Functional analysis of I alpha promoter regions of multiple IgA heavy chain genes

The 13 nonallelic IgA H chain genes of rabbit are differentially expressed in vivo. They can be grouped into those expressed at high levels (Calpha4, Calpha5, Calpha6, Calpha9, Calpha10, Calpha12, and Calpha13), those expressed at low levels (Calpha1, Calpha2, Calpha7, and Calpha11), and those that are not expressed (Calpha3 and Calpha8). We tested whether the differential in vivo expression is due to differential responses of the Ialpha promoters to TGF-beta stimulation. We stimulated the rabbit B cell line 55D1 with TGF-beta and, using single-cell RT-PCR, found that expression of germline (GL) transcripts of alpha3 and alpha8 could not be induced. By luciferase reporter gene assay and EMSA we found that the promoters of the unexpressed isotypes Calpha3 and Calpha8 are defective, thereby explaining the absence of IgA3 and IgA8 in vivo. When comparing the promoter activities of the other isotypes we found that the activities did not reflect the degree of in vivo expression. Instead, the promoters of the isotypes expressed at high or low levels promoted expression of the luciferase gene to a similar degree, except for the Ialpha4 promoter, which had much higher activity. Also the degree to which TGF-beta induced GL expression of the various isotypes in 55D1 B cells did not reflect in vivo expression. However, most of the TGF-beta-stimulated cells expressed GL mRNA of multiple isotypes; no isotype was expressed preferentially. These results suggest that the final switch to a single isotype is regulated in a step subsequent to GL transcription, rather than by induction of GL transcripts by the Ialpha promoter.