Three isoforms of a novel member of the steroid hormone nuclear receptor superfamily related to the retinoic acid receptors have been identified. The three isoforms, referred to as RORal, R0Ra2, and R0Ra3, share common DNA-and putative ligand-binding domains but are characterized by distinct amino-terminal domains generated by alternative RNA processing. An exon encoding a functionally important subregion of the amino-terminal domain of the RORa2 isoform resides on the opposite strand of a cytochrome c-processed pseudogene. Binding site selection using in vitro-synthesized proteins reveals that the RORal and R0Ra2 isoforms bind DNA as monomers to hormone response elements composed of a 6-bp AT-rich sequence preceding a half-site core motif PuGGTCA (RORE). However, RORal and RORa2 display different binding specificities: RORal binds to and constitutively activates transcription from a large subset of ROREs, whereas R0Ra2 recognizes ROREs with strict specificity and displays weaker transcriptional activity. The differential DNA-binding activity of each isoform maps to their respective amino-terminal domains. Whereas truncation of the amino-terminal domain diminishes the ability of RORal to bind DNA, a similar deletion relaxes RORa2-binding specificity to that displayed by RORal. Remarkably, transfer of the entire amino-terminal region of RORal or amino-terminal deletion of RORa2 confers RORE-binding specificities to het^erologous receptors. These results demonstrate that the amino-terminal domain and the zinc finger region work in concert to confer high affinity and specific DNA-binding properties to the ROR isoforms and suggest a novel strategy to control DNA-binding activity of nuclear receptors.

The vertebrate lens is a classical system for examining mechanisms of tissue determination and differentiation, yet little is known about the signaling molecules controlling its development. Here, we report that retinoic acid IRA}, a substance known for its teratogenic effects on the eye and as a natural endogenous morphogenetic agent, acts as a regulator of gene expression in the lens. We have identified a novel type of RA response element (RARE) within the lens-specific mouse 7F-crystallin promoter, consisting of two (A/G)GGTCA motifs in an everted arrangement spaced by 8 nucleotides. This element {TF-RARE) mediates activation of the 7F-crystallin promoter by ligand-activated endogenous lens cell RA receptors (RARs) and confers RA responsiveness when linked to a heterologous promoter. 7F-RARE is bound in vitro by RAR/RXR heterodimers, and both receptors cooperate in vivo to trans-activate this element. These observations demonstrate a direct effect of RA on lens-specific gene expression and reveal a novel role for retinoids in the development and homeostasis of the mammalian eye.

We have used the in vitro motility assay to investigate the effect of caldesmon on the movement of actin-tropomyosin filaments over thiophosphorylated smooth muscle myosin and skeletal muscle heavy meromyosin. Using either motor, incorporation of up to 8 nM caldesmon inhibited filament movement by decreasing the proportion of filaments motile from > 85% to < 30%. There was a minimal effect on filament attachment and a modest decrease in motile filament velocity in this concentration range. The reduction in the proportion of filaments motile could be completely reversed by incorporation of an excess of calmodulin at pCa 4.5. The expressed C-terminal fragment, 606C, which retains caldesmon's inhibitory capacity but does not bind to myosin, decreased the proportion of filaments motile but had no effect on velocity. We conclude that the velocity reduction by whole caldesmon is due to actin-myosin cross-linking. A significant decrease in filament attachment was observed when caldesmon was added to an excess over actin (> 10 nM). In the absence of tropomyosin, addition of an excess of caldesmon caused a similar decrease in the filament density, but there was no effect on the proportion of filaments that were motile. Our results demonstrate that caldesmon can switch actin-tropomyosin from motile to non-motile states without controlling velocity of movement or weak binding affinity and show the inhibitory action of caldesmon in the motility assay to be functionally indistinguishable from that reported for troponin.

The elements regulating lens-specific expression of the mouse yF-crystallin gene were examined. Here we show that mouse yF-crystallin sequences -67 to +45 contain a low basal level of lens-specific promoter activity and that sequences -67 to -25, which are highly conserved among different y-crystallin genes, are able to function as a strong transcriptional activator when duplicated and placed upstream of the TATA box. We also show that nuclear factors from lens and nonlens cells are able to form different complexes with sequences centered at -46 to -36 and demonstrate that binding of the factor from lens cells correlates with lens-specific promoter activity of the mouse -yF-crystallin gene.The vertebrate lens is a transparent spheroidal structure consisting of essentially two cell types: terminally differentiated fiber cells, which are longitudinally arrayed and make up the main body of the lens, and epithelial cells, which are mitotically active and form a monolayer at the anterior surface. In mammals, terminal lens fiber cell differentiation is characterized by expression of the y-crystallins, a group of closely related polypeptides of =20 kDa (3-5, 7, 14, 15). The exact function of these proteins is unknown, although it is generally believed that they play a critical role in determining the optical properties of the transparent lens.In mice, the -y-crystallins are encoded by six closely linked genes located on chromosome 1 (18, 21). The different members of this gene family are coordinately activated at the onset of lens fiber cell differentiation but are temporally and spatially regulated during development, resulting in differences in the relative abundance of individual transcripts in different regions of the lens (2, 17). Insight into the mechanisms regulating expression of the mouse y-crystallins has come primarily from investigations of the mouse yF-crystal- or no activity in vitro, contain sufficient information for imparting lens fiber cell specificity, while homologous and heterologous enhancers are able to act in concert with these sequences to modulate the spatial pattern of gene expression within the lens.In the present study, we have examined the proximal sequences of the mouse -yF-crystallin promoter for determinants of lens specificity. We show that 5'-flanking sequences -67 to +45 contain lens-specific promoter activity and that the interval -67 to -25, which defines the proximal domain, is able to function as a strong transcriptional activator when duplicated and placed upstream of the TATA box. We also demonstrate that lens-specific promoter activity correlates with the ability of sequences centered at -46 to -36 to bind a lens cell nuclear factor and report that this factor, or its binding activity, is not detectable in various cell types of nonlens origin. MATERIALS AND METHODSPlasmid constructions. All recombinant plasmids containing the bacterial chloramphenicol acetyltransferase (CAT) gene (cat) were based on the parent vector pSVoATCAT (12, 16). Plasmid -y226-CAT, which carries the cat...

Previously, we have identified a hormone response element (gamma F-HRE) composed of an everted repeat of the half-site (A/G)GGTCA motif separated by 8 base pairs that mediates retinoic acid (RA) activation of the gamma F-crystallin promoter. Here, we report that this element is bound by the thyroid hormone (T3) receptor in the form of heterodimers with either the retinoid X receptor (RXR) or the retinoic acid receptor (RAR). The T3R/RXR heterodimer binds to this element with high affinity but the transcriptional activity of the T3 receptor on this element is effectively antagonized by RAR alpha. Thus, RAR alpha exerts a dominant effect on the gamma F-HRE-everted repeat by mediating both RA activation and preventing T3 response. Although RAR/T3R heterodimers bind to the gamma F-HRE, they do not appear to be involved in transcriptional regulation since they bind with low affinity, and their ability to bind DNA is dramatically decreased by T3. Repression requires the DNA- and ligand-binding domains of RAR alpha and is consistent with a competitive DNA binding model of repression. However, in vitro binding studies indicate that RAR/RXR heterodimers form less stable interactions with the gamma F-HRE compared with T3R/RXR heterodimers; this suggests that in vivo the binding affinity of RAR/RXR heterodimers may be enhanced by accessory factors.