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

Abstract

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 ORF 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.

Green Algae and the Origins of Multicellularity in the Plant Kingdom

“…In regA (somatic regenerator) mutants, somatic cells form normally after embryogenesis, but then redifferentiate into gonidia; they withdraw their flagella, grow, and subsequently divide into new embryos (Starr 1970;Huskey and Griffin 1979). regA is a nuclear protein that is related to transcriptional repressors and expressed in somatic cells and required to maintain their fate Stark et al 2001). regA belongs to a family of VARL (Volvocine algae regA-like) proteins with 14 members in Volvox and 12 in Chlamydomonas (Duncan et al 2006(Duncan et al , 2007.…”

A Novel Downstream Regulatory Element Cooperates with the Silencing Machinery to Repress EPA1 Expression in Candida glabrata

“…To our knowledge, cis-acting elements located outside promoters, or 39 of the ORF affecting the expression of promoters have not previously been reported in yeast for Pol II promoters, though similar cis-acting elements have been described in yeast for Pol III transcribed genes and in other organisms, some localized inside introns (Errede et al 1987;Martin et al 2001;Stark et al 2001;Calderwood et al 2003;Delaloy et al 2008). In yeast, activators or repressors generally function when their corresponding cis-acting elements are located at distances no greater than 700 bp (Guarente and Hoar 1984;Struhl 1984;Keegan et al 1986) upstream of the start site of transcription.…”

Translational control ofregA, a key gene controlling cell differentiation inVolvox carteri

“…Previous studies had shown that the transcription of regA is regulated primarily by two enhancers and one silencer located in the introns (Stark et al, 2001). But there were also indications that regA might be regulated at the translational level.…”

“…Cells of the V. carteri strain 153-68, which carries non-revertible mutations in both the nitA and regA loci , were co-bombarded with pVcNR15 (Gruber et al, 1996) and various regA constructs that were described before. Integration of regA constructs after transformation was tested by PCR as described by Stark et al (Stark et al, 2001). …”

“…1C) appears, in which the somatic cells appear to differentiate normally at first, but then redifferentiate as fully functional reproductive cells. RegA, the regA gene product, is a protein with features of a transcriptional repressor that is present in somatic nuclei through most of the life cycle, but can never be detected in gonidia Stark et al, 2001). Sixteen putative targets of RegA repression were found to be nuclear genes encoding important chloroplast proteins; this led to the hypothesis that regA blocks reproductive activity in somatic cells by preventing chloroplast biogenesis, and thereby preventing their growth (Meissner et al, 1999).…”