Pax6 Regulation in Retinal Cells by CCCTC Binding Factor

A homeobox transcription factor, Pax6, is one of the nine members of the Pax gene family in vertebrates.1 PAX6 plays essential roles in the development of ocular structures, including the cornea, iris, lens, and retina.2–5 Overexpression of Pax6 results in ectopic compound eyes in Drosophila and well-formed ectopic lenses in Xenopus laevis.6,7 Mutation of Pax6 can cause small eye (Sey) defects in mice and aniridia in the human eye.8–10 In addition, homozygous mutation of the Pax6 gene results in missing eye structures in Drosophila.11 Expression of the Pax6 gene is regulated via two promoters, P0 and P1.12–14 The P0 promoter–initiating transcriptions predominate in the cornea, conjunctival epithelia, lens placode, and retina, and the P1 promoter initiates transcriptions mainly in the lens placode, optic vesicle, and central neural system.14 An ectoderm enhancer (EE), found approximately −3.5 kb upstream of the P0 promoter, plays a key role in enhancing Pax6 transcription.15 There is also a regulatory element in intron 4 of the Pax6 gene to direct expression of P0 and P1 promoters in amacrine cells, ciliary body, and iris.14 However, it is still not clear how the tissue-specific expression of the Pax6 gene is regulated during embryonic eye development. Results of recent studies in our laboratory indicate that the transcription of the Pax6 gene is regulated by CTCF, a nuclear protein and transcription regulator.16,17 CTCF is a zinc finger (ZF) phosphoprotein that interacts with targeting DNA sequences by its DNA-binding domain containing 11 ZFs.18 CTCF protein is composed of 728 amino acids and its molecular mass is 82 kDa. Message RNA encoding CTCF is composed of 4.1-kb nucleotides and contains a long open reading frame (21.8 kb). However, the reported protein size of CTCF is 130 kDa, according to SDS-PAGE. The discrepancy of CTCF protein sizes between the predicted and apparent molecular mass suggests anomalous migration of the protein in SDS-PAGE.19 On the one hand, CTCF downregulates transcriptions of several important genes including c-myc, β-globin, and chicken lysozyme genes.20 On the other hand, CTCF can upregulate expression of the amyloid protein precursor gene through transcriptional control.21 More interesting, CTCF is a major factor that mediates monoallelic expression of imprinted genes, such as IGF2 (insulin-like growth factor-2) and H19 through a DNA methylation-sensitive mechanism.22,23 In fact, CTCF regulates DNA imprinting and controls IGF2 and H19 expression from the paternal allele and maternal allele, respectively. Another recent finding suggests that CTCF is a transacting factor for X-inactivation, silencing of one of two female X chromosomes.24 Expression patterns of CTCF suggest that it may play roles in the eye, because it is not only expressed in a wide range of mature tissues, but is also highly expressed in the eye in the ciliary marginal zone, inner nuclear layer, and anterior lens epithelium.18,25 In the present study, we report Rb cells in which fetal bovine serum (FBS)–induced increases in CTCF expression effectively suppressed Pax6 expression. We used the Pax6-LacZ reporter containing the 4.2-kb DNA fragment isolated from the 5′ flank region of the Pax6 gene upstream from the P0 promoter and its deletion mutants to study in retina-derived Rb cells the interaction of CTCF and Pax6 transcription. Furthermore, we investigated regulation of Pax6 reporter activities by CTCF in Rb cells and in the retina. Overexpression of CTCF in transgenic chicken and mouse revealed the role of CTCF in controlling Pax6 gene expression through transcription regulation.