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Pax6 paired domain mediates direct autoregulation of promoter P1

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posted on 30.12.2011, 17:04 authored by Timothy Grocott, Victoria Frost, Marjorie Maillard, Terje Johansen, Grant N. Wheeler, Lucy J. Dawes, I. Michael Wormstone, Andrew Chantry

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Taken from "The MH1 domain of Smad3 interacts with Pax6 and represses autoregulation of the Pax6 P1 promoter"

Nucleic Acids Research 2007;35(3):890-901.

Published online 23 Jan 2007

PMCID:PMC1807973.

© 2007 The Author(s).

An isolated Pax6 paired domain (PD) inhibits the function of full-length Pax6 in dominant negative manner. HEK-293 cells were transiently transfected with 2 μg of P6CON-Luc in combination with 5 μg of Pax6 or PD. Cells were lysed 48 h post-transfection and luciferase assays were performed. The data represent mean +/−SEM ( = 3). Dominant negative paired domain (PD) inhibits the basal activity of promoter P1 indicating an autoregulatory component. HEK-293 cells were transiently transfected with 2 μg of P1-Luc in combination with 5 μg of PD. Cells were lysed 48 h post-transfection and luciferase assays were performed. The data represent mean +/−SEM ( = 5). Alignment of the human and Pax6 P1 promoters reveals an evolutionary conserved putative paired domain-binding site. Sequences of the human and Pax6 P1 promoters were aligned in the region corresponding to the reporter construct P1-Luc. This alignment was then aligned to the consensus-paired domain binding sequence, P6CON, which is boxed (Note: this is the reverse-complement P6CON sequence as the putative binding site is encoded 5′ to 3′ on the opposite strand). A broken arrow indicates orientation of the P6CON consensus. Nucleotides matching the majority sequence are shaded in grey, while the TATA and CCAAT boxes are shaded in black. Bold letters indicate the location of PCR primers used to generate DNA probes for gel-shift assays, and solid lines indicate primer orientation. Human transcription start site is indicated as +1. Deletion of a putative paired domain binding site in promoter P1 (P1(▵PBS)-Luc) disrupts Pax6 autoregulation. HEK-293 cells were transiently transfected with 2 μg of P1(▵PBS)-Luc in combination with 5 μg of Pax6. Cells were lysed 48 h post-transfection and luciferase assays were performed. The data represent mean +/−SEM ( = 5). Gel mobility shift assay of GST-Pax6-PD and GST-Pax6-HD binding to the P1 promoter. Equal amounts of GST-Pax6-PD and GST-Pax6-HD were used as described in the materials and methods. Probe 1 was amplified using the PCR primers FL-P1-F and FL-P1-R, and Probe 2 using S-P1-F and FL-P1-R as indicated in the P1 sequence shown in C. The actual primer sequences used here are provided in the materials and methods. The migration of the major shifted bands is indicated as (*).

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