BACKGROUND
the caenorhabditis elegans excretory system is composed of four cells: the excretory duct cell, the bi-nucleate excretory gland cell, the excretory pore cell, and the excretory  cell .
each of these cells are descendents of the ab cell lineage  <cit> .
the ec in particular has a unique h-shaped structure consisting of two pairs of bilaterally-symmetrical projections that protrude anteriorly and posteriorly from the central cell body.
the ec forms approximately  <dig> minutes after the first cellular division near the centre of the embryo  <cit> .
subsequently, two processes extend dorso-laterally, which then bifurcate to form the anterior and posterior canal branches.
by the end of the first larval stage, the ec canals have reached their full length relative to the length of the nematode  <cit> .
further growth of the canals is influenced by their attachment to the hypodermis, which promotes extension of the ec canals following the first larval stage  <cit> .
not surprisingly, because of similarities in their structures, the ec and neurons share many developmental cues that dictate elongation, guidance and outgrowth  <cit> .
even though the ec shares developmental cues with neurons, it has a distinct function.
the ec plays a role in maintaining osmotic balance by collecting soluble organic and inorganic metabolic waste substances and expelling these to the environment  <cit> .
for c. elegans, osmoregulation is a critical function due to the continuous and unpredictable stresses placed upon worms in their native soil habitat.
to facilitate the exchange of dissolved material, the fluid-filled ec arms are exposed to pseudocoelomic fluid.
the pseudocoelomic fluid in turn is in contact with most of the cells in c. elegans, presumably having a function analogous with circulatory fluid.
the pseudocoelomic fluid also provides the turgor for the hydrostatic skeleton of c. elegans, which is essential for locomotion.
in addition to maintaining osmotic balance, the excretory system is responsible for secreting hormones  <cit>  and fluids required for molting  <cit> .
notably, the osmoregulatory function of the ec resembles the role of the mammalian kidneys.
thus, characterizing conserved mechanisms that govern ec transcription may provide insight into regulatory circuits that control kidney-specific transcription.
we are interested in the mechanisms that govern transcriptional regulation in the ec of c. elegans.
our laboratory has previously characterized two distinct transcriptional regulatory mechanisms affecting ec gene expression.
one of these mechanisms relies upon binding of the transcriptional regulator dcp- <dig> to the ex- <dig> cis-regulatory element .
together, dcp- <dig> and ex- <dig> drive ec-exclusive expression of pgp- <dig>  an abc transporter-encoding gene  <cit> .
however, dcp- <dig> is a component of the transcriptional inhibitory nucleosomal remodeling and deacetylase  complex, which is typically associated with gene repression  <cit> .
the second mechanism, involves ceh- <dig>  a class iii pou homeobox transcription factor , which binds to a cis-regulatory element called the octamer   <cit> .
we originally characterized the octamer as an element required for ec-expression of the aquaporin-encoding gene aqp- <dig>  of note, ceh- <dig> is also expressed in the excretory cell , thus fulfilling the spatial requirements of a tf responsible for ec-selective transcription.
additionally, ceh- <dig> is detected in the bilaterally symmetric neurons , p.na cells , five rectal cells   <cit> , two tail nerve cells, ventral hypoderm, anterior body wall muscle, body wall muscle cells, and intestine  <cit> .
the broad expression of ceh- <dig> indicates that it likely regulates transcription in a several ectodermal cells as well.
because ceh- <dig> interacts with the octamer to drive aqp- <dig> expression in the ec, we wanted to determine whether the octamer is generally linked to ec-expression.
if so, octamer regulated genes could represent novel 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 transcription.
specifically, we determined whether the octamer is under spatial restrictions within promoter regions, identified genes that require the octamer for ec expression, and identified variants of the octamer that are able to drive ec-expression.
thus, this work identified a set of candidate genes that could be relevant to kidney function in vertebrates.
overall, our data reinforce the role of octamers and presumably their cognate transcription factors such as ceh- <dig> in directing osmoregulatory organ gene expression.
RESULTS
the location of the octamer sequence in the promoter can be flexible
previously, we found evidence that the octamer is spatially restricted in the aqp- <dig> promoter  <cit> .
however, we also determined that the octamer still had the capacity to drive ec-specific expression when placed in close proximity to the atg using the Δpes- <dig> minimal promoter  <cit>  .
thus, we hypothesized that the octamer might have different spatial restrictions in the promoter regions of different genes.
to assess spatial dependence of the octamer within the promoter region, we used the vit- <dig> promoter as a tool.
vit- <dig> encodes a yolk protein that is expressed at high levels in the intestine  <cit> , but is not normally expressed in the ec.
a promoter encompassing  <dig> bps directly upstream of the vit- <dig> atg is sufficient for driving intestinal expression of vit- <dig>  <cit> .
therefore, we used the intestinal expression to assay for promoter function.
we generated a series of promoter constructs by appending tandem octamer sequences to the 5' end of vit- <dig> promoter truncation constructs  .
these constructs were fused to gfp and then injected into wild-type worms to generate transgenic nematodes.
subsequently, we monitored for gfp expression in the intestine and in the ec.
we observed that the octamer was not able to drive ec expression in constructs that contained less than  <dig> bp upstream of the vit- <dig> atg.
however, these constructs retained the ability to drive intestinal expression indicating that the transgene was successfully generated and functional .
placing the octamer at the 5'-end of vit- <dig> promoter constructs larger than  <dig> bp upstream of the atg led to ectopic gfp expression in the ec .
an exception to this was from a transgene containing octamers  <dig> bp upstream of vit-2's atg, which did not drive assayable levels of gfp.
this transgenic strain drove intestinal gfp, but failed to drive gfp in the ec.
the lack of ec-expression indicates that there may be a cis-linked element located between  <dig> bp and  <dig> bp upstream of the vit- <dig> atg that represses ec expression.
alternatively, it is possible that this transgene construct formed a concatemer in vivo that is incompatible with the expression of the gfp reporter.
this second scenario is unlikely because we did observe intestinal expression.
in any case, these data suggest that octamer function may be spatially restricted in some promoters.
taken together, although the above data did not allow us to conclude whether the octamer has an optimal distance from the atg for influencing ec-expression, the data do suggest that functional octamers can be present at various places in different promoters.
many ec-expressed genes are not regulated by octamers
because functional octamers may be located at various distances upstream of the atg, we searched for genes with upstream octamers within  <dig>  bp upstream of their atgs; wormbase .
thus, we identified  <dig>  candidate genes including promoters that contain either the forward and/or the inverse of the octamer .
to assess the function of these octamers, we selected genes from this set that are expressed in the ec  <cit> .
in addition, we assessed the function of the octamer in the promoters of hlh- <dig> and zc <dig> , genes with no known ec-expression.
for these seventeen genes, we tested if regions containing the octamer are required for ec-expression by truncating the promoter from the 5'-end and observing whether the octamer regions affect the level of ec expression .
however, we could not conclude whether the octamers were functional in promoters of y69e1a.
<dig>  f36h <dig> , y48a6b.
<dig>  f29f <dig> , b <dig> , c02b <dig> , h23n <dig> , r13f <dig> , and, y53g8ar.
<dig> because ec expression was lost in worms carrying promoter constructs that still had the octamer, suggesting that there are other cis-linked elements that drive ec-expression of these genes .
amongst the other eight genes, our promoter truncation analysis revealed three genes that are likely dependent upon the octamer containing region for ec expression: zc <dig> , c01b <dig> , and hlh-8/c02b <dig> .
these results are not surprising as previous reports indicate that 12% of ec-expressed genes are predicted to be regulated by the dcp-66/ex- <dig> mechanism  <cit>  additionally, the nuclear hormone receptor nhr- <dig> regulates most of the vacuolar atpase  components in the ec  <cit>  pointing to multiple regulatory mechanisms that specify ec-expression.
the octamer in zc <dig> 's promoter region is located  <dig> bp upstream of the atg.
zc <dig>  is localized to most neurons, intestine, pharynx, and vulva  <cit> .
however, a zc <dig>  promoter construct containing the octamer  drove expression in a different pattern than the original longer promoter.
in this shorter promoter construct, we observed gfp in the ec, anterior neurons, intestine, and rectal epithelia .
the differences between this and the construct with the larger promoter indicate that there is a cis-linked repressor element that modulates ec and rectal epithelial tissue expression in zc <dig> 's promoter.
interestingly, the ec and rectal epithelia expression pattern resulting from the shorter construct overlaps with ceh-6's expression pattern  <cit> .
furthermore, deleting the octamer containing region leads to loss of expression in both the ec and rectal epithelia.
additionally, there must be an independent transcriptional regulatory mechanism that drives zc <dig>  expression in anterior neurons and the intestine .
an octamer is located  <dig>  bp upstream of the atg in c01b <dig> 's promoter region.
a transcriptional reporter construct with a 5'-end  <dig>  bp upstream of the atg leads to expression in the ec , hypoderm, spermatheca, and the anal depressor muscle  <cit> .
however, the ec-expression level is greatly decreased when the promoter is truncated  corresponding to removal of a large portion of the promoter and including an octamer .
interestingly, the orthologs of c01b <dig>  in c. briggsae and c. remanei also have octamers in their promoters.
moreover, the distance of the octamers from the atg are similar , suggesting that this element might also regulate ec-expression of c01b <dig>  orthologs.
the hlh- <dig> promoter contains an octamer located  <dig> bp upstream of the atg.
it was previously shown that hlh- <dig> is expressed in the intestine, anterior neurons, and vulva  <cit> .
a 5' truncation that limits the promoter to only seven bases upstream of the octamer  results in expression localized to the ec and the second pharyngeal bulb .
this change in expression pattern indicates the likely presence of a repressor element that blocks ec and pharyngeal expression.
expression of hlh- <dig> in the ec is completely abolished upon deletion of the octamer-containing fragment.
in sum, our promoter deletion analysis identified octamer-containing promoter regions required for the ec-specific expression for the above three genes.
conserved octamer sequences in promoter regions strongly bias for ec expression
due to the low success rate in finding octamer containing regions associated with ec-expression with the above approach , we turned to phylogenetic footprinting.
using this comparative method, we identified promoters with perfectly conserved octamers between three closely related caenorhabditis species .
this resulted in the identification of  <dig> promoters that contain conserved octamers .
of the  <dig> genes, nineteen genes had previously characterized expression patterns  <cit> .
to obtain a larger sample size, we analyzed the expression patterns of an additional  <dig> candidates .
from these  <dig> promoters, we identified  <dig> that could drive detectable levels of gfp expression, including  <dig>  that were ec-expressed.
this represents a significant enrichment of genes expressed in the ec when compared to a control dataset of  <dig>  expression patterns, within which only  <dig> %  of all genes are expressed in the ec  <cit> .
strikingly, twelve genes  in our set were expressed only in the ec.
this is a vast enrichment over the control set which contains only  <dig> %  genes with ec-exclusive expression  <cit>  .
next we wanted to determine whether their ec-expression was dependent on the octamer containing fragments.
using the same approach as in the previous section , we selected  <dig> promoters that drove ec expression for further analysis .
from this set, we identified four genes that are completely dependent on fragments containing the octamer for ec expression , c05d <dig> ) and two genes that exhibit reduced ec expression upon deletion of the octamer region   .
sequences flanking functional octamers are likely conserved
in the experiments described above, we identified genes that may depend on the octamer for expression in the ec.
thus, we used these promoters to study whether the sequences flanking the octamer are conserved.
we aligned the nine octamer sequences along with  <dig> bp of upstream and downstream flanking sequences.
the resulting alignments were displayed using weblogo  <cit>  .
this analysis revealed that, in general, octamers are flanked by at-rich regions.
more specifically, regions upstream of the octamer are biased towards being a/c-rich and downstream regions tend to be t-rich.
thus, our newly identified collection of ec-expressed genes has allowed us to define additional specificity determinants related to the octamer.
intra-octamer nucleotide substitutions have different effects on ec expression
previously, we observed that the octamer is perfectly conserved within the promoters of aqp- <dig> orthologs among five caenorhabditis species  <cit> .
therefore, we hypothesized that the octamer sequence must be absolutely conserved to drive ec-specific expression.
to examine the octamer in more detail, we manipulated its sequence using site-directed mutagenesis.
we generated variants of the octamer by targeting nucleotides - <dig> bp and - <dig> bp of the octamer in the aqp- <dig> promoter .
every possible single-nucleotide substitution and a dual nucleotide substitution were tested at these sites.
the mutated constructs were fused to gfp to visualize changes in ec-expression.
the 5'-end of every construct was defined as  <dig> bp upstream of the aqp-8's atg because truncation constructs ranging between  <dig>  kb to  <dig> bp upstream of aqp-8's atg provide consistent ec-specific expression patterns  <cit> .
also, because the aqp- <dig> promoter produces consistent levels of ec expression, our reference transgene construct is a  <dig>  kb aqp- <dig> promoter region fused to gfp   <cit> .
at - <dig> bp, a g→a residue change ::gfp) did not alter the expression level or pattern .
however, a g→t change ::gfp led to a significant decrease in the ec expression level .
a g→c substitution ::gfp) led to a complete loss of ec-expression.
at - <dig> bp, a c→a residue substitution in aqp-8promoter::gfp led to decreased ec-expression .
the c→g and c→t substitutions ::gfp and aqp-8promoter::gfp) both led to a complete losses of gfp expression.
finally, replacing the gc pair at - <dig> with an ag pair ::gfp) led to decreased ec-expression .
in all constructs, the gfp signal remained localized to the ec suggesting that some octamer sequence variants have the capacity to influence ec expression.
discussion
in this study, we demonstrate that octamer containing regions are involved in driving ec-expression of several genes.
this builds upon our previous data demonstrating that aqp- <dig> is dependent upon the pou homeobox tf ceh- <dig> and the octamer for ec-expression  <cit> .
in several nematodes, the position of the octamer relative to the atg is fairly well conserved among aqp- <dig> orthologs  <cit> .
this is interesting as some cis-regulatory elements are spatially restricted within promoters.
for example, in c. elegans functional x-box motifs cluster roughly  <dig> bp upstream of atgs to drive neuronal expression  <cit> .
likewise, the gc-box cis-regulatory element has an optimal distance in relation to the tata box.
moving the gc-box away from its optimal distance leads to decreased expression levels of nearby genes even though its cognate tf, sp <dig>  still bind with similar affinities  <cit> .
in addition to spatial restriction relative to atgs, relative positioning between cis-regulatory elements within the same promoter can affect expression.
for instance, the β-actin promoter contains the so-called ccaat and ccargg boxes.
these regulatory elements are binding sites for the tfs nuclear factor y  and serum response factor , respectively.
manipulating the intra-element distance between these two cis-regulatory sequences accordingly affects β-actin message levels  <cit> .
however, in the present study we had also observed that the octamer could be placed at different positions in a heterologous promoter and still drive ec-specific expression.
therefore, unlike the above examples, the octamer does not appear to be as spatially restricted within promoter although there still might be some tight limitations to octamer location.
in this study, we used different strategies to identify novel genes that require upstream octamer containing regions for expression in the ec.
first, we identified genes with octamer sequences in their promoters.
using this strategy we identified a small number of genes that are modulated by upstream octamer-containing fragments.
secondly, we used a more stringent approach to identify octamers by relying on interspecies conservation.
we discovered that the expression patterns of genes in this filtered promoter set had a higher than expected incidence of ec-expression.
in these two screens, we identified nine genes that are likely octamer-modulated.
several of these genes likely have osmoregulatory functions, agreeing with the notion the ec is analogous to the kidney.
in general, the genes we identified fell into four categories: transmembrane channels/pores, hsp <dig> co-chaperones, proteins with unknown functions, and tfs.
the largest group of genes encodes transmembrane channels/pores, indicating that many genes regulated by the octamer participate in substrate transport.
the five channels/pores are:
1) twk- <dig> encodes a c. elegans twik potassium ion channel protein.
in vertebrates, twiks are commonly expressed in neuronal tissues and, to a lesser extent in lungs, skeletal muscle  <cit> , and tubular portions of the kidney   <cit> .
the mammalian twik, task, is sensitive to changes in extracellular ph, indicating that some of these proteins have roles in modulating cellular responses to ph flux  <cit> .
also, because their conductance is osmotically regulated, twiks can influence cellular volume  <cit> .
the c. elegans genome has  <dig> twk genes.
as in other organisms, most of the c. elegans twks are expressed in neurons  <cit> ; however, twk- <dig> is the only twk expressed in the ec  <cit> .
interestingly, another group has demonstrated that twk- <dig> is directly regulated by ceh- <dig>  <cit> .
this independent study strengthens the notion ceh- <dig> is a bona fide regulator of ec expression that likely acts through the octamer in the promoter of twk- <dig>  therefore, it is possible that ceh- <dig> regulation impacts at least some of the octamer-dependent candidates identified here.
2) aqp- <dig>  an aquaporin whose function and regulation have been characterized previously  <cit> .
3) c05d <dig>  is a homolog of the cytochrome b561/ferric reductase sdr- <dig>  in mammals, sdr- <dig> is expressed in the brain  <cit>  and kidney where it aids in iron reabsorption via the accessory transporter, divalent-cation transporter  <dig>   <cit> .
cytochrome b <dig> proteins transport electrons in an ascorbate-dependent manner.
due to the role of sdr- <dig> in ascorbate regeneration, c05d <dig>  could be involved in vitamin c homeostasis and/or oxidative stress responses.
4) r02f <dig>  encodes a solute carrier  protein that is most similar to the mammalian slc <dig> subfamily.
slc <dig> proteins are localized to intracellular and plasma membranes  <cit>  where they function as symporters.
slc <dig> proteins transport small neutral amino acids such as glycine, alanine, and proline.
because slc <dig> proteins affect proton flux, they also contribute to intracellular ph homeostasis  <cit> .
in mammals there are four slc <dig> genes, two of which are expressed in the kidney   <cit> .
5) c01b <dig>  encodes a c. elegans bestrophin  <dig> homolog.
bestrophins are transmembrane proteins that modulate calcium dependent transport of chloride ions across cellular membranes.
bestrophins are enriched in the plasma membranes of epithelial cells where they manage cellular volume  <cit> .
bestrophins are also expressed in exocrine gland tissues , lung, testis and kidney  <cit> .
in these tissues they facilitate trans-epithelial movement of chloride ions leading to water and electrolyte movement  <cit> .
in addition to transmembrane channels and pores, we uncovered several other genes that have less obvious links to osmoregulation and kidney biology.
one of these, zc <dig> , is homologous to thehighly conserved hsp <dig> co-chaperone protein, p <dig>  p <dig> interactions with hsp <dig> to ensure the proper folding and maturation of many proteins including steroid receptors  <cit> , telomerase  <cit> , and proteins that are upregulated in cancers  <cit> .
we also identified m <dig> , a gene with little prior functional data.
m <dig>  is a nematode-specific gene that is mainly composed of hydrophobic amino acids and is therefore likely to be localized to cell membranes and/or forms a globular protein.
finally, we identified two tfs.
the first, f16f <dig>  is a homolog of the mammalian protein lipopolysaccharide-induced tumor necrosis factor-alpha factor   <cit> .
litaf is linked to charcot-marie-tooth  disease, a heritable neuropathy characterized by loss of muscle tissue and touch sensation  <cit> .
in cmt1c, litaf is implicated in protein degradation  <cit> .
litaf also functions in cytokine production  <cit> .
we speculate that because f16f <dig>  is expressed in the ec, a tissue exposed to the environment, it could be involved in innate immune responses.
also, because c. elegans f16f <dig>  is expressed in neurons, it is possible that the nematode could act as a model for cmt1c.
the other tf we identified is hlh- <dig>  a helix-loop-helix tf related to human twist.
twist was originally characterized in drosophila as a gene involved in dorsal-ventral patterning  <cit> .
twist tfs bind e-box cis-regulatory elements.
in c. elegans, hlh- <dig> is important for regulating muscle, intestinal and anal muscle development.
consequently hlh- <dig> mutants exhibit defecation and egg-laying defects  <cit> .
several transcriptional targets of hlh- <dig> are known, including cdh- <dig>  egl- <dig>  c18b <dig> , f08d <dig> , rbc- <dig>  npr- <dig>  dhs- <dig>  sgcb- <dig>  erv- <dig>  m <dig> , r02e <dig> , rev- <dig>  and myo- <dig>  <cit> .
most of these genes are unlikely to be transcriptional targets of hlh- <dig> in the ec as they are not expressed in this cell.
an exception is cdh- <dig>  <cit> , which encodes a widely expressed cadherin, which is also expressed in the ec.
due to the limited number of hlh- <dig> targets in the ec, we can envisage a model where ceh- <dig> plays a role in directing the precise transcriptional outcomes of downstream tfs .
in this role, ceh- <dig> could modulate target genes  specifically in a subset of ectodermal tissues including the ec.
several of the genes that depend on their upstream octamer containing fragments for ec expression are also expressed in additional tissues.
an interesting consequence of assaying the activity of truncated promoters is that loss of the octamer containing fragment sometimes led to loss of expression in multiple tissues including neurons as indicated in the cases of zc <dig> , twk- <dig>  m <dig> , and f16f <dig>  .
this is not surprising as vertebrate orthologs of ceh- <dig> including brn <dig> are involved in neuronal and kidney development  <cit> .
our strategy for studying the above genes involved comparing the expression patterns resulting from promoter truncations that either contain or remove the octamer.
analyzing promoter function by means of these truncations imposes some significant drawbacks; for example, we could not address the function of several candidate octamer elements because removing regions upstream of the octamer led to loss of ec expression.
also, because our promoter truncations deleted the octamer and some flanking regions, we cannot be certain that loss of ec-selective expression is the consequence of removing the octamer.
however, because the 5' ends of the truncations were in general fairly close to the octamer, and because loss of ec-expression correlated with octamer deletion, we believe that these genes are likely dependent on octamers for their expression in the ec.
to address the issue of whether these are indeed functional octamers, one could introduce point mutations within the octamer and assess the resulting consequences on ec-expression.
however, we demonstrated in our mutagenesis experiments that certain point mutations are not sufficient to abolish ec expression in the context of the aqp- <dig> promoter.
another potential drawback from our approach is the fact that much of our study is based on expression patterns arising from transgenic c. elegans strains containing extrachromosomal arrays.
such arrays are susceptible to somatic transgene loss, which results in mosaic reporter expression.
this mosaiscism has the potential to confound our analysis by under-representing the expression pattern.
however, expression patterns resulting from genome-integrated transgenes  are identical to the expression patterns in aqp-8promoter::gfp strains carrying extrachromosomal transgenes.
therefore, mosaic loss of the extrachromosomal transgene array is not likely an issue for analyzing changes in ec expression.
despite the potential shortcomings detailed above, our study revealed a set of genes whose expression likely depends on the octamer for expression in the ec; these genes are excellent ceh- <dig> candidate targets.
in agreement with this notion, twk- <dig>  one of the genes that we identified as dependent on an upstream octamer fragement, has been demonstrated, independently, to be regulated by ceh- <dig>  <cit> .
because our bioinformatic search did not bias the direction of the octamer, we discovered promoters in the forward and inverted orientation can be associated with ec-expression.
octamers in a forward orientation occur in the promoters of aqp- <dig>  m <dig> , twk- <dig>  zc <dig> , hlh- <dig>  c01b <dig> , and c05d <dig> , whereas inverted octamers are present in the promoters of f16f <dig>  and r02f <dig> .
although we could not determine from our small sample set whether the direction of the octamer has a functional consequence, there are possible implications related to direction of the octamer sequence.
for example, octamers upstream of immunoglobulin light and heavy chain genes have directional preferences   <cit> .
in addition, the human pou homeobox gene oct <dig> is auto-regulated by two upstream octamers, which are also situated in inverted orientations.
although each site binds oct <dig> with equal affinity, each of these sites has different effects on oct <dig> expression  <cit> .
it appears that transcriptional auto-regulation is a common mode of regulation among pou tfs  <cit> .
interestingly, we detected an octamer upstream of ceh-6's atg in c. elegans.
likewise, there is an octamer located in the regulatory region of the c. briggsae gene encoding a putative ceh- <dig> ortholog, providing evidence that auto-regulation might be conserved.
however, these octamer are located within a predicted non-coding rna gene   <cit> .
nevertheless, it would be interesting to determine whether ceh- <dig>  like other pou tfs is auto-regulated.
with our set of nine candidate octamers, we had the opportunity to determine whether residues flanking the element are conserved.
globally, the g/c content of c. elegans is 31%  <cit> .
however, the regions flanking the octamers-associated with ec-expression contain a slightly higher g/c content .
our alignments of these octamers revealed that despite the higher g/c content, some positions have preferences for a/t residues.
strikingly, directly 3' to the octamer, an a or t is always present.
the conservation of this residue is consistent with the results of a previous study, which identified oct <dig> binding preferences using a systematic evolution of ligands by exponential environment -based in vitro binding approach  <cit> .
because of the enriched g/c content in octamer adjacent regions, it is likely that the observed preference for a/t at certain positions have relevance.
we tested the effects of targeted-octamer mutations on ec-expression.
we found that several single-base substitutions did not affect the cis-regulatory element's ability to drive expression in the ec.
interestingly, we did not observe a change in expression level or pattern when position five of the octamer was mutated from a purine to purine .
this variant of the octamer was demonstrated to be a binding site for the catfish class iii pou tf, oct <dig>  <cit> .
therefore, this residue change results in an octamer variant which retains the ability to interact with the pous sub-domain binding consensus sequence atattc)  <cit> .
at the same residue position, a thymine replacement , led to weak gfp expression also restricted to the ec.
this sequence was able to bind to oct <dig> in vitro in an electrophoretic mobility shift assay   <cit> .
however, in another study this variant of the octamer was not able to drive reporter expression in human cells  <cit> .
these results, taken together, indicate that this motif variant is a sub-optimal pou tf binding site that can drive weak ec expression in c. elegans.
a mutation at the sixth residue  also led to weak ec-localized expression  <cit> .
this octamer variant is functional in the promoter of the drosophila gene, choline acetyltransferase .
in the chat promoter, atttgaat interacts with the pou homeobox tf, dpou- <dig>  <cit> .
all other single nucleotide substitutions at these two locations led to loss of gfp expression.
however, a double residue replacement of these residues  could still drive expression, as indicated by weak gfp expression in the ec.
there have been no previous reports of this dual nucleotide substitution variant associating with pou tfs and it is therefore a novel pou tf binding site variant.
overall, our mutagenesis assays indicate that the octamer cis-regulatory element could have a range of functional variants in c. elegans.
thus, identifying and characterizing promoters containing these octamer variants may reveal a larger group genes expressed in the ec.
because variants of the octamer can influence ec-expression, we examined the pgp- <dig> promoter region in c. elegans more closely.
previously, it was demonstrated that pgp- <dig> expression is regulated by dcp-66/ex- <dig>  <cit> .
in this report, the tf/cis-regulatory element interaction was confirmed using in vitro and genetic approaches.
loss of either ex- <dig>  or dcp- <dig> resulted in loss of ec expression  <cit> .
we detected an octamer like sequence  that partially overlaps the ex- <dig> .
we also identified this octamer-like sequence in the orthologous regions of c. briggsae.
using the transcriptional element search system   <cit>  to identify predicted cis-regulatory elements, we found that the octamer-like sequence is indeed a potential target for octamer binding proteins.
additionally this sequence binds oct <dig> in vitro  <cit> .
in fact, zhao et al.
reported that promoter constructs encompassing ex- <dig> at - <dig> bp results in strong reporter expression during all developmental stages  <cit> .
they also studied the expression pattern resulting from a promoter region defined by a 5'-end  <dig> bp upstream of the atg, thereby removing three nucleotides from the octamer-like sequence.
although this promoter still drove ec-expression, the intensity of the gfp reporter was greatly decreased in adult and larval worms.
additionally, embryonic expression was almost eliminated.
finally, a construct with a 5'-end  <dig> bp upstream of the atg was not able to drive expression of gfp indicating the necessity of the ex- <dig>  for ec expression.
therefore, not only did this prior study define the role of the ex- <dig> for ec-expression, but it also indirectly provided evidence that the octamer upstream of pgp- <dig> might affect ec-expression.
this suggests that the dcp-66/ex- <dig> and the octamer-directed transcriptional regulatory mechanisms co-operatively modulate the expression of pgp- <dig> in the ec.
this model of concerted and redundant regulation of ec-expression may have relevance in other genes including, possibly, several genes within our phylogenetically defined candidate set.
CONCLUSIONS
overall, we determined that the octamer is likely responsible for the expression of several genes within the ec, an osmoregulatory organ analogous to the kidney.
because one of our candidate genes, twk- <dig>  has been demonstrated to be a bona fide target of ceh- <dig> regulation, it would be interesting to determine whether ceh- <dig> is involved in the regulation of four other candidates.
although our candidates, for the most part, were chosen based upon perfect conservation of the octamer, we determined that several variants of the octamer can drive ec-expression.
the existence of functional octamer variants indicates that future searches for octamer-driven genes should use a loosely defined octamer sequence.
overall, understanding conserved mechanisms of gene regulation that determine appropriate ec expression may provide insight into underlying transcriptional mechanisms that regulate transcription in analogous organs including the kidney.

 the caenorhabditis elegans excretory system is composed of four cells: the excretory duct cell, the bi-nucleate excretory gland cell, the excretory pore cell, and the excretory cell. each of these cells are descendents of the ab cell lineage. the ec in particular has a unique h-shaped structure consisting of two pairs of bilaterally-symmetrical projections that protrude anteriorly and posteriorly from the central cell body. the ec forms approximately minutes after the first cellular division near the centre of the embryo. subsequently, two processes extend dorso-laterally, which then bifurcate to form the anterior and posterior canal branches. by the end of the first larval stage, the ec canals have reached their full length relative to the length of the nematode. further growth of the canals is influenced by their attachment to the hypodermis, which promotes extension of the ec canals following the first larval stage. not surprisingly, because of similarities in their structures, the ec and neurons share many developmental cues that dictate elongation, guidance and outgrowth. even though the ec shares developmental cues with neurons, it has a distinct function. the ec plays a role in maintaining osmotic balance by collecting soluble organic and inorganic metabolic waste substances and expelling these to the environment. for c. elegans, osmoregulation is a critical function due to the continuous and unpredictable stresses placed upon worms in their native soil habitat. to facilitate the exchange of dissolved material, the fluid-filled ec arms are exposed to pseudocoelomic fluid. the pseudocoelomic fluid in turn is in contact with most of the cells in c. elegans, presumably having a function analogous with circulatory fluid. the pseudocoelomic fluid also provides the turgor for the hydrostatic skeleton of c. elegans, which is essential for locomotion. in addition to maintaining osmotic balance, the excretory system is responsible for secreting hormones and fluids required for molting. notably, the osmoregulatory function of the ec resembles the role of the mammalian kidneys. thus, characterizing conserved mechanisms that govern ec transcription may provide insight into regulatory circuits that control kidney-specific transcription. we are interested in the mechanisms that govern transcriptional regulation in the ec of c. elegans. our laboratory has previously characterized two distinct transcriptional regulatory mechanisms affecting ec gene expression. one of these mechanisms relies upon binding of the transcriptional regulator dcp- to the ex- cis-regulatory element. together, dcp- and ex- drive ec-exclusive expression of pgp- an abc transporter-encoding gene. however, dcp- is a component of the transcriptional inhibitory nucleosomal remodeling and deacetylase complex, which is typically associated with gene repression. the second mechanism, involves ceh- a class iii pou homeobox transcription factor, which binds to a cis-regulatory element called the octamer. we originally characterized the octamer as an element required for ec-expression of the aquaporin-encoding gene aqp- of note, ceh- is also expressed in the excretory cell, thus fulfilling the spatial requirements of a tf responsible for ec-selective transcription. additionally, ceh- is detected in the bilaterally symmetric neurons, p.na cells, five rectal cells, two tail nerve cells, ventral hypoderm, anterior body wall muscle, body wall muscle cells, and intestine. the broad expression of ceh- indicates that it likely regulates transcription in a several ectodermal cells as well. because ceh- interacts with the octamer to drive aqp- expression in the ec, we wanted to determine whether the octamer is generally linked to ec-expression. if so, octamer regulated genes could represent novel 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 transcription. specifically, we determined whether the octamer is under spatial restrictions within promoter regions, identified genes that require the octamer for ec expression, and identified variants of the octamer that are able to drive ec-expression. thus, this work identified a set of candidate genes that could be relevant to kidney function in vertebrates. overall, our data reinforce the role of octamers and presumably their cognate transcription factors such as ceh- in directing osmoregulatory organ gene expression.  the location of the octamer sequence in the promoter can be flexible previously, we found evidence that the octamer is spatially restricted in the aqp- promoter. however, we also determined that the octamer still had the capacity to drive ec-specific expression when placed in close proximity to the atg using the Δpes- minimal promoter. thus, we hypothesized that the octamer might have different spatial restrictions in the promoter regions of different genes. to assess spatial dependence of the octamer within the promoter region, we used the vit- promoter as a tool. vit- encodes a yolk protein that is expressed at high levels in the intestine, but is not normally expressed in the ec. a promoter encompassing bps directly upstream of the vit- atg is sufficient for driving intestinal expression of vit-. therefore, we used the intestinal expression to assay for promoter function. we generated a series of promoter constructs by appending tandem octamer sequences to the 5' end of vit- promoter truncation constructs. these constructs were fused to gfp and then injected into wild-type worms to generate transgenic nematodes. subsequently, we monitored for gfp expression in the intestine and in the ec. we observed that the octamer was not able to drive ec expression in constructs that contained less than bp upstream of the vit- atg. however, these constructs retained the ability to drive intestinal expression indicating that the transgene was successfully generated and functional. placing the octamer at the 5'-end of vit- promoter constructs larger than bp upstream of the atg led to ectopic gfp expression in the ec. an exception to this was from a transgene containing octamers bp upstream of vit-2's atg, which did not drive assayable levels of gfp. this transgenic strain drove intestinal gfp, but failed to drive gfp in the ec. the lack of ec-expression indicates that there may be a cis-linked element located between bp and bp upstream of the vit- atg that represses ec expression. alternatively, it is possible that this transgene construct formed a concatemer in vivo that is incompatible with the expression of the gfp reporter. this second scenario is unlikely because we did observe intestinal expression. in any case, these data suggest that octamer function may be spatially restricted in some promoters. taken together, although the above data did not allow us to conclude whether the octamer has an optimal distance from the atg for influencing ec-expression, the data do suggest that functional octamers can be present at various places in different promoters. many ec-expressed genes are not regulated by octamers because functional octamers may be located at various distances upstream of the atg, we searched for genes with upstream octamers within bp upstream of their atgs; wormbase. thus, we identified candidate genes including promoters that contain either the forward and/or the inverse of the octamer. to assess the function of these octamers, we selected genes from this set that are expressed in the ec. in addition, we assessed the function of the octamer in the promoters of hlh- and zc, genes with no known ec-expression. for these seventeen genes, we tested if regions containing the octamer are required for ec-expression by truncating the promoter from the 5'-end and observing whether the octamer regions affect the level of ec expression. however, we could not conclude whether the octamers were functional in promoters of y69e1a. f36h, y48a6b. f29f, b, c02b, h23n, r13f, and, y53g8ar. because ec expression was lost in worms carrying promoter constructs that still had the octamer, suggesting that there are other cis-linked elements that drive ec-expression of these genes. amongst the other eight genes, our promoter truncation analysis revealed three genes that are likely dependent upon the octamer containing region for ec expression: zc, c01b, and hlh-8/c02b. these results are not surprising as previous reports indicate that 12% of ec-expressed genes are predicted to be regulated by the dcp-66/ex- mechanism additionally, the nuclear hormone receptor nhr- regulates most of the vacuolar atpase components in the ec pointing to multiple regulatory mechanisms that specify ec-expression. the octamer in zc's promoter region is located bp upstream of the atg. zc is localized to most neurons, intestine, pharynx, and vulva. however, a zc promoter construct containing the octamer drove expression in a different pattern than the original longer promoter. in this shorter promoter construct, we observed gfp in the ec, anterior neurons, intestine, and rectal epithelia. the differences between this and the construct with the larger promoter indicate that there is a cis-linked repressor element that modulates ec and rectal epithelial tissue expression in zc's promoter. interestingly, the ec and rectal epithelia expression pattern resulting from the shorter construct overlaps with ceh-6's expression pattern. furthermore, deleting the octamer containing region leads to loss of expression in both the ec and rectal epithelia. additionally, there must be an independent transcriptional regulatory mechanism that drives zc expression in anterior neurons and the intestine. an octamer is located bp upstream of the atg in c01b's promoter region. a transcriptional reporter construct with a 5'-end bp upstream of the atg leads to expression in the ec, hypoderm, spermatheca, and the anal depressor muscle. however, the ec-expression level is greatly decreased when the promoter is truncated corresponding to removal of a large portion of the promoter and including an octamer. interestingly, the orthologs of c01b in c. briggsae and c. remanei also have octamers in their promoters. moreover, the distance of the octamers from the atg are similar, suggesting that this element might also regulate ec-expression of c01b orthologs. the hlh- promoter contains an octamer located bp upstream of the atg. it was previously shown that hlh- is expressed in the intestine, anterior neurons, and vulva. a 5' truncation that limits the promoter to only seven bases upstream of the octamer results in expression localized to the ec and the second pharyngeal bulb. this change in expression pattern indicates the likely presence of a repressor element that blocks ec and pharyngeal expression. expression of hlh- in the ec is completely abolished upon deletion of the octamer-containing fragment. in sum, our promoter deletion analysis identified octamer-containing promoter regions required for the ec-specific expression for the above three genes. conserved octamer sequences in promoter regions strongly bias for ec expression due to the low success rate in finding octamer containing regions associated with ec-expression with the above approach, we turned to phylogenetic footprinting. using this comparative method, we identified promoters with perfectly conserved octamers between three closely related caenorhabditis species. this resulted in the identification of promoters that contain conserved octamers. of the genes, nineteen genes had previously characterized expression patterns. to obtain a larger sample size, we analyzed the expression patterns of an additional candidates. from these promoters, we identified that could drive detectable levels of gfp expression, including that were ec-expressed. this represents a significant enrichment of genes expressed in the ec when compared to a control dataset of expression patterns, within which only % of all genes are expressed in the ec. strikingly, twelve genes in our set were expressed only in the ec. this is a vast enrichment over the control set which contains only % genes with ec-exclusive expression. next we wanted to determine whether their ec-expression was dependent on the octamer containing fragments. using the same approach as in the previous section, we selected promoters that drove ec expression for further analysis. from this set, we identified four genes that are completely dependent on fragments containing the octamer for ec expression, c05d ) and two genes that exhibit reduced ec expression upon deletion of the octamer region. sequences flanking functional octamers are likely conserved in the experiments described above, we identified genes that may depend on the octamer for expression in the ec. thus, we used these promoters to study whether the sequences flanking the octamer are conserved. we aligned the nine octamer sequences along with bp of upstream and downstream flanking sequences. the resulting alignments were displayed using weblogo. this analysis revealed that, in general, octamers are flanked by at-rich regions. more specifically, regions upstream of the octamer are biased towards being a/c-rich and downstream regions tend to be t-rich. thus, our newly identified collection of ec-expressed genes has allowed us to define additional specificity determinants related to the octamer. intra-octamer nucleotide substitutions have different effects on ec expression previously, we observed that the octamer is perfectly conserved within the promoters of aqp- orthologs among five caenorhabditis species. therefore, we hypothesized that the octamer sequence must be absolutely conserved to drive ec-specific expression. to examine the octamer in more detail, we manipulated its sequence using site-directed mutagenesis. we generated variants of the octamer by targeting nucleotides - bp and - bp of the octamer in the aqp- promoter. every possible single-nucleotide substitution and a dual nucleotide substitution were tested at these sites. the mutated constructs were fused to gfp to visualize changes in ec-expression. the 5'-end of every construct was defined as bp upstream of the aqp-8's atg because truncation constructs ranging between kb to bp upstream of aqp-8's atg provide consistent ec-specific expression patterns. also, because the aqp- promoter produces consistent levels of ec expression, our reference transgene construct is a kb aqp- promoter region fused to gfp. at - bp, a g→a residue change::gfp) did not alter the expression level or pattern. however, a g→t change::gfp led to a significant decrease in the ec expression level. a g→c substitution::gfp) led to a complete loss of ec-expression. at - bp, a c→a residue substitution in aqp-8promoter::gfp led to decreased ec-expression. the c→g and c→t substitutions::gfp and aqp-8promoter::gfp) both led to a complete losses of gfp expression. finally, replacing the gc pair at - with an ag pair::gfp) led to decreased ec-expression. in all constructs, the gfp signal remained localized to the ec suggesting that some octamer sequence variants have the capacity to influence ec expression. discussion in this study, we demonstrate that octamer containing regions are involved in driving ec-expression of several genes. this builds upon our previous data demonstrating that aqp- is dependent upon the pou homeobox tf ceh- and the octamer for ec-expression. in several nematodes, the position of the octamer relative to the atg is fairly well conserved among aqp- orthologs. this is interesting as some cis-regulatory elements are spatially restricted within promoters. for example, in c. elegans functional x-box motifs cluster roughly bp upstream of atgs to drive neuronal expression. likewise, the gc-box cis-regulatory element has an optimal distance in relation to the tata box. moving the gc-box away from its optimal distance leads to decreased expression levels of nearby genes even though its cognate tf, sp still bind with similar affinities. in addition to spatial restriction relative to atgs, relative positioning between cis-regulatory elements within the same promoter can affect expression. for instance, the β-actin promoter contains the so-called ccaat and ccargg boxes. these regulatory elements are binding sites for the tfs nuclear factor y and serum response factor, respectively. manipulating the intra-element distance between these two cis-regulatory sequences accordingly affects β-actin message levels. however, in the present study we had also observed that the octamer could be placed at different positions in a heterologous promoter and still drive ec-specific expression. therefore, unlike the above examples, the octamer does not appear to be as spatially restricted within promoter although there still might be some tight limitations to octamer location. in this study, we used different strategies to identify novel genes that require upstream octamer containing regions for expression in the ec. first, we identified genes with octamer sequences in their promoters. using this strategy we identified a small number of genes that are modulated by upstream octamer-containing fragments. secondly, we used a more stringent approach to identify octamers by relying on interspecies conservation. we discovered that the expression patterns of genes in this filtered promoter set had a higher than expected incidence of ec-expression. in these two screens, we identified nine genes that are likely octamer-modulated. several of these genes likely have osmoregulatory functions, agreeing with the notion the ec is analogous to the kidney. in general, the genes we identified fell into four categories: transmembrane channels/pores, hsp co-chaperones, proteins with unknown functions, and tfs. the largest group of genes encodes transmembrane channels/pores, indicating that many genes regulated by the octamer participate in substrate transport. the five channels/pores are: 1) twk- encodes a c. elegans twik potassium ion channel protein. in vertebrates, twiks are commonly expressed in neuronal tissues and, to a lesser extent in lungs, skeletal muscle, and tubular portions of the kidney. the mammalian twik, task, is sensitive to changes in extracellular ph, indicating that some of these proteins have roles in modulating cellular responses to ph flux. also, because their conductance is osmotically regulated, twiks can influence cellular volume. the c. elegans genome has twk genes. as in other organisms, most of the c. elegans twks are expressed in neurons; however, twk- is the only twk expressed in the ec. interestingly, another group has demonstrated that twk- is directly regulated by ceh-. this independent study strengthens the notion ceh- is a bona fide regulator of ec expression that likely acts through the octamer in the promoter of twk- therefore, it is possible that ceh- regulation impacts at least some of the octamer-dependent candidates identified here. 2) aqp- an aquaporin whose function and regulation have been characterized previously. 3) c05d is a homolog of the cytochrome b561/ferric reductase sdr- in mammals, sdr- is expressed in the brain and kidney where it aids in iron reabsorption via the accessory transporter, divalent-cation transporter. cytochrome b proteins transport electrons in an ascorbate-dependent manner. due to the role of sdr- in ascorbate regeneration, c05d could be involved in vitamin c homeostasis and/or oxidative stress responses. 4) r02f encodes a solute carrier protein that is most similar to the mammalian slc subfamily. slc proteins are localized to intracellular and plasma membranes where they function as symporters. slc proteins transport small neutral amino acids such as glycine, alanine, and proline. because slc proteins affect proton flux, they also contribute to intracellular ph homeostasis. in mammals there are four slc genes, two of which are expressed in the kidney. 5) c01b encodes a c. elegans bestrophin homolog. bestrophins are transmembrane proteins that modulate calcium dependent transport of chloride ions across cellular membranes. bestrophins are enriched in the plasma membranes of epithelial cells where they manage cellular volume. bestrophins are also expressed in exocrine gland tissues, lung, testis and kidney. in these tissues they facilitate trans-epithelial movement of chloride ions leading to water and electrolyte movement. in addition to transmembrane channels and pores, we uncovered several other genes that have less obvious links to osmoregulation and kidney biology. one of these, zc, is homologous to thehighly conserved hsp co-chaperone protein, p p interactions with hsp to ensure the proper folding and maturation of many proteins including steroid receptors, telomerase, and proteins that are upregulated in cancers. we also identified m, a gene with little prior functional data. m is a nematode-specific gene that is mainly composed of hydrophobic amino acids and is therefore likely to be localized to cell membranes and/or forms a globular protein. finally, we identified two tfs. the first, f16f is a homolog of the mammalian protein lipopolysaccharide-induced tumor necrosis factor-alpha factor. litaf is linked to charcot-marie-tooth disease, a heritable neuropathy characterized by loss of muscle tissue and touch sensation. in cmt1c, litaf is implicated in protein degradation. litaf also functions in cytokine production. we speculate that because f16f is expressed in the ec, a tissue exposed to the environment, it could be involved in innate immune responses. also, because c. elegans f16f is expressed in neurons, it is possible that the nematode could act as a model for cmt1c. the other tf we identified is hlh- a helix-loop-helix tf related to human twist. twist was originally characterized in drosophila as a gene involved in dorsal-ventral patterning. twist tfs bind e-box cis-regulatory elements. in c. elegans, hlh- is important for regulating muscle, intestinal and anal muscle development. consequently hlh- mutants exhibit defecation and egg-laying defects. several transcriptional targets of hlh- are known, including cdh- egl- c18b, f08d, rbc- npr- dhs- sgcb- erv- m, r02e, rev- and myo-. most of these genes are unlikely to be transcriptional targets of hlh- in the ec as they are not expressed in this cell. an exception is cdh-, which encodes a widely expressed cadherin, which is also expressed in the ec. due to the limited number of hlh- targets in the ec, we can envisage a model where ceh- plays a role in directing the precise transcriptional outcomes of downstream tfs. in this role, ceh- could modulate target genes specifically in a subset of ectodermal tissues including the ec. several of the genes that depend on their upstream octamer containing fragments for ec expression are also expressed in additional tissues. an interesting consequence of assaying the activity of truncated promoters is that loss of the octamer containing fragment sometimes led to loss of expression in multiple tissues including neurons as indicated in the cases of zc, twk- m, and f16f. this is not surprising as vertebrate orthologs of ceh- including brn are involved in neuronal and kidney development. our strategy for studying the above genes involved comparing the expression patterns resulting from promoter truncations that either contain or remove the octamer. analyzing promoter function by means of these truncations imposes some significant drawbacks; for example, we could not address the function of several candidate octamer elements because removing regions upstream of the octamer led to loss of ec expression. also, because our promoter truncations deleted the octamer and some flanking regions, we cannot be certain that loss of ec-selective expression is the consequence of removing the octamer. however, because the 5' ends of the truncations were in general fairly close to the octamer, and because loss of ec-expression correlated with octamer deletion, we believe that these genes are likely dependent on octamers for their expression in the ec. to address the issue of whether these are indeed functional octamers, one could introduce point mutations within the octamer and assess the resulting consequences on ec-expression. however, we demonstrated in our mutagenesis experiments that certain point mutations are not sufficient to abolish ec expression in the context of the aqp- promoter. another potential drawback from our approach is the fact that much of our study is based on expression patterns arising from transgenic c. elegans strains containing extrachromosomal arrays. such arrays are susceptible to somatic transgene loss, which results in mosaic reporter expression. this mosaiscism has the potential to confound our analysis by under-representing the expression pattern. however, expression patterns resulting from genome-integrated transgenes are identical to the expression patterns in aqp-8promoter::gfp strains carrying extrachromosomal transgenes. therefore, mosaic loss of the extrachromosomal transgene array is not likely an issue for analyzing changes in ec expression. despite the potential shortcomings detailed above, our study revealed a set of genes whose expression likely depends on the octamer for expression in the ec; these genes are excellent ceh- candidate targets. in agreement with this notion, twk- one of the genes that we identified as dependent on an upstream octamer fragement, has been demonstrated, independently, to be regulated by ceh-. because our bioinformatic search did not bias the direction of the octamer, we discovered promoters in the forward and inverted orientation can be associated with ec-expression. octamers in a forward orientation occur in the promoters of aqp- m, twk- zc, hlh- c01b, and c05d, whereas inverted octamers are present in the promoters of f16f and r02f. although we could not determine from our small sample set whether the direction of the octamer has a functional consequence, there are possible implications related to direction of the octamer sequence. for example, octamers upstream of immunoglobulin light and heavy chain genes have directional preferences. in addition, the human pou homeobox gene oct is auto-regulated by two upstream octamers, which are also situated in inverted orientations. although each site binds oct with equal affinity, each of these sites has different effects on oct expression. it appears that transcriptional auto-regulation is a common mode of regulation among pou tfs. interestingly, we detected an octamer upstream of ceh-6's atg in c. elegans. likewise, there is an octamer located in the regulatory region of the c. briggsae gene encoding a putative ceh- ortholog, providing evidence that auto-regulation might be conserved. however, these octamer are located within a predicted non-coding rna gene. nevertheless, it would be interesting to determine whether ceh- like other pou tfs is auto-regulated. with our set of nine candidate octamers, we had the opportunity to determine whether residues flanking the element are conserved. globally, the g/c content of c. elegans is 31%. however, the regions flanking the octamers-associated with ec-expression contain a slightly higher g/c content. our alignments of these octamers revealed that despite the higher g/c content, some positions have preferences for a/t residues. strikingly, directly 3' to the octamer, an a or t is always present. the conservation of this residue is consistent with the results of a previous study, which identified oct binding preferences using a systematic evolution of ligands by exponential environment -based in vitro binding approach. because of the enriched g/c content in octamer adjacent regions, it is likely that the observed preference for a/t at certain positions have relevance. we tested the effects of targeted-octamer mutations on ec-expression. we found that several single-base substitutions did not affect the cis-regulatory element's ability to drive expression in the ec. interestingly, we did not observe a change in expression level or pattern when position five of the octamer was mutated from a purine to purine. this variant of the octamer was demonstrated to be a binding site for the catfish class iii pou tf, oct. therefore, this residue change results in an octamer variant which retains the ability to interact with the pous sub-domain binding consensus sequence atattc). at the same residue position, a thymine replacement, led to weak gfp expression also restricted to the ec. this sequence was able to bind to oct in vitro in an electrophoretic mobility shift assay. however, in another study this variant of the octamer was not able to drive reporter expression in human cells. these results, taken together, indicate that this motif variant is a sub-optimal pou tf binding site that can drive weak ec expression in c. elegans. a mutation at the sixth residue also led to weak ec-localized expression. this octamer variant is functional in the promoter of the drosophila gene, choline acetyltransferase. in the chat promoter, atttgaat interacts with the pou homeobox tf, dpou-. all other single nucleotide substitutions at these two locations led to loss of gfp expression. however, a double residue replacement of these residues could still drive expression, as indicated by weak gfp expression in the ec. there have been no previous reports of this dual nucleotide substitution variant associating with pou tfs and it is therefore a novel pou tf binding site variant. overall, our mutagenesis assays indicate that the octamer cis-regulatory element could have a range of functional variants in c. elegans. thus, identifying and characterizing promoters containing these octamer variants may reveal a larger group genes expressed in the ec. because variants of the octamer can influence ec-expression, we examined the pgp- promoter region in c. elegans more closely. previously, it was demonstrated that pgp- expression is regulated by dcp-66/ex-. in this report, the tf/cis-regulatory element interaction was confirmed using in vitro and genetic approaches. loss of either ex- or dcp- resulted in loss of ec expression. we detected an octamer like sequence that partially overlaps the ex-. we also identified this octamer-like sequence in the orthologous regions of c. briggsae. using the transcriptional element search system to identify predicted cis-regulatory elements, we found that the octamer-like sequence is indeed a potential target for octamer binding proteins. additionally this sequence binds oct in vitro. in fact, zhao et al. reported that promoter constructs encompassing ex- at - bp results in strong reporter expression during all developmental stages. they also studied the expression pattern resulting from a promoter region defined by a 5'-end bp upstream of the atg, thereby removing three nucleotides from the octamer-like sequence. although this promoter still drove ec-expression, the intensity of the gfp reporter was greatly decreased in adult and larval worms. additionally, embryonic expression was almost eliminated. finally, a construct with a 5'-end bp upstream of the atg was not able to drive expression of gfp indicating the necessity of the ex- for ec expression. therefore, not only did this prior study define the role of the ex- for ec-expression, but it also indirectly provided evidence that the octamer upstream of pgp- might affect ec-expression. this suggests that the dcp-66/ex- and the octamer-directed transcriptional regulatory mechanisms co-operatively modulate the expression of pgp- in the ec. this model of concerted and redundant regulation of ec-expression may have relevance in other genes including, possibly, several genes within our phylogenetically defined candidate set.  overall, we determined that the octamer is likely responsible for the expression of several genes within the ec, an osmoregulatory organ analogous to the kidney. because one of our candidate genes, twk- has been demonstrated to be a bona fide target of ceh- regulation, it would be interesting to determine whether ceh- is involved in the regulation of four other candidates. although our candidates, for the most part, were chosen based upon perfect conservation of the octamer, we determined that several variants of the octamer can drive ec-expression. the existence of functional octamer variants indicates that future searches for octamer-driven genes should use a loosely defined octamer sequence. overall, understanding conserved mechanisms of gene regulation that determine appropriate ec expression may provide insight into underlying transcriptional mechanisms that regulate transcription in analogous organs including the kidney. 
third, we used promoter::gfp fusions to examine the expression patterns of of the genes. we have previously demonstrated that the pou transcription factor ceh- is required for driving aqp- expression in the c. elegans excretory cell, an osmotic regulatory organ that is functionally analogous to the kidney. here, we further characterize octamer driven transcription in c. elegans. 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- 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. to do so, we assayed the effects on excretory cell expression by placing the octamer within the well-characterized promoter of vit- second, using phylogenetic footprinting between three caenorhabditis species, we identified a set of genes that contain conserved upstream octamers in their promoters. this analysis demonstrated that conservation of octamers in promoters increases the likelihood that the gene is expressed in the excretory cell. taken together, we have demonstrated that octamer-containing regions are associated with excretory cell expression of several genes that have putative roles in osmoregulation. this transcriptional regulation occurs through a ceh- binding to a cis-regulatory element called the octamer, which is located in the aqp- promoter.
 we have previously demonstrated that the pou transcription factor ceh- is required for driving aqp- expression in the c. elegans excretory cell, an osmotic regulatory organ that is functionally analogous to the kidney. this transcriptional regulation occurs through a ceh- binding to a cis-regulatory element called the octamer, which is located in the aqp- promoter.  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- second, using phylogenetic footprinting between three caenorhabditis species, we identified a set of genes that contain conserved upstream octamers in their promoters. third, we used promoter::gfp fusions to examine the expression patterns of of the 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.  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- 