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Lance Patrick Doucette, Tim Footz, Michael A Walter; A Method to Evaluate the Roles of FOXC1 and PITX2 in the Regulation of Micro RNAs. Invest. Ophthalmol. Vis. Sci. 2016;57(12):796. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
FOXC1 and PITX2 are transcription factors that cause Axenfeld-Rieger syndrome, a condition often resulting in blindness due to juvenile glaucoma. To expand our knowledge of the transcriptional networks of FOXC1 and PITX2, we are examining their roles in the regulation of micro RNAs (miRNAs), ~20 nucleotide molecules which act as negative regulators of mRNA expression.
To examine the effects of FOXC1 on the regulation of miRNA molecules, we have created four constructs: FOXC1(WT)-HR, FOXC1(S131L)-HR, PITX2(WT)-HR, PITX2(T68P)-HR. These produce an inactive form of FOXC1 and PITX2. These transcription factors can then be activated by the addition of mifepristone. We transiently transfect each of these constructs into a cell line (ODM2). After 36 hours, cells are treated with cyclohexamide, disabling translation, and then activating the exogenous proteins through administration of Mifepristone (Figure 1). The transcription factors will transcribe their targets, and produce miRNAs. This method allows the identification of direct targets of each of these transcription factors. These RNAs are then to be analyzed on a miRNA chip with Oceanridge Biosciences (ORB). This chip will interrogate the relative amounts of 2097 miRNAs.
Western analysis shows that these constructs are able to produce exogenous FOXC1 and PITX2 proteins of appropriate size. Transactivation analysis using a reporter with a PITX2 binding site (SLC13A3 upstream sequencing) and FOXC1 (FOXO1A upstream sequence) show an increase in luciferase signal after treatment with 10nM mifepristone.
This method has been used previously to define the direct transcriptional targets of FOXC1 and PITX2, and we propose to use this method to interrogate the roles of these two proteins in the regulation of miRNAs. This research will further expand our knowledge of these transcriptional networks, and potentially identify miRNAs for targeted therapies.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Schematic of the FOXC1(WT)-HR and FOXC1(S131L)-HR constructs and their mode of action. Reference: Berry FB, et al. FOXC1 is required for cell viability and resistance to oxidative stress in the eye through the transcriptional regulation of FOXO1A. Hum Mol Genet. 2008 Feb 15;17(4):490-505. Epub 2007 Nov 9.
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