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Characterization of the octamer, a cis-regulatory element that modulates excretory cell gene-expression… Mah, Allan K; Tu, Domena K; Johnsen, Robert C; Chu, Jeffrey S; Chen, Nansheng; Baillie, David L Mar 8, 2010

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RESEARCH ARTICLE Open AccessCharacterization of the octamer, a cis-regulatoryelement that modulates excretory cellgene-expression in Caenorhabditis elegansAllan K Mah1,2*, Domena K Tu1, Robert C Johnsen1, Jeffrey S Chu1, Nansheng Chen1, David L Baillie1AbstractBackground: We have previously demonstrated that the POU transcription factor CEH-6 is required for drivingaqp-8 expression in the C. elegans excretory (canal) cell, an osmotic regulatory organ that is functionally analogousto the kidney. This transcriptional regulation occurs through a CEH-6 binding to a cis-regulatory element called theoctamer (ATTTGCAT), which is located in the aqp-8 promoter.Results: Here, we further characterize octamer driven transcription in C. elegans. First, we analyzed the positionalrequirements of the octamer. To do so, we assayed the effects on excretory cell expression by placing the octamerwithin the well-characterized promoter of vit-2. Second, using phylogenetic footprinting between threeCaenorhabditis species, we identified a set of 165 genes that contain conserved upstream octamers in theirpromoters. Third, we used promoter::GFP fusions to examine the expression patterns of 107 of the 165 genes. Thisanalysis demonstrated that conservation of octamers in promoters increases the likelihood that the gene isexpressed in the excretory cell. Furthermore, we found that the sequences flanking the octamers may havefunctional importance. Finally, we altered the octamer using site-directed mutagenesis. Thus, we demonstrated thatsome nucleotide substitutions within the octamer do not affect the expression pattern of nearby genes, butchange their overall expression was changed. Therefore, we have expanded the core octamer to include flankingregions and variants of the motif.Conclusions: Taken together, we have demonstrated that octamer-containing regions are associated withexcretory cell expression of several genes that have putative roles in osmoregulation. Moreover, our analysis of theoctamer sequence and its sequence variants could aid in the identification of additional genes that are expressedin the excretory cell and that may also be regulated by CEH-6.BackgroundThe Caenorhabditis elegans excretory system is com-posed of four cells: the excretory duct cell, the bi-nucle-ate excretory gland cell, the excretory pore cell, and theexcretory (canal) cell (EC). Each of these cells are des-cendents of the AB cell lineage [1]. The EC in particularhas a unique H-shaped structure consisting of two pairsof bilaterally-symmetrical projections that protrude ante-riorly and posteriorly from the central cell body. The ECforms approximately 270 minutes after the first cellulardivision near the centre of the embryo [2]. Subsequently,two processes extend dorso-laterally, which thenbifurcate to form the anterior and posterior canalbranches. By the end of the first larval stage, the ECcanals have reached their full length relative to thelength of the nematode [3]. Further growth of the canalsis influenced by their attachment to the hypodermis,which promotes extension of the EC canals followingthe first larval stage [3]. Not surprisingly, because ofsimilarities in their structures, the EC and neurons sharemany developmental cues that dictate elongation, gui-dance and outgrowth [4].Even though the EC shares developmental cues withneurons, it has a distinct function. The EC plays a rolein maintaining osmotic balance by collecting solubleorganic and inorganic metabolic waste substances andexpelling these to the environment [5]. For C. elegans,* Correspondence: amah@cmmt.ubc.ca1Department Molecular Biology and Biochemistry, Simon Fraser University,8888 University Drive, Burnaby, British Columbia, Canada, V5A 1S6Mah et al. BMC Molecular Biology 2010, 11:19http://www.biomedcentral.com/1471-2199/11/19© 2010 Mah et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.osmoregulation is a critical function due to the continu-ous and unpredictable stresses placed upon worms intheir native soil habitat. To facilitate the exchange ofdissolved material, the fluid-filled EC arms are exposedto pseudocoelomic fluid. The pseudocoelomic fluid inturn is in contact with most of the cells in C. elegans,presumably having a function analogous with circulatoryfluid. The pseudocoelomic fluid also provides the turgorfor the hydrostatic skeleton of C. elegans, which isessential for locomotion. In addition to maintainingosmotic balance, the excretory system is responsible forsecreting hormones [6] and fluids required for molting[7]. Notably, the osmoregulatory function of the ECresembles the role of the mammalian kidneys. Thus,characterizing conserved mechanisms that govern ECtranscription may provide insight into regulatory circuitsthat control kidney-specific transcription.We are interested in the mechanisms that governtranscriptional regulation in the EC of C. elegans. Ourlaboratory has previously characterized two distincttranscriptional regulatory mechanisms affecting EC geneexpression. One of these mechanisms relies upon bind-ing of the transcriptional regulator DCP-66 to the Ex-1cis-regulatory element (CCATACATTA). Together,DCP-66 and Ex-1 drive EC-exclusive expression of pgp-12, an ABC transporter-encoding gene [8]. However,DCP-66 is a component of the transcriptional inhibitorynucleosomal remodeling and deacetylase (NuRD) com-plex, which is typically associated with gene repression[9]. The second mechanism, involves CEH-6, a class IIIPOU homeobox transcription factor (TF), which bindsto a cis-regulatory element called the octamer (ATTTG-CAT) [10]. We originally characterized the octamer asan element required for EC-expression of the aqua-porin-encoding gene aqp-8. Of note, CEH-6 is alsoexpressed in the excretory cell (EC), thus fulfilling thespatial requirements of a TF responsible for EC-selectivetranscription. Additionally, CEH-6 is detected in thebilaterally symmetric neurons (RMDDLR, RMDVLR,AUALR and AVHLR), P.na cells (ventral nerve cord),five rectal cells (B, Y, U, F and K) [11], two tail nervecells, ventral hypoderm, anterior body wall muscle, bodywall muscle cells, and intestine [12]. The broad expres-sion of CEH-6 indicates that it likely regulates transcrip-tion in a several ectodermal cells as well.Because CEH-6 interacts with the octamer to driveaqp-8 expression in the EC, we wanted to determinewhether the octamer is generally linked to EC-expres-sion. If so, octamer regulated genes could representnovel candidates that function in the EC. Furthermore,we also attempted to define the role of the octamer cis-regulatory element as a driver of EC-selective transcrip-tion. Specifically, we determined whether the octamer isunder spatial restrictions within promoter regions,identified genes that require the octamer for EC expres-sion, and identified variants of the octamer that are ableto drive EC-expression. Thus, this work identified a setof candidate genes that could be relevant to kidneyfunction in vertebrates. Overall, our data reinforce therole of octamers and presumably their cognate tran-scription factors such as CEH-6 in directing osmoregu-latory organ gene expression.ResultsThe Location of the Octamer Sequence in the Promotercan be FlexiblePreviously, we found evidence that the octamer is spa-tially restricted in the aqp-8 promoter [10]. However,we also determined that the octamer still had the capa-city to drive EC-specific expression when placed in closeproximity to the ATG using the Δpes-10 minimal pro-moter [10] (note that we refer to the ATG becausemany transcriptional start sites are poorly characterizedin C. elegans). Thus, we hypothesized that the octamermight have different spatial restrictions in the promoterregions of different genes. To assess spatial dependenceof the octamer within the promoter region, we used thevit-2 promoter as a tool. vit-2 encodes a yolk proteinthat is expressed at high levels in the intestine [13], butis not normally expressed in the EC. A promoterencompassing 247 bps directly upstream of the vit-2ATG is sufficient for driving intestinal expression of vit-2 [14]. Therefore, we used the intestinal expression toassay for promoter function. We generated a series ofpromoter constructs by appending tandem octamersequences to the 5’ end of vit-2 promoter truncationconstructs (258 bp to 700 bp upstream of the ATG)(Figure 1A). These constructs were fused to GFP andthen injected into wild-type worms to generate trans-genic nematodes. Subsequently, we monitored for GFPexpression in the intestine and in the EC.We observed that the octamer was not able to driveEC expression in constructs that contained less than448 bp upstream of the vit-2 ATG. However, these con-structs retained the ability to drive intestinal expressionindicating that the transgene was successfully generatedand functional (Figure 1B). Placing the octamer at the5’-end of vit-2 promoter constructs larger than 448 bpupstream of the ATG led to ectopic GFP expression inthe EC (Figure 1C). An exception to this was from atransgene containing octamers 652 bp upstream of vit-2’s ATG, which did not drive assayable levels of GFP.This transgenic strain drove intestinal GFP, but failed todrive GFP in the EC. The lack of EC-expression indi-cates that there may be a cis-linked element locatedbetween 600 bp and 652 bp upstream of the vit-2 ATGthat represses EC expression. Alternatively, it is possiblethat this transgene construct formed a concatemerMah et al. BMC Molecular Biology 2010, 11:19http://www.biomedcentral.com/1471-2199/11/19Page 2 of 13in vivo that is incompatible with the expression of theGFP reporter. This second scenario is unlikely becausewe did observe intestinal expression. In any case, thesedata suggest that octamer function may be spatiallyrestricted in some promoters. Taken together, althoughthe above data did not allow us to conclude whether theoctamer has an optimal distance from the ATG forinfluencing EC-expression, the data do suggest thatfunctional octamers can be present at various places indifferent promoters.Many EC-expressed Genes are not regulated by OctamersBecause functional octamers may be located at variousdistances upstream of the ATG, we searched for geneswith upstream octamers within 1,200 bp upstream oftheir ATGs; WormBase (WS195). Thus, we identified1,340 candidate genes including promoters that containeither the forward and/or the inverse of the octamer(ATGCAAAT). To assess the function of these octa-mers, we selected genes from this set that are expressedin the EC [14,15]. In addition, we assessed the functionof the octamer in the promoters of hlh-8 and ZC395.10,genes with no known EC-expression. For theseseventeen genes, we tested if regions containing theoctamer are required for EC-expression by truncatingthe promoter from the 5’-end and observing whetherthe octamer regions affect the level of EC expression(Additional file 1). However, we could not concludewhether the octamers were functional in promoters ofY69E1A.6, F36H1.2, Y48A6B.8, F29F11.6, B0334.4,C02B8.6, H23N18.3, R13F6.3, and, Y53G8AR.3 becauseEC expression was lost in worms carrying promoterconstructs that still had the octamer, suggesting thatthere are other cis-linked elements that drive EC-expres-sion of these genes (Additional file 1). Amongst theother eight genes, our promoter truncation analysisrevealed three genes that are likely dependent upon theoctamer containing region for EC expression: ZC395.10,C01B12.3, and hlh-8/C02B8.4. These results are not sur-prising as previous reports indicate that 12% of EC-expressed genes are predicted to be regulated by theDCP-66/Ex-1 mechanism [8] Additionally, the nuclearhormone receptor NHR-31 regulates most of the vacuo-lar ATPase (vATPase) components in the EC [16] point-ing to multiple regulatory mechanisms that specify EC-expression.Figure 1 Effects of the octamer at various distances upstream of a gene’s translational start site. We placed the octamer upstream ofvarious 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 promoterfragment (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 largerthan and including the -448 bp (shown)construct had GFP expression in the EC (E) in addition to the expected expression in the intestine. Theexception 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.Mah et al. BMC Molecular Biology 2010, 11:19http://www.biomedcentral.com/1471-2199/11/19Page 3 of 13The octamer in ZC395.10’s promoter region is located120 bp upstream of the ATG. ZC395.10 is localized tomost neurons, intestine, pharynx, and vulva [14,15].However, a ZC395.10 promoter construct containing theoctamer (5’-end 135 bp upstream of the ATG and in theforward orientation) drove expression in a different pat-tern than the original longer promoter. In this shorterpromoter construct, we observed GFP in the EC, ante-rior neurons, intestine, and rectal epithelia (Figure 2A).The differences between this and the construct with thelarger promoter indicate that there is a cis-linked repres-sor element(s) that modulates EC and rectal epithelialtissue expression in ZC395.10’s promoter. Interestingly,the EC and rectal epithelia expression pattern resultingfrom the shorter construct overlaps with CEH-6’sexpression pattern [3]. Furthermore, deleting the octa-mer containing region leads to loss of expression inboth the EC and rectal epithelia. Additionally, theremust be an independent transcriptional regulatorymechanism that drives ZC395.10 expression in anteriorneurons and the intestine (Figure 2B).An octamer is located 1,055 bp upstream of the ATGin C01B12.3’s promoter region. A transcriptional reporterconstruct with a 5’-end 2,853 bp upstream of the ATGleads to expression in the EC (Figure 2C), hypoderm,spermatheca, and the anal depressor muscle [14,15].However, the EC-expression level is greatly decreasedwhen the promoter is truncated (879 bp upstream of theATG) corresponding to removal of a large portion of thepromoter and including an octamer (Figure 2D). Interest-ingly, the orthologs of C01B12.3 in C. briggsae and C.remanei also have octamers in their promoters. More-over, the distance of the octamers from the ATG aresimilar (1,108 bp and 1,100 bp upstream of the ATGrespectively), suggesting that this element might also reg-ulate EC-expression of C01B12.3 orthologs.The hlh-8 promoter contains an octamer located 582bp upstream of the ATG. It was previously shown thathlh-8 is expressed in the intestine, anterior neurons, andvulva [14,15]. A 5’ truncation that limits the promoterto only seven bases upstream of the octamer (589 bpupstream of the ATG) results in expression localized tothe EC and the second pharyngeal bulb (Figure 2E).This change in expression pattern indicates the likelypresence of a repressor element(s) that blocks EC andpharyngeal expression. Expression of hlh-8 in the EC iscompletely abolished upon deletion of the octamer-con-taining fragment. In sum, our promoter deletion analysisidentified octamer-containing promoter regions requiredfor the EC-specific expression for the above three genes.Figure 2 Analysis of excretory cell expression-dependence on upstream octamers. We identified several genes that require the octamerfor 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 therectal 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 rectalepithelial 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 bpupstream 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 theEC and the pharynx (P). Exposure times are indicated on the images.Mah et al. BMC Molecular Biology 2010, 11:19http://www.biomedcentral.com/1471-2199/11/19Page 4 of 13Conserved Octamer sequences in Promoter RegionsStrongly Bias for EC ExpressionDue to the low success rate in finding octamer contain-ing regions associated with EC-expression with theabove approach (only 3/17), we turned to phylogeneticfootprinting. Using this comparative method, we identi-fied promoters with perfectly conserved octamersbetween three closely related Caenorhabditis species (C.elegans, C. briggsae, and C. remanei). This resulted inthe identification of 165 promoters that contain con-served octamers (Additional file 2).Of the 165 genes, nineteen genes had previously char-acterized expression patterns [14,15]. To obtain a largersample size, we analyzed the expression patterns of anadditional 88 candidates (Additional file 3). From these107 promoters, we identified 64 that could drive detect-able levels of GFP expression, including 25 (39%) thatwere EC-expressed. This represents a significant enrich-ment of genes expressed in the EC when compared to acontrol dataset of 1,885 expression patterns, withinwhich only 10.2% (193/1,885) of all genes are expressedin the EC [14,15]. Strikingly, twelve genes (19%) in ourset were expressed only in the EC. This is a vast enrich-ment over the control set which contains only 0.3% (6/1,885) genes with EC-exclusive expression [14,15] (sig-nificance P < 0.01 as determined by 1-tailed Z-test).Next we wanted to determine whether their EC-expression was dependent on the octamer containingfragments. Using the same approach as in the previoussection (5’ serial promoter truncations), we selected 21promoters that drove EC expression for further analysis(Additional file 4). From this set, we identified fourgenes that are completely dependent on fragments con-taining the octamer for EC expression (M176.5, aqp-8/K02G10.7, twk-36/R12G8.2), C05D12.1) and two genesthat exhibit reduced EC expression upon deletion of theoctamer region (R02F2.8, and F16F9.1) (Additional file 4;Figure 3) (note: the aqp-8 promoter was identified in thephylogenetic analysis, but was not subject to truncationanalysis as it’s octamer has been previously characterized[10]).Sequences Flanking Functional Octamers areLikely ConservedIn the experiments described above, we identified genesthat may depend on the octamer for expression in theEC. Thus, we used these promoters to study whetherthe sequences flanking the octamer are conserved. Wealigned the nine octamer sequences along with 15 bp ofupstream and downstream flanking sequences. Theresulting alignments were displayed using WebLogo [17](Figure 4; note that the reverse complementary sequencewas used if the octamer was inverted). This analysisrevealed that, in general, octamers are flanked by AT-rich regions. More specifically, regions upstream of theoctamer are biased towards being A/C-rich and down-stream regions tend to be T-rich. Thus, our newly iden-tified collection of EC-expressed genes has allowed us todefine additional specificity determinants related to theoctamer.Intra-octamer Nucleotide Substitutions Have DifferentEffects on EC ExpressionPreviously, we observed that the octamer is perfectlyconserved within the promoters of aqp-8 orthologsamong five Caenorhabditis species [10]. Therefore, wehypothesized that the octamer sequence must be abso-lutely conserved to drive EC-specific expression. Toexamine the octamer in more detail, we manipulated itssequence using site-directed mutagenesis. We generatedvariants of the octamer by targeting nucleotides -264 bpand -263 bp of the octamer in the aqp-8 promoter (boldresidues ATTTGCAT). Every possible single-nucleotidesubstitution and a dual nucleotide substitution wereFigure 3 The level of EC expression is decreased upon loss of the octamer in the region upstream of C05D12.1. The octamer is located205 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 leadsstrong expression localized to the EC. B, A -141 bp 5’ truncation leads to a lower level of expression, but still localized to the EC.Mah et al. BMC Molecular Biology 2010, 11:19http://www.biomedcentral.com/1471-2199/11/19Page 5 of 13tested at these sites. The mutated constructs were fusedto GFP to visualize changes in EC-expression. The 5’-end of every construct was defined as 276 bp upstreamof the aqp-8’s ATG because truncation constructs ran-ging between 1.6 kb to 272 bp upstream of aqp-8’sATG provide consistent EC-specific expression patterns[10]. Also, because the aqp-8 promoter produces consis-tent levels of EC expression, our reference transgeneconstruct is a 1.6 kb aqp-8 promoter region fused toGFP (Figure 5A) [10].At -264 bp, a G®A residue change (aqp-8promoter(-264G®A)::GFP) did not alter the expression level or pat-tern (Figure 5B). However, a G®T change (aqp-8promoter(-264G®T)::GFP led to a significant decrease in the ECexpression level (Figure 5C). A G®C substitution (aqp-8promoter(-264G®C)::GFP) led to a complete loss of EC-expression. At -263 bp, a C®A residue substitution inaqp-8promoter(-263C®A)::GFP led to decreased EC-expres-sion (Figure 5D). The C®G and C®T substitutions(aqp-8promoter(-263C®G)::GFP and aqp-8promoter(-263C®T)::GFP) both led to a complete losses of GFP expression.Finally, replacing the GC pair at -264 with an AG pair(aqp-8promoter(-264GC®AG)::GFP) led to decreased EC-expression (Figure 5E). In all constructs, the GFP signalremained localized to the EC suggesting that some octa-mer sequence variants have the capacity to influence ECexpression.DiscussionIn this study, we demonstrate that octamer containingregions are involved in driving EC-expression of severalgenes. This builds upon our previous data demonstrat-ing that aqp-8 is dependent upon the POU homeoboxTF CEH-6 and the octamer for EC-expression [10]. Inseveral nematodes, the position of the octamer relativeto the ATG is fairly well conserved among aqp-8 ortho-logs [10]. This is interesting as some cis-regulatory ele-ments are spatially restricted within promoters. Forexample, in C. elegans functional X-box motifs clusterroughly 100 bp upstream of ATGs to drive neuronalexpression [18]. Likewise, the GC-box cis-regulatory ele-ment has an optimal distance in relation to the TATAbox. Moving the GC-box away from its optimal distanceleads to decreased expression levels of nearby geneseven though its cognate TF, Sp1, still bind with similaraffinities [19]. In addition to spatial restriction relativeto ATGs, relative positioning between cis-regulatory ele-ments within the same promoter can affect expression.For instance, the b-actin promoter contains the so-called CCAAT and CCArGG boxes. These regulatoryelements are binding sites for the TFs nuclear factor Y(NF-Y) and serum response factor (SRF), respectively.Manipulating the intra-element distance between thesetwo cis-regulatory sequences accordingly affects b-actinmessage levels [20]. However, in the present study wehad also observed that the octamer could be placed atdifferent positions in a heterologous promoter and stilldrive EC-specific expression. Therefore, unlike theabove examples, the octamer does not appear to be asspatially restricted within promoter although there stillmight be some tight limitations to octamer location.In this study, we used different strategies to identifynovel genes that require upstream octamer containingregions for expression in the EC. First, we identifiedgenes with octamer sequences in their promoters. Usingthis strategy we identified a small number of genes thatare modulated by upstream octamer-containing frag-ments. Secondly, we used a more stringent approach toidentify octamers by relying on interspecies conserva-tion. We discovered that the expression patterns ofgenes in this filtered promoter set had a higher thanexpected incidence of EC-expression. In these twoscreens, we identified nine genes that are likely octamer-modulated. Several of these genes likely have osmoregu-latory functions, agreeing with the notion the EC is ana-logous to the kidney. In general, the genes we identifiedFigure 4 Alignment of octamer and flanking regions of octamers responsible for EC expression reveals that flanking residues are A-Trich. 15 bp upstream and 15 bp downstream flanking regions of the functional octamers were used for the WebLogo alignment http://weblogo.berkeley.edu/.Mah et al. BMC Molecular Biology 2010, 11:19http://www.biomedcentral.com/1471-2199/11/19Page 6 of 13fell into four categories: transmembrane channels/pores,Hsp90 co-chaperones, proteins with unknown functions,and TFs.The largest group of genes encodes transmembranechannels/pores, indicating that many genes regulated bythe octamer participate in substrate transport. The fivechannels/pores are:1) twk-36 encodes a C. elegans TWIK potassium ionchannel protein. In vertebrates, TWIKs are commonlyexpressed in neuronal tissues and, to a lesser extent inlungs, skeletal muscle [21], and tubular portions of thekidney (proximal tubule, ascending limbs, distal convo-luted tubules, and medullary collecting duct) [22]. Themammalian TWIK, TASK, is sensitive to changes inextracellular pH, indicating that some of these proteinshave roles in modulating cellular responses to pH flux[23]. Also, because their conductance is osmoticallyregulated, TWIKs can influence cellular volume [24].The C. elegans genome has 42 twk genes. As in otherorganisms, most of the C. elegans twks are expressed inneurons [25]; however, twk-36 is the only twk expressedin the EC [25]. Interestingly, another group has demon-strated that twk-36 is directly regulated by CEH-6 [26].This independent study strengthens the notion CEH-6is a bona fide regulator of EC expression that likely actsthrough the octamer in the promoter of twk-36. There-fore, it is possible that CEH-6 regulation impacts atleast some of the octamer-dependent candidates identi-fied here.2) aqp-8, an aquaporin whose function and regulationhave been characterized previously [10,27].3) C05D12.1 is a homolog of the cytochrome b561/ferric reductase SDR-2. In mammals, SDR-2 is expressedin the brain [28] and kidney where it aids in iron reab-sorption via the accessory transporter, divalent-cationtransporter 1 (DCT-1) [29]. Cytochrome b561 proteinstransport electrons in an ascorbate-dependent manner.Due to the role of SDR-2 in ascorbate regeneration,C05D12.1 could be involved in vitamin C homeostasisand/or oxidative stress responses.Figure 5 Effects of octamer mutagenesis on expression levels. We performed substitutions of residues within the octamer in aqp-8’spromoter region and assayed for changes in EC-expression. We targeted the -264G and -263C residues. The expression patterns were either notaffected, diminished, or completely lost. A, aqp-8promoter::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.Mah et al. BMC Molecular Biology 2010, 11:19http://www.biomedcentral.com/1471-2199/11/19Page 7 of 134) R02F2.8 encodes a solute carrier (SLC) protein thatis most similar to the mammalian SLC36 subfamily.SLC36 proteins are localized to intracellular and plasmamembranes [30] where they function as symporters.SLC36 proteins transport small neutral amino acidssuch as glycine, alanine, and proline. Because SLC36proteins affect proton flux, they also contribute to intra-cellular pH homeostasis [30]. In mammals there arefour SLC36 genes, two of which are expressed in thekidney (SLC36A1 and SLC36A2) [31].5) C01B12.3 encodes a C. elegans Bestrophin 3 homo-log. Bestrophins are transmembrane proteins that modu-late calcium dependent transport of chloride ions acrosscellular membranes. Bestrophins are enriched in theplasma membranes of epithelial cells where they managecellular volume [32]. Bestrophins are also expressed inexocrine gland tissues (e.g. pancreas, lacrimal and salivaryglands), lung, testis and kidney [33]. In these tissues theyfacilitate trans-epithelial movement of chloride ions lead-ing to water and electrolyte movement [33].In addition to transmembrane channels and pores, weuncovered several other genes that have less obviouslinks to osmoregulation and kidney biology. One ofthese, ZC395.10, is homologous to thehighly conservedHsp90 co-chaperone protein, p23. p23 interactions withHsp90 to ensure the proper folding and maturation ofmany proteins including steroid receptors [34], telomer-ase [35], and proteins that are upregulated in cancers[36]. We also identified M176.5, a gene with little priorfunctional data. M176.5 is a nematode-specific gene thatis mainly composed of hydrophobic amino acids and istherefore likely to be localized to cell membranes and/orforms a globular protein.Finally, we identified two TFs. The first, F16F9.1 is ahomolog of the mammalian protein lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF; a.k.a.SIMPLE/Small Integral Membrane Protein of Lysosome/Late Endosome) [37]. LITAF is linked to Charcot-Marie-Tooth (CMT1C) disease, a heritable neuropathy charac-terized by loss of muscle tissue and touch sensation [38].In CMT1C, LITAF is implicated in protein degradation[39]. LITAF also functions in cytokine production [40].We speculate that because F16F9.1 is expressed in theEC, a tissue exposed to the environment, it could beinvolved in innate immune responses. Also, because C.elegans F16F9.1 is expressed in neurons, it is possiblethat the nematode could act as a model for CMT1C.The other TF we identified is hlh-8, a helix-loop-helixTF related to human TWIST. TWIST was originallycharacterized in Drosophila as a gene involved in dorsal-ventral patterning [41]. TWIST TFs bind E-box cis-reg-ulatory elements. In C. elegans, HLH-8 is important forregulating muscle, intestinal and anal muscle develop-ment. Consequently hlh-8 mutants exhibit defecationand egg-laying defects [42]. Several transcriptional tar-gets of HLH-8 are known, including cdh-4, egl-15,C18B12.6, F08D12.7, rbc-1, npr-10, dhs-5, sgcb-1, erv-46,M60.6, R02E4.1, rev-1, and myo-3 [43]. Most of thesegenes are unlikely to be transcriptional targets of HLH-8 in the EC as they are not expressed in this cell. Anexception is cdh-4 [14,15], which encodes a widelyexpressed cadherin, which is also expressed in the EC.Due to the limited number of HLH-8 targets in the EC,we can envisage a model where CEH-6 plays a role indirecting the precise transcriptional outcomes of down-stream TFs (e.g. hlh-8). In this role, CEH-6 could modu-late target genes (e.g. cdh-4) specifically in a subset ofectodermal tissues including the EC.Several of the genes that depend on their upstreamoctamer containing fragments for EC expression arealso expressed in additional tissues. An interesting con-sequence of assaying the activity of truncated promotersis that loss of the octamer containing fragment some-times led to loss of expression in multiple tissuesincluding neurons as indicated in the cases of ZC395.10,twk-36, M176.5, and F16F9.1 (Additional file 4). This isnot surprising as vertebrate orthologs of CEH-6 includ-ing Brn1 are involved in neuronal and kidney develop-ment [44,45].Our strategy for studying the above genes involvedcomparing the expression patterns resulting from pro-moter truncations that either contain or remove theoctamer. Analyzing promoter function by means ofthese truncations imposes some significant drawbacks;for example, we could not address the function of sev-eral candidate octamer elements because removingregions upstream of the octamer led to loss of ECexpression. Also, because our promoter truncationsdeleted the octamer and some flanking regions, we can-not be certain that loss of EC-selective expression is theconsequence of removing the octamer. However,because the 5’ ends of the truncations were in generalfairly close to the octamer, and because loss of EC-expression correlated with octamer deletion, we believethat these genes are likely dependent on octamers fortheir expression in the EC. To address the issue ofwhether these are indeed functional octamers, one couldintroduce point mutations within the octamer and assessthe resulting consequences on EC-expression. However,we demonstrated in our mutagenesis experiments thatcertain point mutations are not sufficient to abolish ECexpression in the context of the aqp-8 promoter.Another potential drawback from our approach is thefact that much of our study is based on expression pat-terns arising from transgenic C. elegans strains contain-ing extrachromosomal arrays. Such arrays aresusceptible to somatic transgene loss, which results inmosaic reporter expression. This mosaiscism has theMah et al. BMC Molecular Biology 2010, 11:19http://www.biomedcentral.com/1471-2199/11/19Page 8 of 13potential to confound our analysis by under-represent-ing the expression pattern. However, expression patternsresulting from genome-integrated transgenes (aqp-8pro-moter::GFP) are identical to the expression patterns inaqp-8promoter::GFP strains carrying extrachromosomaltransgenes. Therefore, mosaic loss of the extrachromo-somal transgene array is not likely an issue for analyzingchanges in EC expression. Despite the potential short-comings detailed above, our study revealed a set ofgenes whose expression likely depends on the octamerfor expression in the EC; these genes are excellent CEH-6 candidate targets. In agreement with this notion, twk-23, one of the genes that we identified as dependent onan upstream octamer fragement, has been demonstrated,independently, to be regulated by CEH-6 [26].Because our bioinformatic search did not bias thedirection of the octamer, we discovered promoters inthe forward and inverted orientation can be associatedwith EC-expression. Octamers in a forward orientationoccur in the promoters of aqp-8, M176.5, twk-36,ZC395.10, hlh-8, C01B12.3, and C05D12.1, whereasinverted octamers are present in the promoters ofF16F9.4 and R02F2.8. Although we could not deter-mine from our small sample set whether the directionof the octamer has a functional consequence, thereare possible implications related to direction of theoctamer sequence. For example, octamers upstream ofimmunoglobulin light and heavy chain genes havedirectional preferences (ATGCAAAT and ATTTG-CAT respectively) [46]. In addition, the human POUhomeobox gene Oct1 is auto-regulated by twoupstream octamers, which are also situated in invertedorientations. Although each site binds Oct1 with equalaffinity, each of these sites has different effects onOct1 expression [47].It appears that transcriptional auto-regulation is acommon mode of regulation among POU TFs [47-49].Interestingly, we detected an octamer upstream of ceh-6’s ATG in C. elegans. Likewise, there is an octamerlocated in the regulatory region of the C. briggsae geneencoding a putative CEH-6 ortholog, providing evidencethat auto-regulation might be conserved. However, theseoctamer are located within a predicted non-coding RNAgene (class RNAz) [50]. Nevertheless, it would be inter-esting to determine whether CEH-6, like other POUTFs is auto-regulated.With our set of nine candidate octamers, we had theopportunity to determine whether residues flanking theelement are conserved. Globally, the G/C content of C.elegans is 31% [51]. However, the regions flanking theoctamers-associated with EC-expression contain aslightly higher G/C content (38%). Our alignments ofthese octamers revealed that despite the higher G/Ccontent, some positions have preferences for A/Tresidues. Strikingly, directly 3’ to the octamer, an A or Tis always present. The conservation of this residue isconsistent with the results of a previous study, whichidentified Oct1 binding preferences using a SystematicEvolution of Ligands by Exponential Environment(SELEX)-based in vitro binding approach [52]. Becauseof the enriched G/C content in octamer adjacentregions, it is likely that the observed preference for A/Tat certain positions have relevance.We tested the effects of targeted-octamer mutationson EC-expression. We found that several single-basesubstitutions did not affect the cis-regulatory element’sability to drive expression in the EC. Interestingly, wedid not observe a change in expression level or patternwhen position five of the octamer was mutated from apurine to purine (ATTTGCAT®ATTTACAT). Thisvariant of the octamer was demonstrated to be a bind-ing site for the catfish class III POU TF, Oct2 [53].Therefore, this residue change results in an octamervariant which retains the ability to interact with thePOUS sub-domain binding consensus sequence (TG(C/A)ATattc) [54]. At the same residue position, a thy-mine replacement (ATTTGCAT®ATTTTCAT), led toweak GFP expression also restricted to the EC. Thissequence was able to bind to Oct1 in vitro in an elec-trophoretic mobility shift assay (EMSA) [55]. However,in another study this variant of the octamer was notable to drive reporter expression in human cells [56].These results, taken together, indicate that this motifvariant is a sub-optimal POU TF binding site that candrive weak EC expression in C. elegans. A mutation atthe sixth residue (ATTTGCAT®ATTTGAAT) alsoled to weak EC-localized expression [10]. This octamervariant is functional in the promoter of the Drosophilagene, Choline Acetyltransferase (ChAT). In the ChATpromoter, ATTTGAAT interacts with the POUhomeobox TF, dPOU-19 [57]. All other single nucleo-tide substitutions at these two locations led to loss ofGFP expression. However, a double residue replace-ment of these residues (ATTTGCAT®ATTTAGAT)could still drive expression, as indicated by weak GFPexpression in the EC. There have been no previousreports of this dual nucleotide substitution variantassociating with POU TFs and it is therefore a novelPOU TF binding site variant. Overall, our mutagenesisassays indicate that the octamer cis-regulatory elementcould have a range of functional variants in C. elegans.Thus, identifying and characterizing promoters con-taining these octamer variants may reveal a largergroup genes expressed in the EC.Because variants of the octamer can influence EC-expression, we examined the pgp-12 promoter region inC. elegans more closely. Previously, it was demonstratedthat pgp-12 expression is regulated by DCP-66/Ex-1 [8].Mah et al. BMC Molecular Biology 2010, 11:19http://www.biomedcentral.com/1471-2199/11/19Page 9 of 13In this report, the TF/cis-regulatory element interactionwas confirmed using in vitro and genetic approaches.Loss of either Ex-1 (located 238 bp upstream pgp-12’sATG) or DCP-66 resulted in loss of EC expression [1].We detected an octamer like sequence (ATTTCCAT)that partially overlaps the Ex-1 (-241 bp). We also iden-tified this octamer-like sequence in the orthologousregions of C. briggsae. Using the Transcriptional Ele-ment Search System (TESS) [58] to identify predictedcis-regulatory elements, we found that the octamer-likesequence is indeed a potential target for octamer bind-ing proteins. Additionally this sequence binds Oct1 invitro [52]. In fact, Zhao et al. reported that promoterconstructs encompassing Ex-1 at -241 bp results instrong reporter expression during all developmentalstages [8]. They also studied the expression patternresulting from a promoter region defined by a 5’-end238 bp upstream of the ATG, thereby removing threenucleotides from the octamer-like sequence. Althoughthis promoter still drove EC-expression, the intensity ofthe GFP reporter was greatly decreased in adult and lar-val worms. Additionally, embryonic expression wasalmost eliminated. Finally, a construct with a 5’-end 228bp upstream of the ATG was not able to drive expres-sion of GFP indicating the necessity of the Ex-1 (andthe octamer-like sequence) for EC expression. There-fore, not only did this prior study define the role of theEx-1 for EC-expression, but it also indirectly providedevidence that the octamer upstream of pgp-12 mightaffect EC-expression. This suggests that the DCP-66/Ex-1 and the octamer-directed transcriptional regulatorymechanisms co-operatively modulate the expression ofpgp-12 in the EC. This model of concerted and redun-dant regulation of EC-expression may have relevance inother genes including, possibly, several genes within ourphylogenetically defined candidate set.ConclusionsOverall, we determined that the octamer is likelyresponsible for the expression of several genes withinthe EC, an osmoregulatory organ analogous to the kid-ney. Because one of our candidate genes, twk-36, hasbeen demonstrated to be a bona fide target of CEH-6regulation, it would be interesting to determine whetherCEH-6 is involved in the regulation of four other candi-dates. Although our candidates, for the most part, werechosen based upon perfect conservation of the octamer,we determined that several variants of the octamer candrive EC-expression. The existence of functional octa-mer variants indicates that future searches for octamer-driven genes should use a loosely defined octamersequence. Overall, understanding conserved mechanismsof gene regulation that determine appropriate ECexpression may provide insight into underlyingtranscriptional mechanisms that regulate transcriptionin analogous organs including the kidney.MethodsNematode strains and maintenanceC. elegans strains were maintained at 20°C on nematodegrowth media (NGM) plates inoculated with E. coliOP50. All manipulations were conducted using standardprocedures [59]. For the list of promoter::reporter con-structs used in this study, refer to Additional file 5.Generation of transgene constructs and strainsDNA constructs were generated using fusion PCR aspreviously described [60]. Promoter-containingsequences were fused upstream of the GFP-codingregion in the pPD95.67 GFP-coding cassette. The octa-mer::vit-2promoter::GFP chimeric constructs were gener-ated by PCR as follows. The forward PCR primerscontain three tandem repeats of the octamer at the 5’end of a vit-2-promoterspecific sequence. The right pri-mer of the vit-2 chimeric promoter constructs remainedconsistent between strains (vit2reverse -AGT CGA CCTGCA GGC ATG CAA GCT CGA CCT GAT GGCTGA ACC G). The chimeric promoters were fused tothe GFP-coding region in the pPD95.67. The mutagen-ized octamer constructs were generated by substitutingtarget nucleotides in the forward PCR primer.All C. elegans microinjections were conducted oneither an Olympus BH2-HLSH or a Zeiss 47 3016 invertmicroscope. The PCR constructs were injected into thesyncitial region of the gonad. The final concentrationsof the injection mix are 30 ng/μl of the target constructalong with 100 ng/μl of the marker construct, pCeh361(dpy-5(+)) [61], into the target strain dpy-5(e907)(CB907). Transgenic F1s (Dpy-5 rescued) were individu-ally plated. Wild type F2 lines were selected to establishthe transgenic lines. When available, we analyzed a sec-ond independently segregating transgenic line.Identification of all C. elegans genes with upstreamoctamersAll genes containing an octamer (ATTTGCAT or ATG-CAAAT) within 1,200 bp upstream of a protein-codinggene in C. elegans were identified in WormBase, WS195.Identification of all genes with interspecies conservedupstream octamer1,000 bp upstream of the ATG of all orthologous genegroups in the nematodes:C. elegans, C. briggsae, and C.remanei (WormBase WS195), were searched for the pre-sence of octamers. A C. elegans promoter was consid-ered if its C. briggsae and C. remanei counterparts bothcontain one or more upstream octamers. Sequencesflanking the ATGs from these three CaenorhabditisMah et al. BMC Molecular Biology 2010, 11:19http://www.biomedcentral.com/1471-2199/11/19Page 10 of 13species and the predicated motifs were loaded into aMySQL database using the GFF3 format http://www.sequenceontology.org/gff3.shtml. The comparative ana-lysis was performed in programs written in Perl. Weused the Bio::DB::GFF Perl module [62].MicroscopyAll GFP-expression analyses were conducted on a ZeissAxioscope equipped with a QImaging camera and theappropriate GFP optical filter sets. Worms were immo-bilized with 100 mM sodium azide (in water) immedi-ately prior to imaging. All images were captured at400× with identical camera and fluorescence settingsfor all images (exposure times are indicated in the Fig-ures) using QCapture software. The GFP images fromeach transgenic strain are representative of theirpopulations.Additional file 1: 5’ deletion of regions containing upstreamoctamers. Promoter regions that caused EC-expression were truncatedin a 5’-manner. The constructs were either truncated in an octamer-targeted manner or in an unbiased manner. The 5’ ends of the PCRprimer and octamer locations are relative to the genes’ ATGs. The stagesof expression are designated as E: embryonic, L: larval, and A: adult. Theexpression intensity levels are designated as L: low, M: medium, or H:high.Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2199-11-19-S1.XLS ]Additional file 2: Genes with promoters containing conservedoctamers. Through comparisons of promoter regions betweenCaenorhabditis species, we found 165 genes in C. elegans that haveconserved octamers.Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2199-11-19-S2.XLS ]Additional file 3: Expression patterns of genes containinginterspecies conserved octamers in their upstream regions. Weanalyzed the expression patterns of 107 genes that have upstreamoctamers. 64 promoters led to assayable levels of the GFP reporter. 25 ofthese had EC-expression with 13 expressing in the EC-exclusively.Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2199-11-19-S3.XLS ]Additional file 4: Testing of upstream octamers associated with ECexpression. We selected a subset of promoters to analyze fromAdditional file 2. These promoters were demonstrated to influence EC-expression. The third column represents the direction of the octamersequence. Forward: ATTTGCAT, Reverse: ATGCAAAT. Upstream regionsthat drove EC expression were truncated from the 5’ end in an octamer-targeted manner. The 5’ end of the PCR primer and octamer location arerelative to the genes’ ATGs. The stages of expression are designated as E:embryonic, L: larval, and A: adult. The expression intensity levels aredesignated as L: low, M: medium, or H: high.Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2199-11-19-S4.XLS ]Additional file 5: Transgenic strains used in this study. Thetransgenic strains used in this study are encompassed in this list.Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2199-11-19-S5.XLS ]AcknowledgementsThe authors thank the members of our laboratories (DLB and NSC) forproductive discussions. A. K. Mah is supported by an NSERC doctoralscholarship. J. S. Chu is supported by an NSERC doctoral scholarship. N. S.Chen is supported by a grant from NSERC Canada and a Faculty start-upfund provided by Simon Fraser University. D. L. Baillie is a Canada ResearchChair in Genomics and is supported by grants from NSERC and CIHR.Additionally, we would like to thank Stefan Taubert for productivecomments on the manuscript.Author details1Department Molecular Biology and Biochemistry, Simon Fraser University,8888 University Drive, Burnaby, British Columbia, Canada, V5A 1S6.2Department of Medical Genetics, Centre for Molecular Medicine andTherapeutics, University of British Columbia, 950 West 28th Avenue,Vancouver, British Columbia, Canada V5Z H4H.Authors’ contributionsAKM carried out the molecular genetic studies, conceived the study, andwrote the manuscript. DKT carried out the microinjections. RCJ helped todraft the manuscript. JSC performed the bioinformatics portion of the study.NC participated in the design of the study. DLB participated in the designand coordination of the study. All authors read and approved the finalmanuscript.Received: 6 July 2009Accepted: 8 March 2010 Published: 8 March 2010References1. 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Schug JOG: Current Protocols in Bioinformatics 1996, Chapter 2.6.59. Brenner S: The genetics of Caenorhabditis elegans. Genetics 1974,77(1):71-94.Mah et al. BMC Molecular Biology 2010, 11:19http://www.biomedcentral.com/1471-2199/11/19Page 12 of 1360. Hobert O: PCR fusion-based approach to create reporter gene constructsfor expression analysis in transgenic C. elegans. Biotechniques 2002,32(4):728-730.61. Thacker C, Sheps JA, Rose AM: Caenorhabditis elegans dpy-5 is a cuticleprocollagen processed by a proprotein convertase. Cell Mol Life Sci 2006,63(10):1193-1204.62. Stein LD, Mungall C, Shu S, Caudy M, Mangone M, Day A, Nickerson E,Stajich JE, Harris TW, Arva A, et al: The generic genome browser: abuilding block for a model organism system database. Genome Res 2002,12(10):1599-1610.doi:10.1186/1471-2199-11-19Cite this article as: Mah et al.: Characterization of the octamer, a cis-regulatory element that modulates excretory cell gene-expression inCaenorhabditis elegans. BMC Molecular Biology 2010 11:19.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitMah et al. BMC Molecular Biology 2010, 11:19http://www.biomedcentral.com/1471-2199/11/19Page 13 of 13


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