We have been studying crystallins, the abundant water-soluble proteins responsible for the optical properties of the eye lens, as a model of tissue-specific gene expression. This year we have published and accomplished the following: ? ? We have continued to examine the regulation of expression of mouse small heat shock protein (shsp)/alpha B-crystallin and the related, closely linked and divergently transcribed shsp myotonic dystrophy protein kinase binding protein (MKBP/HSPB2) genes. Unlike alpha B-crystallin which is expressed abundantly in the lens and heart and moderately in several tissues, the MKBP/HSPB2 gene is expressed at low levels in muscle and heart, and not in the lens. Tissue-specific alpha B-crystallin gene expression is directed by an enhancer situated approximately in the middle of the 1 kb intergenic region. We now showed that the glucocorticoid receptor (GR) and Sp1 bind enhancer elements E1 and E3, respectively, and differentially affect the divergently arranged promoters. Mutation of 2 E-boxes between the enhancer and the MKBP/HSPB2 gene decreased alphaB-crystallin promoter activity without affecting the nearby MKBP/HSPB2 promoter in transfected cells and transgenic mice. We have also shown in transfection experiments that the alpha B-crystallin promoter is activated by the aryl hydrocarbon receptor (AhR), which binds the E2 enhancer element. Together these results indicated that these linked genes evolved shared and promoter-preferred cis-control elements within the intergenic sequence that are context dependent.? ? We have continued our collaborative project with Dr. Eviatar Nevo (University of Haifa, Israel) on the blind mole rat. This rodent develops an eye during embryogenesis that regresses and has a degenerate lens fragment in the adult. We showed earlier that the truncated (-661/+43) alpha B-crystallin promoter/enhancer fragment of the mouse and blind mole rat (-668/+45) are similar but not identical. Luciferase reporter transgenes driven by either a mole rat or a mouse alpha B-crystallin promoter/enhancer are expressed similarly in the heart of transgenic mice. However, the truncated mole rat promoter/enhancer fragment is at least than 10 times more active than the mouse promoter/enhancer fragment in skeletal muscle and barely active in the lens of transgenic mice. The mole rat promoter does function during early embryonic in the transgenic mouse, but turns off later in development, suggesting the existence of a developmental switch for shsp/alpha B-crystallin gene expression. We concluded that the shsp/alpha B-crystallin promoter/enhancer underwent adaptive changes corresponding to the subterranean evolution of the blind mole rat. As in mice, the HSPB2 gene is situated head to head approximately 1 kb upstream of the mole rat alpha B-crystallin gene. We have now established that the entire intergenic DNA sequence of the mole rat alpha B-crystallin locus has little lens but high muscle activity in transgenic mice as the truncated promoter/enhancer fragment. We also showed that a 2 nucleotide mutation just 3 to the enhancer of the mouse alpha B-crystallin gene results in the loss of lens and gain of muscle activity of the mouse promoter, however the reciprocal mutations in the homologous mole rat sequence has no effect. These results strengthen the importance of context dependency of cis-control elements. Finally, cell lines stably transformed with the mouse or mole rat alpha B-crystallin promoter/enhancer-reporter construct resemble the transgenic mouse results and provide evidence that the former is more responsive than the latter to transcription factors used for crystallin gene expression in the lens. ? We have continued investigating alpha B-crystallin gene expression in zebrafish. Zebrafish have 2 alpha B-crystallin genes which we have cloned. The alphaB1-crystallin gene is more specialized for lens expression than is the alpha B2-crystallin gene. However, there is negligible if any alphaB1-crystallin gene expression in the lens until approximately 8 days post fertilization. By contrast, the alphaB2 crystallin gene is expressed in many tissues, including the lens, throughout zebrafish development. AlphaB1-crystallin gene expression increases, while alphaB2-crystallin gene expression is constant in the lens during zebrafish development. In mice two genes, Dixdc1 and Heat shock protein B2 (HSPB2), are tandemly linked in opposite orientation to the single alphaB-crystallin gene locus. In zebrafish, the alpha B1-crystallin and Dixdc1 genes are linked in opposite orientation on chromosome 15, and the alpha B2-crystallin and predicted HSPB2 genes are linked in opposite orientation on chromosome 21. These chromosomal environments are consistent with the two zebrafish alpha B-crystallin genes being co-orthologous to the single mouse alpha B-crystallin gene. ? ? The roles of aldehyde dehydrogenase 1a1 (ALDH1a1) and ALDH3a1 were studied collaboratively with Drs. Vasilis Vasiliou, and Bronwyn Bateman (University of Colorado Health Sciences Center, Denver) using double ALDH3a1 and ALDH1a1 knockout mice created earlier. The lenses of the ALDH1a1/ALDH3a1 null mice are susceptible to UV-induced opacities. The absence of ALDH1A1 in the lens probably contributes to opacification by denying the lens sufficient detoxification potential of toxic oxidative aldehydes. However, the absence of ALDH3A1 probably contributes to susceptibility to lens opacification by causing a reduced UV filtering capacity of the cornea, because the wild type cornea is rich in ALDH3A1 and the wild type lens lacks ALDH3A1. The double knockout mice provide a useful tool to study protection of lens clarity by lens ALDH1A1 enzyme function and by corneal ALDH3A1 by non-enzymatic functions.? ? The beta-catenin gene was conditionally deleted in the mouse lens and retina using the Cre/lox system, resulting in abnormal lens morphogenesis due to defective cell adhesion. In contrast, loss-of-function of beta-catenin in the nasal ectoderm leads to the formation of ectopic lentoid bodies expressing crystallins. These data suggest that beta-catenin plays a dual role in lens development: as a cytoskeletal component it affects lens morphogenesis, and as part of the canonical Wnt signaling pathway it suppresses lens cell fate in the nasal ectoderm. ? ? We extended our earlier molecular characterization of scallop ALDH1A9/omega-crystallin. Previously we reported on the basis of gel filtration properties that this mollusc crystallin differs from its vertebrate homologs by being a dimeric instead of a tetrameric protein. We have now established by multi-angle laser light scattering that scallop omega-crystallin is a tetramer. Moreover, phylogenetic analysis demonstrated that it groups with the mitochondrial ALDH2 and ALDH1B1 proteins rather than with the cytoplasmic ALDH1A1; the crystallin remains in the cytoplasm due to the absence of a mitochondrial leader sequence. Its anomalous gel filtration behavior suggests interesting adaptive structural modifications worthy of further study.? ? Finally, cubomedusan jellyfish contain the most basal sophisticated, lens-bearing eyes known. Previously we demonstrated that there are 3 distinct jellyfish lens crystallins (J1-3), that J1 and J3 are expressed in the lens and extracellular tissues, and that the J1 and J3 promoters are activated by the transcription factor PaxB. We have now cloned J2, which is a novel protein; the J2-crystallin gene is expressed in lens and nonlens tissues of the jellyfish and its promoter is activated by PaxB. These results support the idea that crystallin recruitment of multifunctional proteins was driven by convergent changes involving Pax (and other) transcription factors in the promoters of non-homologous genes within and between species.
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