Functional analysis of human mutations in homeodomain transcription factor PITX3

Satoru Sakazume,Yoshiki Iwamoto,Elena V Semina,Elena Sorokina

doi:10.1186/1471-2199-8-84

Abstract

BackgroundThe homeodomain-containing transcription factor PITX3 was shown to be essential for normal eye development in vertebrates. Human patients with point mutations in PITX3 demonstrate congenital cataracts along with anterior segment defects in some cases when one allele is affected and microphthalmia with brain malformations when both copies are mutated. The functional consequences of these human mutations remain unknown.ResultsWe studied the PITX3 mutant proteins S13N and G219fs to determine the type and severity of functional defects. Our results demonstrate alterations in DNA-binding profiles and/or transactivation activities and suggest a partial loss-of-function in both mutants with the G219fs form being more severely affected. No anomalies in cellular distribution and no dominant-negative effects were discovered for these mutants. Interestingly, the impairment of the G219fs activity varied between different ocular cell lines.ConclusionThe G219fs mutation was found in multiple families affected with congenital cataracts along with anterior segment malformations in many members. Our data suggest that the presence/severity of anterior segment defects in families affected with G219fs may be determined by secondary factors that are expressed in the developing anterior segment structures and may modify the effect(s) of this mutation. The S13N mutant showed only minor alteration of transactivation ability and DNA binding pattern and may represent a rare polymorphism in the PITX3 gene. A possible contribution of this mutation to human disease needs to be further investigated.

Highlights

The homeodomain-containing transcription factor PITX3 was shown to be essential for normal eye development in vertebrates
An additional PITX3 mutant was included in our study, K111E, which was constructed to carry a mutation at position 50 of the homeodomain that changes a lysine into glutamic acid
The K111E alteration is identical to the K88E mutation in PITX2 homeodomain that was shown to have severe DNA-binding and transactivation defects and demonstrated a dominant-negative effect [33,34]

Summary

Methods

DNA constructs The human PITX3, PITX3_S13N, and PITX3_G219fs cDNAs were cloned into the pcDNA3.1 MycHisC expression vector containing the T7 promoter and an in-frame C-terminal c-Myc epitope (Invitrogen). Transfection and luciferase assays B3 human lens epithelial cells were obtained from ATCC (CRL-11421TM) and cultured in medium as suggested by the supplier. Human corneal stromal cells were a generous gift of Dr Watsky (University of Tennessee) [57] these cells were propagated in the DMEM medium containing 10% FBS. The B3 human lens epithelial cells and human corneal stromal cells were cultured in 6-well tissue culture plates. The manufacturer's protocols were followed (Invitrogen); in short, 1.5 μg of reporter DNA, 1.5 μg of effectors DNA, 0.5 μg of pcDNA_lacZ (for normalization of transfection efficiency), 1.75 μl of reagent Plus and 5.25 μl of LipofectamineLTX or 2000 (Invitrogen) were added to every well in 0.5 ml of OPTI-MEM medium. Each experiment was performed in three replicates and transfections were independently repeated at least three times

Results

Discussion

Conclusion