Identification of DVA Interneuron Regulatory Sequences in Caenorhabditis elegans

Background: The identity of each neuron is determined by the expression of a distinct group of genes comprising its terminal gene battery. The regulatory sequences that control the expression of such terminal gene batteries in individual neurons is largely unknown. The existence of a complete genome sequence for C. elegans and draft genomes of other nematodes let us use comparative genomics to identify regulatory sequences directing expression in the DVA interneuron. Methodology/Principal Findings: Using phylogenetic comparisons of multiple Caenorhabditis species, we identified conserved non-coding sequences in 3 of 10 genes (fax-1, nmr-1, and twk-16) that direct expression of reporter transgenes in DVA and other neurons. The conserved region and flanking sequences in an 85-bp intronic region of the twk-16 gene directs highly restricted expression in DVA. Mutagenesis of this 85 bp region shows that it has at least four regions. The central 53 bp region contains a 29 bp region that represses expression and a 24 bp region that drives broad neuronal expression. Two short flanking regions restrict expression of the twk-16 gene to DVA. A shared GA-rich motif was identified in three of these genes but had opposite effects on expression when mutated in the nmr-1 and twk-16 DVA regulatory elements. Conclusions/Significance: We identified by multi-species conservation regulatory regions within three genes that direct expression in the DVA neuron. We identified four contiguous regions of sequence of the twk-16 gene enhancer with positive and negative effects on expression, which combined to restrict expression to the DVA neuron. For this neuron a single binding site may thus not achieve sufficient specificity for cell specific expression. One of the positive elements, an 8-bp sequence required for expression was identified in silico by sequence comparisons of seven nematode species, demonstrating the potential resolution of expanded multi-species phylogenetic comparisons

Reconstructing a metazoan genetic pathway with transcriptome-wide epistasis measurements

RNA-sequencing (RNA-seq) is commonly used to identify genetic modules that respond to perturbations. In single cells, transcriptomes have been used as phenotypes, but this concept has not been applied to whole-organism RNA-seq. Also, quantifying and interpreting epistatic effects using expression profiles remains a challenge. We developed a single coefficient to quantify transcriptome-wide epistasis that reflects the underlying interactions and which can be interpreted intuitively. To demonstrate our approach, we sequenced four single and two double mutants of Caenorhabditis elegans. From these mutants, we reconstructed the known hypoxia pathway. In addition, we uncovered a class of 56 genes with HIF-1–dependent expression that have opposite changes in expression in mutants of two genes that cooperate to negatively regulate HIF-1 abundance; however, the double mutant of these genes exhibits suppression epistasis. This class violates the classical model of HIF-1 regulation but can be explained by postulating a role of hydroxylated HIF-1 in transcriptional control

Expression of wild-type and mutated <i>twk-16</i> constructs.

<p>Summary of expression of wild-type and mutated <i>twk-16</i> constructs in transgenic <i>C. elegans</i> lines. The total number of animals scored is in parentheses with YFP expressing animals shown as a percentage of the total under the corresponding regions of the nervous system. Lines were produced with PCR fusion constructs with the expression vector shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054971#pone-0054971-g002" target="_blank">Figure 2B</a>. Lines are denoted as wild-type (WT) or mutated (Mut) followed by the size (bp) of the experimental sequences. The experimental sequences in WT195 to 5′3′Mut85 are diagramed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054971#pone-0054971-g007" target="_blank">Figure 7A, 7B</a>) and were as follows: WT195 with WT24 sequences; Mut195 with mutations of WT24 sequences; WT85 with WT24 sequences; Mut85 with mutations of WT24; 5′Mut 85 with mutations of 5′ 17 bp of WT85; 3′Mut85 with mutations of 3′ 15 bp of WT85; and 5′3′Mut85 with mutations of both 5′ 17 bp and 3′ 15 bp of WT85. The experimental sequences used in constructs WT53 to Mut8 are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054971#pone-0054971-g005" target="_blank">Figure 5B</a>.</p

Photomicrographs of the expression of <i>twk-16</i> constructs in transgenic lines.

<p>The photomicrographs are arranged from left to right in three columns of six photomicrographs. <b>DVA expression of wild-type </b><b><i>twk-16</i></b><b> intron constructs.</b> Panels <b>A–F</b> are photomicrographs of the tail region of transgenic L4-adult <i>C. elegans</i> generated with the experimental sequences shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054971#pone-0054971-g005" target="_blank">Figure 5A</a>. Yellow lines indicate DVA neurons expressing YFP. The constructs used to generate the transgenics in each panel were: <b>A</b>. WT2000∶500 bp of the 5′region of <i>twk-16</i> gene, the first exon and entire 1.4-kb first intron with twk-16.cs1 and twk-16.cs2. <b>B</b>. WT500: twk-16.cs1 and flanking sequence producing both DVA and DVC expression. <b>C</b>. WT300: twk-16.cs1 and flanking sequence <b>D</b>. WT195: twk-16.cs1 and flanking sequence. <b>E</b>. WT113: twk-16.cs1 and flanking sequence. <b>F</b>. WT85: twk-16.cs1 and short flanking sequences. <b>Expression of wild-type </b><b><i>twk-16</i></b><b>.cs1 constructs.</b> Panels <b>G–L</b> are photomicrographs of the tail of transgenic L4-adult <i>C. elegans</i> animals with the following constructs: <b>G</b>. WT85∶53 bp of twk-16.cs1 with 17 bp 5′ and 12 bp 3′ of flanking sequence. <b>H</b>. WT53∶53-bp fragment of twk-16.cs1 in wild-type orientation. <b>I</b>. WT53R in reverse orientation (3′-5′) to the expression vector. <b>J</b>. WT29: the 5′ 29 bp of WT53. <b>K</b>. WT24: the 3′ 24 bp of WT53 <b>L</b>. Vector: no experimental sequence and PCR expression vector Δ<i>pes-10</i>::4X NLS::YFP::<i>unc-54</i>::<i>unc-119</i>. <b>Expression of mutated </b><b><i>twk-16</i></b><b>.cs1 constructs.</b> Panels <b>M–R</b> are photomicrographs of the tail region of transgenic L4-adult <i>C. elegans</i> animals made with the following constructs containing mutations (Mut2-Mut8) of the 53-bp fragment (WT53) of the twk-16.cs1 region: <b>M</b>. Mut5. <b>N</b>. Mut2. <b>O</b>. Mut2. <b>P</b>. Mut3. <b>Q</b>. Mut6. <b>R</b>. Mut8. L4 Vulva expression in Mut2 transgenic is shown in Panel O with a yellow line identifying the vulva. Scale bars are specific to each column of six photomicrographs and = 20 µm.</p

Mutation analysis. A. Analysis of the <i>twk-16</i> intron and enhancer.

<p>Deletion analysis of the <i>twk-16</i> intron with 73 bp twk-16.cs1 (cs1) and 259 bp twk-16.cs2 (cs2) denoted in red with flanking sequences in black and not to scale. The approximate sizes of the wild-type (WT) sequences are denoted by numbers from WT2000 to WT53. WT2000 was a plasmid construct and contains 500 bp 5′ of exon 1, exon 1 and 1.4 kb of the first intron containing both cs1 and cs2. WT700 contains 38 bp of flanking sequences 5′ to cs1 and 244 bp of flanking sequences 5′ to cs2 and 102 bp of 3′ flanking sequences. WT350 contains 55 bp of flanking sequences 5′ to cs2 and 45 bp of 3′ flanking sequences. WT500 contains 202 bp of flanking sequences 5′ to cs1 and 223 bp of 3′ flanking sequences. W300 contains 114 bp of flanking sequences 5′ to cs1 and 121 bp of 3′ flanking sequences. WT195 contains 114 bp of 5′ flanking sequence to cs1 and 8 bp of 3′ flanking sequence. WT113 contains 23 bp of 5′ flanking sequences to cs1 and 17 bp of 3′ flanking sequence. WT85 contains 4 bp of 5′ flanking sequences to cs1 and 8 bp of 3′ flanking sequence. WT85 contains WT53 with 17 bp of 5′ flanking sequence and 15 bp of 3′ flanking sequence. WT53 contains 53 bp of the 73 bp twk-16.cs1 region. <b>B. </b><b>Mutational analysis of WT53.</b> The wild-type sequence is denoted by black type with the mutations of WT53 shown in red type. Conserved sequences identified by the seven species MUSSA comparison are in blue type with blue underlining. WT29 and WT24 are generated by cleavage of WT53 within the Mut3 region. DVA or broad neuronal expression is denoted by+or – in the box to the right of each construct.</p

Schematic images of <i>C. elegans</i> tail ganglion and dorsal rectal ganglion neurons.

<p>The labeled ganglion are the Pre-anal ganglion (PA), Lumbar ganglion (LG) and Dorsal Rectal Ganglion (DRG). The individual neurons comprising the DRG are DVB, DVA and DVC in the black box. The gut is in pink and the rectum shown in darker brown. The images were derived from <a href="http://www.WormBase.org" target="_blank">www.WormBase.org</a>, by Christopher Grove (Caltech).</p

Conserved regions driving expression in DVA.

<p>Panels <b>A–D</b> are photomicrographs of the tail region of transgenic L4-adult <i>C. elegans</i>. DVA expression is denoted by a yellow line identifying the DVA neuron. The gene name is followed by the conserved region numbered by its position relative to the first exon. <b>A</b>. DVA expression of the 190 bp conserved region 2 of <i>nmr-1</i> (nmr-1.cs2). <b>B</b>. DVA expression of the 308 bp fragment containing conserved region 1 of <i>twk-16</i> (twk-16.cs1). <b>C</b>. DVA expression of the 180 bp conserved region 3 of <i>fax-1</i> (fax-1.cs3). <b>D</b>. DVA expression of the 322 bp conserved region 4 of <i>fax-1</i> (fax-1.cs4). Scale bar = 20 µm.</p

Figure 8

<p><b>A. Seven species comparison of </b><b><i>twk-16</i></b><b> enhancer and model.</b> The WT53 element is in black type and highly conserved bases identified by seven species MUSSA analysis in blue type and underlined. The experimental sequences of Mut2-Mut8 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054971#pone-0054971-g005" target="_blank">Figure 5B</a>) containing mutations of WT53 sequence are shown in red type and wild-type sequences of WT29 and WT24 in black type. Neuronal expression is denoted by+or – under DVA or Broad. MUSSA comparison of 3′-ward WT53 or WT53-like sequences from the <i>twk-16</i> genes or homolog’s of seven nematode species: <i>C. elegans</i>, <i>C. briggsae</i>, <i>C. remanei</i>, <i>C. brenneri</i>, <i>C. japonica</i>, <i>C. angaria</i> and <i>Heterorhabditis bacteriophora</i>. WT53 and WT53-like sequences are in uppercase; adjacent 3′-ward residues are in lowercase. Conserved bases shared by all seven species are in blue type. <b>B. Model of the 85 bp </b><b><i>twk-16</i></b><b> enhancer.</b> The WT85 sequence showing the A-D regions: A region 17 bp (purple); the D region 15 bp (purple); B region 29 bp (green); and C region 24 bp (orange). UniPROBE predicted TF binding sites for homeodomain TF’s (denoted by HOX) and ETS family TF’s (ETS) are shown below as colored sequence corresponding to the WT85 sequence. The GA-rich motif (green) in the B region with green arrow denoting the GA-rich motif is on the minus strand. Below the A-D regions is a summary of effects on neuronal expression as+or - of the four regions in rows labeled as DVA or Broad. The model diagram shows the A-D regions as letters with the same color scheme as the above WT85 sequence. Lines with arrowheads designate a positive effect on expression and the lines ending with a vertical line designate a negative effect on DVA or Broad neuronal expression.</p

Expression of wild-type <i>twk-16</i> intron constructs.

<p>Summary of expression in transgenic <i>C. elegans</i> lines. Lines are denoted as wild type (WT) followed by a number with approximate size (bp) of the <i>twk-16</i> experimental sequence. The total number of animals scored is in parentheses with YFP expressing animals shown as a percentage of the total under the corresponding regions of the nervous system. All constructs (except plasmid WT2000) were made by PCR fusion with the expression vector shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054971#pone-0054971-g002" target="_blank">Figure 2B</a>. The constructs used to generate the transgenic lines were: WT2000 with 500 bp 5′ of exon 1, exon 1 and the 1.4-kb first intron. The experimental sequences used were the following constructs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054971#pone-0054971-g005" target="_blank">Figure 5A</a>): WT700 with twk-16.cs1 and twk-16.cs2 regions and flanking sequence; WT350 with twk-16.cs2 region and flanking sequence; WT500 with twk-16 cs.1 region and flanking sequences; WT300 with twk-16.cs1 region and flanking sequence; WT195 with twk-16.cs1 region and flanking sequence; WT113 with twk-16.cs1 region and flanking sequence; WT85 with twk-16.cs1 and short flanking sequence; WT53 with 53 bp of twk-16.cs1; and WT53R is the reverse complement of WT53. The sub-fragments of WT53 are the 5′ 29b bp of WT53 (WT29) and the 3′ 24 bp of WT53 (WT24) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054971#pone-0054971-g005" target="_blank">Figure 5B</a>). The only individual neuron scored was DVA. (F signifies faint YFP expression).</p

A. Conserved regions analyzed for DVA expression.

<p>Relative location of conserved regions identified by MUSSA from the <i>acr-15</i>, <i>fax-1</i>, <i>nmr-1</i> and <i>twk-16</i> genes. Conserved regions are depicted as red boxes below the corresponding gene and denoted as cs1-cs4 based on their relative position from the first exon in black. The intergenic regions are shown as a black line with the size (kb) above. Neuronal expression is shown in the vertical oriented box as+or - under DVA or Broad. The parentheses and asterisk (53 bp*) following the 73 bp twk-16 cs1 region denotes that the 53 bp fragment of the 73 bp cs1 region expressed in DVA and Broadly. The 73 bp twk-16 cs1 region does not show expression. <b>B. Expression vector</b>. The features of the PCR expression vector are denoted by colors: experimental sequences (red); Δpes-10 (purple); nuclear localization signal (NLS) (blue); YFP (yellow); and derived from the Fire Vector pPD122.53. The <i>unc-119</i> promoter and <i>unc-119</i> mini-gene are in green. Experimental sequences were fused by PCR to this expression vector to form a single PCR product.</p