CIS-REGULATORY CONTROL OF THE SM50 GENE, AN EARLY MARKER OF SKELETOGENIC LINEAGE SPECIFICATION IN THE SEA-URCHIN EMBRYO

The SM50 gene encodes a minor matrix protein of the sea urchin embryo spicule. We carried out a detailed functional analysis of a cis-regula tory region of this gene, extending 440 bp upstream and 120 bp downstr eam of the transcription start site, that had been shown earlier to co nfer accurate skeletogenic expression of an injected expression vector . The distal portion of this fragment contains elements controlling am plitude of expression, while the region from -200 to +105 contains spa tial control elements that position expression accurately in the skele togenic lineages of the embryo. A systematic mutagenesis analysis of t his region revealed four adjacent regulatory elements, viz two copies of a positively acting sequence (element D) that are positioned just u pstream of the transcription start site; an indispensable spatial cont rol element (element C) that is positioned downstream of the start sit e; and further downstream, a second positively acting sequence (elemen t A). We then constructed a series of synthetic expression constructs. These contained oligonucleotides representing normal and mutated vers ions of elements D, C, and A, in various combinations. We also changed the promoter of the SM50 gene from a TATA-less to a canonical TATA bo x form, without any effect on function. Perfect spatial regulation was also produced by a final series of constructs that consisted entirely of heterologous enhancers from the CyIIIa gene, the SV40 early promot er, and synthetic D, C, and A elements. We demonstrate that element C exercises the primary spatial control function of the region we analyz ed. We term this a 'locator' element. This differs from conventional ' tissue-specific enhancers' in that while it is essential for expressio n, it has no transcriptional activity on its own, and it requires othe r, separable, positive regulatory elements for activity. In the normal configuration these ancillary positive functions are mediated by elem ents A and D. Only positively acting control elements were observed in the SM50 regulatory domain throughout this analysis.