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P. J. Gage, M. Qian, C. Kuang; Determining the Mechanism by Which PITX2 Regulates Expression of the Downstream Effector Dkk2. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4004.
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© ARVO (1962-2015); The Authors (2016-present)
Dkk2 is required downstream of the homeodomain transcription factor PITX2 to regulate canonical Wnt/β-catenin signaling within the developing anterior segment. However, the underlying mechanism(s) by which PITX2 regulates Dkk2 expression are unknown. The purpose of the present work is to map the PITX2-responsive sequences within Dkk2 and to determine the underlying mechanism(s).
Chromatin immunoprecipitation (ChIP) with anti-PITX2 antibodies was used to test for physical association of PITX2 with sequences 5’ and 3’ of the Dkk2 transcriptional start site. Dkk2 promoter sequences and their derivatives cloned into the pGL3-basic luciferase reporter plasmid were tested for PITX2-responsiveness by co-transfection into CHO cells. Additional biochemical approaches will be employed as indicated.
PITX2 physically associates with sequences located 5’ and 3’ from the Dkk2 transcriptional start site. A 4-kb Dkk2 fragment encompassing 3.5-kb of 5’-flanking sequences and 0.5-kb of sequences 3’ from the transcriptional start site confers PITX2-responsiveness on a luciferase reporter plasmid. Based on deletion and mutation analysis, the PITX2-responsive sequences are located 3’ to the transcriptional start, within the non-coding first exon. Surprisingly, this region contains no recognizable PITX2 binding sites based on previous studies. Further mutational analyses are ongoing to identify the minimal sequences required for PITX2-responsiveness.
PITX2 activates Dkk2 expression through sequences located within the first exon of the Dkk2 gene. Surprisingly, this region contains no recognizable PITX2 binding sites based on previous data. This suggests that PITX2 activation of Dkk2 may occur through a previously unrecognized sequence motif or though a novel mechanism. Either result will provide important new insight into the function of PITX2 in eye development and glaucoma (NIH RO1 EY014126 & P30 EY007003, Research to Prevent Blindness Unrestricted Grant)
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