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Identification of pax6-dependent gene regulatory networks in the mouse lens.

Wolf LV, Yang Y, Wang J, Xie Q, Braunger B, Tamm ER, Zavadil J, Cvekl A - PLoS ONE (2009)

Bottom Line: Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis.Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development.This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
Lineage-specific DNA-binding transcription factors regulate development by activating and repressing particular set of genes required for the acquisition of a specific cell type. Pax6 is a paired domain and homeodomain-containing transcription factor essential for development of central nervous, olfactory and visual systems, as well as endocrine pancreas. Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis. Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development. Using DNA microarrays, we have identified 559 genes expressed differentially between 1-day old mouse Pax6 heterozygous and wild type lenses. Of these, 178 (31.8%) were similarly increased and decreased in Pax6 homozygous embryonic telencephalon [Holm PC, Mader MT, Haubst N, Wizenmann A, Sigvardsson M, Götz M (2007) Loss- and gain-of-function analyses reveals targets of Pax6 in the developing mouse telencephalon. Mol Cell Neurosci 34: 99-119]. In contrast, 381 (68.2%) genes were differently regulated between the lens and embryonic telencephalon. Differential expression of nine genes implicated in lens development and homeostasis: Cspg2, Igfbp5, Mab21l2, Nrf2f, Olfm3, Spag5, Spock1, Spon1 and Tgfb2, was confirmed by quantitative RT-PCR, with five of these genes: Cspg2, Mab21l2, Olfm3, Spag5 and Tgfb2, identified as candidate direct Pax6 target genes by quantitative chromatin immunoprecipitation (qChIP). In Mab21l2 and Tgfb2 promoter regions, twelve putative individual Pax6-binding sites were tested by electrophoretic mobility shift assays (EMSAs) with recombinant Pax6 proteins. This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs. Collectively, the present studies represent an integrative genome-wide approach to identify downstream networks controlled by Pax6 that control mouse lens and forebrain development.

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EMSAs with five Pax6-binding sites confirmed by qChIP in Mab21l2 and Tgfb2 loci.P6CON, Mab21l2 (sites 1 and 5), and Tgfb2 (site 6, 8 and 11/12) formed specific complexes with recombinant Pax6 GST-PD and GST-PD/HD proteins. The individual autoradiograms were exposed for 2.5, 15, 15, 10, 21 and 10 hours, respectively. 50 ng (approximately 50∶1 molar ratio with the radioactive probe) P6CON ds oligonucleotide competitor was added as indicated to demonstrate specificity of individual complexes.
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pone-0004159-g007: EMSAs with five Pax6-binding sites confirmed by qChIP in Mab21l2 and Tgfb2 loci.P6CON, Mab21l2 (sites 1 and 5), and Tgfb2 (site 6, 8 and 11/12) formed specific complexes with recombinant Pax6 GST-PD and GST-PD/HD proteins. The individual autoradiograms were exposed for 2.5, 15, 15, 10, 21 and 10 hours, respectively. 50 ng (approximately 50∶1 molar ratio with the radioactive probe) P6CON ds oligonucleotide competitor was added as indicated to demonstrate specificity of individual complexes.

Mentions: Direct binding of Pax6 in Mab21l2 and Tgfb2 genes in lens chromatin suggests two novel regulatory mechanisms for embryonic eye development (see Discussion). Therefore, we wanted to localize the putative Pax6-binding sites within the regions occupied by Pax6 in lens chromatin. Using the 20 base pair Pax6 PD “consensus” sequence [36] and a 20 bp Pax6-specific derivative of the 17 base pair PHO (PD/HD) Drosophila paired “consensus” [37] described in Methods and in Fig. S10, we identified five putative Pax6-binding sites corresponding to the “peak” regions identified by ChIP-on-chip in Mab21l2 and seven Pax6-binding sites in Tgfb2 loci, respectively. Based on these 12 predicted binding sites, we prepared 11 probes for EMSAs (see Fig. S10 and Table S4). As candidate sites 11 and 12 in the Tgfb2 Pax6-binding region were close to each other, a single probe was used. The P6CON probe was used to determine the optimal concentration range of GST-Pax6 recombinant proteins, PD and PD/HD, needed to detect specific protein-DNA complexes as we described earlier [15], [38]. Five of eleven probes tested (probe 1, 5, 6, 8 and 11/12, Fig. S10) generated specific protein-DNA complexes that were reduced in the presence of an excess of P6CON cold oligonucleotide competitor, as shown in Fig. 7. Each probe was incubated with similar amounts of recombinant GST-Pax6 proteins; however, the exposure times ranged from 10 to 21 hours, compared to the 2.5 hour exposure needed to visualize Pax6-binding to the “optimal” P6 CON probe. These results identified at least one Pax6-binding site from major ChIP-on-chip “peaks” and showed that the binding affinities of the natural Pax6-binding sites were lower compared to the P6CON. In addition, two probes, site 1 and 5, showed increased affinity towards Pax6-PD/HD compared to PD alone, showing that the internal HD modulates the DNA-binding properties of the PD as described for structurally similar Pax3 proteins [39]. The identification of six “false” positives further underscores the need to improve prediction of Pax6-binding sites in regulatory regions of its candidate direct targets [15], [18], [20], [40]


Identification of pax6-dependent gene regulatory networks in the mouse lens.

Wolf LV, Yang Y, Wang J, Xie Q, Braunger B, Tamm ER, Zavadil J, Cvekl A - PLoS ONE (2009)

EMSAs with five Pax6-binding sites confirmed by qChIP in Mab21l2 and Tgfb2 loci.P6CON, Mab21l2 (sites 1 and 5), and Tgfb2 (site 6, 8 and 11/12) formed specific complexes with recombinant Pax6 GST-PD and GST-PD/HD proteins. The individual autoradiograms were exposed for 2.5, 15, 15, 10, 21 and 10 hours, respectively. 50 ng (approximately 50∶1 molar ratio with the radioactive probe) P6CON ds oligonucleotide competitor was added as indicated to demonstrate specificity of individual complexes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2612750&req=5

pone-0004159-g007: EMSAs with five Pax6-binding sites confirmed by qChIP in Mab21l2 and Tgfb2 loci.P6CON, Mab21l2 (sites 1 and 5), and Tgfb2 (site 6, 8 and 11/12) formed specific complexes with recombinant Pax6 GST-PD and GST-PD/HD proteins. The individual autoradiograms were exposed for 2.5, 15, 15, 10, 21 and 10 hours, respectively. 50 ng (approximately 50∶1 molar ratio with the radioactive probe) P6CON ds oligonucleotide competitor was added as indicated to demonstrate specificity of individual complexes.
Mentions: Direct binding of Pax6 in Mab21l2 and Tgfb2 genes in lens chromatin suggests two novel regulatory mechanisms for embryonic eye development (see Discussion). Therefore, we wanted to localize the putative Pax6-binding sites within the regions occupied by Pax6 in lens chromatin. Using the 20 base pair Pax6 PD “consensus” sequence [36] and a 20 bp Pax6-specific derivative of the 17 base pair PHO (PD/HD) Drosophila paired “consensus” [37] described in Methods and in Fig. S10, we identified five putative Pax6-binding sites corresponding to the “peak” regions identified by ChIP-on-chip in Mab21l2 and seven Pax6-binding sites in Tgfb2 loci, respectively. Based on these 12 predicted binding sites, we prepared 11 probes for EMSAs (see Fig. S10 and Table S4). As candidate sites 11 and 12 in the Tgfb2 Pax6-binding region were close to each other, a single probe was used. The P6CON probe was used to determine the optimal concentration range of GST-Pax6 recombinant proteins, PD and PD/HD, needed to detect specific protein-DNA complexes as we described earlier [15], [38]. Five of eleven probes tested (probe 1, 5, 6, 8 and 11/12, Fig. S10) generated specific protein-DNA complexes that were reduced in the presence of an excess of P6CON cold oligonucleotide competitor, as shown in Fig. 7. Each probe was incubated with similar amounts of recombinant GST-Pax6 proteins; however, the exposure times ranged from 10 to 21 hours, compared to the 2.5 hour exposure needed to visualize Pax6-binding to the “optimal” P6 CON probe. These results identified at least one Pax6-binding site from major ChIP-on-chip “peaks” and showed that the binding affinities of the natural Pax6-binding sites were lower compared to the P6CON. In addition, two probes, site 1 and 5, showed increased affinity towards Pax6-PD/HD compared to PD alone, showing that the internal HD modulates the DNA-binding properties of the PD as described for structurally similar Pax3 proteins [39]. The identification of six “false” positives further underscores the need to improve prediction of Pax6-binding sites in regulatory regions of its candidate direct targets [15], [18], [20], [40]

Bottom Line: Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis.Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development.This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
Lineage-specific DNA-binding transcription factors regulate development by activating and repressing particular set of genes required for the acquisition of a specific cell type. Pax6 is a paired domain and homeodomain-containing transcription factor essential for development of central nervous, olfactory and visual systems, as well as endocrine pancreas. Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis. Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development. Using DNA microarrays, we have identified 559 genes expressed differentially between 1-day old mouse Pax6 heterozygous and wild type lenses. Of these, 178 (31.8%) were similarly increased and decreased in Pax6 homozygous embryonic telencephalon [Holm PC, Mader MT, Haubst N, Wizenmann A, Sigvardsson M, Götz M (2007) Loss- and gain-of-function analyses reveals targets of Pax6 in the developing mouse telencephalon. Mol Cell Neurosci 34: 99-119]. In contrast, 381 (68.2%) genes were differently regulated between the lens and embryonic telencephalon. Differential expression of nine genes implicated in lens development and homeostasis: Cspg2, Igfbp5, Mab21l2, Nrf2f, Olfm3, Spag5, Spock1, Spon1 and Tgfb2, was confirmed by quantitative RT-PCR, with five of these genes: Cspg2, Mab21l2, Olfm3, Spag5 and Tgfb2, identified as candidate direct Pax6 target genes by quantitative chromatin immunoprecipitation (qChIP). In Mab21l2 and Tgfb2 promoter regions, twelve putative individual Pax6-binding sites were tested by electrophoretic mobility shift assays (EMSAs) with recombinant Pax6 proteins. This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs. Collectively, the present studies represent an integrative genome-wide approach to identify downstream networks controlled by Pax6 that control mouse lens and forebrain development.

Show MeSH