An attempt to assay the state of determination by using transfected genes as probes in transdifferentiation of neural retina into lens

Hybrid genes coding for chloramphenicol acetyltransferase (CAT) with a non-specific retroviral, lens-specific delta-crystallin or lens-specific alpha-crystallin promoters were constructed to transfect the transdifferentiating (lentoidogenic) and non-transdifferentiating (non-lentoidogenic) cultures of chicken embryonic neural retina for assaying the state of determination towards lens differentiation. The expression occurred only when CAT genes with lens-specific promoters were transfected to the cultures maintained in the conditions permissive to lentoidogenesis. The expression of these exogenous, lens-specific CAT genes began at stages of culturing that were earlier than the expression of endogenous crystallin. Presumably, there are two steps in the transdifferentiation of neural retina into lens; acquisition of capacity to express crystallin genes and derepression of the endogenous crystallin genes.     

Tissue-specific expression of the chicken alpha A-crystallin gene in cultured lens epithelia and transgenic mice

The present experiments show that the single gene for the lens-specific protein alpha A-crystallin of chickens and mice uses a different subset of cis- and trans-acting regulatory elements for expression in transfected embryonic chicken lens epithelial cells. A chicken alpha A-crystallin-chloramphenicol acetyltransferase (CAT) fusion gene required 162 base pairs whereas the murine alpha A-crystallin-CAT fusion gene required only 111 base pairs of 5'-flanking sequences for efficient tissue-specific expression in the transfected chicken lens cells. Gel retardation and competition experiments were performed using embryonic chicken lens nuclear extract and oligodeoxynucleotides identical to the 5'-flanking region of the chicken (-170/-111) and murine (-111/-88 and -88/-55) alpha A-crystallin gene. The results indicated that these homologous promoters use different nuclear factors for function. Methylation interference analysis identified a dyad of symmetry (CTGGTTCCCACCAG) at position -153 to -140 in the chicken alpha A-crystallin promoter which binds one or more lens nuclear factors. Gel mobility shift experiments using nuclear extracts of brain, reticulocytes, and muscle of embryonic chickens or HeLa cells suggested that the factor(s) binding to the chicken alpha A-crystallin gene promoter sequences are not lens specific. Despite differences in the functional and protein-binding properties of the alpha A-crystallin gene promoter of chickens and mice, expression of the chicken alpha A-crystallin-CAT fusion gene in transgenic mice was lens specific, consistent with a common underlying mechanism for expression of the alpha A-crystallin gene in chickens and mice.     

Structure and expression of the scallop Omega-crystallin gene. Evidence for convergent evolution of promoter sequences.

Omega-crystallin of the scallop lens is an inactive aldehyde dehydrogenase (1A9). Here we have cloned the scallop Omega-crystallin gene. Except for an extra novel first exon, its 14-exon structure agrees well with that of mammalian aldehyde dehydrogenases 1, 2, and 6. The -2120/+63, -714/+63, and -156/+63 Omega-crystallin promoter fragments drive the luciferase reporter gene in transfected alphaTN4-1 lens cells and L929 fibroblasts but not in Cos7 cells. Putative binding sequences for cAMP-responsive element-binding protein (CREB)/Jun, alphaACRYBP1, AP-1, and PAX-6 in the Omega-crystallin promoter are surprisingly similar to the cis-elements used for lens promoter activity of the mouse and chicken alphaA-crystallin genes, which encode proteins homologous to small heat shock proteins. Site-specific mutations in the overlapping CREB/Jun and Pax-6 sites abolished activity of the Omega-crystallin promoter in transfected cells. Gel shift experiments utilizing extracts from the alphaTN4-1, L929, and Cos7 cells and the scallop stomach and oligonucleotides derived from the putative binding sites of the Omega-crystallin promoter showed complex formation. Gel shift experiments showed binding of recombinant Pax-6 and CREB to their respective sites. Our data suggest convergent evolutionary adaptations that underlie the preferential expression of crystallin genes in the lens of vertebrates and invertebrates.     

Characterization of squid crystallin genes. Comparison with mammalian glutathione S-transferase genes.

Previous experiments have indicated that the crystallins of the squid lens (S-crystallins) are evolutionarily related to glutathione S-transferases (GST) (EC 2.5.1.18). Here we confirm by peptide sequencing that the crystallins of the lens of the squid Ommastrephes sloani pacificus comprise a family of GST-like proteins. Squid lens extracts showed 400 times less GST activity than those of liver using 1-chloro-2,4-dinitrobenzene as a substrate, suggesting that the abundant GST-like crystallins lack enzymatic activity. Four different cDNAs (pSL20-1, pSL18, pSL11, and pSL4) showed 20-25% similarity in homologous regions with mammalian GST polypeptides. pSL20-1, pSL18, and pSL4 each encode an S-crystallin with a unique internal peptide that is unrelated to mammalian GSTs or any other sequence in GenBank. The S-crystallin family is encoded in a minimum of 9-10 genes, and the exon-intron structures of at least two of these (SL20-1 and SL11) are similar to those of the mammalian GST genes. The SL20-1 gene has six exons, with the its unique internal peptide encoded precisely in exon 4; the SL11 gene lacks a unique internal peptide and has five exons. Experiments using bacterial chloramphenicol acetyltransferase as a reporter gene showed that at least 84 and 111 base pairs of 5'-flanking sequence are needed for function of the SL20-1 and SL11 promoters, respectively, in a transfected rabbit lens epithelial cell line (N/N1003A). Within these regions each has a putative TATA box and an upstream AP-1 site overlapping with antioxidant responsive-like elements, which are regulatory elements in the rat GST Ya and quinone reductase genes responsive to oxidative stress.     

A complex array of positive and negative elements regulates the chicken alpha A-crystallin gene: involvement of Pax-6, USF, CREB and/or CREM, and AP-1 proteins.

The abundance of crystallins (> 80% of the soluble protein) in the ocular lens provides advantageous markers for selective gene expression during cellular differentiation. Here we show by functional and protein-DNA binding experiments that the chicken alpha A-crystallin gene is regulated by at least five control elements located at sites A (-148 to -139), B (-138 to -132), C (-128 to -101), D (-102 to -93), and E (-56 to -41). Factors interacting with these sites were characterized immunologically and by gel mobility shift experiments. The results are interpreted with the following model. Site A binds USF and is part of a composite element with site B. Site B binds CREB and/or CREM to enhance expression in the lens and binds an AP-1 complex including CREB, Fra2 and/or JunD which interacts with USF on site A to repress expression in fibroblasts. Sites C and E (which is conserved across species) bind Pax-6 in the lens to stimulate alpha A-crystallin promoter activity. These experiments provide the first direct data that Pax-6 contributes to the lens-specific expression of a crystallin gene. Site D (-104 to -93) binds USF and is a negative element. Thus, the data indicate that USF, CREB and/or CREM (or AP-1 factors), and Pax-6 bind a complex array of positive and negative cis-acting elements of the chicken alpha A-crystallin gene to control high expression in the lens and repression in fibroblasts.