Cloning of the Ly-6A (Sca-1) gene locus and identification of a 3′ distal fragment responsible for high-level γ-interferoninduced expression in vitro

The Ly-6A and Ly-6E allelic genes encode the Sca-1 protein, which is one of the most widely used markers in haematopoietic stem cell isolation procedures. Identification of the specific gene regulatory elements that direct haematopoietic stem cell specific expression of Sca-1 is of current interest for purposes of stem cell manipulation. Both the Ly-6E and Ly-6A alleles have been examined for regions containing DNase I hypersensitive sites thought to be indicative of transcriptional regulatory elements. In these previous studies, the Ly-6E allele with its flanking regulatory sequences was cloned, and the region responsible for high-level gamma -interferon (gamma -IFN)-induced expression was localized to a 3' distal sequence containing two strong DNase1 hypersensitive sites. Because the Ly-6A allele is thought to provide higher levels of expression in haematopoietic stem cells, we isolated over 25 kb of the Ly-6A gene and flanking regulatory regions. We show here that sequences analogous to those in the Ly-6E allele are responsible for high-level gamma -IFN-induced expression in vitro. Furthermore, we show that this 3' distal Ly-6A fragment directs high-level gamma -IFN-induced expression from a heterologous promoter, suggesting that it is a potent enhancer that could be useful for expression in haematopoietic stem cells.