1.1 Rhabditidae genomes encode far more NHRs than insects or mammals
While the Drosophila
genome contains only 18 nhr
genes and the human genome contains 48 such genes, sequence homology identifies more that 284 nhrs
in the C. elegans
genome (Antebi, 2006
; Haerty et al., 2008
; King-Jones and Thummel, 2005
; Sluder et al., 1999
), 232 in C briggsae
(reported by (Haerty et al., 2008
)) and 256 in C. remanei
(Haerty et al., 2008
). Of the 284 nhr
genes in C. elegans
, only 13 (Robinson-Rechavi et al., 2003
; Robinson-Rechavi et al., 2005
) can be regarded as highly conserved nuclear receptors. The remaining C. elegans nhr
genes encode receptors that are categorized as supplementary nuclear receptors (supnrs), the majority of which appear to have been derived from successive duplications of a single ancestral gene related to vertebrate HNF-4 (Robinson-Rechavi et al., 2005
). A few of these supnrs have been ascribed functions. For example, the HNF-4-related NHR encoded by nhr-49
is a key regulator of metabolic genes in response to fasting and feeding (Van Gilst et al., 2005a
) while supnr nhr-60
plays a role in epidermal cell development (Simeckova et al., 2007
). Characterizing the pattern of expression and functions of additional supnrs is an important step in understanding the evolutionary pressures that have led to the explosion of these receptors in Rhabditidae genomes. In this study, we have chosen to focus on a cluster of seven supplementary nhr
genes for an analysis of transcription, fasting response, and function.
1.2. The genomic organization of a nhr gene cluster localized on chromosome V
Seven nhr genes are organized as a direct tandem repeat within a 17 kb region of Chromosome V represented on the cosmids R07B7.13 and C13C4.3 (V: 12,092,022 – 12,109,114; WS207). The genes localized in this region are in close proximity to each other and include the following nhrs starting from the conventional chromosomal left end: nhr-206, nhr-208, nhr-207, nhr-209, nhr-154, nhr-153 and nhr-136 ().
Genomic organization and sequence similarity of seven clustered NHRs
In order to characterize the mRNAs transcribed from this cluster, we prepared cDNAs from mixed cultures of C. elegans wild type (N2) worms and performed polymerase chain reactions (PCR) with primers derived from each gene as identified by the GeneFinder program. To capture the 5’ mRNA ends, we included the SL1 and SL2 splice leader primers or used rapid amplification of cDNA ends (RACE). PCR or RACE products were cloned into pCR4 and/or PCRII vectors and sequenced. This strategy confirmed the expression of mRNAs for all seven genes in the cluster with exon-intron boundaries as depicted in WormBase (WS207).
An alignment of amino acid sequences of all NHRs from this cluster was generated by the Clustal program (Larkin et al., 2007
) (). The analysis shows that the strongest homology between receptors is within the DNA binding domains (DBD) and at the C-terminal regions of the receptors spanning the putative ligand binding domain (LBD). Outside of the DBD and LBD, these NHRs show striking sequence diversity, although Blast searches identified genes of this cluster as the closest homologues in the C. elegans
genome. The genomic organization and sequence similarities strongly suggest that these genes arose by successive duplications with subsequent sequence divergence. The phylogenic and cladistic analyses indicated that the genes in the cluster can be divided to two subgroups, the first containing the genes most related to nhr-209
and the second related to a common ancestor of nhr-153
(). The analysis also indicated that the later cluster is more ancient and that genes nhr-206
were formed by more recent duplications. All seven NHRs within this cluster belong to Class I C. elegans
nuclear hormone receptors, based on their P-box sequences (Van Gilst et al., 2002
). P-box sequence relatedness is in agreement with the overall homology estimated by Blast: NHR-206, NHR-208 and NHR-207, have the P box sequence CNGCKA and form a small set of receptors in subgroup 12 whereas NHR-209, NHR-154, NHR-153 and NHR-136 have the P box sequence CNGCKT and form subgroup 8 (Van Gilst et al., 2002
Evolutionary relatedness and intron-exon similarities among the seven clustered nhrs
We compared the sequences of the clustered C. elegans receptors with their closest homologues in C. briggsae and found that the receptors with P box sequence CNGCKT are evolutionarily conserved, including identical P-box sequences. Comparison of the C. elegans and C. briggsae genomic sequences indicates the following relationships: the ortholog of NHR-209 is CBP 20617 (CBG 23379), the ortholog of NHR-154 is CBP 24460 (CBG23380), the ortholog of NHR-153 is CBP24461 (CBG23381) and the ortholog of NHR-136 is CBP 24462 (CBG23383). The clear orthology defines these genes as Cbr-nhr-209, Cbr- nhr-154, Cbr-nhr-153 and Cbr-nhr-136. The amino aid sequences of this set of conserved NHRs in C. elegans are 66% to 76% identical to the orthologous receptors in C. briggsae (NHR-209, 66%; NHR-154, 76%; NHR-153, 69 %, NHR-136, 75%). We also found exon size conservation among orthologs in these two species (). The sequences and the sizes of the introns are not conserved, nor are the sequences of the intergenic regions. In addition, the first three receptors in the cluster (NHR-206, -208 and -207) do not have orthologs in C. briggsae.
1.3 nhr-207 and nhr- 209 are organized in an operon
The intergenic regions between genes of this nhr
gene cluster vary from 270 to 1277 bp in length. Short distances between genes transcribed in the same direction in C. elegans
are suggestive of an operon in which co-transcribed mRNAs are processed into individual coding messages via trans-splicing (Blumenthal, 2005
; Blumenthal et al., 2002
). Preceding nhr-206
, Wormbase identifies a gene R07B7.12a
that is transcribed from the same strand, raising the possibility that is also part of an operon including the nhrs
encodes a large, predicted extracellular protein unrelated to NHRs. In order to distinguish between individually transcribed genes and genes expressed from operons, we prepared cDNA from mixed stages of N2 animals and performed multiple PCRs with primers specific for Splice Leader 1 (SL1) and Splice Leader 2 (SL2) in combination with gene-specific, reverse strand primers. Approximately 75% of genes in C. elegans
are trans-spliced to SL1 and when present, this leader sequence is indicative of transcription as a single gene or the first gene in an operon. SL2 trans-splicing predominantly occurs on the second, and subsequent, genes in an operon. We identified trans-splicing with SL1 for nhr-154
resulting in a 13 bp long 5’UTR (tatagtggcagcc), consistent with WormBase information. Trans-splicing with SL2 was detected for nhr-209
resulting in a 257 bp long 5’UTR. Trans-splicing of nhr-209
has not previously been reported and demonstrates it is co-transcribed with one or more flanking genes oriented on the same coding strand. Surprisingly, trans-splicing to SL1 or SL2 was not detected for any of the remaining genes of this nhr
cluster, although the expression of the predicted mRNAs was readily amplified using gene specific primers derived from the predicted sequences. Our results demonstrate that nhr-207
are part of a novel two-gene operon while all other nhrs
within this cluster are transcribed individually.
1.4. nhr cluster gene expression during C. elegans and C. briggsae development
We prepared cDNA from individual stages of C. elegans and C. briggsae animals as follows: embryos, larval stages L1, L2, L3, L4 and young adults. Quantitative Reverse Transcriptase PCR (RT-qPCR) was done using SYBR Green technology. The ama-1 gene in C. elegans, encoding the large subunit of RNA Polymerase II and the ortholog of ama-1 in C. briggsae (CBG05355) were used for normalization of the data.
All seven C. elegans
genes of the studied cluster, and all four C. briggsae
homologs, were expressed during all developmental stages. C. elegans
were detected in hundreds of units (), while the evolutionarily conserved genes (nhr-209
) were detected at much higher relative levels (10,000 to 20,000 units) (). All C. elegans
genes, with the exception of nhr-154
, and all C. briggsae
genes showed relatively stable expression during development under normal growth conditions. In contrast, nhr-154
was expressed at high levels in embryos, L1s, and young adults, but decreased during mid-larval stages (). It is possible that the rise in expression in the adult stage sample is due to embryos within gravid adults, rather than adult expression per se (see below). The expression values detected for C. brigssae
were similar to that observed for their C. elegans
counterparts () with a slight dip in mid-larval stages. These results demonstrate that the seven nhrs
of the cluster are expressed throughout development and that the recent expansion in C. elegans
-related genes was accompanied by a change in expression levels. Our results are largely consistent with whole genome expression array data from early embryogenesis (Yanai and Hunter, 2009
), demonstrating detectable expression of most genes in the cluster in both species. An exception was nhr-209
for which we found qPCR evidence for embryonic expression while Yanai and Hunter detected essentially no early embryonic expression; late embryonic onset of expression would be consistent with both results
The developmental profile of clustered nhr gene expression
1.5. The expression of gfp-fusion reporter genes shows partially overlapping patterns for the clustered nhrs
In order to determine in which tissues and cells the individual receptors are expressed, we prepared transgenic lines carrying putative promoters of each gene fused with the gene encoding green fluorescence protein (GFP) (). At least three independent lines for each of two different promoter constructs were generated for each nhr gene in the cluster. A “short” promoter construct used a 500 – 600 bp segment immediately upstream of the coding region of each gene. A second “long” promoter construct used a 1,000–2,000 bp segment upstream of the coding region that often included part of the upstream, flanking gene. One exception to this strategy was nhr-153, for which only a 968 bp segment upstream of the coding region was used with the reporter gene. Five of the seven nhr genes tested showed gfp expression: nhr-206, nhr-208, nhr-207, nhr-154, and nhr-153. Transgenes for nhr-209 and nhr-136 failed to yield reporter gene expression. As detailed above, nhr-209 is the second gene of an operon. Consequently, we tried several different promoter regions for this reporter transgene, including one with the putative promoter and entire coding region of the upstream flanking gene (nhr-207), all without success. The lack of expression of the nhr-136 reporter transgenes in 12 independent lines was unexpected because of its relatively high expression when assayed by RT-qPCR. It is possible that this gene is regulated by a distant enhancer located far upstream, or downstream, of the coding region.
Promoter segments used for GFP reporter gene analysis
A. Expression of the evolutionarily conserved subgroup of clustered nhrs at standard laboratory conditions
The results of all GFP reporter transgenes are summarized in . As noted above, only two of the evolutionarily conserved nhrs within this cluster yielded reporter gene expression.
Table 1 Summarized results of reporter gene expression for C. elegans clustered nhrs. The intensity of expression was scored from low (+) to high (+++) in each cell or tissue type. Absence of expression is indicated (−). If specific developmental stages (more ...)
The expression of nhr-154
GFP reporter transgenes was first detected in the 2-fold stage embryo within the developing pharynx and in precursors of several unidentified head neurons. By the three-fold stage of embryogenesis, expression was seen in the pharynx and throughout the intestine, a pattern reminiscent of the developmental transcription factor pha-4
(Smith and Mango, 2007
; Updike and Mango, 2006
) (). In L1 and L2 stages, the reporter gene expression was strong in the pharyngeal muscles (anterior and posterior bulbs, predominantly), in unidentified head neurons, the intestine, and in the intestinal-rectal valve or sphincter cell (). Consistent with our RT-qPCR analysis, GFP reporter gene expression decreased in subsequent larval stages (L3, L4) so that by the adult stage only pharyngeal expression persistence is reproducibly observed. This suggested that the peak of expression in adults detected by RT-qPCR might be due to the embryos inside gravid adults in these samples (). No obvious differences in pattern were seen between the two promoter lengths tested. The expression of nhr-154
GFP reporter was not observed in male specific structures.
Examples of expression patterns for five of the seven clustered nhrs in C. elegans for which GFP reporter genes resulted in a signal in transgenic lines
The expression of nhr-153 GFP reporter transgenes was first detected in the 2-fold stage embryo in pharyngeal and intestinal cells (). In all larval stages, the reporter gene expression was very strong in the posterior bulb of pharynx and in all intestinal cells as well as the intestinal-rectal valve or rectal gland cells. Expression was also seen in several unidentified neurons near the posterior pharyngeal bulb and in the tail (). In males, very strong reporter expression was observed in some of the ray-associated neurons ().
B. Expression of recently duplicated clustered nhrs at standard laboratory conditions
The expression of nhr-206
GFP reporter transgenes starts during the comma stage of embryogenesis, and is initially seen in four unidentified cells localized in the head region. By the 2-fold stage, embryonic expression is observed in the pharynx with weaker expression in intestine (). This expression pattern continues throughout all larval stages and in adults with pronounced anterior pharyngeal expression. The reporter genes were also strongly expressed in rectal gland cells, the anal sphincter, and in epidermal cells in the tail (). Weaker expression was also observed in the vulva and spermatheca. In males, expression was visible in male specific neurons of the tail and rays (). The pattern of expression is very similar to that described by Reece-Hoyes and coworkers in their high throughput screen (Reece-Hoyes et al., 2007
The expression of nhr-208 GFP reporters started in embryos at the 1.5-fold stage within the pharynx, intestinal sphincter, and epidermal cells in the tail. By the 3-fold stage of embryogenesis, additional expression was observed in rectal gland and surrounding cells (). During all larval stages, strong expression of the transgenes was visible in pharyngeal and unidentified head neurons, the pharyngeal-intestinal valve cell, the posterior part of the intestine, the intestinal sphincter, two rectal gland cells, the intestinal-rectal valve cell, and the epidermal hyp10 cell (). In males, the expression was seen in several rays (6–8) and other male specific neurons (). This pattern was similar to that described by Reece-Hoyes and coworkers (using a 653 bp long promoter), although our reporters did not result in expression in the excretory cell and vulva.
The expression of nhr-207 GFP reporters began in 1.5-fold embryos in pharyngeal and epidermal cells (). In 3-fold stage embryos, expression was observed in pharyngeal neurons, intestinal cells, the intestinal-rectal valve, and the sphincter. This pattern of expression was present during all larval stages and in adults (). In larvae and in adults, additional expression was observed in the pharyngeal-intestinal valve () and spermathecae. In males, the expression was seen in male specific neurons, including rays (). Our results overlap those reported by Reece-Hoyes and coworkers (using a 340 bp long promoter).
To summarize, the functional nhr GFP fusion reporter genes had many common features. Onset of expression was similar in all cases (comma to 2-fold embryos) with common expression in the intestine, the intestinal-rectal valve, the sphincter, and unidentified head neurons. All but one (nhr-154:gfp) also showed male tail specific neuron expression, including within a common subset of the rays. The recently duplicated nhrs (nhr-206, -208, -207) showed a strong bias of expression in the intestine at the posterior end and two had additional expression in the vulva and spermatheca. We concluded that this gene cluster shares many common sites of expression, although there are clear differences in the patterns between the evolutionarily conserved versus the recently expanded nhrs within this cluster.
1.6. The expression of clustered nuclear hormone receptors responds to fasting
The function of several C. elegans
NHRs have been linked to metabolism (Magner and Antebi, 2008
; Pohludka et al., 2008
; Van Gilst et al., 2005a
; Van Gilst et al., 2005b
). For example, our previous work on NHR-40 demonstrated developmental phenotypes that were dependent on their nutritional status and environmental conditions such as temperature (Brozova et al., 2006
; Pohludka et al., 2008
). Since the clustered nhr
s we focused on in the current study belong to the same subgroup of NHRs as NHR-40, we tested if their expression was similarly affected by alterations in feeding status. To eliminate developmental effects, we used L1s synchronized by hatching in the absence of food; such animals arrest development until fed.
We assayed the expression of the clustered nhrs
by RT-qPCR in L1 populations of C. elegans
that had been hatched and starved for either six or 54 hours using the expression of ama-1
to normalize the data. The expression of C. elegans nhr-206
showed a dramatic up regulation (5 to10-fold) under both fasting conditions. There also was a slight increase (2-fold) in expression of nhr-153
during fasting (). As controls, we assayed the expression of genes that are known to be increased (acs-2
), decreased (fat-7
) or unaffected (nhr-49
) by fasting (Van Gilst et al., 2005b
). As expected, acs-2
was up-regulated approximately 2-fold by fasting, fat-7
decreased approximately 5 to 10-fold and the expression of nhr-49
did not change in our experimental protocols (). Analysis of the expression of C. briggsae
orthologs after six hours of starvation showed that only Cbr-nhr-209
increased (2-fold) in fasting larvae ().
The expression of seven clustered C. elegans nhrs during fasting was analyzed by RT-qPCR
The substantial up-regulation of several of the clustered nhrs in response to starvation, as detected by RT-qPCR, suggested corresponding GFP reporter genes might behave similarly. Indeed, the elevated expression of GFP fusion transgenes was clearly visible and was tissue restricted, as seen for the intestinal cell expression for nhr-206, nhr-208 and nhr-207 (). We quantitated changes in expression using densitometric analyses of images captured at constant settings in conditions of feeding versus fasting for the two major sites of expression for the transgenes; the pharynx and the intestine. The analyses confirmed the elevated expression in gfp fusion genes in intestinal cells of transgenic animals expressing nhr-206, nhr-208 and nhr-207 and in pharynx of animals expressing gfp fusion genes of nhr-153 and nhr-154 (). These results confirmed the RT-qPCR results and demonstrated that the recent expansion of nhr genes within this cluster in C. elegans was coupled to altered transcriptional responses, showing dramatic up-regulation in response to fasting that is restricted to intestinal cells. The evolutionarily conserved nhrs of the cluster showed more modest increases that were predominantly seen in the pharynx.
Responses of GFP reporter genes to fasting and feeding conditions
1.7. Inhibition of clustered nhrs, either alone or in combination, does not affect viability
We studied the effect of gene inhibition for all seven C. elegans
by RNA-mediated interference (RNAi) induced by soaking of larvae in solutions of dsRNA as well as by microinjection into the gonads of parental hermaphrodites. All genes were inhibited individually as well as simultaneously in combinations of two, three or four genes or all seven genes together. We did not observed any morphological phenotypes as a result of RNAi, but there was a slight increase (up to ~5%) of embryonic lethality when all evolutionarily conserved genes were inhibited simultaneously (see Supplementary Table 1
). We concluded that none of the genes on their own was essential for viability, consistent with redundancy of function among many nhr
family members and their common roles in metabolic regulation in response to changing environmental conditions.
In conclusion, in an effort to further understand the evolutionary pressures leading to the explosive expansion of NHRs in nematodes, we studied set of tightly clustered nhrs in C. elegans. Our results demonstrate that four of the clustered nhrs are evolutionarily conserved between C. elegans and C. briggsae, sharing identical DNA binding P-box domains between orthologs as well as exon-intron structures. Recent duplications of one of these genes has led to the expansion of this cluster in C. elegans to include three additional nhrs that are not present in C. briggsae, providing an example of the continued evolutionary flux of this gene family. We found that all genes of these clusters in both species are expressed and GFP reporters demonstrate conserved temporal and spatial patterns for some aspects of expression. The conserved expression patterns, coupled with high sequence conservation among orthologs, strongly argue that these nhrs are functional in the organism. However, RNAi knockdown of these seven nhr genes, either alone or in combinations, failed to reveal any essential function, suggesting these genes act redundantly and/or that they are involved in processes that are not vital for development and viability. Interestingly, the most recently duplicated genes within the cluster that are present in C. elegans are distinct from their evolutionarily conserved paralogs in their expression. Each gene in this triplet is dramatically responsive to fasting, whereas the evolutionarily conserved quartet of genes is not or only slightly so. In addition, the triplet and quartet of nhrs show different tissue responses to fasting. This data demonstrates that cluster expansion in C. elegans was accompanied by an alteration in gene regulation at the level of metabolic response and cell type specificity. Our results support the concept that nhrs are a dynamically fluxing set of transcription factors that display plasticity in regulation, allowing them to rapidly adapt to novel metabolic and developmental roles.