USF IN THE LYTECHINUS SEA-URCHIN EMBRYO MAY ACT AS A TRANSCRIPTIONAL REPRESSOR IN NON-ABORAL ECTODERM CELLS FOR THE CELL LINEAGE-SPECIFIC EXPRESSION OF THE LPS1 GENES

Expression of the aboral ectoderm-specific LpS1 gene in Lytechinus was used to study lineage-specific transcriptional regulation during sea urchin development. Band shift assays using anti-USF antibody showed t hat a USF-like protein bound the USF core sequence 5'-CACGTG-3' in the promoter of the LpS1 gene. DNA constructs consisting of a wild-type L pS1 promoter and the same LpS1 promoter with a mutated USF binding sit e fused to the bacterial chloramphenicol acetyltransferase reporter ge ne were tested. The mutation in the USF binding site caused an increas e in chloramphenicol acetyltransferse activity. We selected a clone th at encodes USF, LvUSF, from a gastrula-stage cDNA library representing Lytechinus variegatus. Transactivation experiments, in which LvUSF RN A or a DNA construct consisting of the LvUSF cDNA clone fused to the L ytechinus pictus metallothionein promoter coinjected with the wild typ e or mutated LpS1 promoter-chloramphenicol acetyltransferase gene cons truct, showed that chloramphenicol acetyltransferase activity from the wild-type construct was repressed, while the construct mutated at the USF binding site was active. The same wild-type and mutated LpS1 prom oter DNA fragments ligated to the green fluorescent protein reporter g ene were used to examine spatial expression. The reporter gene constru cts containing the mutated USF binding site were expressed inappropria tely in all cell types including the gut and oral ectoderm in gastrula and larva stage embryos, while the wild-type constructs were expresse d primarily in the aboral ectoderm. USF was expressed in all cells of the early embryo and in all tissues except the aboral ectoderm in late r embryos. The data are consistent with a model depicting Lytechinus U SF, as a temporal and spatial regulator by repressing LpS1 gene transc ription in non-aboral ectoderm cells. (C) 1996 Academic Press Limited