Interaction of nuclear proteins with a putative mouse CD8α gene regulatory element

T-T cell hybrids produced by fusing BW5147 cells with CD8-positive CTLs were found to shut off transcription of the CD8α gene. Analysis of stable transfectants of BW5147 with CD8α gene constructs containing varying amounts of 5′ flanking sequence revealed an element (called L2a) located approximately 5 kb upstream which appeared to be the target of negative regulation in BW5147 hybrids. EMSA analyses using the L2a element as probe and nuclear extracts from various cell lines revealed two major retarded bands. A strong band of higher mobility (band 1) was observed with thymocyte nuclear extract and generally correlated with CD8 expression by T cell lines. Interestingly, this band was not observed in the presence of antiserum directed against SATB1, a thy mus-specific nuclear protein which binds to AT-rich DNA sequences shown to associate with the nuclear matrix (matrix associated regions or MARs). The sequence of the L2a element has similarities with known MARs. A band of lower mobility in EMSA assays (band 2) was observed using nuclear extracts from both CD8⁺ and CD8^- cell lines of various origins. Studies using the missing nucleotide approach, DNasc I protection analysis, and mutant and deleted L2a elements suggest that the proteins which give rise to bands 1 and 2 compete for binding to overlapping sites on the L2a element. A model is proposed in which the L2a element functions as a MAR and the proteins eivine rise to retarded bands 1 and 2 reeulate CD8α eene transcriotion in a positive and negative fashion, respectively.