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MIP/Aquaporin 0 represents a direct transcriptional target of PITX3 in the developing lens.

Sorokina EA, Muheisen S, Mlodik N, Semina EV - PLoS ONE (2011)

Bottom Line: In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation.Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter.Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Children's Research Institute, Medical College of Wisconsin and Children's Hospital of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
The PITX3 bicoid-type homeodomain transcription factor plays an important role in lens development in vertebrates. PITX3 deficiency results in a spectrum of phenotypes from isolated cataracts to microphthalmia in humans, and lens degeneration in mice and zebrafish. While identification of downstream targets of PITX3 is vital for understanding the mechanisms of normal ocular development and human disease, these targets remain largely unknown. To isolate genes that are directly regulated by PITX3, we performed a search for genomic sequences that contain evolutionarily conserved bicoid/PITX3 binding sites and are located in the proximity of known genes. Two bicoid sites that are conserved from zebrafish to human were identified within the human promoter of the major intrinsic protein of lens fiber, MIP/AQP0. MIP/AQP0 deficiency was previously shown to be associated with lens defects in humans and mice. We demonstrate by both chromatin immunoprecipitation and electrophoretic mobility shift assay that PITX3 binds to MIP/AQP0 promoter region in vivo and is able to interact with both bicoid sites in vitro. In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation. Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter. Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos. Therefore, our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and that the alteration of MIP/AQP0 expression is likely to contribute to the lens phenotype in cataract patients with PITX3 mutations.

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Electrophoretic mobility shift assays (EMSA) demonstrate interaction between PITX3 and bcd1 and bcd2 sites.EMSA performed with bcd1 and bcd2 oligonucleotides. DNA-PITX3 complexes are indicated with a full arrow; supershifts are shown with an arrowhead. ab = antibody, NE = Nuclear extracts, wt = wild type.
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pone-0021122-g002: Electrophoretic mobility shift assays (EMSA) demonstrate interaction between PITX3 and bcd1 and bcd2 sites.EMSA performed with bcd1 and bcd2 oligonucleotides. DNA-PITX3 complexes are indicated with a full arrow; supershifts are shown with an arrowhead. ab = antibody, NE = Nuclear extracts, wt = wild type.

Mentions: We first performed electrophoretic mobility shift assay (EMSA) to examine whether these putative bicoid sequences are able to bind PITX3 in vitro. Nuclear extracts were isolated from human lens epithelial cells transiently transfected with either a PITX3 expression plasmid or an empty pcDNA vector and incubated with labeled oligonucleotides containing the TAATCC motif and 13-bp of flanking sequences on either side of each bicoid site. The samples derived from PITX3-enriched nuclear extracts produced clearly visible shifts with both probes which were not observed with samples prepared from mock-transfected cells (Figure 2). The PITX3-DNA complexes were further verified by addition of PITX3 polyclonal antibody, which resulted in reduction of the intensities of the shifted bands and formation of supershifts (Figure 2). In addition to this, the specificity of binding was confirmed by EMSA analysis using modified oligonucleotides carrying a 2-nt mutation within the bicoid sites: the TAATCC sequence was replaced with TAATTT in both probes to abolish PITX3 binding [27]. Mutations in the bicoid sites resulted in the disappearance of protein-DNA complexes, confirming that these bands are the product of specific PITX3-DNA interactions (Figure 2).


MIP/Aquaporin 0 represents a direct transcriptional target of PITX3 in the developing lens.

Sorokina EA, Muheisen S, Mlodik N, Semina EV - PLoS ONE (2011)

Electrophoretic mobility shift assays (EMSA) demonstrate interaction between PITX3 and bcd1 and bcd2 sites.EMSA performed with bcd1 and bcd2 oligonucleotides. DNA-PITX3 complexes are indicated with a full arrow; supershifts are shown with an arrowhead. ab = antibody, NE = Nuclear extracts, wt = wild type.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3117865&req=5

pone-0021122-g002: Electrophoretic mobility shift assays (EMSA) demonstrate interaction between PITX3 and bcd1 and bcd2 sites.EMSA performed with bcd1 and bcd2 oligonucleotides. DNA-PITX3 complexes are indicated with a full arrow; supershifts are shown with an arrowhead. ab = antibody, NE = Nuclear extracts, wt = wild type.
Mentions: We first performed electrophoretic mobility shift assay (EMSA) to examine whether these putative bicoid sequences are able to bind PITX3 in vitro. Nuclear extracts were isolated from human lens epithelial cells transiently transfected with either a PITX3 expression plasmid or an empty pcDNA vector and incubated with labeled oligonucleotides containing the TAATCC motif and 13-bp of flanking sequences on either side of each bicoid site. The samples derived from PITX3-enriched nuclear extracts produced clearly visible shifts with both probes which were not observed with samples prepared from mock-transfected cells (Figure 2). The PITX3-DNA complexes were further verified by addition of PITX3 polyclonal antibody, which resulted in reduction of the intensities of the shifted bands and formation of supershifts (Figure 2). In addition to this, the specificity of binding was confirmed by EMSA analysis using modified oligonucleotides carrying a 2-nt mutation within the bicoid sites: the TAATCC sequence was replaced with TAATTT in both probes to abolish PITX3 binding [27]. Mutations in the bicoid sites resulted in the disappearance of protein-DNA complexes, confirming that these bands are the product of specific PITX3-DNA interactions (Figure 2).

Bottom Line: In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation.Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter.Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Children's Research Institute, Medical College of Wisconsin and Children's Hospital of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
The PITX3 bicoid-type homeodomain transcription factor plays an important role in lens development in vertebrates. PITX3 deficiency results in a spectrum of phenotypes from isolated cataracts to microphthalmia in humans, and lens degeneration in mice and zebrafish. While identification of downstream targets of PITX3 is vital for understanding the mechanisms of normal ocular development and human disease, these targets remain largely unknown. To isolate genes that are directly regulated by PITX3, we performed a search for genomic sequences that contain evolutionarily conserved bicoid/PITX3 binding sites and are located in the proximity of known genes. Two bicoid sites that are conserved from zebrafish to human were identified within the human promoter of the major intrinsic protein of lens fiber, MIP/AQP0. MIP/AQP0 deficiency was previously shown to be associated with lens defects in humans and mice. We demonstrate by both chromatin immunoprecipitation and electrophoretic mobility shift assay that PITX3 binds to MIP/AQP0 promoter region in vivo and is able to interact with both bicoid sites in vitro. In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation. Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter. Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos. Therefore, our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and that the alteration of MIP/AQP0 expression is likely to contribute to the lens phenotype in cataract patients with PITX3 mutations.

Show MeSH
Related in: MedlinePlus