Identification of Transcription Start Sites and Preferential Expression of Select CB₂ Transcripts in Mouse and Human B Lymphocytes

Abstract

Marijuana cannabinoids, the endocannabinoids, and cannabinoid cell receptors have been shown to play important roles in immune regulation particularly as potent modulators of anti-inflammatory cytokines. The predominant cannabinoid receptor involved in this immune regulation is cannabinoid receptor 2 (CB₂), which is predominantly expressed in B lymphocytes. However, the promoter region and mechanisms of CB₂ gene regulation are unknown in this immune cell type. Utilizing a combination of bioinformatics, 5′ rapid amplification of cDNA ends (5′ RACE), real-time reverse transcription-polymerase chain reaction, DNA sequencing, and luciferase reporter assays, we show that human B cells express one CB₂ transcript while mouse B cells express three CB₂ transcripts, with specific transcript selection occurring during B cell activation by lipopolysaccharide. Alignment of our sequenced RACE products to either the mouse or human genome, along with the GenBank submitted mRNA sequences, revealed that the transcripts we isolated contained previously unidentified transcriptional start sites (TSS). In addition, expression construct testing of the genomic region containing the TSSs of the mouse CB₂ exon 1 transcripts showed an eightfold increase of promoter activity over baseline. These data show for the first time that human B cells use only one TSS for CB₂ while mouse B cells use multiple TSSs and that the mouse TSSs are in a genomic area with promoter activity, thus suggesting the location of the gene promoter region. Defining these TSSs also provides clues to the various gene regulatory factors involved in the expression of CB₂ during B cell activation.