Characterization of a novel Dictyostelium discoideum prespore-specific gene, PspB, reveals conserved regulatory sequences

Abstract

While Dictyostelium discoideum has been studied as a developmental system for decades, and many regulatory proteins have been cloned, the molecular mechanisms of cell-type-specific gene expression are poorly understood. In this paper we characterize a novel prespore gene, PspB, and undertake a comparative analysis of the regulatory regions in prespore-specific D. discoideum promoters. Sequence alignment of the PSPB gene product with other prespore-specific proteins identifies a conserved, repeated 12 amino acid cysteine-containing motif that may be involved in spore coat function or assembly. Analysis of the PspB promoter identifies two domains essential for developmentally induced promoter activity. The first region includes two CA-rich elements (CAEs) that we show to be functionally homologous to the cAMP-inducible elements previously identified in the SP60 (cotC) promoter. The PspB CAEs compete with the SP60 (cotC) CAEs for binding in vitro to a developmentally regulated nuclear activity. We identify this activity as G-box Binding Factor, a developmentally induced transcription factor. The PspB CAEs and adjacent nucleotides direct a very low level of prespore-enriched expression, but high levels of cell-type-specific expression requires a second promoter region: a 46-bp AT-rich sequence that does not resemble the CAEs or any other previously described late gene promoter elements. Comparison of the PspB AT element with regulatory regions of the SP60 (cotC), SP70 (cotB), and D19 (pspA) promoters reveals an extensive consensus sequence. We suggest that these AT-rich sequences may represent a common regulatory element (or elements) required for prespore gene activation.