Two enhancers and one silencer located in the introns of regA control somatic cell differentiation in Volvox carteri

The regA gene plays a central role in germ-soma differentiation of Volvox carteri by suppressing all reproductive functions in somatic cells. Here we show that the minimal promoter of regA consists of only 42 bp immediately upstream of the transcription start site, and that it contains no discernible regulatory elements. However, introns 3 and 5 are both required for regA expression in somatic cells, and intron 7 is essential for silencing regA in gonidia (asexual reproductive cells). A regA gene lacking intron 7 rescues the normal phenotype of mutant somatic cells, but also results in gonidia that reproduce only weakly and soon die out. The same phenotype is observed when a regA gene containing intron 7 is placed under control of a constitutive promoter, suggesting that the silencing activity of intron 7 is promoter specific. Intron 7 is unusual in that it contains a potential ORE that is in frame with exons 7 and 8, and some transcripts are produced in which intron 7 is retained. However, a regulatory role for the intron 7 translation product can be ruled out, because a construct in which intron 7 must be translated, and one in which it cannot be translated, both result in wild-type development of both cell types. Furthermore, intron 7 is unable to act in trans to silence regA, but is able to exert its normal effect when placed in a different location within the gene. Therefore, it appears that intron 7 functions in gonidia as a classical cell-type-specific and promoter-specific enhancer, of the inhibitory type that is often referred to as a silencer.