Transcription factors SMA-3 and SMA-9 bind both overlapping and distinct genomic sites

(a) Cumulative probability distribution graph of the distances between the centroids (base pair position located centrally within each peak) of nearest neighbor SMA-3 and SMA-9 ChIP-seq peaks. The black line represents actual inter-peak distances, whereas the green line represents a hypothetical randomized dataset. The horizontal dotted line indicates the point in the curve at which half of the peak pairs fall. (b) Same cumulative probability distribution as in (a), but focused on distances less than 500 bps. The centroids of nearly half of all SMA-3/SMA-9 neighboring pairs fall within 500 bps of each other. (c) Histogram of the interpeak distances (actual data in black, randomized data in green), as well as the ChIP-seq peak widths (SMA-3 in red, SMA-9 in blue). Most peaks are larger in size than most interpeak centroid distances, indicating substantial peak overlap. (d) Size proportional Venn diagram showing the number of SMA-3 and SMA-9 peaks that either overlap with one another or remain independent. Although most SMA-3 peaks overlap with SMA-9 peaks, more than half of SMA-9 peaks are located independent from SMA-3 peaks.

Identification of direct transcriptional targets of SMA-3 and SMA-9

(a) Principal Component Analysis (PCA) over two dimensions (PC1 and PC2) for RNA-seq datasets for wild type (in black), sma-3(wk30) (in red), and sma-9(ok1628) (in blue). The percent of variance for each component is indicated. The three biological replicates for each genotype are well clustered. (b) Volcano plot of RNA-seq FDR values versus log2 fold change (FC) expression for individual genes (squares) in sma-3 mutants relative to wild type. The direct targets identified by BETA are indicated with red squares; the negative log2 FC values demonstrate that SMA-3 promotes the expression of these genes. Non-target genes nevertheless showing differential expression are indicated with green squares. (c) Strategy for integrating SMA-3 ChIP-seq and mutant RNA-seq data to identify directly regulated targets. (d) Volcano plot of RNA-seq FDR values versus log2 fold change expression for individual genes (squares) in sma-9 mutants relative to wild type. The direct targets identified by BETA are indicated with blue squares; the combination of positive and negative log2FC values demonstrates that SMA-9 promotes the expression of some of these genes and inhibits the expression of others. Non-target genes nevertheless showing differential expression are indicated with green squares. (e) Strategy for integrating SMA-9 ChIP-seq and mutant RNA-seq data to identify directly regulated targets.

Significant overlap in directly regulated DEGs of SMA-3 and SMA-9

(a) Strategy for integrating SMA-3 and SMA-9 ChIP-seq and mutant RNA-seq data to identify common versus unique directly regulated targets. (b-f) Cartoon representations of the different types of direct target genes, their neighboring SMA-3 and/or SMA-9 binding sites, and the effect of those sites on that gene’s expression. The red circle labeled “3” represents SMA-3 binding sites, whereas the blue circle labeled “9” represents SMA-9 binding sites. Arrows represent that the wild type transcription factor promotes the expression of the neighboring DEG (gray), whereas T-bars indicate that it inhibits the expression of the DEG. Types of regulation include (b) SMA-3 alone promoting DEG expression, (c) SMA-3 and SMA-9 combined promoting expression, (d) SMA-3 and SMA-9 showing antagonistic regulation of expression, (e) SMA-9 alone promoting DEG expression, and (f) SMA-9 alone inhibiting DEG expression. Example DEGs and tables of annotation clusters for gene ontology terms for those DEGs (via DAVID, with accompanying statistical EASE score) are shown under the cartoon demonstrating each type of regulation.

Genetic interactions between SMA-3 and SMA-9

(a) Mean body length of L4 animals (measured head to tail in microns). Dots indicate the size of individual animals. ****P<0.0001, **P<0.01 One way ANOVA with Tukey’s multiple comparison test. (b) Mean mRNA levels for the indicated target gene (X-axis) for the indicated genotypes (sma-3, sma-9, or the double mutant combination) relative to the level in wild type. Expression values are in log2 fold change (FC). Individual genotype mRNA levels for each gene were first normalized to actin mRNA levels in that genotype. *P<0.05 Two way ANOVA with Tukey’s multiple comparison test.

Multiple SMA-3 and SMA-9 target genes regulate body size

(a) Mean body length of L4 animals (normalized to wild type) for the indicated mutants. Dots indicate the size of individual animals. Asterisks above each column indicate one way ANOVA with Dunnett’s multiple comparison test against wild type (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05). Asterisks over pairwise comparison bars indicate one way ANOVA with Sídák’s multiple comparison test. The dotted line indicates the size value falling two standard deviations below the mean of wild type. (b) Glass’ effect size (the difference between the mean of the mutant and the mean for wild type, divided by the standard deviation of wild type) for the indicated mutants. The dotted line indicates an effect size of one. (c,d) Mean body size and Glass’ effect size for RNAi knockdowns of the indicated gene in the rrf-3 RNAi sensitive strain. “EV” indicates the empty RNAi vector as a negative control. Asterisks above each column indicate one way ANOVA with Dunnett’s multiple comparison test against EV (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05). For all graphs, red, blue, and purple columns indicate values for sma-3 (mutant or RNAi knockdown), sma-9 (mutant or RNAi knockdown), and their double mutant combination, respectively. Green bars indicate genotypes for which the mutant or RNAi knockdown resulted in a statistically significant reduction (P<0.05) with a Glass’ effect size of one or greater.

DBL-1 signaling promotes body size through collagen secretion

(a) Cartoon illustrating a cross section through the nematode body (dorsal oriented up). Rings of cuticular collagen annuli (magenta), secreted by the underlying hypodermal cell layer (tan) surround the body. Lateral alae containing collagen, secreted by the underlying seam cells (gray), run orthogonal along the length of the body. (b) Cartoon illustrating a cross section through a portion of the nematode body (lateral oriented up) underneath a microscope coverglass. Horizontal green bars indicate the cuticular versus hypodermal plates captured by confocal microscopy in panels c-e. (c) ROL-6::wrmScarlet fluorescence detected in annuli and alae in the cuticular layer, as well as in nuclear envelopes in the underlying hypodermal layer in wild-type animals. The horizontal red line indicates the specific xz cross section shown below the cuticular and hypodermal plane panels. The bar indicates 5 microns. (d) ROL-6::wrmScarlet in sma-3 mutants, visualized as per wild type. Patches of cuticular surface show diminished levels of ROL-6:wr:mScarlet, whereas the protein is detected in the hypodermal layer just under these patches (yellow arrowheads), suggesting a failure to deliver collagen to the surface cuticle (easily visualized in the xz panel). (e) Mutants for sma-9 show the same phenotype as sma-3. (f) Quantification of ROL-6::wrmScarlet fluorescence in the hypodermal layer of indicated mutants. Dots indicate the fluorescence of individual animals. Asterisks over pairwise comparison bars indicate one way ANOVA with Sídák’s multiple comparison test (***P<0.001, **P<0.01). (g) A similar analysis for RNAi knockdowns of the SMA-3 target gene dpy-11, as well as four known ER secretion factors as comparative controls. Asterisks above each column indicate one way ANOVA with Dunnett’s multiple comparison test against wild type (***P<0.001, **P<0.01, *P<0.05).

ROL-6::wrmScarlet accumulates in the ER of sma-3 mutants

ROL-6::wrmScarlet (magenta) and ssGFP::KDEL (an ER marker, shown here in yellow) shown separately (a-d, g-j) or merged (e-f, k-l) in either (a-f) wild type or (g-l) a sma-3 mutant. (a,c,e,g,i,k) shows the interface between the cuticle and the hypodermis, as visualized by SIM super resolution microscopy. (b,d,f,h,j,l) shows the hypodermal layer at a focal plane centered around the nuclear envelope. In wild type, most ROL-6::wrmScarlet is delivered into the cuticle. In sma-3 mutants, lower levels of ROL-6::wrmScarlet are present in the cuticle and rapidly bleached under the SIM laser even under low power, whereas abundant ROL-6::wrmScarlet colocalized with the ER ssGFP::KDEL marker (yellow). We noted that ER reticulation in sma-3 was thinner and skeletonized compared to wild type, perhaps suggesting reduced secretory throughput. (m) Quantification of ROL-6::wrmScarlet fluorescence in the hypodermal layer of tunicamycin treated versus untreated nematodes. (n) Mean body length of L4 animals (normalized to untreated) for tunicamycin treated versus untreated nematodes. (m,n) Dots indicate the values for individual animals. Asterisks over pairwise comparison bars indicate a student t test (****P<0.0001).

DAVID annotation clusters of gene ontology terms for identified SMA-3 and SMA-9 targets.

Tables of annotation clusters for gene ontology terms for direct target DEGs (via DAVID, with accompanying statistical EASE score) are shown for (a) SMA-3 and (b) SMA-9.

Target genes required for exaggerated growth in lon-2 mutants

(a) Mean body length of L4 animals (normalized to EV) for the indicated RNAi knockdowns in the rrf-3; lon-2 double mutant. “EV” indicates the empty RNAi vector as a negative control. Dots indicate the size of individual animals. Asterisks above each column indicate one way ANOVA with Dunnett’s multiple comparison test against EV (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05). The dotted line indicates the size value falling two standard deviations below the mean of EV (b) Glass’ effect size (the difference between the mean of the RNAi knockdown and the mean for EV, divided by the standard deviation for EV) for the indicated gene undergoing knockdown. The dotted line indicates an effect size of one. Green bars indicate genotypes for which the RNAi knockdown resulted in a statistically significant reduction (P<0.05) with a Glass’ effect size of one or greater. (c) The average of the Glass’ effect size between the rrf-3 wild-type background and the rrf-3 lon-2 mutant background (individual dots for each are shown). For (c), yellow bars indicate genotypes for which the RNAi knockdown resulted in a statistically significant reduction (P<0.05) with a Glass’ effect size of one or greater in both the wild-type and the lon-2 mutant background. For all graphs, red and blue columns indicate values for sma-3 and sma-9 RNAi knockdown, respectively.

DPY-11 promotes body size through collagen secretion

(a) ROL-6::wrmScarlet fluorescence detected in annuli and alae in the cuticular layer, as well as in nuclear envelopes in the underlying hypodermal layer in wild-type animals exposed to an empty vector for RNAi knockdown. (b-f) ROL-6::wrmScarlet in animals exposed to RNAi knockdown for the indicated gene. (b) In animals knocked down for dpy-11, little ROL-6::wrmScarlet makes it to the cuticle, instead accumulating intracellularly in the hypodermis. (c-f) In animals knocked down for known ER secretory factors, patches of cuticular surface show diminished levels of ROL-6::wrmScarlet, whereas the protein is detected in the hypodermal layer just under these patches (yellow arrowheads), similar to what is observed in sma-3 and sma-9 mutants, and consistent with a failure to deliver collagen to the surface cuticle. The bar indicates 5 microns.

Tunicamycin treatment mimics the effect of sma-3 and sma-9 mutations on collagen secretion

(a) ROL-6::wrmScarlet fluorescence detected in annuli and alae in the cuticular layer, as well as in nuclear envelopes in the underlying hypodermal layer in untreated wild-type animals. (b) ROL-6::wrmScarlet in animals exposed to tunicamycin. Patches of cuticular surface show diminished levels of ROL-6::wrmScarlet, whereas the protein is detected in the hypodermal layer just under these patches (yellow arrowheads), similar to what is observed in sma-3 and sma-9 mutants, and consistent with a failure to deliver collagen to the surface cuticle. In addition, collagen in the lateral alae is disorganized, suggesting secretion is impaired in the seam cells. The bar indicates 5 microns.