Involvement of AP-2 in Regulation of theR-FABPGene in the Developing Chick Retina

Abstract

Little is known regarding the molecular pathways that underlie the retinal maturation process. We are studying the regulation of the retinal fatty-acid-binding protein (R-FABP) gene, highly expressed in retinal precursor cells, to identify DNA regulatory elements and transcriptional factors involved in retinal development. Although the upstream sequence of the R-FABP gene is extremely GC rich, CpG methylation in this region is not implicated in the regulation of this gene because the 5 flanking DNA remains unmethylated with tissue differentiation when there is a dramatic decrease in R-FABP transcript levels. Using a combination of DNase I hypersensitivity experiments, gel shift assays, and DNase I footprinting, we have found three sites of DNA-protein interaction within 205 bp of 5 flanking DNA in the undifferentiated retina and four sites in the differentiated retina. DNA transfection analysis indicates that the first two footprints located within 150 bp of 5 flanking DNA are required for high levels of transcription in primary undifferentiated retinal cultures. The first footprint includes a putative TATA box and Sp1 binding sites while the second footprint contains a consensus AP-2 DNA binding site. Supershift experiments using antibodies to AP-2 and methylation interference experiments indicate that an AP-2-like transcription factor present in both late-proliferative-stage retina and differentiated retina binds to the upstream region of the R-FABP gene. A combination of data including the expression profile of AP-2 during retinal development and DNA transfection analysis using constructs mutated at critical residues within the AP-2 binding site suggests that AP-2 is a repressor of R-FABP transcription.The fatty-acid-binding protein (FABP) family consists of a number of structurally related proteins with characteristic cellular, tissue, and developmental distribution patterns. Members of this family include heart FABP, intestinal FABP, adipocyte lipid-binding protein, myelin P 2 , cellular retinoicacid-binding proteins, and cellular retinol-binding proteins (1, 51). Some FABPs, such as adipocyte FABP and myelin P 2 , are restricted to one tissue or organ type while others are more broadly expressed. For example, intestinal FABP is found in both intestine and stomach while liver FABP is present in liver, intestine, kidney, and stomach (reviewed in reference 51). Roles proposed for these proteins include the uptake, intracellular solubilization, storage, and/or delivery of fatty acids and retinoids (1). A role in signal transduction has also been proposed for heart FABP and adipocyte lipid-binding protein via phosphorylation of a tyrosine residue at position 19 by the insulin receptor (8, 41). Furthermore, mammary-derived growth inhibitor (also called heart FABP [46]) and liver FABP appear to be involved in the differentiation of mammary epithelial cells and in the control of hepatocyte cell proliferation, respectively (35,36,60). Some FABPs are located in both the nucleus and the cytoplasm, suggesting that the...

Expression of AP‐2δ in the developing chick retina

“…We have previously shown that AP-2␣ and AP-2␤ are specifically expressed in the amacrine and horizontal cell lineages of the developing chick retina (Bisgrove et al, 1997). Here, we describe a third member of the AP-2 family, AP-2␦, which has an even more restricted distribution pattern in the developing chick retina.…”

“…Five AP-2 genes have been isolated in mice and humans: AP-2␣, AP-2␤, AP-2␥, AP-2␦, and AP-2⑀ (Williams et al, 1988;Moser et al, 1995;Bosher et al, 1996;Chazaud et al, 1996;Zhao et al, 2001a;Feng and Williams, 2003;Tummala et al, 2003). In chicken, two AP-2 genes have been isolated to date, AP-2␣ and AP-2␤ (Bisgrove et al, 1997;Shen et al, 1997;Bisgrove and Godbout, 1999). AP-2 proteins have a highly conserved C-terminal half, which contains the central and helix-span-helix domains involved in DNA binding and dimerization.…”

Cloning and characterization of the Drosophila homologue of the AP-2 transcription factor

“…AP-2 expression during mouse embryogenesis was observed around the eye in areas of developing neuroepithelial sensory structures (Mitchell et al, 1991Moser et al, 1995, and AP-2a null mice exhibit a displaced optic cup with dysmorphic neural and pigmented retinal layers of the eye developing medially (Schorle et al, 1996). Recently, it was shown that AP-2 is a regulator of the retinal fatty-acid-binding protein, which is involved in the retinal maturation process (Bisgrove et al, 1997). DAP-2 expression within the region of the brain/optical lobe zone raises the question whether AP-2 is also involved in the development of the visual system.…”

Conditional Deletion of Activating Protein 2α (AP-2α) in the Developing Retina Demonstrates Non-Cell-Autonomous Roles for AP-2α in Optic Cup Development

“…In the carboxy-terminal half of the protein, all family members share a basic DNA-binding region, which is followed by a helix-span-helix dimerization motif through which the AP-2 proteins can form homodimers, or heterodimers with other family members (18,41,44,65,66,71). Both AP-2␣ and AP-2␤ have been linked to fatty acid-related ocular development (3,4,72). During chick retinogenesis, AP-2␣ and AP-2␤ have been shown to act as transcriptional repressors of retinal fatty acid binding protein (R-FABP), a homologue of mammalian brain FABP that is strongly expressed in the developing chick retina (3,4).…”