wild-type’ isoform [38], it is thought that there is not antagonistic action between Pax6 and Pax6(5a) because they have their own set of target genes during development [39]. Moreover, PAX6(5a) is unable to bind to the sites for Pax6, and Pax6 binds to the sites for PAX6(5a) as a monomer displaying less efficiency [40].…”

Section: Discussionmentioning

confidence: 99%

“…However, a functional interaction between Pax6 and Pax6(5a) has been found to synergistically enhance the activation of the crystalline promoter in mouse [41]. Moreover, in the 3T3 cell line, which stably expresses Pax6 or Pax6(5a), Pax6-upregulated genes are associated with cell growth and adhesion, while Pax6(5a)-upregulated genes are correlated with cell motion and migration [39]. In this study, Pax6 and Pax6(5a) display a synergistic expression pattern.…”

Section: Discussionmentioning

confidence: 99%

The Expression of Pax6 Variants Is Subject to Posttranscriptional Regulation in the Developing Mouse Eyelid

et al. 2013

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Pax6 is a pivotal transcription factor that plays a role during early eye morphogenesis, but its expression and function in eyelid development remain unknown. In this study, the expression patterns of Pax6 mRNA and protein were examined in the developing mouse eyelid at embryonic days 14.5, 15.5, and 16.5. The function of Pax6 in eyelid development was determined by comparing it to that in the eyes-open-at-birth mutant mouse. In the normally developing eyelid, Pax6 and Pax6(5a) mRNA levels were low at E14.5, increased at E15.5, and then declined at E16.5, accompanied by a change in the Pax6/Pax6(5a) ratio. Pax6 protein was mainly located in the mesenchyme and conjunctiva. It was expressed at low levels in the epidermis at E14.5, severely reduced at E15.5, but re-expressed in the keratinocyte cells of the periderm at E16.5. In contrast, Pax6 and the Pax6/Pax6(5a) ratio were considerably higher with strong nuclear expression in the mutant at E15.5. Next, we examined the relationship of Pax6 to epidermal cell proliferation, migration, and the associated signalling pathways. The Pax6 protein in the developing eyelid was negatively correlated with epidermal cell proliferation but not migration, and it is in contrast to the activation of the EGFR-ERK pathway. Our in vivo data suggest that Pax6 expression and the Pax6/Pax6(5a) ratio are at relatively low levels in the eyelid, and acting as a transcription factor, Pax6 is required for the initiation of eyelid formation and for differential development of the keratinised cells in the closed eyelid. The Pax6 protein is likely to be controlled by the EGFR-ERK pathways. An abnormal increase in Pax6 expression and the Pax6/Pax6(5a) ratio due to alteration of the pathway activity could suppress epidermal cell proliferation leading to the eyes-open-at-birth defect. This study offers insight into the function of the Pax6 protein in eyelid development.

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3T3 Cell Lines Stably Expressing Pax6 or Pax6(5a) – A New Tool Used for Identification of Common and Isoform Specific Target Genes

Cited by 32 publications

References 81 publications

Pax6 Interactions with Chromatin and Identification of Its Novel Direct Target Genes in Lens and Forebrain

Pax6 Interactions with Chromatin and Identification of Its Novel Direct Target Genes in Lens and Forebrain

A novel mutation of PAX6 identified in a Chinese twin family with congenital aniridia complicated with nystagmus

The Expression of Pax6 Variants Is Subject to Posttranscriptional Regulation in the Developing Mouse Eyelid

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