Crystallin genes: lens specificity of the murine alpha A-crystallin gene.

The abundant soluble proteins of the eye lens, the crystallins, are encoded by several gene families which are developmentally regulated in the embryonic lens. We have studied the expression of the murine alpha A-crystallin gene. Transfection experiments using the pSVO-CAT vector and explanted lens epithelia from embryonic chickens demonstrated proximal (-88 to -60) and distal (-111 to -85) regulatory sequences which interact when the alpha A-crystallin promoter is activated in the lens cells. Transgenic mouse experiments showed that the sequence between positions -366 to +46 of the alpha A-crystallin promoter can drive foreign genes selectively in the lens. A fusion gene consisting of this alpha A-crystallin promoter sequence and the T-antigen gene of SV40 produced a lens tumor in transgenic mice. Thus, crystallin promoters provide a useful model for tissue-specific gene expression and permit targeting the expression of foreign genes to a highly differentiated tissue during development. ImagesFIGURE 3.FIGURE 6.FIGURE 7.FIGURE 8. AFIGURE 8. B


Introduction
The function of the transparent lens is to focus the light that enters the eye onto the retina. The clarity of the lens and its appropriate refractive index are attained by accumulation of structural proteins called crystallins (1,2). The crystallins compriseapproximately 90% ofthe water-soluble protein of the mature lens. There are four major crystallin gene families, namely, a, ,3, y, and 8 (3,4). a and ,B are present in all vertebrates; by contrast, -y is absent from birds and reptiles, where it is replaced by B-crystallins. The crystallin gene families differ in genetic organization and coding information. Each crystallin gene family contains two (a, 8) or more (,B, -y) members that code for related polypeptides. The members of the 8 and -y crystallin gene family are linked (although one y is separate), whereas the two a-crystallin genes are on separate chromosomes (5); the genes are either on separate chromosomes or are distantly linked. Both the structures and coding sequences of the crystallin genes have been highly conserved during evolution.
The diagram in Figure 1 illustrates that the crystallin *Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD   families are distributed differently in the lens. In the rat, a-crystallin is present in the epithelial and fiber cells, ,B-crystallin is found in the equatorial and central fiber cells, and -y-crystallin is confined to the central fiber cells. The spatial distributions of the lens crystallins reflect the differential expression of the crystallin genes during development (2,6,7). Not only are the various families of the crystallin genes expressed differently in the lens, but the individual members of the crystallin gene families are regulated independently during lens cell differentiation. We are interested in understanding the molecular basis for the tissue specificity and developmental regulation of the crystallin genes.  (8). Solid bars: exons. Exon "ins" is present in aA'n8 mRNA and absent from atA2 mRNA. Exon 3, present in both mRNAs, is not indicated. Dotted bars: coding sequences. Pr: DNA fragment containing initiation site of transcription but no coding sequences, inserted in expression vectors and tested in explanted lens epithelia and in transgenic mice. Photograph of an explanted lens epithelium from a 14day-old chicken embryo, used in transient expression assays (15). The explant is about 1 mm in diameter.

Activation of the Murine aA-Crystallin Gene Promoter in Explanted Chicken Lens Epithelia
The a-crystallin family contains the aA and aB-crystallin genes (5,(8)(9)(10). These genes are situated on different chromosomes in the human (5). The murine oaAcrystallin gene is located on chromosome 17 (11,12) and codes for two polypeptides, aA2 and aAm', that are produced by alternative RNA splicing (8). The splicing process that eliminates the insert exon occurs with higher efficiency. This alternative splicing reaction is not developmentally regulated (13). We have been studying the 5' flanking sequences (Pr region) involved in the regulation of transcription of this gene (Fig. 2).
In order to identify DNA sequences that regulate transcription of the murine aA-crystallin gene, DNA fragments containing 5' flanking sequences of different lengths were fused to the bacterial gene for chloramphenicol acetyltransferase (CAT). The CAT gene is not present in eukaryotic cells and can be tested by a very sensitive enzymatic assay (14). Initially, two constructs were made. In the first construct a DNA fragment containing 366 base pairs of 5' flanking sequence and 46 base pairs of exon 1 (Fig. 2) of the murine oaA-crystallin gene was inserted upstream from the CAT gene in the pSVO-CAT vector (14). The second construct was similar except that only 88 base pairs of oaA-crystallin 5' flanking sequence were used. The resulting plasmids were called poLA3662-CAT and paA882-CAT, respectively. The subscript "a" signifies that the plasmids contain the murine DNA fragment in the same orientation as in the original gene (15).
For testing the functional ability of the putative aAcrystallin promoter, we used explanted embryonic chicken lens epithelia that are able to differentiate in vitro (7). The plasmids were introduced into the explants by the calcium phosphate method (15) and CAT assays were performed 3 days later. This system has been very useful for studying crystallin gene regulatory signals (15,16).
A primer extension experiment showed that transcription of the aA-CAT gene starts 46 bp upstream of the acA-crystallin-CAT junction in the hybrid gene in the explanted chicken lens epithelia. This site of initiation of transcription of the hybrid gene was the same  (14). Their ability to activate CAT gene expression in transient assays in lens epithelia (15)    6 C and + 46 that appear to interact with one another in order to produce an active aA-crystalln promoter in the hybrid aA-crystallin-CAT gene. The proximal element requires the sequence between positions 88 and -60, and the distal element requires the sequence between positions -85 and -111. The latter can function in either orientation (16). These experiments also suggested that the sequence between positions -111 and -366 may contain one or more additional regulatory elements.
Crystallin promoters function in a species-independent fashion (4). It is particularly interesting that crystallin promoters can function in lens cells ofheterologous species, even when the species from which the lens cells were derived do not contain the crystallin gene being tested. For example, the chicken 51-crystallin promoter can function in mouse lens cells (17,18), which do not contain endogenous 8-crystallin genes, and the mouse -y2-crystallin promoter can function in embryonic chicken y2 lens cells (19), which do not contain endogenous -y-crystallin genes.
To date, no functional consensus regulatory sequence has been detected in the 5' flanking sequence ofdifferent crystallin genes, as has been found for the pancreatic genes (20). In the otA-crystallin gene, the 5' flanldng sequence of the mouse (8) and hamster (10) is highly conserved; however, much of this similarity is lost in the chicken (21). In the mouse, rat, and human y-crystallin gene family there are several sequences upstream from the TATA box that are highly conserved (19,(22)(23)(24)(25). Except for the 81-crystallin gene (26,27), no other crystallin gene contains the 5'CCAAT3' sequence in their 5' flanking region (5,8,10,19,(22)(23)(24)(25)(28)(29)(30). The CCAAT sequence has been found to interact with a specific trans-acting factor (31)(32)(33). At present, we are performing in vitro binding experiments with lens nuclear extracts to help us identify cis-acting crystallin regulatory sequences that interact with trans-acting factors in the lens.

Activation of the Murine aLA-Crystallin Gene Promoter in Transgenic Mice
From the experiments described above, we knew that a DNA fragment containing 366 bp upstream and 46 bp downstream from the cap site of the murine aA-crystallin gene contains an active tissue-specific promoter when tested in a transient expression assay in explanted chicken lens epithelia. We were curious to learn whether these sequences would still function with tissue specificity when introduced into the mouse genome. To this end, we created transgenic mice containing the aA-crystallin-CAT hybrid gene. The pronuclei of fertilized FVB/N mouse eggs were microinjected with the aAcrystallin-CAT DNA (34) (Fig. 5A), and the eggs were transplanted into surrogate mothers (35)(36)(37).
Two transgenic lines were obtained that contained the aA-crystallin-CAT construct stably integrated into the germline. These mice transmitted the newly acquired gene in a Mendelian fashion. When different tissues of an adult F1 transgenic mouse from each line were analyzed, CAT activity was found only in the eye (34). When the eye was dissected further, CAT activity was confined to the epithelia and fibers of the lens (34).
A developmental study showed that the appearance of CAT activity in aA-crystallin-CAT embryonic lenses followed closely the appearance of aA-crystallin (Fig.  6). Thus, this relatively short aA-crystallin 5' flanking sequence contains sufficient infornation to direct the expression of a foreign gene to the lens at the same (or closely similar) time as that when the endogenous aAcrystallin gene is expressed. In other experiments using CAT-fusion genes with promoters whose expression is not normally restricted to the lens, CAT activity was found in different tissues of transgenic mice (38,39). We conclude therefore that the lens specificity is conveyed by the aA-crystallin promoter.
Our results suggested that we would be able to target the expression of other genes to the lens by fusing them to the 415 bp DNA fragment containing the active promoter of the murine aA-crystallin gene, possibly altering lens phenotype. We chose to study the effect of an oncogene on lens differentiation by directing its expression to the lens. The oncogene we used was that for the SV40 large T-antigen (40)(41)(42). The hybrid gene injected contained the aA-crystallin promoter fused to the SV40 early region lacking its promoter and enhancer (Fig.  5B).
Seven Fo transgenic mice were obtained carrying the aA-crystallin-T-antigen fusion gene. All presented the same phenotype when their eyes opened, i.e., white, opaque lenses (43). The cellular differentiation of the lens was completely disturbed; the elongation of lens epithelial cells into fibers was prevented and only round mononucleated cells were observed. a and P-crystallins were still present, but -y-crystallin was greatly reduced (Fig. 7). The aberrant lens cells were mitotically active and in 2 to 3 months produced a vascularized lens mass with the growth characteristics of a tumor. This lens tumor was invasive and ultimately broke through the lens capsule and filled the eye cavity (Fig. 8B). The presence of SV40 large T-antigen appeared to be the cause of this process, since immunofluorescence experiments demonstrated this antigen was present in the nuclei of the lens cells. In experiments of others, transgenic mice carrying the SV40 early region containing the SV40 enhancer developed tumors in the choroid plexus (44)(45)(46). The coding sequences of the SV40 early region fused to the insulin or the elastase promoter of the rat produced tumors in the pancreas of transgenic mice (47,48). Although no naturally occurring tumors have been reported in the lens of any vertebrate, we have been able to produce a lens tumor in transgenic mice by using a crystallin promoter to direct the expression of an oncogene to the lens. Table 1 summarizes the characteristics of the strains of transgenic mice obtained with the aA-crystallin-CAT and aA-crystallin-SV4OT fusion genes.

Future Directions
We have demonstrated that it is possible to target gene expression to the lens with the aA-crystallin promoter by using recombinant DNA techniques. Since the different crystallin gene families are expressed in different parts of the lens (Fig. 1), we presume that the use of regulatory signals from different crystallin genes will permit the expression of foreign genes to be directed to specific regions of the lens. This invaluable tool opens new directions in the study of lens differentiation, with implications for both basic and medical advances in lens research. We are presently using transient expression tests, in vitro binding tests, and transgenic mice to investigate further at the molecular level the cis-and trans-acting regulatory elements responsible for the activation of crystallin promoters in the lens.