Requirement for intron structures in activating the <i>Cd8a</i> locus.

Abstract

During differentiation of CD4⁺CD8⁺ double-positive (DP) thymocytes into the CD4^-CD8⁺ single-positive (CD8SP) thymocytes committed to the cytotoxic T cell lineage, <i>Cd8a</i> transcription is temporally terminated after positive selection and is subsequently reinitiated, a process known as coreceptor reversal. Despite the identification of a transcriptional enhancer in the <i>Cd8a</i> gene that directs reporter transgene expression specifically in CD8SP thymocytes, the molecular mechanisms controlling reactivation of the <i>Cd8a</i> gene are not fully understood. Here, we show that, after positive selection, hCD2 reporter expression from the <i>Cd8a</i> locus, which was generated by insertion of hCD2 cDNA into the first exon of the <i>Cd8a</i> gene, requires the incorporation of intron sequences into the <i>hCD2</i> transcript. The presence of polyadenylation signals after hCD2 cDNA inhibited hCD2 expression in mature CD8⁺ T cells, whereas hCD2 expression in DP thymocytes recapitulated the <i>Cd8a</i> expression. Incorporation of the endogenous short intron structure and heterologous intron structure of the <i>Cd4</i> locus restored hCD2 expression in mature CD8⁺ T cells in a variegated manner. Interestingly, stage-specific DNA demethylation was impaired in <i>Cd8a</i> reporter alleles that failed to express hCD2 in CD8⁺ T cells, and intron sequences lacking RNA splicing signals still restored hCD2 expression. These observations indicate that "intron-mediated enhancement" is involved in a stage-specific reactivation of the <i>Cd8a</i> locus harboring hCD2 cDNA. However, the <i>Cd8a</i> gene was transcribed in mature CD8⁺ T cells, albeit at a lower level, from a mutant <i>Cd8a</i> locus lacking intron structures, suggesting that protein-coding sequences in transcripts affect sensitivity to intron-mediated enhancement.