ABSTRACT
Pioneer factors such as Zelda help initiate zygotic transcription in Drosophila early embryos, but whether other factors support this dynamic process is unclear. Odd-paired (Opa), a zinc-finger transcription factor expressed at cellularization, controls transition of genes from pair-rule to segmental patterns along the anterior-posterior axis. Finding that Opa also regulates late expression through enhancer sog_Distal, along the dorso-ventral axis, we hypothesized that Opa acts as a general timing factor. Chromatin-immunoprecipitation (ChIP-seq) confirmed Opa in vivo binding to sog_Distal but also identified widespread binding throughout the genome, comparable to Zelda. Furthermore, chromatin assays (ATAC-seq) demonstrate that Opa, like Zelda, influences chromatin accessibility genome-wide, suggesting both are pioneer factors with common as well as distinct targets. Lastly, embryos lacking opa exhibit widespread, late patterning defects spanning both axes. Collectively, these data suggest Opa, a general timing factor and likely a late-acting pioneer factor, heralds in a secondary wave of zygotic gene expression.
INTRODUCTION
The transition from dependence on maternal transcripts deposited into the egg to newly transcribed zygotic transcripts is carefully regulated to ensure proper development of early embryos. During the maternal-to-zygotic transition (MZT), maternal products are cleared and zygotic genome activation occurs (rev. in Hamm and Harrison, 2018; Vastenhouw et al., 2019). In Drosophila embryos, the first 13 mitotic divisions involve rapid nuclear cycles (nc), that include only a short DNA replication S phase and no G2 phase, and the nuclei are not enclosed in separate membrane compartments but instead present in a joint cytoplasm (Foe and Alberts, 1983). This streamlined division cycle likely relates to the fast development of Drosophila embryos, permitting rapid increase in cell number before gastrulation in a matter of a few hours. Dynamic gene expression is initiated during the early syncytial stage, as early as nc7, and continues to the cellularized blastoderm stage (Ali-Murthy and Kornberg, 2016; Lott et al., 2011). Gene expression patterns may be transient or continuous, lasting through gastrulation or beyond (Kvon et al., 2014). This process is controlled by a specific class of transcriptional factors (TF), called pioneer factors, that bind to closed chromatin cis-regulatory regions to create accessible binding sites for additional transcription factors during development (Iwafuchi-Doi and Zaret, 2014). A pivotal factor in Drosophila embryogenesis is the pioneer TF Zelda (Zld), a ubiquitous, maternal transcription factor that binds to promoters of the earliest zygotic genes and primes them for activation (Harrison et al., 2011, 2010; Liang et al., 2008). It was unknown whether a similar regulation exists in other animals, until Pou5f1 (homolog of the mammalian Oct4) was shown to act in an analogous manner to Zld in that it controls zygotic gene activation in vertebrates (Leichsenring et al., 2013).
The exact molecular mechanism that supports widespread activation of zygotic gene expression is not known, but several regulatory mechanisms have been proposed. One model suggests that a decrease in histone levels occurs over time that provides an opportunity for the pioneer factors that herald zygotic gene expression to successfully compete for DNA, in order to access and activate transcription (Hamm and Harrison, 2018; Shindo and Amodeo, 2019). For example, Zld is a pivotal activator of the MZT as it increases accessibility of chromatin at enhancers, to facilitate binding of other transcriptional activators to these DNA sequences and thereby allow initiation of zygotic gene expression (Harrison et al., 2011; Liang et al., 2008; Nien et al., 2011). Zld binds nucleosomes and is considered a pioneer factor (McDaniel et al., 2019). Loss of Zld leads to a large-scale, global decrease in zygotic gene expression as many enhancer regions remain inaccessible and thus non-functional (Schulz et al., 2015). By facilitating chromatin accessibility, Zld has been shown to influence the ability of morphogen transcription factors, Bicoid and Dorsal, to support morphogen gradient patterning (Foo et al., 2014; Xu et al., 2014). While Zld is clearly pivotal for supporting MZT, some genes continue to be expressed even in its absence (Nien et al., 2011). In particular, as chromatin accessibility in the early embryo has recently been shown to be a dynamic process (Blythe and Wieschaus, 2016), it is possible that Zld contributes in a stage-specific manner. It is likely, therefore, that other pioneer factors also exist that contribute to zygotic genome activation.
Furthermore, the embryo undergoes a widespread state change after the 14th nuclear division, and this developmental milestone is classified as the midblastula transition (MBT) (Foe and Alberts, 1983; Shermoen et al., 2010). The cycle slows dramatically and nuclei become cellularized relating to when embryonic programs of morphogenesis and differentiation initiate. Cell membranes emerge to encapsulate nuclei, forming a single layered epithelium. In addition, at nc14, developmental changes relating to DNA replication occur; namely a lengthened S-phase and the introduction of G2 phase. MBT is also associated with clearance of a subset of maternally provided mRNAs, large-scale transcriptional activation of the zygotic genome, and an increase in cell cycle length (Tadros and Lipshitz, 2009; Yuan et al., 2016). We hypothesized that other late-acting pioneer factors manage the MBT in addition or in place of Zld.
An attractive research target in the field of developmental biology is the transcription factor Zinc finger in the cerebellum (ZIC human ortholog); its role in early developmental processes has been established across major animal models and also linked to human developmental pathology (rev. in Aruga and Millen, 2018; Houtmeyers et al., 2013). However, the founding member of the Zic family is the Drosophila gene odd-paired (opa) (Aruga et al., 1996; Hursh and Stultz, 2018). opa is a broadly expressed gene of relatively long transcript length (∼17 kB) that initiates expression at mid-nuclear cycle 14 (Benedyk et al., 1994; Cimbora and Sakonju, 1995) and recently was shown to function as a timing factor in early embryos controlling segmentation (Clark and Akam, 2016). Opa protein has a DNA binding domain containing five Cys2His2-type zinc fingers, and shares homology with mammalian Zic1, 2, and 3 transcription factors. It is categorized as an atypical pair-rule gene, as it is expressed in a broad domain within early embryos rather than the 7-stripe pattern typical of other pair-rule genes. Its pair-rule phenotype relates to its ability to act as a timing factor to control the transition of genes with pair-rule patterns to segmental expression pattern (i.e. from 7-to 14-stripes) (Clark and Akam, 2016). opa is a widely expressed gene and has additional functions throughout development. For example, opa mutant embryos die before hatching, exhibiting aberrant segmentation, but mutant embryos also exhibit defects in larval midgut formation (Cimbora and Sakonju, 1995). During midgut formation, it regulates expression of a pivotal receptor tyrosine kinase required for proper morphogenesis of the visceral mesoderm (Mendoza-García et al., 2017). In addition, at later stages, Opa supports temporal patterning of intermedial neural progenitors of the Drosophila larval brain (Abdusselamoglu et al., 2019). Therefore, opa supports a number of patterning roles throughout development, several relating to temporal patterning.
In addition, previous studies had suggested that Opa can influence the activity of other transcription factors to promote gene expression. A well characterized target of Opa in the early embryo is sloppy-paired 1 (slp1), a gene exhibiting a segment polarity expression pattern, for which two distinct enhancers have been identified that are capable of responding to regulation by Opa and other pair-rule transcription factors (Cadigan et al., 1994; Prazak et al., 2010). One of these, the slp1 DESE enhancer, mediates both Runt-dependent activation and repression (Hang and Gergen, 2017). Opa is clearly a central player in slp1 regulation by supporting Run’s role as activator at DESE, but its uniform expression pattern does not provide positional information as other pair-rule gene inputs. Furthermore, our recent study showed that Runt regulates the spatiotemporal response of another enhancer, sog_Distal (also known as sog_Shadow; Hong et al., 2008; Ozdemir et al., 2011) to support its expression in a broad stripe across the dorsal-ventral (DV) axis on both sides of the embryo (Koromila and Stathopoulos, 2019). Using a combination of fixed and live imaging approaches, our analysis suggested that Run’s role changes from repressor to activator, over time in the context of sog_Distal; late expression requires Run input as activator. These analyses of slp1 DESE and sog_Distal regulation supported the view that Opa might provide temporal input into both these enhancers.
The current study was initiated to investigate whether Opa supports late expression through the sog_Distal enhancer. Previous studies had not linked Opa to the regulation of DV patterning. Nevertheless, through mutagenesis experiments coupled with live in vivo imaging, we provide evidence that Opa does regulate expression of the sog_Distal enhancer and that its role is indeed late-acting, occurring in embryos at mid-nc14 onwards whereas the enhancer initiates expression at nc10. Furthermore, using both chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-seq) as well as single-embryo Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), our whole-genome data support the view that Opa supports patterning in the embryo by serving as a general timing factor, and possibly a pioneer factor, that broadly influences zygotic transcription at around the time that embryo begin cellularization to support the late phase of the maternal-to-zygotic transition.
RESULTS
Opa regulates the sog_Distal enhancer demonstrating a role for this pair-rule gene in DV axis patterning
In a previous study, we created a reporter in which the 650 bp sog_Distal enhancer sequence was placed upstream of a heterologous promoter from the even-skipped gene (eve.p), driving expression of a compound reporter gene containing both tandem array of MS2 sites and the gene yellow including its introns (Koromila and Stathopoulos, 2017). The MS2 cassette contains 24 repeats of a DNA sequence encoding an RNA stem-loop when transcribed. The stem-loop is recognized by an MCP-GFP fusion protein, as MCP is a phage protein able to bind the RNA-stem loop whereas GFP provides a strong green signal that can be monitored within the nucleus of Drosophila embryos at the site of nascent transcript production associated with the transgene (Bothma et al., 2014). This compound reporter gene allows assay of spatiotemporal gene expression live, through MS2-MCP imaging, or in fixed samples through in situ hybridization to gene yellow. We used this sog_Distal reporter to assay gene expression in the early embryo, and output was analyzed using a previously defined computational approach to analyze spatiotemporal dynamics (Koromila and Stathopoulos, 2019). Expression through this enhancer initiates at nc10 and continues into gastrulation. In this study, late expression through this enhancer was assayed from nc13 into nc14. Furthermore, nc14 was assayed in four intervals each representing ∼10 min of developmental time: nc14a, nc14b, nc14c, and nc14d, due to its longer length (i.e. ∼45 min compared to ∼15 min for nc13).
In our previous study, mutation of the single Run site in the sog_Distal enhancer led to expansion of expression early (i.e. nc13 and nc14a) but loss of the pattern late (i.e. nc14c and nc14d) (Koromila and Stathopoulos, 2019). These results suggested that Run’s role switches from that of repressor to activator in the context of sog_Distal enhancer (Koromila and Stathopoulos, 2019). Other studies had suggested that Run can function as either repressor or activator depending on context, as the response of different enhancers to Run is influenced by the presence or absence of other specific transcription factors (Hang and Gergen, 2017; Prazak et al., 2010; Swantek and Gergen, 2004). In particular, Opa is critically required for the Runt-dependent activation of slp1 (Swantek & Gergen, 2004). We hypothesized that Opa also influences Run’s role in the context of the sog_Distal enhancer and thus that Opa functions to support late expression, when Run was identified as providing activating input (Koromila and Stathopoulos, 2019).
Surprisingly, the sog Distal 650 bp sequence contains five matches to the 12bp Opa binding site consensus based on vertebrate Zic1/Zic3 when one mismatch is allowed (JASPAR; Figure 1F, G). Upon mutation of these five sites through introduction of 2-4 bp changes to each sequence (i.e. sogD_ΔOpa), we found reporter expression as assayed by MS2-MCP live in vivo of nascent transcription was relatively normal up to stage nc14b but, subsequently, exhibited a decrease that is apparent at nc14c (Figure 1B; Movie 1). Using a previously described quantitative analysis pipeline (Koromila and Stathopoulos, 2019), we quantified the MS2-MCP signal in embryos containing either the wildtype sog_Distal or sogD_ΔOpa reporters, confirming that Opa expression is greatly reduced at nc14c for the mutant reporter compared to wildtype (Figure 1 - supplement figure 1E). A similar loss of late expression only (i.e. nc14c onwards) was obtained when even a single Opa site is mutated (Figure 1 - figure supplement 1A-C) comparable to when the Run site is mutated (Koromila and Stathopoulos, 2019). These results support the view that Opa promotes expression through sog_Distal from nc14c onwards at least in part by serving to switch Run from repressor to activator (see Discussion).
In this and all other subsequent figures lateral views of embryos are shown with anterior to the left and dorsal side up, unless otherwise noted.
(A, B) Stills from representative movies of the two indicated sog_Distal MS2-yellow reporter variants (A) sog_Distal (A) or sogD_DOpa (B) in which 5 predicted Opa binding sites were mutated as shown in (G) to detect transcription via MS2-MCP-GFP (Koromila and Stathopoulos, 2019) imaging at three representative time points: nc13, nc14a, and nc14c. Blue dots for each construct indicate presence of GFP+ dots, representing nascent transcripts labeled by the MS2-MCP system; thresholding was applied and remaining signals identified by the Imaris Bitplane software, for visualization purposes only. Nuclei were labelled by Nup-RFP (Lucas et al., 2018). Scale bar represents 50μm.
(C, D) Anti-Opa (C) and anti-Zld (D) antibody staining of early wild-type embryos at nc13, nc14a and nc14c.
(E) Integrative Genomics Viewer (IGV) genome browser track of the sog locus showing Zld ChIP-seq data for embryos of stage 5 (GEO dataset GSM763061: nc13-nc14) in comparison with Opa ChIP-seq for embryos of two stages: 3h (stage 4/5) and 4h (stage 6/7) (see Methods). Zld and Opa 3h ChIP-seq samples are of overlapping timepoints, whereas Opa 4h ChIP-seq sample is later. Peaks of predicted binding are detected at the sog_Distal enhancer and weak binding is detected at the sog_Intronic enhancer (Markstein et al., 2002) and another putative enhancer in the third intron. Gray box marks the sog_Distal enhancer location that is, bound by both Zld and Opa (3h) early, but not bound late (Opa 4h).
(F) JASPAR consensus binding site for Opa based on mammalian Zic proteins identified using SELEX (Khan et al., 2018; Sen et al., 2010).
(G) Five matches to the Jaspar consensus binding site for Opa (F) are present within the 650bp sog_Distal enhancer region; 12 bp matches shown (black) allowing for 1 mismatch to consensus. These predicted Opa sites were mutated by introduction of changes (blue bases above sequence) predicted to eliminate binding creating sogD_Δopa (B; see Methods). Bases in bold (7bp) indicate matches to the Opa de novo motifs identified by ChIP-seq analysis (see I,J). For sake of comparison to consensus sequence, a subset of sites are shown as reverse complement.
(H) Consensus binding site for mus musculus Zic3/Opa homolog identified using ChIP-seq (Lim et al., 2010).
(I-K) Sequence logo representations of consensus binding site identified as the most significant site in the Opa 3 h(I) 3h, Opa 4h (J), or Zld (K) ChIP-seq datasets defined by HOMER de novo motif analysis (Central motif enrichment P-values 1e-566, 1e-354, and 1e-3283, respectively).
The timing of Opa expression supports a role for this factor in driving expression of sog_Distal at mid-nc14, approximately at the time of the MBT. Using an anti-Opa antibody (Mendoza-García et al., 2017), we examined spatiotemporal dynamics associated with Opa protein in the early embryo through analysis of localization in a time series of fixed embryos. Opa expression is absent at nc13, first observed at nc14a, and encompasses its mature full pattern approximately half-way into nc14, at nc14c (Figure 1C). The timing of Opa onset of expression correlates with the timing of loss of late expression from the sog_Distal reporter, observed when Opa sites are mutated (Figure 1C, compare with 1B). On the other hand, the ubiquitous, maternal transcription factor Zld is detected throughout this time period including at nc13 (Figure 1D). Loss of Zld input to sog_Distal through mutagenesis of Zld sites, contrastingly, leads to decreased spatial extent of the reporter pattern (sog_Shadow; Yamada et al., 2019) rather than an overall loss of expression as observed when Opa binding sites are mutated (Figure 1B).
These results suggested that Opa may function as a timing factor, to regulate expression of sog_Distal, specifically, mid nc14 and later. Recent studies have demonstrated that the opa gene is generally important for the temporal regulation of segmental patterning in Drosophila as well as in Tribolium (Clark and Akam, 2016; Clark and Peel, 2018). However, as our results suggest a role for Opa in the regulation of sog expression, which relates to DV patterning, we hypothesized Opa’s role extends beyond control of segmentation along the AP axis to patterning of the embryo, in general.
Use of anti-Opa antibody to conduct assay of in vivo occupancy to DNA through ChIP-seq analysis
To examine the in vivo occupancy of Opa in early Drosophila embryos, we conducted chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq). Anti-Opa antibody was used to immunoprecipitate chromatin obtained from two embryo samples of average age 3h (roughly stages 4-6) or 4h (roughly stages 5-7) (see Methods). Focusing on the Opa 3h ChIP-seq dataset, the MACS2 peak caller was used to identify 16,085 peaks, representing an estimate of the number of genomic positions that are occupied by Opa in vivo. 200 bp-regions associated with these peaks were analyzed using the HOMER program (Heinz et al., 2010) to identify overrepresented sequences that relate to transcription factor binding sites (see Methods). The most significant hit, present in over 19% of all peaks, is a 7 bp core sequence exhibiting partial overlap with the 12 bp Opa JASPAR site (Figure 1I, compare with 1F) as well as sharing significant homology with the binding site observed for mammalian homolog Zic transcription factors (e.g. see Figure 1H; Lim et al., 2010); however, this site exhibits less overlap with consensus binding site defined for Opa in vitro by systematic evolution of ligands by exponential enrichment (SELEX) (Sen et al., 2010). A second motif exhibiting extended homology with the JASPAR Opa consensus was also identified through analysis of the 3h ChIP-seq dataset, but this extended site is present at lower abundance (Figure 2 - supplemental figure 1B). In the sog_Distal enhancer sequences, the five initial matches made to the JASPAR Opa binding site consensus also match the Opa de novo ChIP-seq derived consensus in 6 of the 7 bases. However, there is a notable mismatch in the 3’ most position; while the de novo Opa consensus at 3h does not include A in this position, both the JASPAR site and de novo Opa consensus derived for the 4h ChIP-seq timepoint do (Figure 1F, J, compare with 1G). It is possible that the predominant Opa de novo motifs derived from the ChIp-seq datasets represent high affinity sites, whereas the five Opa sites found in sog_Distal represent lower-affinity, cooperating sites; alternatively, the de novo Opa consensus derived from ChIP-seq may relate to a particular heterodimeric complex that is present only in vivo in the embryo at the assayed stages.
We hypothesized that Opa might also support expression of sog_Distal late, specifically, following mid-nc14. Independent chromatin immunoprecipitation experiments have analyzed Zld in vivo binding at a similar stage (i.e. nc13-nc14 early is roughly equivalent to 3h) and detected widespread binding of Zld throughout the genome including at the sog_Distal sequence (Figure 1E) (Foo et al., 2014; Harrison et al., 2011). We compared Opa binding to sog_Distal as well as to other regions through comparison of Opa 3h or 4h ChIP-seq experiments. Opa ChIP-seq detects Opa occupancy at the sog locus, including at the sog_Distal enhancer sequence, in the 3h but not the 4h time point (Figure 1E). At gastrulation, sog_Distal enhancer output changes from a broad lateral stripe to a thin stripe expressed at the midline; it is possible that at this stage, expression of sog_Distal is no longer directly dependent on Opa.
Assay of overrepresented sites associated with Opa ChIP-seq peaks
Region overlap analysis was used to identify overlapping regions of occupancy for Opa (3h) and Zld using the respective ChIP-seq datasets obtained independently but assaying embryos of similar stage (see Methods). Each ChIP-seq experiment identified 16,085 peaks of occupancy representing locations in the genome that are occupied by either Opa or Zld. Opa and Zld have 6087 peaks in common (Opa-Zld overlap), whereas 9998 regions were bound by Opa alone (Opa-only) and 10781 regions were bound by Zld alone (Zld-only) (Figure 2D). When the Opa and Zld binding site consensus sequences, generated de novo by assay of the full ChIP-seq datasets (i.e. Figure 1I,K), are used to analyze binding in each of these classes, it is clear that these motifs are differentially enriched in the Opa-only (Figure 2A), Zld-only (Figure 2B), and Opa-Zld overlap regions (Figure 2C) (also see Figure 2 - supplemental figure 1).
(A-C) Aggregation plots showing enrichment of top de novo motifs identified from Opa (3h) peaks and Zld peaks surrounding. (A) Opa_only bound regions (after exclusion of Zld-only and Opa-Zld overlap peaks); (B) Zld-only bound regions (after exclusion of Opa and Opa-Zld overlap peaks); and (C) for Opa-Zld overlap regions.
(D) Venn diagram of ChIP-seq data from Drosophila embryos during cellularization showing the number of peaks called using MACS2 through analysis of: Opa ChIP-seq (blue) and Zld ChIP-seq (orange) data. Opa and Zld experiments used embryos of 2.5-3.5h in age (stage 5) or nc13/nc14 early, which are overlapping timepoints. Opa-only peaks (*); Opa-Zld overlap peaks (**); Zld-only peaks (***).
(E-G) Sequence logo representations of 2-3 most abundant motifs identified using HOMER de novo motif analysis within the three corresponding sets of peaks (A: Opa-only, B: Zld-only, C: Opa-Zld overlap peaks). As expected, motifs exhibiting match to the JASPAR Opa and Zld consensus sequences are found. In addition, motif exhibiting match to Dref (E), Caudal (Cad; F), and Trl/GAF (G) were also uncovered in particular datasets. Sequence logo height indicates nucleotide frequency; corresponding Percentage of peaks containing match to motifs also shown for each set, as indicated. P-values representing the significance of motifs’ enrichment compared with the genomic background are as follows: 1) Opa-only peaks: Opa motif enrichment P-value=1e-522; Dref motif enrichment P-value=1e-119, 2) Zld-only peaks: Zld motif enrichment P-value=1e-2833, Caudal motif enrichment P-value=1e-63, 3) Opa-Zld overlap peaks: Zld motif enrichment P-value=1e-240; insulator Trl/GAF motif enrichment P-value=1e-76; Opa motif enrichment P-value=1e-43 (see also Figure 2 - supplemental table 1).
The HOMER sequence analysis program was used to identify overrepresented motifs within these three classes of peaks: Opa-only, Zld-only, or Opa-Zld overlap; in order to identify associated motifs that might provide insight into the differential or combined functions of Opa and Zld. As expected, the top motifs in each class matched the Opa or Zld consensus sequences with 16% of the Opa-only peaks containing an Opa site; 55% of the Zld-only peaks containing a Zld site; and 5% and 15% of the Opa-Zld overlap peaks containing an Opa or Zld site, respectively (Figure 2E-G). In addition, the second-most significant site identified in each class of called peaks corresponds to Dref (6%) for Opa-only; Caudal (Cad; 14%) for Zld-only; and Trl/GAF (11%) for Opa-Zld overlap (Figure 2E-G). Dref (DNA replication-related element-binding factor) is a BED finger-type transcription factor shown to bind to the sequence 5′-TATCGATA (Hirose et al., 1993), a highly conserved sequence in the core promoters of many Drosophila genes (Ohler et al., 2002); whereas Cad encodes a homeobox transcription factor that is maternally provided but forms a concentration-gradient enriched at the posterior (Mlodzik et al., 1985) and exhibits preferential activation of DPE-containing promoters (Shir-Shapira et al., 2015). These sites and other additional, significant co-associated sites were uncovered when all called peaks within samples [i.e. Opa (3h), Opa (4h), and Zld] were analyzed by HOMER including a highly significant unknown motif associated with both Opa samples (Figure 1 - figure supplement 2).
Collectively, these results support the view that Zld acts early whereas Opa acts later, and that distinct sets of transcription factors likely help each of these factors to support their different roles (see Discussion).
Opa ChIP-seq peaks are associated with late-acting enhancers driving expression along both axes
A direct comparison of the Opa (3h) and Zld ChIP-seq occupancy at stage 5, through aggregation plots, suggests that the two transcription factors can bind either simultaneously to the same enhancers (e.g. Figure 2C) or independently to distinct enhancers (e.g. Figure 2A,B). Furthermore, the respective sites appear explanatory for the observed in vivo occupancy to DNA sequences as the matches to the consensus sequences correlate with the center of the peak (Figure 2A-C). Furthermore, the widespread binding of Opa in the genome supports the view that this factor functions broadly to support gene expression. Within the total set of 16085 Opa chromatin immunoprecipitation peaks associated with the 3h timepoint, we found, surprisingly, that Opa is associated with genes expressed along both axes: anterior-posterior (AP; Figure 3A-D) and dorsal-ventral (DV; Figure 3E-G), including but not limited to genes involved in segmentation (e.g. slp1 and oc: Figure 3C,D) as predicted by previous studies (e.g. Clark and Akam, 2016; Prazak et al., 2010). We also found evidence in occupancy trends for Opa and Zld that suggest these factors both influence the timing of enhancer action.
(A-H) IGV browser tracks of genes expressed along either the AP (A-D) or DV (E-H) axes showing single Opa (blue) and Zld (orange) ChIP-seq replicates (as indicated). Anti-Opa antibody was used to immunoprecipitate chromatin isolated from embryos ∼3 hours in age (see Methods). Published Zld ChIP-seq data for stage 5 embryos is shown as a point of comparison (GSM763061: nc13-nc14; Harrison et al., 2011). Gray boxes indicate regions with significant occupancy by both Opa and Zld as detected by ChIP peaks, which can be located at either promoter and/or distal regions (A, C-E, G, H). Light blue boxes indicate regions with significant Opa-only binding at promoter and/or distal regions (B, F).
(I, J) Heatmaps produced by deepTools (see Methods) were used to plot histone H3K4me3 (I) and H3K4me1 (J) signal intensities around different ChIP-seq regions (Zld-only, Opa-Zld overlap, and Opa-only bound ChIP-Seq peaks) at two different timepoints nc14a and nc14c (stage 5). Heatmaps are centered on ChIP-seq peak summits. Key indicates histone signal intensities (deepTools normalized RPKM with bin size 10) around different ChIP-seq regions.
Enhancers enriched for Opa-occupancy tend to be active starting at nc14, at the earliest, whereas earlier acting enhancers tend to be bound by Zld alone or Zld and Opa together (Figure 3A-G). Opa binding (3h) is associated with enhancers that are initiated in nc14: hb_stripe (Figure 3A; Koromila and Stathopoulos, 2017; Perry et al., 2012), slp1_DESE (Figure 3C; Prazak et al., 2010), oc_Prox (Figure 3D; Chen et al., 2012; Perry et al., 2011), and ind_moduleA (Figure 3G; Stathopoulos and Levine, 2005a). Additionally, Opa binding is also associated with enhancers that are activated later at gastrulation: hb_HG4-7 (Figure 3A; Hirono et al., 2012), eve_LE (Figure 3B; Fujioka et al., 1996), sim_VT40842 (Figure 3E; Kvon et al., 2014), and rho_SHA (Figure 3F; Pearson and Crews, 2014; Rogers et al., 2017). In particular, the hb_HG4-7 enhancer is not bound by Zld early but is bound by Opa increasingly from 3h to 4h timepoint (Figure 3A); in contrast, the hb_stripe enhancer, which is active earlier at nc14, is strongly bound by Zld and also exhibits a concomitant reduction in occupancy of Opa from the 3h to 4 h timepoint. Similarly, the eve_LE enhancer is bound by Opa, with little input from Zld, and is also late-acting relative to all other even-skipped (eve) stripe enhancers, as it mediates the refinement of all seven eve stripes after they are initially positioned (Figure 3B; Fujioka et al., 1996). The SHA enhancer associated with the gene rhomboid (rho_SHA) is bound by Opa predominantly and drives expression in the midline at gastrulation (Figure 3F); in contrast, the rho_NEE enhancer is bound by both Opa as well as Zld supports rho expression earlier in the cellularized blastoderm (Ip et al., 1992). Collectively, these results show that Opa binds to enhancers known to function at nc14 (stage 5) or later, at or following gastrulation (e.g. stage 6/7), and, in contrast, enhancers that receive input from Zld-alone or Opa+Zld tend to be active earlier. However, we can not dismiss a role for Zld at later stages, as the VT40842 enhancer associated with the gene single-minded (sim) is active later (stage 6) but is bound by both Opa and Zld at an earlier stage (Figure 3E).
To assay whether Opa binding changes in time, we compared Opa occupancy in the two ChIP-seq samples representing two timepoints, 3h (Opa_early) and 4h (Opa_late). There are 9995 peaks in common, 5859 peaks specifically associated with Opa_early, and 6657 peaks specifically associated with Opa_late suggesting changing targets over time (Figure 3 - supplemental figure 1C,D). For example, sog_Distal and hb_stripe enhancers are Opa-target only bound at the early (3h) timepoint (Figure 1E and 3A); whereas, in turn, Supervillin (Svil) is an Opa-target bound predominantly at the late (4h) timepoint (Figure 3H). However, the position of peaks, generally, does not appear to change much over time, as most Opa peaks continue to be localized in the same positions at both timepoints (Figure 3 - supplemental figure 1B).
Furthermore, Opa associates with promoters whether or not Zld is co-associated. While Zld was shown to preferentially associate to promoters at earlier time points (nc8; Harrison et al., 2011), we found that binding of Zld to Zld-only enhancers occurs in more distal regions at stage 5 (Figure 3 - supplemental figure 1A). It is possible that once Opa is expressed it preferentially associates with promoter regions and either competes and/or co-regulates with Zld (see Discussion).
H3K4me3 and H3K4me1 histone marks at nc14 are enriched at regions occupied by Opa
Previous genomics studies have demonstrated that particular histone marks correlate with active enhancers at different developmental stages in the early embryo. For example, there is a dramatic increase in the abundance of histone modifications at the MZT, coinciding with zygotic genome activation (Schulz and Harrison, 2019). We investigated whether Opa-only, Zld-only, and Opa-Zld overlap regions (Figure 2D) exhibit differences in chromatin marks that might support our working hypothesis that Opa-associated regions are active later than Zld-only regions. For the purposes of this analysis, ten published ChIP-seq datasets relating to histone/histone modifications were investigated to assay coincidence of any marks with Opa and/or Zld-bound regions identified by our analysis (see Methods). Only H3K4me3 and H3K4me1 were found to exhibit a difference in Opa versus Zld bound peaks (Figure 3I,J). Both histone marks are first detectable at the MBT, while absent prior to nc14a; whereas their associated genes are considered to be activated at later stages, for example, at MBT following early zygotic activation (Chen et al., 2013; Li et al., 2014).
Heatmap modules of deepTools (see Methods) were used to calculate and plot histone H3K4me3 and H3K4me1 signal intensities assayed at two timepoints, nc14a and nc14c, at different sets of ChIP-seq defined regions: Opa-only; Zld-only; or Opa-Zld overlap. Our analysis shows that Zld-bound regions are underrepresented for H3K4me3, as shown previously (Li et al., 2014), as well as exhibit less enrichment of H3K4me1 at both time points relative to Opa or Opa-Zld overlap bound regions (Figure 3I,J). To our surprise, Opa-Zld overlap and Opa-only bound regions are more enriched for both H3K4me3 and H3K4me1, compared to the Zld-only bound regions (Figure 3I,J). The unique histone methylation pattern exhibited by these classes of peaks, in particular, indicates that Opa targets are likely subject to later developmental regulation. The higher levels of H3K4me1 in the Opa-bound peaks could potentially also be connected to a poised state of late-acting enhancers (Koenecke et al., 2017) or because these regions correspond to other cis-regulatory elements, promoters or insulators, for example (Bonn et al., 2012; Rada-Iglesias et al., 2011) (see Discussion).
Global changes in chromatin accessibility result upon knock-down of Opa
We hypothesized that Opa functions as a pioneer factor to regulate temporal gene expression starting at nc14 in the celluarizing blastoderm. To test this view, we investigated whether Opa functions to regulate chromatin accessibility. We used ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) to investigate the state of chromatin accessibility in embryos that had been depleted of opa in comparison to wild type. To start, embryos were depleted of Opa by expression of a short hairpin (sh) RNAi construct (Staller et al., 2013) that we show is effective in reducing opa transcript levels when expressed at high levels maternally and zygotically in embryos (Figure 4A,B). In a side by side comparison, we found that opa RNAi embryos exhibit phenotypes similar to opa1 zygotic mutants (Figure 4 - supplemental figure 1B, C). However, opa expression is retained, though slightly reduced, in embryos in which zld levels are knocked-down, using a similar approach, through zld RNAi (i.e. sh zld; Figure 4C). This results suggests that opa expression is not completely under Zld regulation, and indicates these factors could have separable roles (see Discussion).
(A-C) In situ hybridization using opa riboprobe (cyan) in either wildtype (wt; A), opa RNAi (sh opa; B), or zld RNAi (sh zld; C) embryos (n=3-5 per genotype) at stage 5.
(D) Aggregation plot for wt chromatin accessibility (ATAC-Seq) signals ±2kb from the peak of Opa and Zld ChIP-seq called peaks. Signals were generated using deepTools (Ramírez et al., 2014) with RPKM method for normalization (see Methods).
(E-H) UCSC dm6 genome browser tracks of representative loci showing single replicates of nc14B (E-G) and nc14D (H) ATAC-seq. Examples of late enhancer regions that significantly lose accessibility, compared to wt, either in sh opa-only (blue; E, F, H) or in both sh opa and sh zld (orange) (G) enhancer regions are defined by gray boxes. Plots show mean normalized read coverage of the replicates.
To provide insight into the potentially different roles of these two transcriptional factors, Opa and Zld, that support gene expression, ATAC-seq analysis was conducted on individual, single embryos either control (wt), opa RNAi (sh opa), or zld RNAi (sh zld) and the results compared. To start, we determined the relative accessibility indices for Opa-only, Zld-only, or Opa-Zld overlap regions at stage 5 (i.e. Figure 2D) using deepTools (Ramírez et al., 2014) with RPKM method for normalization (see Methods). Regions occupied by both Opa and Zld through ChIP-seq (i.e. Opa-Zld overlap) had on average 2-2.5 fold higher ATAC-seq signal (i.e. accessibility) than those that were bound by either Zld or Opa alone as detected by ChIP-seq (i.e. Zld-only or Opa-only) (Figure 4D). In summary, occupancy of both Opa and Zld is a better indicator of open chromatin regions than either factor alone. As Zld has been documented to function as a pioneer factor, that helps to make chromain accessible, these results suggest that Opa may also function in this role.
Chromatin accessibility as identified through single-embryo ATAC-seq was also examined at particular enhancers that are bound by Opa. In these Opa-bound regions, three trends were observed: (i) regions require Opa for accessibility (e.g. Figure 3E, compare with 1E; Figure 3F, compare with 3A; Figure 4 - supplemental figure 2G); (ii) regions require both Opa and Zld for accessibility (Figure 4G, compare with Figure 3B); and (iii) regions require Zld, but not Opa, for accessibility (Figure 4 - supplemental figure 1F). These results suggest that Opa can influence chromatin accessibility, but not all regions that are bound by Opa require this factor for accessibility. It is possible that at Opa-Zld overlap regions, in which both factors are bound, either Opa or Zld can suffice to support accessibility. Furthermore, in studies of Zld accessibility using a distinct method, FAIRE-seq, it was determined that, while Zld is clearly important for facilitating chromatin accessibility in the early embryo, it is not the only factor that supports this function; some chromatin regions remain accessible in zld mutants (Harrison et al., 2010).
Opa-only occupied peaks require Opa to support their accessibility at mid-nc14
Co-occupancy of Opa and Zld to DNA regions correlates with increased chromatin accessibility compared to either factor alone (Figure 4D), suggesting Opa is able to influence chromatin accessibility. Furthermore, we found that in some cases Opa supports chromatin accessibility even in regions in which little or no Zld is bound (e.g. eve_LE; Figure 4G) and that Opa is expressed in zld mutants, though at reduced levels (Figure 4C). We hypothesized that Opa relates to the unidentified, Zld-independent pioneer activity.
We took a whole-genome approach towards assaying Opa’s function, and examined how chromatin accessibility is affected in Opa RNAi embryos compared to wildtype, when regions were separately analyzed based on Opa and Zld binding patterns: Opa-only, Zld-only, and Opa-Zld overlap (Figure 5A-D; also see Figure 3D). No obvious change in global accessibility was identified for the Zld-only or Opa-Zld overlap regions (Figure 5A-C); whereas, in contrast, the Opa-only regions exhibit a decreasing trend in accessibility upon Opa RNAi (sh opa, compare with control, wt; Figure 5D). As a control, we conducted the same type of analysis comparing wildtype to zld RNAi embryos, and found that all the regions were decreased upon knock-down of zld (sh zld Figure 5 - supplemental figure 1C-E). These results demonstrate that while Opa does regulate chromatin accessibility, we cannot exclude an accessory role for Zld. It is possible that these factors coordinate to regulate chromatin accessibility and/or that Zld affects Opa activity indirectly; for example, by regulating an Opa-cofactor.
(A-D) Aggregated signals and heatmaps of late nc14 normalized ATAC-seq signal from wt and sh opa mutant embryos for Zld-only (B; red trace, A), Zld &Opa overlap (C; black trace, A) and Opa-only (D; blue trace, A) ChIP-seq regions (see Figure 2D). Each row of the heatmap is a genomic region, centered to peaks of accessibility signals. The accessibility is summarized with a color code key representative of no accessibility (red) to maximum accessibility (blue). Another plot, on top of the heatmap, shows the mean signal at the genomic regions, which were centered to peaks of accessibility signals (A).
(E-G) In situ hybridization using riboprobes to hb (blue) and sog (red), as well as anti-Dorsal staining (to highlight ventral regions) of wt and opa RNAi (sh opa) Drosophila embryos at indicated stages.
(I) Schematic illustrating model supported by our results, which is that Opa, a general timing factor and likely a late-acting pioneer factor, heralds in a secondary wave of zygotic gene expression, following and coordinate with Zelda, to support the maternal-to-zygotic transition.
To determine whether these global effects on chromatin accessibility that we had observed in opa mutants have consequence on patterning, we examined gene expression in mutant embryos assaying for patterning phenotypes. We conducted in situ hybridizations to wildtype and opa RNAi embryos using riboprobes to detect endogenous transcripts for genes sog and sna expressed along the DV axis, and hb expressed along the AP axis. zld RNAi mutants were examined, in comparison; as all sog and hb genes are known to be affected in zld mutants, whereas sna is delayed in expression in zld mutants but recovers by nc14 (Liang et al., 2008; Nien et al., 2011). In addition, embryos were examined at two timepoints, nc13 and nc14b, corresponding to time points before and after Opa is expressed (e.g. Figure 1C). Even early, at nc13, the zld mutant embryos exhibit loss of expression for all three genes examined, hb, sna, and sog, supporting the view that Zld is pivotal for early expression (Figure 5E). In contrast, in opa mutants, at nc13, little difference was observed; opa is not expressed at nc13, therefore mutants would not be expected to affect patterning at this stage (Figure 5F). Later, at nc14b, the opa mutants do exhibit expression defects. sog is diminished in opa mutant relative to wildtype (Figure 5G); and the hb pattern found in opa mutants at nc14b is associated with an earlier pattern encompassing the full anterior cap (Koromila and Stathopoulos, 2017). It appears that the hb_stripe enhancer’s anterior stripe is expressed at lower levels in the opa mutant (Figure 5H). In contrast, the expression of sna in opa mutants appears normal (Figure 4 - supplemental figure 1D), which is the expected result as no effect on chromatin accessibility at the sna locus is observed (Figure 4 - supplemental figure 2A).
DISCUSSION
In summary, our experiments indicate that Opa is a late-acting timing factor, and likely pioneer factor, which regulates gene expression across the entire embryo, along DV as well as AP axes. Previously, Opa, a non-canonical, broadly-expressed pair-rule gene, was linked only to AP axis patterning but our initial analysis of the sog_Distal enhancer, which is active along the DV axis, led us to investigate a more general role for this timing factor. We show that opa mutants also exhibit broad DV patterning changes in addition to previously identified AP patterning phenotypes. Opa likely acts directly to support this gene expression relating to patterning as Opa chromatin immunoprecipitation (ChIP) demonstrates widespread genomic binding, including at the sog_Distal enhancer. Additional data from single-embryo ATAC-seq provide insight into the mechanism by which Opa supports gene expression as it contributes to chromatin accessibility at a subset of regions, those occupied by Opa but not by Zld. Zld appears to predominantly support early chromatin accessibility in embryos and, subsequently, once Opa is expressed in mid-nc14 then the two factors presumably work together to support this role. However, zygotic genes (or particular enhancers) associated with mid-nc14 to gastrulation, or later, appear to be preferentially bound by Opa. Therefore, we suggest that Opa acts following the pioneer factor Zld, to also influences timing of activation at a whole-genome level through actively increasing late-acting enhancer accessibility (Figure 5G). Furthermore, Opa-mediated chromatin accessibility (Opa-only), and not just binding in general (shared by Zld), is correlated with late histone methylation. It suggests that Opa’s regulatory impact may include the control of epigenetic marks as part of its developmental program. In addition to supporting gene expression in a general manner by making chromatin accessible, Opa also presumably influences the activity of other transcription factors such as Run, co-bound to enhancers, in a mechanism that is not completely understood (Koromila and Stathopoulos, 2019; Prazak et al., 2010).
Through analysis of over-represented sites in the Opa ChIP-seq called peaks using the program HOMER, we identified a number of sites co-associated with Opa that provide additional insight into its function. For example, a motif matching the Dref consensus binding site was identified in Opa ChIP-seq peaks. Dref plays multiple roles during Drosophila development, as it is a multi-functional protein that has been linked to expression of signaling pathway components, being highly enriched in promoters, as well as to regulation of chromatin organization including insulator function and chromatin remodeling (rev. in Tue et al., 2017). In particular, as Dref has been linked to insulator function (Gurudatta et al., 2013) and Zld has been shown to be associated with locus-specific TAD boundary insulation (Hug et al., 2017), a future area of interest would be to examine whether temporal progression of gene regulatory networks is supported not only by the sequential action of pioneer factors that affect chromatin accessibility to affect gene activation but also affects gene expression by actively influencing chromatin conformation.
Our results also suggest that binding of Opa to DNA is enriched near promoters. Regions of accessibility are established sequentially, where enhancers are opened in advance of promoters and insulators (Blythe 2016). Opa may influence gene expression timing by affecting accessibility at promoters, possibly, in combination with Dref. Alternatively, the Trl/GAF site was associated with all classes of peaks; while present in the Opa-only and Opa-Zld overlap regions, it was enriched in the Zld-only peaks. Other studies have shown that Trl/GAF acts coordinately but separately from Zld to support chromatin accessibility in regions that do not require Zld (Moshe and Kaplan, 2017). Furthermore, Trl/GAF and Dref were shown to be associated with early versus late embryonic expression (stage 5 and later), respectively (Darbo et al., 2013; Hochheimer et al., 2002; Schulz et al., 2015). As Dref was found associated with Opa peaks but also independently linked to late embryonic expression, these results collectively support the view that Opa, like Dref, is also a late-acting factor.
More generally, the triggering of temporal waves of gene regulation in response to chromatin accessibility changes is a potentially widespread mechanism used to control developmental progression. A key future interest is to understand how the expression of genes that act as triggers, such as opa, are regulation. To start, the opa transcript is over 17 kB in length, and this relatively long transcript size may contribute to its expression in late nc14 as previously studies have suggested that transcripts of this length are difficult (or impossible) to transcribe in earlier nuclear cycles of short interphase length (i.e. nc13, etc) (Sandler et al., 2018; Shermoen and O’Farrell, 1991). opa expression is also regulated by the nuclear-cytoplasmic (N/C) ratio, whereas other genes like snail are not sensitive to N/C ratio but appear instead to be regulated by a maternal clock (Lu et al., 2009). It has been hypothesized that the initiation of genes sensitive to N/C ratio is sensitive to repression, which is diluted out with increasing N/C ratio possibly to concentrate an activator (e.g. Hamm and Harrison, 2018). We propose that genes of different timing classes may be preferentially regulated by different pioneer factors (Opa versus Zld, for example). Opa appears to be one timing factor acting at the MBT, but we propose that other factors also exist. For instance, opa mutants do not exhibit cellularization defects or obvious change in length of the cell cycle. It is possible that Opa regulates expression of a large number of genes at the MBT but not all genes that initiate/alter gene expression at this stage. Furthermore, opa expression may be regulated by maternal repression, that slowly decreases in effectiveness with dilution through increasing N/C ratio.
Opa’s expression in the embryonic trunk, but exclusion from the termini, suggest that additional late-acting factors may serve a similar role at the embryonic termini. For example, a recent study suggests that the transcription factor Orthodenticle (Otd) functions in a feed-forward relay from Bicoid to support expression of genes at the anterior of embryos (Datta et al., 2018). Furthermore, in our analysis of sites enriched in the Zld ChIP-seq dataset, we identified that the binding site for Cad transcription factor is enriched in this set. Cad is localized in a gradient that emanates from the posterior pole (Mlodzik et al., 1985). It is possible that these factors, Otd and Cad, function to support the late-activation of genes expressed at the anterior and posterior termini, possibly also functioning as pioneer factors to support action of particular late-acting enhancer in these domains where Opa is not present.
In addition, studies have shown that Zld influences morphogen gradient outputs (Foo et al., 2014), and perhaps Opa does, as well, during the secondary wave of the MZT. A late-acting morphogen gradient in the early embryo, at the time of Opa expression, is Decapentaplegic (Dpp) mediated activation of BMP signaling (Sandler and Stathopoulos, 2016; rev. in Stathopoulos and Levine, 2005b). Broadly-expressed repressors can influence temporal action of enhancers using distinct mechanisms to affect spatial gene expression patterns (e.g. Koromila and Stathopoulos, 2019), and we suggest that temporally-regulated activators such as late-acting, timing factor Opa may also support additional nuanced roles in the temporal regulation of morphogen gradient outputs. For example, our previous study showed that BMP/TGF-βtarget genes exhibit different modes of transcriptional activation, with some targets exhibiting a slower response (Sandler and Stathopoulos, 2016).
Opa is conserved as it shares homology with the Zic family of mammalian transcription factors (Aruga et al., 1996; Hursh and Stultz, 2018). Zic family members have roles in neurogenesis, myogenesis, skeletal patterning, left-right axis formation, and morphogenesis of the brain (Grinberg and Millen, 2005). In addition, Zic family members have been shown to be involved in the maintenance of pluripotency in ES cells (Lim et al., 2007). In particular, Zic3 shares significant overlap with the Oct4, Nanog, and Sox2 transcriptional networks and is important in maintaining ES cell pluripotency by preventing differentiation of cells into endodermal lineages. While we have focused on the role of Opa as an activator of gene expression, it is also possible that Opa acts to limit differentiation paths of cells.
MATERIALS AND METHODS
Fly stocks and husbandry
All flies were reared under standard conditions at 23°C and yw background was used as wild type, unless otherwise noted. opa1/TM3,Sb (BDGP#3212) mutant flies were rebalanced with TM3 ftz-lacZ marked balancer. Additionally, embryos were depleted of maternal and zygotic Opa by maternal expression of a short hairpin (sh) RNAi construct directed to the opa gene (i.e. sh opa). UAS-shRNA-opa females [TRiP.HMS01185_attP2/TM3, Sb1 - Bloomington Drosophila Stock Center (BDSC), stock #34706] were crossed to MTD-Gal4 males (BDSC#31777). F1 MTD-Gal4/UAS-shRNA-opa females were crossed back to shRNA-opa males (#34706), and F2 embryos collected and assayed by in situ for opa mutant phenotypes. Both sh opa and classical opa1 mutant embryos exhibit loss of opa gene expression, as detected by in situ hybridization using an opa riboprobe, as described (Koromila and Stathopoulos, 2017). In addition, other phenotypes of mutant embryos (i.e. sh hairpin and classical mutant) were compared and confirmed to be very similar to each other. Both male and female embryos were examined; sex was not determined but assumed to be equally distributed.
Cloning
The sog_Distal sequences with mutated Opa or Run binding sites (i.e. sogD_Δopa, sogD_Δopa4 and sogD_Δrun yellow reporters) were chemically synthesized and ligated into the eve2promoter-MS2.yellow-attB vector using standard cloning methods as previously described (Koromila and Stathopoulos, 2019, 2017). Site-directed transgenesis was carried out using a D. melanogaster stock containing attP insertion site at position ZH-86Fb (Bloomington stock #23648). Two constructs with five and one Opa binding sites mutated (sogD_Δopa and sogD_Δopa4, respectively) were generated to check sog_Distal’s expression levels upon different levels of the activator Opa at nc14a and later. Mutated site sequences and their wt equivalent fragments are listed here:
sogD_Δrun: tgcggtt > tAcgAtt
sogD_Δopa: aaattcccacca > aTGttcTcacca (1), gcgcccctttta > gcgcATctttta (2), cttttcccacgc > cttttcTcaTTc (3), caacgcccgcca > caacgcATgcca (4), gaatacccacga > ATGtacTcacga (5)
sog_DistalΔopa4: caacgcccgcca > caacgcATgcca
In situ hybridizations, immunohistochemistry, and image processing
Enzymatic in situ hybridizations were performed with antisense RNA probes labeled with digoxigenin, biotin or FITC-UTP to detect reporter or in vivo gene expression. sna, hb, sog (both regular and sog intronic), opa and yellow intronic riboprobes were used for multi-plex fluorescent in situ hybridization (FISH). For immunohistochemistry we used anti-Opa antibody (Mendoza-García et al., 2017) and anti-Zld antibody (rabbit) that is was raised for this study (1:200). Images were taken under the same settings, 26-30 Z-sections through the nuclear layer at 0.5-μm intervals, on a Zeiss LSM 880 laser-scanning microscope using a 20x air lens for fixed embryos.
Live imaging, data acquisition and analysis
In order to monitor the various sog_Distal reporters described above in live embryos, female virgins of line yw;Nucleoporin-RFP;MCP-NoNLS-GFP were crossed with MS2 reporter-containing males. Confocal live imaging on the Zeiss LSM 880 as well as imaging optimization, segmentation, and data quantification were conducted as previously described (Koromila and Stathopoulos, 2019).
ChIP-seq procedure
Opa-ChIP was performed as described previously (Mendoza-García et al., 2017) using chromatin prepared from 100mg of pooled collection of 3h (average of 2.5-3.5 h) and 4h (average of 3.5-4.5 h) embryos (yw) with anti-Opa serum. The precipitated DNA fragments were ligated with adaptors and amplified by ten cycles of PCR using NEBNext Ultra II DNAlibrary Prep Kit for Illumina (NEB) to prepare libraries for DNA sequence determination using Illumina HiSeq2500 and single-end reads of 50bp. The libraries were quantified by Qubit and Bio-Analyzer (Agilent Bioanalyzer 2100).
ATAC-seq procedure
Embryos embryos produced from either wild-type (D. melanogaster.ZH-86Fb) or mutant sh opa and sh zld were collected on agar plates. Individual embryos were selected from plates, and nuclear morphology was observed live under a compound microscope at 20x magnification. Temperatures for sample collection were maintained at 25°C to minimize variation in cell cycle timing. Under these conditions, cell cycle times were indistinguishable. The staging of the samples started at 3-min intervals from the onset of anaphase of the previous cell cycle. Each embryo was hand-selected and hand-dechorionated for the analysis. Prepared libraries were subject to single-end sequencing, of 50bp reads, using an Illumina HiSeq2500. Fragmentation and amplification of ATAC-seq libraries were performed essentially as described previously (Blythe and Wieschaus, 2016; Buenrostro et al., 2015).
Next generation sequencing bioinformatics
An initial analysis of the Opa-ChIP-seq data conducted by Y.I. led to the identification of the 7bp motif shown for Opa shown in Figure 1I.
The raw fastq data (50bp single-end) for Opa ChIP-seq libraries, and for wt, sh zld and sh opa ATAC-seq libraries were generated from the Illumina HiSeq2500 platform. The raw data for Zld ChIP-seq (GSM763061) and histone H3K4me1/H3K4me3 (GSE58935) were downloaded from the Gene Expression Omnibus (GEO) database. Trimmomatic-0.38 tool (Bolger et al., 2014) was used to remove Illumina adapter sequence before alignment to the Drosophila dm6 reference genome assembly with the Bowtie2 alignment program (Langmead and Salzberg, 2012). Alignment BAM files were subject to further sorting and duplicate removal using the Samtools package (Li et al., 2009). Reads mapped to chr2L, chr2R, chr3L, chr3R, chr4, chrX were kept and biological replicate BAM files were merged for downstream analysis. ChIP-seq and ATAC-seq signal trace files were generated using the bamCoverage function of deepTools (Ramírez et al., 2014), with RPKM r normalization and 10bp for the genomic bin size.
Both IP and input data were used for ChIP-seq peak calling. For calling of transcription factor binding sites, a workflow using bdgcmp and bdgpeakcall modules of the MACS2 peak caller (Zhang et al., 2008) was utilized. Peak calling was performed using ChIP data against input data (a proxy for genomic background). Genomic regions with q-value less than 10-5 were defined as ChIP-seq peak regions. Opa and Zld peak regions were combined and overlapping peaks were merged. Combined regions that overlapped both Opa and Zld peaks were defined as Opa-Zld overlap regions; regions overlapping with either Opa or Zld peaks were defined as Opa-only and Zld-only regions respectively. Further de novo motif analysis was performed on different ChIP-Seq regions using the HOMER program (Heinz et al., 2010) with default parameters and with options –size 200 and -mask. The most enriched de novo motifs identified from Opa ChIP-seq peaks and from Zld ChIP-seq peaks were queried against the Opa-Zld overlap, Opa-only and Zld-only regions for comparison and for generating aggregation plots. Average ATAC-seq signals around different ChIP-seq regions were also calculated using the annotatePeaks.pl module of HOMER, with the -size 4000 -hist 10 options used for aggregation plots. Also different ChIP-seq regions were annotated and linked to the nearest gene transcription start sites. Functional gene annotation was performed using DAVID v6.7 (https://david.ncifcrf.gov/home.jspcitation). In addition, computeMatrix and plotHeatmap modules of deepTools were used to calculate and plot normalized histone mark and ATAC-seq signal intensities around different ChIP-seq regions. DNA sequence logos were plotted using the seqLogo R package. Region overlap analysis was performed using the NeuVenn module of Neutools (doi: https://doi.org/10.1101/429639). Unless noted otherwise, R was used to generate aggregation and violin plots.
For the individual loci ATAC-seq data that are depicted in Figure 4: mapped reads were normalized similarly to a published method for better visualization (Blythe and Wieschaus, 2016): First, to define the background, 150 bp peaks were called from the original data using a very sensitive mode of HOMER (-localSize 50000-minDist 50 -size 150 -fragLength 0) to capture most of the non-background regions. These 150 bp peaks were extended from the center to form 20000 bp “signal zones”. Outside these signal zones are “background zones”. Next, to sample the background noise, 100000 150bp random regions were generated. Those 150bp random regions that completely fell into the “background zones” were regarded as “background regions”. The mean and standard deviation for the background noise were calculated from positive RPM scores of each nucleotide in these regions (ypbkg) based on log-normal distribution. Finally, RPM scores for the whole genome were centered and scaled based on the mean and standard deviation calculated, using 1 as pseudocount:
AUTHOR CONTRIBUTIONS
A.S. and T.K conceived the project and planned the experimental approach. A.S. directed the project. T.K performed wet experiments except ChIP-seq, which was performed by Y.I. with support of the Caltech genomics core. The computational approach was overseen by F.G., P.H., and T.K.; F.G. performed all computational analysis except normalization of ATAC-seq data for visualization of individual loci, which was performed by P.H. An initial, independent analysis of the Opa-ChIP-seq data was conducted by Y.I. Quantitative analysis of imaging data was performed by T.K. Data were analyzed by A.S., T.K., and F.G. The manuscript was written by A.S. and T.K. with input and editing help from F.G., Y.I, P.H., L.P. and J.P.G.
COMPETING INTERESTS
No competing interests declared.
DATA AND CODE AVAILABILITY
GEO accession numbers: ChIP-seq and ATAC-seq (GSE140722).
The codes for Opa ChIP-seq and ATAC-seq processing (alignment and peak calling) were uploaded to github: https://github.com/gaofan83/Stathopoulos_Lab
ACKNOWLEDGEMENTS
We are grateful to Igor Antoshechkin and Henry Amrhein at the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology for sequencing, the lab of Josh Dubnau for assistance with Bioanalyzer samples, David Carlson and the Institute for Advanced Computational Science at the Stony Brook University, Leslie Dunipace and Frank Macabenta for assistance with experiments and comments on the manuscript, and the entire Stathopoulos lab for helpful discussions. This study was supported by funding from NIH R35GM118146 and R03HD097535 to A.S., the Bioinformatics Resource Center at the Beckman Institute of Caltech to F.G. and L.P., and the Stony Brook University College of Arts and Sciences to J.P.G.
REFERENCES
