Faculty Collaboration Database

MIP/Aquaporin 0 represents a direct transcriptional target of PITX3 in the developing lens. PLoS One 2011;6(6):e21122

Date

06/24/2011

Pubmed ID

21698120

Pubmed Central ID

PMC3117865

DOI

10.1371/journal.pone.0021122

Scopus ID

2-s2.0-79959200303 (requires institutional sign-in at Scopus site)   25 Citations

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.

Author List

Sorokina EA, Muheisen S, Mlodik N, Semina EV

Authors

Elena V. Semina PhD Chief, Professor in the Ophthalmology and Visual Sciences department at Medical College of Wisconsin
Elena A. Sorokina Research Scientist I in the Ophthalmology and Visual Sciences department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Aquaporins
Base Sequence
Cell Line
Chromatin Immunoprecipitation
DNA Primers
Electrophoretic Mobility Shift Assay
Eye Proteins
Homeodomain Proteins
Humans
In Situ Hybridization
Lens, Crystalline
Mice
Promoter Regions, Genetic
Reverse Transcriptase Polymerase Chain Reaction
Transcription Factors
Transcription, Genetic
Zebrafish