GENES ANCESTRAL TO THE THYROID/STEROID RECEPTOR FAMILY

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In one line of work we continued with nuclear hormone receptors from Caenorhabditis elegans which we had previously cloned. With one of them, CHR 3 we showed both with antibody staining and with beta galactoside ligated to the promoter enhancer region of CHR3, that in early embryogenesis it localized to the nucleus of many cells, and in late embryogenesis and during development in epidermal cells of the worm. In these cells the protein product is found in the nucleus. Since transgenic animals are easily developed by injection of RNA or DNA into the ovarial syncytium of the worm, and expression can be monitored with either green fluorescent protein or beta galactosides, we were able to show that full promoter/enhancer activity required the first 1.6kb upstream of the start site, as well as a portion of the first intron. We were also able to show using an RNAi construct (in these worms, double stranded RNA (RNAi) is a more effective inhibitor of a gene's effect than antisense RNA) that inhibiting CHR 3 function resulted in a significant mortality in the worms, and those that survived had incompletely detached cuticle. The reverse experiment in which CHR3 was over expressed could be performed using a heat shock promoter. These worms showed blisters under the cuticle. We conclude that CHR3 is required for proper development and in particular for molting. We cannot be sure whether the effect is related to one or more of the 150 genes the worm has for collagens, or whether it is related to a proteolytic function. It is of interest that DHR3, a close drosophila homologue is related to metamorphosis in that insect. We are currently trying to characterize a mutant line we have one of the most primitive of chordates but has been shown to synthesize thyroxine. Dr. Carosa was able using the PCR technique to isolate a nuclear hormone receptor quite similar to vertebrate TRs. Curiously, it did not bind T4 or T3. This was probably due to a long sequence in the ligand binding domain that is unrelated to any other LBD. This gene, ciNR1 was only expressed in the developing embryo and the larva and not in the adult. Expression of CiNR 1 in COS 7 cells caused down regulation of a reporter gene containing a TRE. urthermore, when the same cells were transfected with a chicken TR, CiNR 1 inhibited its expression in a dominant negative fashion. This required the LBD. We are currently screening other ciona libraries for further nuclear hormone receptors and have begun work on receptors in scallops.