Characterization of the octamer, a cis-regulatory element that modulates excretory cell gene-expression in Caenorhabditis elegans

Abstract

Background: We have previously demonstrated that the POU transcription factor CEH-6 is required for driving aqp-8 expression in the C. elegans excretory (canal) cell, an osmotic regulatory organ that is functionally analogous to the kidney. This transcriptional regulation occurs through a CEH-6 binding to a cis-regulatory element called the octamer (ATTTGCAT), which is located in the aqp-8 promoter.

Results: Here, we further characterize octamer driven transcription in C. elegans. First, we analyzed the positional requirements of the octamer. To do so, we assayed the effects on excretory cell expression by placing the octamer within the well-characterized promoter of vit-2. Second, using phylogenetic footprinting between three Caenorhabditis species, we identified a set of 165 genes that contain conserved upstream octamers in their promoters. Third, we used promoter::GFP fusions to examine the expression patterns of 107 of the 165 genes. This analysis demonstrated that conservation of octamers in promoters increases the likelihood that the gene is expressed in the excretory cell. Furthermore, we found that the sequences flanking the octamers may have functional importance. Finally, we altered the octamer using site-directed mutagenesis. Thus, we demonstrated that some nucleotide substitutions within the octamer do not affect the expression pattern of nearby genes, but change their overall expression was changed. Therefore, we have expanded the core octamer to include flanking regions and variants of the motif.

Conclusions: Taken together, we have demonstrated that octamer-containing regions are associated with excretory cell expression of several genes that have putative roles in osmoregulation. Moreover, our analysis of the octamer sequence and its sequence variants could aid in the identification of additional genes that are expressed in the excretory cell and that may also be regulated by CEH-6.

Figure 1

Effects of the octamer at various distances upstream of a gene's translational start site. We placed the octamer upstream of various lengths of vit-2 promoter fragment to assay the ability of the octamer to drive EC-expression at different places within promoters. A, Octamers were appended onto the 5' end of decreasing vit-2 promoter-regions. The number represents the 5'-position of the vit-2 promoter fragment (distance upstream indicated). B, All vit-2 constructs less than and including the -392 bp (shown) construct failed to result in EC-expression although each of these constructs still have reporter expression in the intestine (I). C, Constructs with vit-2 upstream regions larger than and including the -448 bp (shown)construct had GFP expression in the EC (E) in addition to the expected expression in the intestine. The exception was the vit-2 promoter construct with the -652 bp 5'-end which failed to drive EC expression. * Both fluorescent images (B and C) were captured using 2 second exposure times.

Figure 2

Analysis of excretory cell expression-dependence on upstream octamers. We identified several genes that require the octamer for proper levels of EC-expression. A, A -135 bp 5'-truncation of ZC395.10's upstream region drives expression in the EC (EC) along with the rectal epithelia (RE) and intestine (I). B, A -105 bp 5'-truncation of ZC395.10's region can still drive expression in the intestine, but EC and rectal epithelial expression is lost. C, A -2,853 bp 5'-end C01B12.3 drives relatively strong EC expression. D, Truncating the C01B12.3 promoter to 879 bp upstream of the ATG leads to a drop in the EC expression level. E. A -589 bp 5'-truncation of C02B8.4's upstream region drives expression in the EC and the pharynx (P). Exposure times are indicated on the images.

Figure 3

The level of EC expression is decreased upon loss of the octamer in the region upstream of C05D12.1. The octamer is located 205 bp upstream of the ATG of C05D12.1. Loss of the octamer leads to a decrease in the GFP expression level. A, A -247 bp 5' truncation leads strong expression localized to the EC. B, A -141 bp 5' truncation leads to a lower level of expression, but still localized to the EC.

Figure 4

Alignment of octamer and flanking regions of octamers responsible for EC expression reveals that flanking residues are A-T rich. 15 bp upstream and 15 bp downstream flanking regions of the functional octamers were used for the WebLogo alignment http://weblogo.berkeley.edu/.

Figure 5

Effects of octamer mutagenesis on expression levels. We performed substitutions of residues within the octamer in aqp-8's promoter region and assayed for changes in EC-expression. We targeted the -264G and -263C residues. The expression patterns were either not affected, diminished, or completely lost. A, aqp-8^promoter::GFP reference strain. B, a -264G→A change led to no change in expression level. C, D, E, Mutations in the form of -264G→T, -263C→A, and -264GC→AG all lead to appreciable loss of GFP expression.