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E. Sorokina, S. Muheisen, N. Mlodik, E. Semina; Mip Represents a Potential Direct Downstream Target of Pitx3 Transcription Factor. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2791. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Pitx3 is a bicoid-type homeodomain-containing transcriptional factor which is expressed during lens development. Mutations in PITX3 cause autosomal-dominant cataracts and anterior segment dysgenesis in humans. Major intrinsic protein (MIP) is expressed in the developing lens fibers and it is necessary for maintenance of lens transparency. MIP deficiency is also known to cause congenital cataracts in humans. Using multi-species sequence alignment software for discovery of regulatory elements containing paired bicoid sites, we identified an element in the MIP promoter that was predicted to interact with PITX3. Therefore, MIP was examined as a potential direct downstream target of PITX3.
Chromatin immunoprecipitation assay was performed using PITX3 and FLAG-tag specific antibodies followed by PCR with promoter-specific primers. Wild-type and mutant MIP promoters were cloned into pGL3-basic luciferase reporter vector (Promega) as well as pG1 GFP reporter vector for in vivo analysis. Reporter transactivation assays with wild type and mutant promoters were performed in B3 lens epithelial and HEK293 cells. In situ hybridization was used to compare mip1 expression in wt and pitx3 morphant embryos.
By immunoprecipitation of chromatin-bound PITX3-flag from B3 cells that were transiently transfected with PITX3-flag construct, we were able to observe enrichment of the precipitated DNA by fragments corresponding to MIP promoter. This enrichment was not observed in cells that were transfected with an empty vector. Replacement of -52 TAATCC bicoid sequence with TAATTT, which is not able to bind PITX3 in EMSA, abolished this transactivation while a similar mutation at -514 bicoid site did not result in any significant change. Effects of these mutations on GFP expression in developing zebrafish embryos as well as analysis on mip1 expression in pitx3 morphants is currently under study
We demonstrate using standard ChIP-PCR assay that PITX3 occupies proximal MIP promoter in B3 human lens epithelial cells. Bicoid site at the position-52 from transcriptional initiation point appears to be responsible for this binding. Further investigation into a role of PITX3 in regulation of MIP expression is ongoing.
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