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V. Canto Soler, H. Huang, K. J. Wahlin, M. McNally, S. Romero, R. Adler; CTCF Is a Negative Regulator of Pax6 Expression in Retinal Cells in-vitro. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4480.
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Pax6 downregulation is necessary for retinal progenitor cells to differentiate as photoreceptors (Toy et al, 2002). The zinc-finger transcription factor CTCF could downregulate PAX6 in retinal cells as it does in corneal and retinoblastoma cells (Li T al, 2004; 2006), since Pax6 and CTCF show complementary patterns of expression in the embryonic retina (Canto-Soler et al, 2005). We have tested predictions from this hypothesis through CTCF gain-of-function experiments in cultured chick embryo retinal cells.
The coding region of chicken CTCF was amplified by RT-PCR, modified by primer extension to include an HA-tag, cloned into an expression vector, and confirmed by sequencing. Western blots were used to detect endogenous CTCF expression in chick retina and brain and vector-driven CTCF expression in transfected 293 cells. Low density cultures of dissociated ED8 chick retinal cells were co-transfected with nuclear GFP plus either CTCF- or ßgal- plasmids, fixed 24 or 72 hrs later, processed for PAX6 immunofluorescence, and photographed. Pax6 intensity in GFP(+) cells was quantitated using ImageJ. Statistics were done with the t-test.
Chicken CTCF cDNA showed 83 % and CTCF protein 93 % homology to their mouse, rat and human counterparts. Western blot analysis of embryonic chick retina and brain showed the main 130kD CTCF isoform; several minor isoforms were only observed in retina. All the bands were also observed in blots of CTCF-transfected 293 cells. Pax6 intensity in CTCF- transfected cultured retinal progenitor cells was similar to that in controls 24 hours after transfection, but after 72 hrs Pax6 intensity was significantly lower in CTCF- transfected cells than in controls. Ongoing follow- up studies include in vitro experiments with CTCF constructs modified in both the N and C terminus, and gain- and loss-of-function experiments in ovo.
These results show that CTCF overexpression downregulates PAX6 expression in cultured chick retinal cells, and are consistent with the hypothesis that CTCF may have a role in regulating retinal cell differentiation by controlling Pax6 expression in retinal progenitor cells.
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