New Results

Promoter architecture and sex-specific gene expression in the microcrustacean Daphnia pulex revealed by large-scale profiling of 5′-mRNA ends

R. Taylor Raborn, Ken Spitze, Volker P. Brendel, Michael Lynch
doi: https://doi.org/10.1101/047894

Abstract

Large-scale TSS profiling produces a high-resolution, quantitative picture of transcription initiation and core promoter locations within a genome. However, application of TSS profiling to date has largely been restricted to a small set of prominent model systems. We sought to characterize the cis-regulatory landscape of the water flea Daphnia pulex, an emerging model arthropod that reproduces both asexually (via parthenogenesis) and sexually (via meiosis). We performed CAGE with RNA isolated from D. pulex within three developmental states: sexual females, asexual females, and males. Identified TSSs were utilized to generate a ‘Daphnia Promoter Atlas’-a catalog of active promoters across the surveyed states. We carried out de novo motif discovery using CAGE-defined TSSs and identified eight candidate core promoter motifs; this collection includes canonical promoter elements (e.g. TATA, Initiator) in addition to others lacking obvious orthologs. A comparison of promoter activities found evidence for considerable state-specific differential gene expression between states. Our work represents the first global definition of transcription initiation and promoter architecture in crustaceans. The Daphnia Promoter Atlas presented here provides a valuable resource for comparative study of cis-regulatory regions in arthropods, as well as for investigations into the circuitries that underpin meiosis and parthenogenesis.

Introduction

All biological processes, including development, differentiation, and maintenance of homeostasis, rely upon precise, coordinate regulation of gene expression. A key early step in gene expression is transcription initiation at the core promoter, a short genomic region containing the transcription start site (TSS) (Kadonaga 2012). During initiation, sequences within core promoters recruit general transcription factors (GTFs), which is followed by binding of RNA polymerase II (RNAPII) and formation of the pre-initiation complex (PIC) (Cosma 2002). Identifying the locations and composition of promoters is fundamental for understanding the basis for gene expression regulation. Recent work (Frith et al. 2008, Hoskins et al. 2011) demonstrates that core promoters are structurally more diverse than previously appreciated. This diversity is thought to reflect large numbers of developmental programs and regulatory strategies (Lenhard et al. 2012), but the precise rules and mechanisms underlying promoter function remain unclear.

Genome-scale TSS profiling has identified promoters in a number of metazoans (FANTOM Consortium and the RIKEN PMI and CLST (DGT) 2014, Lenhard et al. 2012). CAGE (Cap Analysis of Gene Expression) (Kodzius et al. 2006, Kurosawa et al. 2011), the most prominent TSS profiling method, identifies core promoter positions at high resolution. This approach revealed that most genes do not possess a single TSS, but instead exhibit sets of closely spaced TSSs that will be referred to as Transcription Start Regions (TSRs) in the following. While the largest number of TSS profiling studies have been performed in mammalian (human and mouse) systems (Djebali et al. 2008, FANTOM Consortium and the RIKEN PMI and CLST (DGT) 2014), CAGE has also been performed in non-mammalian metazoans, including fruit fly (Hoskins et al. 2011), nematode (Nepal et al. 2013), and zebrafish (Haberle et al. 2014). Overall, these studies indicate that the majority of core promoters in metazoan genomes lack TATA elements (Lenhard et al. 2012), an unanticipated finding given previously established models for transcription initiation. At least two major promoter classes are evident. In human and mouse, the largest class is known as CpG island promoters (CPI) (Saxonov et al. 2006, Lenhard et al. 2012). These promoters are located near CpG islands and are generally of high GC-content and depleted for TATA elements. Sequences in the other major promoter class, called “low-CpG”, exhibit low GC-content and are enriched for TATA boxes. This latter class of promoter is consistent with conventional models of promoter structure, such as TATA-dependent transcription initiation (Kadonaga 2012).

Characterization of CAGE-defined promoters in a wider taxonomic context uncovered two distinct patterns of TSS distributions within a given promoter (Carninci et al. 2006, Hoskins et al. 2011, Lenhard et al. 2012). “Peaked” promoters exhibit CAGE signal from a narrow genomic region surrounding a single prominent TSS, whereas “broad” promoters instead feature multiple TSSs distributed across a wide (30 bp and longer) genomic region (Kadonaga 2012, Lenhard et al. 2012). These TSS distribution patterns appear to coincide with the aforementioned (mammalian) classes of promoter architecture: peaked promoters are highly associated with the low-CpG promoter class, whereas broad TSS distributions tend to be found at high-CpG promoters. Peaked and broad promoters also regulate separate functional gene classes: genes with peaked promoters tend to be developmentally regulated or tissue-specific, while genes with broad promoters tend to be housekeeping genes exhibiting constitutive expression (Lenhard et al. 2012). Recent work using CAGE from a variety of mammalian cell types unexpectedly detected widespread enrichment of TSSs at enhancers (Andersson et al. 2014). The new class of RNA defined by this work, enhancer RNAs (eRNAs), are short, transient, RNAPII-derived transcripts generated at active enhancer regions. While enhancers appeared to be distinguishable from promoters on the basis of transcript stability and bidirectionality (Andersson et al. 2014), subsequent reports suggest that enhancers and promoters possess common properties, including motif composition and activity (Arner et al. 2015).

Despite recent progress, considerable gaps remain in the understanding of promoter architecture across metazoan diversity. To date, high-resolution TSS profiling has been reported in just two arthropod species, both closely-related drosophilids: D. melanogaster (Hoskins et al. 2011) and D. pseudoobscura (Chen et al. 2014). Promoter profiling in a broader set of taxa is necessary to establish robust comparative genomic analyses of cis-regulatory regions in metazoa. To address this need, we performed TSS profiling using CAGE in the water flea Daphnia pulex. A freshwater microcrustacean with a cosmopolitan distribution, D. pulex is notable for its ability to reproduce both sexually and asexually, high levels of heterozygosity, and relatively large effective population sizes (Ne) compared to other broadly dispersed arthropods (Tucker et al. 2013, Haag et al. 2009). D. pulex serves as a key model system throughout the biological sciences, from ecosystem ecology to molecular genetics. By mapping TSSs for D. pulex from active promoters within the three developmental states of sexual females, asexual females, and adult (sexual) males, we sought to characterize the architecture of core promoters in D. pulex and also explore meiosis‐ and sex-specific gene regulatory programs. We successfully identified TSSs at high resolution across the entire genome, defining promoters for all genes expressed under the experimental conditions. We then performed computational de novo motif discovery using this set of set of mapped TSSs, obtaining consensus sequences of canonical core promoter elements, including TATA and Initiator (Inr). The quantitative tag counts from the CAGE datasets allowed us to identify differentially-expressed genes within each of the three states surveyed, including those regulated in a sex-specific manner. The resultant D. pulex promoter atlas extends our knowledge of metazoan cis-regulation into Crustacea, a taxonomic expansion that will also serve as a public resource for functional and comparative genomics.

Results

Profiling 5′ mRNA ends characterizes the global landscape of transcription initiation

Interrogation of capped 5′-ends of mRNAs identifies the locations and patterns of transcription initiation within a genome. Through biochemical capture of these 5′ transcript ends (see Methods), CAGE ultimately generates short, strand-specific sequences (CAGE tags), the 5′-ends of which correspond to the first base of the associated mRNA. Sequenced CAGE tags (47bp in length) were aligned to the genome (Figure 1A, panel i). The coordinate corresponding to the 5′ aligned base of each aligned read is defined as a CAGE-detected TSS (CTSS; Figure 1A, panel ii). Multiple CAGE tags mapping to identical CTSS coordinates provide a quantitative measure of the abundance of mRNA ends that originated from that position. Individual CTSSs supported by sufficient numbers of CAGE tags (significant CTSSs; abbreviated sCTSSs; see Methods) occurring in close proximity in the genome were clustered to yield transcription start regions (TSRs) that correspond to genomic intervals that coincide with transcriptionally active promoters (Figure 1A, panel iii). Finally, when CAGE data from multiple conditions or tissues were compared, we define TSRs that agree (i.e. overlap) in all cases as “consensus promoters” (Figure 1A, panel iv).

Figure 1:
Figure 1:

TSS profiling in D. pulex using CAGE. A. Schematic of CAGE annotations. i) Individual sequenced CAGE tags (represented by short, horizontal black lines) are aligned to the genome in a strand-specific manner, ii) defining distinct CTSSs (represented by dark blue vertical lines). iii) CTSSs with CAGE tag support above 2 tpm are spatially clustered into TSRs (indicated with red lines). CTSSs (gray vertical lines) below the 2 tpm threshold are ignored during this clustering step and are not included in the eventual TSRs. iv) TSRs with evidence across three states are classified as consensus promoters. B. A summary of the developmental stages surveyed in this study. We sequenced CAGE-adapted cDNA libraries originating in i) sexual females, ii) males, and iii) asexual females. The life cycle of D. pulex is summarized (left panel), showing the parthenogenic (ameiotic) and sexual (meiotic) cycles. A representative visualization of CAGE tag densities for a single promoter region across the three states is presented at right. The illustration of the Daphnia life cycle in the left panel is adapted from an illustration by Dita B. Vizoso (Freiburg University) in (Ebert 2005), and is used with permission. C. Proportions of CAGE annotations by genomic location. Locations of all aligned CAGE tags, CTSSs and TSRs by genome segment are shown, including 1kb upstream of the CDS (orange), 1kb downstream of CDS (red), within the CDS (light yellow), CDS introns (light blue) and within intergenic (i.e. exclusive to the other categories) regions (dark blue).

A promoter atlas in Daphnia pulex

D. pulex can reproduce asexually, through ameiotically-produced eggs that develop directly, and sexually, through diapausing eggs. We generated CAGE datasets from three distinct adult states of D. pulex (Figure 1B; leftmost panel): males, parthenogenetic females (hereafter asexual females), and pre-ephippial females (hereafter sexual females). These states were chosen to potentially identify distinct genes and gene networks associated with meiosis, parthenogenesis, and sex-specificity. We sequenced eight libraries, generating 1.82×108 CAGE reads overall (Table 1), of which 1.22×8 (67.0%) mapped successfully to the current version (JGIv1.1) of the D. pulex assembly (Colbourne et al. 2011). After normalization (see Methods), replicates for each state were highly correlated (Pearson coefficient >0.97; Figure S1 and S2). We then applied a computational analysis pipeline to identify CTSSs, TSRs and consensus promoters (Figure 1A) from CAGE reads across each of the three states (See Methods).

Figure S1:
Figure S1:

Correlation between within our CAGE experiment. A matrix containing pairwise comparisons of individual CAGE libraries (n=8) is shown. Multiple individual scatterplots are presented (lower-left), which compares CAGE tag count per CTSS within each comparison. In the upper-right portion of the matrix the Pearson correlation coefficient of each individual comparison is shown. Individual experimental samples are colored and labeled along the diagonal of the matrix.

Figure S2:
Figure S2:

Multi-dimensional scaling (MDS) plot of the CAGE samples within this experiment. Distances between each sample are reported in terms of leading log-fold-changes between each pair of samples. The identity of each CAGE sample is labeled directly on the plot.

View this table:
Table 1:

Summary of CAGE libraries in this study. The value at the end of each library name refers to the biological replicate number.

We evaluated our CAGE definitions in their entirety by considering their locations within the D. pulex genome. Among CTSSs (n=2,332,582) pooled across all states, we observe that a sizable fraction (67.5%) were located within 1 kb of a CDS, while 9.88% were present in the first 1 kb downstream of a stop codon (Figure 1C), an observation also reported in D. melanogaster (Hoskins et al. 2011). When CAGE tags are considered individually (rather than unique CTSSs alone), we report a substantially larger percentage (82.3%) located within the first 1 kb upstream of the translation start site of coding genes, while only a small fraction (1.95%) were located downstream of annotated CDSs (Figure 1C). From this we conclude that CTSSs supported by many CAGE reads are more likely to be positioned upstream of coding genes than those supported by fewer reads.

Similar numbers of TSRs (between 11,289 and 11,558) are identified within the three individual states, totaling 12,662 unique TSRs overall (Table 2). The majority of identified promoters (83.1%) were positioned within the first 1 kb upstream of coding genes, indicating general but incomplete agreement with the current D. pulex gene annotation (Figure 1C). This work represents a comprehensive, sex-specific promoter atlas in adult D. pulex, the first of its kind in crustaceans.

View this table:
Table 2:

Summary of CAGE evidence generated in this study.

Promoter shape, base composition, and expression class

The property of the distribution of TSSs is known to be key descriptor of the structure and composition of the underlying promoter in metazoans (Rach et al. 2009, Hoskins et al. 2011). We evaluated CAGE tag distributions at consensus promoters (n=10,580) using two criteria. The first is width, which is defined as the length of the genomic segment occupied by all CTSSs within a TSR or consensus promoter. We observe an ample range of widths (2–163 bp), including a small number (1104; 10.4%) of TSRs with widths >30 bp (Figure 2A). Overall, We observe a median width of 5 bp, and a mean width of 12 bp for all consensus promoters. We applied a second metric, promoter shape, which measures the stability of the CAGE tag distribution at a TSR. For example, a TSR with a sharp distribution of CAGE tags surrounding a single major CTSS would be considered peaked, whereas a TSR with numerous distinct CTSSs supported by roughly equivalent numbers of CAGE tags would be broad. We applied the Hoskins Shape Index (SI) (Hoskins et al. 2011) to measure shape across all consensus promoters. We also observe a wide range of consensus promoter shapes (Figure 1A, inset); the observed median and mean SI values were ‐0.42 and ‐0.54, respectively.

Figure 2:
Figure 2:

A. Distributions of consensus promoter width and shape in the D. pulex promoter atlas. A histogram representing the distribution of calculated consensus promoter (n=10,580) widths is shown in orange (outer figure). Each bin width represents 5 bp. Inset: Consensus promoter shapes have a bimodal distribution. A histogram representing the shapes (measured with the Shape Index (SI)) of all consensus promoters (n=10,580) is shown in white, with each bin indicating 0.1 of a SI. The densities of broad (coral) and peaked (royal blue) consensus promoter shapes were fitted from the overall distribution of SI values (see Methods). B. Distinct dinucleotide preferences at transcription initiation sites in D. pulex. The dinucleotide frequencies at CTSS ([-1,+1]; aqua) are compared to background (coral). CTSSs show a two-fold or greater preference for the dinucleotides CA, GA, GC and GT, and are similarly depleted for AA, AT and TT. C. Representative examples of canonical CAGE tag distribution patterns observed in D. pulex consensus promoters. Peaked consensus promoters (above) exhibit narrow CAGE tag distributions, whereas broad consensus promoters (below) are typified by more a dispersed distribution of CAGE tags. D. Consensus promoter expression correlates with shape more strongly than width. Consensus promoter expression (measured according to total number of CAGE tags) is plotted against TSR width in base pairs (bp). Peaked (SI >1), broad (SI <-1.5) and unclassified (all other) consensus promoters are identified by green, red, and blue circles, respectively. E. Broad consensus promoters have greater expression than peaked consensus promoters. A significantly greater number of CAGE tags are observed in broad consensus promoters relative to peaked consensus promoters (*p <0.0005; Tukey’s HSD). Box-and-whisker plots representing the distributions of the consensus promoter expression in three shape classes (broad: red, peaked: green, and unclassified: blue) are shown.

Two distinct promoter classes have been proposed in mouse, human and Drosophila, defined according to the shape of empirical (generally CAGE-based) 5′-end distributions (Carninci et al. 2006, Hoskins et al. 2011, Kadonaga 2012). We reasoned that if two distinct classes of promoter exist in D. pulex, then the shapes we observe should be bimodally-distributed. We fit the distribution of consensus promoter shapes using an expectation-maximization (EM) algorithm (see Methods), and see strong support for a twocomponent mixture model (Figure 2A, inset), consistent with broad and peaked consensus promoter shapes. This result provides evidence for the existence two classes of promoter in D. pulex and is consistent with previous findings. We classified consensus promoters into categories according to SI, peaked (n=738), broad (n=1318) or unclassified (see Methods). An example of a peaked and broad consensus promoters found within our CAGE dataset is shown in Figure 2C. We then asked if promoter expression (the abundance of CAGE tags associated with a consensus promoter) varied by promoter shape class (see Methods). We find that broad TSRs have significantly higher expression [p <0.0003710] than peaked and unclassified TSRs (Figures 2D and 2E). However, we do not observe a similar relationship between expression and promoter width (data not shown). This suggests that, in D. pulex, shape is more reflective of promoter properties than width.

Dinucleotide preferences of D. pulex TSSs

Global studies of transcription initiation across metazoan diversity identified distinct dinucleotide compositions at the TSS (Frith et al. 2008, Nepal et al. 2013). We investigated dinucleotide preferences in D. pulex, measuring the dinucleotide frequencies present within the [-1,+1] interval relative to CTSSs. We observe a strong preference for CA, GA, GC, GG, and GT relative to background (p <0.01; see Methods) and considerable depletion for AT-rich dinucleotides AA, AT and TT (p <0.02, 0.01 and 0.01, respectively; Figure 2B).

De novo discovery of consensus promoter elements in D. pulex

Core promoter elements and their motif consensus sequences have been identified in D. melanogaster (Ohler et al. 2002, Down et al. 2007, Kadonaga 2012), mammals (i.e. human and mouse) (Carninci et al. 2006, FANTOM Consortium and the RIKEN PMI and CLST (DGT) 2014) and other metazoan model organisms: worm, C. elegans (Saito et al. 2013), and zebrafish, D. rerio (Nepal et al. 2013, Haberle et al. 2014).

Cis-regulatory motifs of any kind in D. pulex are unknown, so we sought to identify core promoter elements using the CAGE data generated in this study. To accomplish this, we performed de novo motif discovery using CAGE evidence (see Methods), applying sequence windows corresponding to core promoters ([-50,+50]). This procedure revealed a set of eight core promoter elements in D. pulex (Figure 3). To evaluate their similarity to known core promoter elements, we performed sequence alignment of each position weight matrix (PWM) against two motif sets: the complete JASPAR database (Portales-Casamar et al. 2009) and a curated list of 14 non-redundant core promoter motifs in D. melanogaster. We find two motifs within our set with strong sequence identity to the most well-characterized metazoan core promoter elements. The motif Dpm2, which has the consensus TATAWAA, has significant identity to the TBP-binding motif consensus in JASPAR (MA0108.1_TBP, e-value = 6.19×10-9) in addition to the TATA element of D. melanogaster (E-value = 7.49×10-10). The TATA-like Dpm2 was observed in 9.48% of promoters. The motif Dpm3, with the consensus NCAGTY, has significant sequence similarity to the Initiator (Inr) element (consensus TCAKTY) (E-value = 6.097×10-6) of D. melanogaster and is found at 12.04% of promoters.

Figure 3:
Figure 3:

De novo discovery of core promoter elements in D. pulex. The D. pulex core promoter motifs identified in this study are listed. For each identified motif (n=8) we show a logo representing the PWM of each motif, its frequency relative to regions surrounding major CAGE peaks (-200,+50) (see Methods), observed motif enrichment E-value, and the E-value of the most similar motif within the JASPAR database (Portales-Casamar et al. 2009). The motif enrichment E-value represents the probability that a motif of equal length would be discovered in an equivalent number of randomly-derived sequences with the same underlying nucleotide frequencies with equal or lower likelihood.

In addition to TATA and Inr, we report a variety of motifs within our set of D. pulex core promoter elements (Figure 3). Dpm5 (consensus TGGCAACNYYG), exhibits significant similarity to (E-value = 5.76×10-8) to the “Ohler8” motif in D. melanogaster (Ohler et al. 2002). All of the remaining motifs match significantly with at least one motif in the JASPAR database. Among these, three motifs exhibit similarity to well-characterized transcription factor binding sites (TFBSs): Dpm4 (consensus ARATGGC) matches the CTCF motif in JASPAR (MA0139.1_CTCF) (E-value = 5.51×10-5), Dpm6, (CGCTAGA) matches the ABF transcription factor binding site consensus (MA0266.1_ABF2) (E-value = 5.51×10-6) (Portales-Casamar et al. 2009), and the motif Dpm5 (consensus CARCGTTGCC) exhibits a significant match to the TFBS consensus of RFX1 (MA0365.1) (E-value = 2.12×6).

Motif co-occurrence at promoters

After completing de novo discovery of core promoter elements in D. pulex (Figure 3), we sought to characterize the overall motif composition of promoters within the Daphnia promoter atlas. We used the consensus sequences of each of the eight motifs in the Daphnia promoter set and searched within a sequence window of [-200,+50] surrounding the midpoint of all annotated promoters. Using this information, we constructed a co-occurrence matrix for all identified promoter motifs, asking as to the overall coincidence of motifs within promoter regions. Several patterns of motif co-occurrence are observed among the Dpm motifs (Figure 4A). We find that TATA (Dpm2)-containing promoters are not enriched for other identified Daphnia motifs and are depleted for Dpm4 and Dpm5. Inr (Dpm3) promoters are enriched for Dpm6 and have fewer Dpm4 and Dpm5 motifs than expected. Dpm4 while strongly enriched for Dpml also exhibits significant enrichment for Dpm5 and Dpm6. We observe strong co-occurrence between Dpm6 and Dpm7. Three motifs, Dpml, Dpm6 and Dpm7 have greater than expected frequencies of co-occurrence. Of note, none of the other core promoter elements are co-enriched with (Dpm2), and two (Dpm4, Dpm5) are depleted. This line of evidence suggests that TATA-containing promoters do not frequently act in combination with the other identified elements.

Figure 4:
Figure 4:

The co-occurrence and distribution of identified D. pulex core promoter motifs within promoter regions. A. Heatmap of co-occurrence frequencies among identified D. pulex motifs. The log of each p-value is plotted within the heatmap. The frequency distributions of Dpm2 and Dpm3 (B), Dpml and Dpm4, (C) and Dpm5 and Dpm6 (D) relative to identified promoters (TSRs) are shown. (The distributions of Dpm7 and Dpm8 are not shown.) E. Current model of core promoter composition in D. pulex derived from the evidence in this study. A cartoon illustration of the Daphnia core promoter motifs that exhibit strong positional distributions are shown, with their approximate locations relative to the TSS (+1). F. Model representing the positions and consensus sequences of canonical core promoter elements between D. pulex and D. melanogaster. The four major core promoter elements in D. melanogaster are displayed, along with their typical positions relative to the TSS (+1). The consensus sequence of each element, if present, is shown for D. melanogaster (Dm; red) and D. pulex (Dp; dark blue). Note that an individual core promoter may have none, all, or some of the elements listed in the illustration. Graphic adapted from (Butler and Kadonaga 2002). G. Comparison of promoter shape between TATA and Inrcontaining promoters and those lacking TATA. The box-and-whisker plots representing the distributions of calculated shape index (SI) values for consensus promoters with Inr (coral), TATA (green) and those lacking TATA (blue) are shown. Initiator (**) and TATA-containing (*) consensus promoters possess a significantly more peaked shape (p <0.001) than TATA-less promoters.

Positional enrichment of identified D. pulex core promoter elements

Many characterized core promoter elements are known to occur at specific locations relative to the TSS (+1). To determine the spatial characteristics of each of the D. pulex motifs, we evaluated their positional distributions relative to CTSSs and found that four of the eight Dpm motifs exhibit positional enrichment. We observe strong positional enrichment of Dpm2 (TATA-like) and Dpm3 (Inr-like) relative to D. pulex promoters (Figure 4B), with peaks at ‐30 and +1, respectively, consistent with the positions of TATA and Inr within other metazoans (Kadonaga 2012). Dpml exhibits a modest peak at approximately +50, while Dpm5 is enriched between ‐50 and ‐40 (Figure 4B and Figure 4C). Dpm4 shows an irregular distribution within promoters, with two distinct peaks near ‐50 and +10 (Figure 4D). We do not observe a positional enrichment for motifs Dpm6, Dpm7 and Dpm8 (Figure 4D and data not shown). Taken together, this positional information allows us to construct an initial working model of the known core promoter elements in D. pulex (Figure 4E), and draw a comparison between canonical core promoter elements in D. pulex and D. melanogaster (Figure 4F).

Patterns of transcription initiation are known to relate to underlying promoter architecture in Drosophila (Rach et al. 2009, Hoskins et al. 2011) and mammals (Kadonaga 2012), so we asked whether possession of the two major core promoter elements Inr and TATA (Dpm2 and Dpm3, respectively) is associated with TSR shape in D. pulex. Using the Shape Index (as previously described) to measure the focus and dispersion of CTSSs within a promoter we find that both Inr‐ and TATA-containing consensus promoters are significantly more peaked overall than TATA-less promoters <0.001 (Figure 4G), consistent with our expectations and the evidence in other metaozan model organisms including D. melanogaster (Rach et al. 2009, Hoskins et al. 2011).

Differential expression of D. pulex promoters

The abundance of CAGE tags that map to a putative promoter region provides quantitative measurement of the extent of transcription initiation at that site; this is capable of estimating expression of the associated genes (Murata et al. 2014, Balwierz et al. 2009), so we sought to identify differentially-expressed genes across the three states surveyed by our CAGE experiment. We used our defined set of consensus promoters (Table 2; n=10,665) and compared the normalized quantities of CAGE reads within a given state. Consensus promoter expression (i.e. the abundance of CAGE tags present at a consensus promoter in a given state) was measured using the number of mapped CAGE tags within the promoter and were represented in units of tags per million (tpm). An illustration of tag abundance within consensus promoters across the three states surveyed in this study is presented in Figure 5A. We carried out differential expression analysis across all libraries using limma (Ritchie et al. 2015), applying the mean-variance relationship of log-tpm (see Methods). During our analysis we compared promoter expression between each state separately (e.g., sexual females vs. asexual females, etc.) in addition to the following comparisons: males vs. both females, sexual vs. asexual females, comprising five comparisons in total. We observe that an average of 1359 consensus promoters were differentially-expressed within each comparison: an average of 690 promoters exhibited significantly increased activity and 669 promoters had significantly decreased activity (Figure 5B). We observe the greatest number of differentially-expressed promoters (n=1206; upregulated, n=1052; downregulated) in the comparison between males and asexual females. Differentially expressed consensus promoters exhibit a complex topology of enrichment patterns across all three states; representative comparisons for asexual females are shown in Figure 5C and Figure 5D. Heatmaps of differentially-expressed promoters from other comparisons are presented in Figure S3.

Figure S3:
Figure S3:

Additional heatmaps of differentially-expressed (p <0.01) consensus promoters are shown. A. Males vs. sexual females. B. Males vs. asexual females. C. Males vs. females (i.e. asexual females and sexual males). D. Asexuals vs. sexuals (i.e. males and sexual females).

Figure 5:
Figure 5:

Differential expression analysis of D. pulex consensus promoters. A. Representation of consensus promoter expression among the states surveyed in this study. A scatterplot of consensus promoter expression (in tpm) within all three states measured within our study is shown, with the value for asexual females (x-axis) plotted against sexual females (y-axis). Corresponding expression values for males are represented according to a color gradient in log-scale. A small number of consensus promoters (n=145) that lie outside the area of the are not shown. B. Venn diagram of differentially-expressed promoters within the three states surveyed. C. Mean-average (MA) plot of consensus promoter expression of within asexual compared to sexual females. Mean average expression of consensus promoters (x-axis) is plotted against the log fold-change (FC) of the ratio of the expression of consensus promoters between asexual females and sexual females (y-axis). Differentially-expressed consensus promoters (p <0.01 are represented by red dots; all others are colored in black. Upper and lower blue lines on the plot indicate the log(FC) of 2 and ‐2, respectively. D. Heatmap of the expression of differentially-expressed (p <0.01) consensus promoters between asexual females and sexual females. E. Heatmap grid of relative expression of consensus promoters of D. pulex meiosis genes within two selected comparisons: males vs. females and asexual females vs. sexuals. Cells are shaded according to the calculated t-statistic of a given comparison. Instances of significant differential expression (p <0.01) are labeled with two asterisks (**).

Differentially-expressed promoters are enriched for endocrine and environmental response functions

We investigated the set of differentially-expressed promoters between each state, asking if the members of each respective gene set were enriched for common functions. We carried this out using the Gene Ontology (GO), using GO terms associated with the gene adjacent to each differentially-expressed consensus promoter. We observe significantly enriched GO categories for every comparison (data not shown). Results for the differentially-expressed genes between asexual and sexual females are summarized in Figure S5. Among asexual females, enriched categories among upregulated genes include nitrogen compound metabolic process (GO:0006807; p <1.2×10-7). In sexual females (Figure S5), we observe enrichment of several GO categories, including (hormone activity (GO:0003735; p <0.014) and organic cyclic compound metabolic process (GO:1901360; p <2.9×10-6).

Differential upregulation of promoters of meiosis genes in asexual (parthenogenetic) females

We then asked whether there was evidence of enrichment of specific pathways within differentially-expressed promoters. Among genes upregulated in asexual females (vs. sexual females) (Figure 5B), we detect enrichment for pathways associated with cell cycle progression and oocyte meiosis (Figure S6), including cell cycle (04110; p<1.57×10-5), p53 signaling pathways (04115;p<3.80×10-3) and oocyte meiosis (04114;6.88×10-3). Upon inspection of the genes associated with these terms, we observe substantial overlaps with annotated meiotic genes in D. pulex. From the differentially expressed genes associated with the cell cycle KEGG pathway (Figure S6), 5 out of 9 (Cdc20, CycA, CycB, CycE and Cdk2; 55.6%) are functionally designated as “meiotic” by at least one study (Schurko et al. 2009). Additionally, 3 of 7 upregulated genes within the Oocyte meiosis category (Cdc20, Cdk2 and CycE) are annotated in meiosis within D. pulex (Schurko et al. 2009), with two others (Plk1 (Pahlavan et al. 2000) and AurA (Crane et al. 2004)) being directly implicated in meiosis in other model systems. Given their positions within gene networks, upregulation of these genes would be expected to have a negative regulatory impact on meiotic progression overall. Relative expression of the detected promoters of meiosis genes among two comparisons: males vs. females and asexual females vs sexuals (i.e. males and sexual females), respectively, are shown (Figure 5E).

We investigated the set of upregulated genes in the (facultatively) asexual females within our study, asking about the extent of the concordance between the differentially-upregulated genes and scaffolds known to be physically linked to obligate asexuality (Tucker et al. 2013). Considering the genomic locations of differentially-upregulated genes, we unexpectedly find that a fraction (4/15 genes) are located on scaffolds linked to “asexual” chromosomes. This list includes Cdk2 (scaffold_77/ChrVIII), Tim-C (scaffold_76/ChrVIII), Plk1-C (scaffold_9/ChrIX) and HDAC (scaffold_13/ChrIX). We also note that two of the 15 genes, CycE (scaffold_163) and,β-TrCP (scaffold_169) are located on short scaffolds that were not previously tested (Tucker et al. 2013).

Dramatic, sex-specific differential expression of a hemoglobin gene

In evaluating the differentially-expressed consensus promoter data (Figure 5A), we note several genes that are dramatically upregulated in a single condition. Among these is the 2-domain hemoglobin protein subunit (ID:315053) gene on scaffold 13. We observe approximately 400-fold more CAGE tags at the promoter of this gene within sexual females than the other two states (males and asexual females) (Figure 6A and 6B), indicating considerable apparent state-specific upregulation of hemoglobin. The striking abundance of CAGE tags at the consensus promoter in sexual females (20,791 tpm) represents just over 2% of all sequenced CAGE tags within that state. An illustration of the core and proximal promoter region of the gene is shown in Figure 6C, including the consensus promoter region and major CTSS identified by this study. The core promoter contains a TATA box (5’-TATATA-3’) at ‐27. We looked in the proximal promoter region for the juvenoid response element (JRE; 5’-CTGGTTA-3’) identical to the one reported in D. magna (Gorr et al. 2006), but did not find one. We anticipate that future investigation will identify the precise cognate cis-regulatory elements within this region. An additional example of sex-specific expression is shown in Figure S4, where upregulation of the consensus promoter for the gene encoding the egg protein vitellogennin among asexual females is presented.

Figure S4:
Figure S4:

State-specific upregulation of the gene of the precursor egg protein vitellogennin (VTG) in asexual females. The number of aligned CAGE tags observed upstream of the VTG gene (ID:322419) are plotted within the genomic region that surrounds the VTG gene (scaffold_47:116142-127656) for all three states. The number of CAGE tags at each position are represented by the y-axis of each plot, respectively.

Figure 6:
Figure 6:

Extreme upregulation observed at the putative promoter of a hemoglobin gene in D. pulex sexual females. A. Mapped CAGE tags from each of the three surveyed states to an annotated hemoglobin gene (ID:315053) on scaffold 13 are shown. The frequency of CAGE tags observed at each genomic coordinate (x-axis) are indicated by the y-axes of each plot. Note that larger y-axis scales are applied for the sexual females plot due to the dramatically higher number of mapped CAGE tags observed at the same locus. B. Consensus promoter expression (number of CAGE tags in tpm) at the same genomic locus as Part A across all three states is presented in the left panel; in the right panel only the values for males and asexual females are shown to provide perspective. The standard error of the mean of all replicates is shown for each individual plot. C. Schematic illustration of the core and proximal promoter region of the hemoglobin gene (ID:315053). The major CTSS (+1) is identified by the blue arrow, and the TATA consensus sequence is represented by the red rectangle. The purple line represents the consensus promoter region identified by CAGE. The genomic coordinates for the sequences (all on Scaffold 13) are shown in black. Note that the sequence for the negative strand is shown; the illustration was flipped to improve legibility. The drawing was not made to scale.

Discussion

In this study, we performed CAGE (Kodzius et al. 2006, Takahashi et al. 2012b) to map 5′-mRNA ends and identify active promoters within the ubiquitous aquatic microcrustacean Daphnia pulex, providing a taxonomic extension to the picture of metazoan promoter architecture. We report an average of 11,448 TSRs across the three conditions, 12,662 unique TSRs, and 10,580 consensus promoters. This D. pulex promoter atlas provides the first comprehensive collection of cis-regulatory elements within Crustacea.

We measured the occurrence of our CAGE-derived annotations with sites within the D. pulex genome, finding that they are generally located in positions consistent with promoter regions. The observation of CTSSs downstream of coding regions is consistent with the findings in D. melanogaster, where 17% of CAGE peaks were detected within annotated 3'UTR regions (Hoskins et al. 2011). The possible functions of CTSSs observed in CDSs and downstream of coding genes are challenging to interpret: they could represent the biochemical background of CAGE (Hoskins et al. 2011) or could alternatively represent bona fide RNA Pol II-derived transcripts. The latter case would suggest conflict with existing gene annotations, which can be resolved as more transcriptome analysis is performed in D. pulex. Approximately 82% of total aligned CAGE tags map upstream of annotated protein-coding genes (Figure 1C), a similar figure to that reported in Drosophila embryos (86%) (Hoskins et al. 2011). The overall incidence of TSRs upstream of coding genes (83%) mirrors that of CAGE tags (82.3%), suggesting that most TSRs in our dataset are positioned in locations consistent with the promoters of coding genes. The collection of TSRs (17%) located elsewhere is likely to contain a number of bona fide promoters.

The total number of unique TSRs defined here, 12,662, is close to the total of 12,454 promoters reported in D. melanogaster (Hoskins et al. 2011). This result may indicate a greater similarity in the number of protein coding genes between D. pulex and D. melanogaster than is presently predicted by the present genome annotation. The existing gene count for D. pulex (30,907) (Colbourne et al. 2011) is considerably larger than the approximately 17,000 currently annotated in D. melanogaster. The high depth of sampling and variety of stages measured in this study would be expected to reveal a similar ratio of active TSRs to annotated genes to what was observed in D. melanogaster (Hoskins et al. 2011). However, given the limited functional genomic evidence in D. pulex currently available, we cannot unequivocally conclude how many of the TSRs we report are, in fact, “true” promoters beyond evaluating their relationship to the current gene annotation. As it currently stands, this reality may lend greater weight to those TSRs that are found upstream of annotated coding genes. Further functional genomic (e.g. RNA-seq) analysis will be helpful to reconcile these existing discrepancies. We propose that the promoter atlas presented here be utilized to form an important component of a new, improved gene annotation in D. pulex.

We explored the properties of the consensus promoters within our D. pulex promoter atlas. The distribution of consensus promoter widths observed are consistent with what is seen in D. melanogaster (Figure 2A) (Hoskins et al. 2011, Chen et al. 2014). A proportion of the consensus promoter widths are long, including 1104 (10.4%) with widths longer than 30 bp (Figure 2A). This value is also similar to the amount observed (10.8%) in D. melanogaster (Hoskins et al. 2011). Promoters with long widths have also been observed in human, mouse (Carninci et al. 2006), and more recently, C. elegans (Saito et al. 2013). The distribution of consensus promoter shapes (Figure 2A, inset) indicates that both broad and peaked transcription initiation patterns are observed at D. pulex promoters. The observation that shape distribution is bimodal (Figure 2A, inset) agrees with previous models of promoter classes and provides rationale for the classification of promoters according to shape. We found that broad promoters exhibited higher promoter expression than did peaked promoters (Figure 2E), but we did not observe the same relationship between width and expression (data not shown). This suggests that shape is a more faithful representation of CTSS distribution and TSR properties than breadth alone. Our finding that broad promoters have higher promoter expression agrees with the available evidence in other species. In D. melanogaster, promoter width was positively associated with CAGE tag count (the equivalent to “expression” as defined here) (Hoskins et al. 2011). In D. melanogaster and elsewhere, broad promoters are associated with higher expression and genes with constitutive expression (Lenhard et al. 2012). While we did not directly address the relationship between promoter class and gene function in this study, such a comparison will be possible using these data, particularly as the functional annotation (i.e the Gene Ontology) of D. pulex genes improves.

We observe a strong preference for specific dinucleotides (CA, GA, GC, GG and GT) at CTSSs (Figure 2B). These results are partly in line with what is known elsewhere; the CA dinucleotide is located at [-1,+1] in Initiator (Inr)-containing promoters (Kadonaga 2012), and purines (A and G) are enriched at the TSS in metazoans, where studied (Nepal et al. 2013, Sandelin et al. 2007, Fitzgerald et al. 2006). However, three of the four over-represented dinucleotides (GA, GC, and GG) have guanines at ‐1, which is observed less commonly in metazoans. D. melanogaster, the most closely related species for which CAGE data are available (Hoskins et al. 2011, Chen et al. 2014), is enriched for YR at [-1,+1]; no enrichment of dinucleotides with G at ‐1 is reported. In human, where core promoters tend to be GC-rich (Fitzgerald et al. 2004; 2006), YR, but no GN dinucleotides, are enriched at initiation sites (Frith et al. 2008, Sandelin et al. 2007).

Our data suggest the overall nucleotide preferences of D. pulex are unusual compared of other metazoans that have been similarly surveyed. We observe the CA dinucleotide at approximately 12% of CTSSs, which is identical to canonical YR code at initiation sites and agrees with the sequence of Initiator (Inr) at the [-1,+1] position (Butler and Kadonaga 2002). By contrast, the other four enriched dinucleotides reported here are observed less frequently at the initiation sites in other metazoans. This may suggest the presence of one or more alternative initiators in D. pulex. We exclude the trivial explanation, 5′ guanine addition bias sometimes observed in CAGE studies (Carninci et al. 2006), for the observed GN enrichment because these were corrected for by our analysis pipeline (see Methods).

Our de novo discovery revealed eight distinct enriched motifs that we call the D. pulex core promoter set (Dpml-Dpm7; Figure 3). Of the eight D. pulex core promoter elements, three have significant sequence identity with a core promoter element in D. melanogaster. We find correspondence to major metazoan core promoter elements: Dpm2, with the consensus TATAWAA, displays similarity to the TATA element in Drosophila (TATAAA), and the consensus of the putative Inr motif Dpm3 (NCAGT) has significant identity to the Initiator motif (Inr) of fruit fly, which is NCAKTY (Ohler et al. 2002) (Figure 4F). The putative TATA Dpm2 and Inr Dpm3 are enriched between ‐30 and +1 (Figure 4B), respectively, consistent with their positions elsewhere within metazoans (Juven-Gershon and Kadonaga 2010). This strongly suggests that we have identified the TATA and Initiator motifs in D. pulex. The motif Dpm5 (TGGCAAC), observed at 15.3% of promoters, bears significant identity to the Ohler8 motif (-YGGCARC-) in D. melanogaster (Ohler et al. 2002). Dpm5 is enriched at approximately +50 (Figure 4D); the D. melanogaster Ohler8 motif has an equivalent, but more modest, peak at the same position (Down et al. 2007). The cis-regulatory role of Ohler8 is unknown, but it has been validated separately on several occasions since its initial discovery (Fitzgerald et al. 2006, Hoskins et al. 2011). In our study, the Ohler8-like Dpm5 motif was observed in a smaller fraction of promoters than observed in D. melanogaster (15.3% vs. 23.2%) (Ohler et al. 2002).

The remainder of the Daphnia promoter motif set is less well-characterized. The five other motifs within our D. pulex core promoter set, Dpml, Dpm6, Dpm7 and Dpm8 (Figure 3), lack similarity to any member of the core promoter list in D. melanogaster. Two of these exhibit a degree of positional enrichment relative to the TSS. Dpml is enriched broadly between approximately ‐40 and ‐75. Dpm4 exhibits a sharp positional enrichment at ‐10, and a second, wider distribution surrounding ‐50. No positional enrichment was observed among Dpm6, Dpm7, and Dpm8 (Figure 4D and data not shown), suggesting that they lack location preferences within core promoter regions.

The core promoter motif discovery described in this study is the first comprehensive glimpse into the cis-regulatory repertoire of D. pulex, and indeed for any crustacean. We observe strong cognates to core promoter elements in more well-studied metozoan genomes, including D. melanogaster. Collectively, these data support a model for the composition of the D. pulex core promoter (Figure 4E). Comparisons between our D. pulex core promoter model and the established model in D. melanogaster highlight the similarity of the reported TATA and Inr elements between the two species, but also underscores the absence of two canonical fly core promoter elements (BRE and DPE) (Butler and Kadonaga 2002) in our set of core promoters (Figure 4F). A finely-tuned motif discovery approach that selects only specific promoter classes (e.g. only Inr-containing promoters) is necessary as it would be more suited for discovery of BRE and DPE, which are less abundant than TATA and Inr.

In total, 3 of 8 Dpm motifs identified by our study lack obvious homologs in Drosophila. While we cannot propose precise functions for these putative core promoter elements, the overall positional enrichment and motif co-occurrence data (Figure 4A-4D) suggests that core promoters in D. pulex may group into TATA and TATA-less categories. In D. melanogaster, promoters that contain TATA, Inr, and a small number of other elements (including Pause Button, which we do not find in our set) are very likely to exhibit a peaked shape (Hoskins et al. 2011). By contrast, broad promoters are depleted for TATA and Inr (Rach et al. 2009, Hoskins et al. 2011); in mammals, they are associated with CpG Islands (Lenhard et al. 2012). Our finding that TATA and Inr-containing promoters have a more peaked shape than TATA-less promoters (Figure 4G) is consistent with this model. A complete characterization of the relationship between core promoter (i.e. Dpm) motif composition (especially TATA and Inr) and TSR shape and expression will require further analysis of the evidence generated in this study.

D. pulex is an important model in which to study the maintenance of sexual and asexual reproduction (Hebert 1981, Tucker et al. 2013); we analyzed the genes associated with differentially-expressed promoters observed between asexual females and sexual females (Figure 5C and 5D) and both sexuals (sexual females and adult males; Figure S3). Our observation of strong enrichment cell-cycle pathways (KEGG IDs: 04110 and 04115) among genes upregulated in asexual females (Figure S6) was unexpected. Upon closer inspection, we find strong overlap between genes in these categories and those belonging to two enriched meiosis-related pathways (Progesterone-mediated oocyte maturation (04914) and Oocyte meiosis (04114); a number have been annotated as meiotic in D. pulex (Schurko et al. 2009). The observation of upregulated meiosis genes in asexual females (Figure 5E) was surprising, but is consistent with what is known about the functions of some of the genes in question. The most compelling of these examples is Cdc20 (ID:326123; NCBI_GN0_7600067), which is more than two-fold upregulated (169.4tpm to 76.2tpm) in asexual females. In mammals, Cdc20 acts with the APC to trigger progression through prophase during Meiosis I (Homer et al. 2009). Increased expression of Cdc20 would be expected to hasten the exit from Meiosis I-like cell-division. Cdc20 misexpression is known to disrupt Meiosis I; mice hypomorphic for Cdc20 were shown to be infertile (or nearly so) due to chromosomal lagging and mis-alignment during Meiosis I (Jin et al. 2010).

Figure S5:
Figure S5:

Gene Ontology (GO) categories that are enriched among genes whose consensus promoters are significantly (p <0.01) i) upregulated in asexual females and ii) upregulated in sexual females within our study. Gene Ontology IDs and Pathway Names are shown.

Figure S6:
Figure S6:

KEGG pathways enriched among the genes of consensus promoters upregulated within asexual females (vs. sexual females). Meiosis-related pathways are shaded in gray, along with number of genes expected and observed within each pathway, and the corresponding odds ratio and p-value.

Figure S7:
Figure S7:

Subgraphs of the most enriched GO terms found in differentially-expressed genes found sexual and asexual females. Rectangles represent the five most significantly enriched GO terms, and are colorcoded from least significant (yellow) to most significant (red). Circular nodes represent GO terms within the GO semantic hierarchy. General information about each node is printed within each node, including the GO ID, a brief descriptor, the calculated p-value and the number of genes containing each individual term. A. GO Molecular Function (MF) categories enriched in asexual females. B. GO Biological Process (BP) categories enriched in asexual females. C. GO Molecular Function (MF) categories enriched in sexual females. D. GO Biological Process (BP) categories enriched in asexual females.

Although we lack comparable sources of expression data in Daphnia, the apparent increase in Cdc20 expression we observe here parthenogeneisis is consistent with current model of parthenogenic oogenesis in D. pulex, which is known to consist of abortive Meiosis I followed by a normal, Meiosis II-like division (Hiruta et al. 2010). We posit that the apparent differential regulation of meiosis and cell-cycle genes observed here is evidence for the transcriptional changes to meiosis that accompany parthenogenesis in D. pulex. However, it must be emphasized that additional molecular and cytological work will be required to appropriately address this question.

Finally, the identity and genomic position of several genes upregulated in asexual females on scaffolds associated with the evolution of asexuality (Figure 5E) is worth noting. Among these are Cdc20 (scaffold 76/ChrVIII) and HDAC (scaffold 13/ChrIX), two genes that were recently shown to be strongly upregulated in cyclic parthenogenesis (relative to obligate parthenogenesis) in Bdelloid rotifers (Hanson et al. 2013).

Taken together, our large-scale analysis of transcription initiation in the microcrustacean D. pulex provides the first glimpse of cis-regulation and core promoter architecture in Crustacea. We find that D. pulex exhibits similar features of promoter architectures relative to fly and mammals, including peaked promoters associated with TATA and Inr and constitutively-expressed broad promoters. We also detect major constituents of its core promoter that lack an obvious ortholog in fly, suggesting some degree of novelty within the core promoter of D. pulex. It is intended that the data presented here, including the D. pulex promoter atlas described here, serve as a resource for future investigations within D. pulex, and comparative genomic analysis across metazoan diversity. We anticipate that, using this resource, comparisons between D. pulex and the fruit fly and fellow arthropod D. melanogaster, which are '600My diverged (Hedges et al. 2006), will be of particular utility.

Methods

Focal genotype and maintenance of individuals

The Daphnia pulex genotype used in this work was isolated from Portland Arch Pond (Warren County, Indiana, USA; geographic coordinates: 40.2096°, ‐87.3294°) and is identified as PA13-42 (hereafter PA42). The PA42 clone originates from a well-characterized natural population (Lynch et al. 1989). D. pulex individuals from the PA42 clone are cyclical parthenogens, meaning that they are capable of reproducing both asexually through eggs that develop directly or sexually through diapausing eggs. All individuals used in this study were the result of asexual reproduction. Females were maintained in 3L containers containing COMBO media (Kilham et al. 1998) (diluted 1:1 with water) at 20°C and fed Scenedesmus at approximately 100,000 cells/mL. New offspring were removed and placed in new containers daily. Asexual females, pre-ephipial (sexual) females, and males were isolated from culture on separate occasions using strainers of differential sizes and visual identification under a dissecting microscope. Males can be visually distinguished from females based on the criteria of enlarged atennules and flattened ventral carapace margin. The current reproductive mode of females can be determined by phenotyptic differences in yolk-filled ovaries: females currently reproducing asexually have more “bulbous” ovaraies that tend to be more green in color, while females currently reproducing sexually have blackish yolks of reduced size and a smoother external margin.

RNA isolation and quantification

Whole D. pulex individuals (approximately 50–75) were collected from fresh cultures from each of the three aforementioned states. Collections were homogenized manually using a small pestle in microcentrifuge tubes containing lysis buffer. Isolation of total RNA was performed using solid phase extraction (Bioline, Inc). Samples were snap-frozen in liquid nitrogen and stored at ‐80°C. RNA samples were quantified and evaluated for quality and using the Bioanalyzer 2100 (Agilent Technologies).

CAGE library preparation and sequencing

A multiplexed CAGE library was constructed as described (Takahashi et al. 2012a) from 5μg total RNA sample using the nAnT-iCAGE protocol (Murata et al. 2014) (K. K. DNAForm, Yokohama, Japan). Briefly, total RNA was reverse transcribed using a random “N6 plus base 3” primer (TCTNNNNNN), using SuperScript III reverse transcriptase (Thermo Fisher). Following oxidation (with sodium peroxide) and biotinylation of the m7G cap structures, 1st-strand-complete mRNA:cDNA hybrids were bound with streptavadin beads, pulled down with a magnet, and released. This was followed by ligation of the 5′ linker, which includes the 3nt barcode (e.g. iCAGE_01 N6 5′-CGACGCTCTTCCGATCTACCNNNNNN‐ 3′) followed by 3′ linker ligation. Finally, 2nd-strand synthesis was performed using the nAnT-iCAGE 2nd primer (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTT-3′), creating the final dsDNA product. For a more detailed protocol, please see the following (Murata et al. 2014).

qRT-PCR evaluation of CAGE libraries

Prior to sequencing, relative mRNA:rRNA ratios were measured for each CAGE library using quantitative reverse-transcriptase PCR (qRT-PCR) with SYBR Green I (Life Technologies). The control gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was selected, (Forward Primer: 5′-ACCACTGTCCATGCCATCACT-3′, Reverse Primer: 5′-CACGCCACAACTTTCCAGAA-3′) and was measured against 18S ribosomal mRNA (Forward Primer: 5′-CCGGCGACGTATCTTTCAA-3', Reverse Primer: 5′-CACGCCACAACTTTCCAGAA-3'). Biological replicates of each of the three states were reflected in the final CAGE library (n=3 for both female groups, n=2 for males). Finally, the completed CAGE library was sequenced using Illumina HiSeq2000 (single-end, 50bp reads) at the University of California, Berkeley Genome Sequencing Laboratory (Berkeley, CA, USA).

CAGE processing, alignment, and rRNA filtering

All CAGE-adapted sequence reads (1.82×108) were demultiplexed (http://hannonlab.cshl.edu/fastx_toolkit/index.html), creating eight separate fastq files corresponding to the original CAGE libraries. All CAGE-adapted sequences (47bp) from each library were aligned separately using bwa (Li and Durbin 2009) to the D. pulex assembly vl.1 (JGI) (Colbourne et al. 2011). Prior to downstream analysis, CAGE alignments (in. bam format) were subjected to a filtering step (rRNAdust; http://fantom.gsc.riken.jp/5/sstar/Protocols:rRNAdust) to remove rRNA sequences (28S, 18S, and 5S). The SAM flags of identified rRNA reads in the alignment were changed to “unmapped”. Overall, 1.22×8 CAGE reads (67.0% of the total) mapped successfully (Table 1), and these were utilized in subsequent analyses. Evaluations of CAGE alignments and pooling of multiple libraries was performed using Samtools (Li et al. 2009). The distribution of CAGE tags within the D. pulex genome was determined using BEDtools (Quinlan 2014). Non-overlapping genomic intervals were created using BEDtools from the Joint Genome Institute’s (JGI) Frozen Gene Catalog annotation (“FrozenGeneCat-alog20110204.gff3”) located at http://genome.jgi.doe.gov/Dappu1/Dappu1.download.html. Further computational methods are described in Supplemental Methods.

Data Access

CAGE sequence data in this manuscript have been deposited in NCBI's Gene Expression Omnibus (Edgar et al. 2002) (http://www.ncbi.nlm.nih.gov/geo) and will be made public immediately after publication of the submitted manuscript.

Author Contributions

RTR co-conceived the idea, performed the computational analyses and experimental work and wrote the paper. KS performed experimental and culturing work and contributed to the paper. VPB developed the idea, contributed to the paper and edited the paper. ML conceived the idea and edited the paper.

Disclosure Declaration

The authors declare that they have no competing interests.

Acknowledgments

We are grateful to Peter Cherbas and Sen Xu for critical comments to the manuscript, and to Teresa Crease for assistance with aspects of the methodology. We would like to thank Kim Young for her work culturing Daphnia collections. We thank Xiangyu Yao for contributing to our motif discovery workflow. This work was supported by a grant-in-aid to ML from the NIH (Identifier: 1R01GM101672-01A1). This work used the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 Instrumentation Grants S10RR029668 and S10RR027303.

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Posted April 28, 2016.
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Promoter architecture and sex-specific gene expression in the microcrustacean Daphnia pulex revealed by large-scale profiling of 5′-mRNA ends
R. Taylor Raborn, Ken Spitze, Volker P. Brendel, Michael Lynch
bioRxiv 047894; doi: https://doi.org/10.1101/047894
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