Brian Kinney

Shubham Sahu

Natalia Stec

Kelly Hills-Muckey

Dexter W Adams

Matt Jaremako, Leemor Jing Wang

Wolfgang Keil
email: wolfgang.keil@curie.fr

Christopher M Hammell
email: chammell@cshl.edu

Kelly Hills

Matt Jaremako

Joshua Leemor

-Tor

Circadian rhythm orthologs drive pulses of heterochronic miRNA transcription in C. elegans

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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(Fig. 1A). To visualize lin-4 transcription, we generated a ~4kb transgene harboring the lin-4 precursor RNA followed by 24 copies of a synthetic MS2 hairpin upstream of the polyadenylation sequences and the upstream regulatory information required to drive normal lin-4 transcription (11) (fig. S1A) (21). This transgene fully rescues the developmental phenotypes associated with the lin-4 null allele (lin-4(e912)) (fig. S1). We also drove ubiquitous expression of MCP fused to GFP (MCP-GFP) to detect MS2-tagged RNAs, and a histone H2B subunit (HIS-58) fused to mCherry to locate nuclei (fig. S1A).

Examination of L3-staged transgenic animals revealed that dynamic lin-4 expression (MCP-GFP foci) occurred from each lin-4::MS2 locus in somatic tissue types known to transcribe lin-4 (Fig. 1B). MCP-GFP foci were never observed in developing embryos (n > 50) or in starvation arrested L1 larva (n = 23); consistent with the activation of lin-4 transcription after the initiation of larval development (4).

Next, we used our microfluidics platform to perform live spinning-disk confocal microscopy of developing C. elegans larvae harboring lin-4::24xMS2/MCP-GFP system (11,22). We focused on quantifying expression dynamics in hypodermal cells (seam, hyp7 and vulval precursor cells (VPCs))

where the timing of transcriptional activity within a given larval stage can be accurately assessed in relation to the occurrence of precisely timed stage-specific cell division patterns (1). We first screened for periods of lin-4 transcriptional activity by imaging at 15min time intervals from early first larval stage (L1) to mid L4 stages (~60h) (n>10). This revealed that lin-4::24xMS2 transcription was highly pulsatile, with a single transcriptional episode of ~45-90min in each cell during each larval stage, followed by extremely long periods of inactivity (>8hrs at 20°C) (Fig. 1C). Transcriptional activation across cells within the entire hypodermis was highly concordant, exhibiting similar transcriptional on and off times for all lin-4::24xMS2 loci (Fig. 1E). For instance, during the mid-L3 stage, the appearance of lin-4::24xMS2 expression throughout the hypodermis generally occurred within minutes of the first VPC divisions (P3.p or P4.p) and were completed by the divisions of P5.p-P7.p (n = 15) (Fig. 1C).

Based on these observations, we performed short-term imaging time courses (<6h) at 4min intervals in staged larvae to capture transcriptional epochs at high temporal resolution. During transcriptional episodes, we detected near synchronous accumulation of MCP-GFP foci at each hypodermal lin-4::24xMS2 locus for 60-90 minutes (Fig. 1D-E, Suppl. Movie 1,2) (>15 animals). We found no signs of "bursty" transcription (23) as loci generally stayed on for the entire transcriptional episode (Suppl. Movie 1,2). These features were independent of the number of lin-4::24xMS2 loci per nucleus as cell types that undergo endoreduplication (i.e., hyp7 cells) exhibited MCP-GFP foci dynamics indistinguishable from diploid cells (Fig. 1D). The dynamic features of lin-4::24xMS2 expression in hypodermal cells were similar across different developmental stages (Fig. 1E) suggesting that the same regulatory programs controlling lin-4 transcription were repeated at each larval stage. Finally, the pulsatile nature of transcriptional activation occurred in a broad array of additional cell types that normally express lin-4. These include pharyngeal muscles and intestinal cells in which the phase of transcriptional induction is shifted later compared to patterns observed in hypodermal cells and the duration of pulses are extended (fig. S2).

NHR-85 Rev-Erb and NHR-23 ROR interact with the regulatory regions that control repetitive lin-4 transcription

We previously demonstrated that highly pulsatile transcription from the lin-4 locus is dependent on the upstream lin-4 PCE (pulse control element) (11). To identify candidate TFs that bind this conserved regulatory sequence, we performed a yeast-one-hybrid screen using the entire 514bp lin-4 PCE as bait and identified three TFs (BLMP-1, NHR-23 and NHR-85) (Fig. 2A). BLMP-1 controls the amplitude of lin-4 expression and functions as a pioneer factor to decompact the lin-4 locus throughout development (11). nhr-85 and nhr-23 encode two nuclear hormone receptors (NHRs) that are the closest nematode orthologs of human circadian TFs Rev-Erb and ROR, respectively (Fig. 2B). Analysis of publicly available ChIP-seq data indicated that all three proteins interact in vivo with lin-4 regulatory sequences, which contain putative consensus binding sites for each TF (Fig. 2A). Furthermore, NHR-85 Rev-Erb , NHR-23 ROR and BLMP-1 binding sites are highly overlapping and enriched in the promoters of numerous cyclically expressed protein coding genes and heterochronic miRNAs (fig. S3) (table S1 andS2).

NHR-85 Rev-Erb and NHR-23 ROR heterodimerize and bind cooperatively lin-4 regulatory elements

Nuclear hormone receptors often bind cooperatively as homo-or hetero-dimeric complexes at closely spaced cis-regulatory DNA elements (24)(25)(26). Several features of NHR-85 Rev-Erb , NHR-23 ROR and the lin-4 PCE suggest that this may also be the case for lin-4 transcription. First, NHR-85 Rev-Erb and NHR-23 ROR share significant sequence homology within their ZnF C4-type DNA-binding domains implicated in DNA interactions and dimerization, suggesting they may function at similar binding sites (Fig. 2C) (27). Second, we found that NHR-85 Rev-Erb and NHR-23 ROR could bind to form heterodimers in yeast two-hybrid assays, which could be reconstituted in vitro with a binding affinity of 5.8 +/-2.2 nM KD (Fig. 2D,E). Third, sequences within the PCE element contain direct repeats of a GGTCA that are contained in predicted binding sites of NHR-85 Rev-Erb and NHR-23 ROR (Fig. 2F andG). Fourth, while NHR-23 ROR alone could bind a DNA fragment at high TF/DNA molar ratios (fig. S4A,B), we found that 6 DNA binding assays containing concentrations of NHR-23 ROR that are insufficient to bind the PCE alone are dramatically stimulated by the addition of NHR-85 Rev-Erb (Fig. 2H,I). NHR-85 Rev-Erb alone was unable to reproducibly bind any lin-4 PCE DNA fragments (fig. S4C). Finally, the cooperative binding of NHR-85 Rev-Erb and NHR-23 ROR to lin-4 enhancer fragments requires the presence of both conserved GGTCA direct repeats, suggesting these NHRs bind closely spaced regulatory elements as a heterodimer (Fig. 2H). Previous studies indicate that LIN-42 Period , the C. elegans ortholog of the circadian Period protein, negatively regulates the expression of lin-4 throughout post-embryonic development (7,12,28). Given the roles of mammalian Period, ROR, Rev-Erb in controlling circadian gene regulation in mouse and human, we examined potential regulatory relationships between the C. elegans orthologs of these genes in the context of lin-4 transcription. Single-cell expression analysis indicates that nhr-85 and nhr-23 mRNAs are enriched in hypodermal and glial cells while lin-42 is broadly expressed (Fig. 3A) (29). nhr-85 transcription initiates from an L1-stage arrest with a pulse of transcription that is followed by a monotonic expression pattern at subsequent larval stages (Fig. 3B). In contrast, nhr-23 and lin-42 mRNAs exhibit phased, high-amplitude oscillatory patterns of expression (Fig. 3B).

NHR-85

We next explored the temporal dynamics of NHR-85 Rev-Erb , NHR-23 ROR and LIN-42 Period protein by quantifying the expression of endogenously-tagged alleles of each gene during the L4 stage, in which the evenly-spaced, stereotyped morphological changes in vulval morphogenesis can be directly correlated with developmental age (30). Consistent with previous characterization of NHR-23 ROR and LIN-42 Period expression patterns determined using reporter transgenes (31,32), each of our endogenous reporters was expressed dynamically in hypodermal cells with a single peak of expression that roughly matches the phased expression of their mRNAs (Fig. 3C). In contrast to the flat expression of nhr-85 transcripts in L2-L4 stages of development, we found that NHR-85 Rev- Erb ::GFP expression was highly dynamic, indicating substantial post-transcriptional regulation of NHR-85 Rev-Erb expression. Specifically, peak expression of NHR-85 Rev-Erb ::GFP occurs at ecdysis (shortly before NHR-23 ROR ::mScarlet onset), becomes undetectable by the L4.3 stage of development, and resumes at the L4.6 stage in an antiphasic manner to the expression pattern of LIN-42 Period ::YFP (Fig. 3C). Highly similar phased expression patterns of these proteins are also maintained in L4-staged vulval tissues (Fig. 3D) as well as in L3-staged hypodermal cells and VPCs (Fig. 4).

The temporal overlap in NHR-85 Rev-Erb and NHR-23 ROR expression correlates with lin-4 transcriptional pulses

The overlap in NHR-85 Rev-Erb ::GFP and NHR-23 ROR ::mScarlet expression during the L4 stage lasts 60-90min (L4.2-L4.3) (Fig. 3D), the approximate duration of lin-4 transcription in hypodermal cells and VPCs at this same stage (Fig. 1D,E) (30). To compare the timing of these events, we examined MCP-GFP localization in vulval cells during the L3 stage, where the rapid, stereotyped vulval cell division patterns (22) enable precise determination of the timing of lin-4::24xMS2 transcription and TF dynamics. We found a striking correspondence between NHR-85 Rev-Erb and NHR-23 ROR co-expression and lin-4::24xMS2 transcription (Fig. 4). Specifically, while the expression of both nuclear receptors is phased, the transient expression of the lin-4::24xMS2 transgene only occurs during the brief period when both NHR proteins are expressed in VPCs (Fig. 4A). This almost perfect correspondence is also maintained in hypodermal cells during this transcriptional cycle where both NHR-85 Rev-Erb expression and MCP-GFP foci are diminished near the completion of the first round of VPC divisions (Fig. 1E and3D). Importantly, the timing of NHR-85 Rev-Erb downregulation is also concurrent with onset of LIN-42 Period expression in VPCs and hypodermal cells (Fig. 3D and4A).

The phased expression patterns of all TFs and the dynamics of MCP-GFP foci accumulation are also conserved in the L4 stage of development (Fig. 4B). This indicates that the timing and duration of lin-4 transcriptional pulses are likely encoded by the dynamic expression patterns of these TFs (Fig. 4C).

LIN-42

Period regulates NHR-85 Rev-Erb levels to modulate the amplitude and duration of lin-4 transcription Mutations in lin-42 Period produce late temporal patterning defects including precocious expression of adult-specific genes in the L4 stage (Fig. 5A) (6,7,33). Mammalian Period orthologs have been shown to interact with multiple NHRs and modulate their transcriptional activity (34). To determine if LIN-42 Period could physically interact with other C. elegans NHRs, we systematically probed LIN-42 Period interactions with a majority of the of C. elegans NHRs (243 of 284) using twohybrid assays. We identified 66 C. elegans NHRs that could interact with LIN-42 Period in yeast (fig. S5A,B), including orthologs of Rev-Erb(NHR-85)(Fig. 5B), HNF4a(NHR-14, NHR-69) and PPARa(NHR-119), indicating evolutionary conservation of physical interactions between Period orthologs and related NHRs (Fig. S5A-B) (34). We mapped the domains of LIN-42 Period that are required for NHR-85 Rev-Erb binding and found that a minimal 51aa fragment present in both major LIN-42 Period isoforms is sufficient to mediate interactions (Fig. 5A). This domain is distinct from the interaction motifs implicated in mammalian Per2 and Rev-Erb (34). Neither LIN-42 Period isoform interacts with NHR-23 ROR in two-hybrid assays (Fig. 5A).

Given the physical interaction between NHR-85 Rev-Erb and LIN-42 Period , we asked whether NHR-85 Rev-Erb expression was required for the precocious phenotypes seen in lin-42(lf) mutants (lin-42(n1089)). We found that lin-42(lf) heterochronic phenotypes are partially ameliorated by removing nhr-85 function. Specifically, precocious expression of col-19::GFP in both seam and hyp7 cells observed in lin-42(lf) mutants is suppressed by nhr-85 deletion; leaving weak expression in seam cells in double mutants, while precocious deposition of adult alae was not suppressed (Fig. 5A). To examine whether LIN-42 Period regulates NHR-85 Rev-Erb temporal dynamics, we compared the dynamics of NHR-85 Rev-Erb ::GFP (and NHR-23 ROR ::mScarlet) expression in wild-type and lin-42(lf) mutants. We found that the expression of NHR-85 Rev-Erb is altered in two ways by a lin-42(lf) mutation: First, expression of NHR-85 Rev-Erb ::GFP is ~2.3x more abundant at the beginning of the L4 stage in lin-42 mutants when compared to wild-type animals (Fig. 5C andD). Importantly, the periodic dampening of NHR-85 Rev-Erb expression at the L4.2 stage observed in wild-type animals (Fig. 3C,D) is altered in lin-42(lf) mutants and NHR-85 Rev-Erb ::GFP expression perdures into the L4.4 stage (Fig. 5C,D).

Mutations in lin-42 do not alter the onset or duration of NHR-23 ROR ::mScarlet accumulation in hypodermal or vulval cells.

Our results suggest that timing of lin-4 transcriptional pulses is defined by the brief coexpression of NHR-85 Rev-Erb and NHR-23 ROR (Fig. 4C). We hypothesized that the corresponding increase in lin-4 expression levels previously observed in lin-42(lf) mutants (6, 7) likely result from alterations in lin-4 transcription associated with mis-regulation of NHR-85 Rev-Erb expression. To test this, we examined expression of MCP-GFP foci throughout the L3 stage and found that, while a majority of wild-type seam cells fire a single time (76%; 66 foci), the percentage of seam cells that exhibit detectable lin-4 transcription is dramatically increased in lin-42 mutants (100%; 58 foci). This indicates that lin-42 normally dampens lin-4 transcriptional pulses in wild-type animals. In addition to elevating the likelihood lin-4::24xMS2 transcription is above a threshold sufficient to generate measurable MCP-GFP foci, lin-42(lf) mutations lead to an increase in the intensity of MCP-GFP foci indicting that LIN-42 Period normally also limits Pol II loading at the lin-4 locus (Fig. 5E). Time course experiments revealed that overall duration of detectable transcription in seam cells is ~2.2 times longer in lin-42 mutants compared to wild-type (Fig. 5E). Consistent with our hypothesis that the duration of lin-4 transcription is dependent on the duration of NHR-85 Rev-Erb expression, transcriptional onset of lin-4 occurs earlier in lin-42 mutants and inappropriately extends through both the first and second Pn.p divisions (Fig. 5F). 9

While nhr-85(0) mutants do not miss-express col-19::GFP (Fig. 5A), high-resolution imaging experiments monitoring VPC divisions and lin-4::24xMS2 expression indicate that two features of developmental timing are altered in nhr-85(0) mutants. Transcriptional pulses of lin-4::24xMS2 are both robust and concordant in adjacent wild-type VPCs (Fig. 5G). In contrast, MCP-GFP foci in nhr-85(0) mutants begin to accumulate at the same relative phase of L3-stage VPC development but are dimmer and only transiently observed (Fig. 5G). Second, under isogenic imaging conditions, the rapid and highly coordinated VPC divisions observed in wild-type animals is altered in nhr-85(0) mutants with some P5.p and P7.p dividing hours after the first P6.p division (Fig. 5G). These results indicate that NHR-85 functions to enhance the robustness of temporally regulated processes during development and that some level of lin-4 transcription occurs without NHR-85 Rev-Erb , perhaps driven by NHR-23 ROR alone. Consistent with the hypothesis that NHR-23 and NHR-85 function together to control the normal temporal regulation, RNAi-mediated depletion of nhr-23 activity in wild-type animals using RNAi conditions that do not induce developmental arrest result in mild heterochronic phenotypes (Fig. S6). The penetrance of these phenotypes is enhanced when nhr-23 is depleted in nhr-85(0) animals, indicating that both NHRs also function cooperatively in temporal patterning (Fig. S7).

Discussion

Most genetic oscillators are composed of auto-regulatory transcriptional/translational feedback loops where transcription of core clock components are self-regulated (35) Here, we show that C. elegans larvae employ a GRN involving multiple circadian-related genes to couple lin-4 transcriptional pulses to the larval molting cycle (Fig. 5H). The complement of 10 shared components between the human and C. elegans GRNs combined with major differences in their regulatory architecture is remarkable. Primary among these differences is the absence of genes encoding Clock and Bmal1 orthologs in the C. elegans genome. We propose here that the worm orthologs of Rev-Erb and ROR, which function in humans in a purely auxiliary capacity to stabilize the expression of core circadian clock components, compose the central transcription factors of the hypodermal developmental clock (Fig. 5H). In contrast to their antagonistic roles in the circadian clock, C. elegans NHR-85 Rev-Erb and NHR-23 ROR heterodimerize and work cooperatively to promote transcriptional pulses (Fig. 5H). Importantly, we demonstrate that cooperativity between NHR-85 Rev- For this analysis, peaks from NHR-85 Rev-Erb , NHR-23 ROR and BLMP-1 ChIP-seq experiments were assigned to the nearest proximal protein coding genes (+3kb upstream and 300bp downstream) (Tables S1 andS2). (B) NHR-85 Rev-Erb , NHR-23 ROR and BLMP-1 binding sites are enriched in the promoters of genes that exhibit oscillatory expression patterns. Calculations were derived from population-based RNA-seq time course data sets (8) that partition the expression of postembryonically expressed genes into three types: (i) transcripts with their transcriptional periodicity tied to the molting cycle (osc; 18.9%; 2,718 of 14,378 total genes) (ii) genes with rising expression throughout larval development (rising; 14.2%) and (iii) genes with flat expression profile (flat; 66.9%) (Fig. 2E). Using these data sets, we compared the numbers of cyclically expressed genes that were (version: 7.8.12.0). Animals were anesthetized on 5% agarose pads containing 10mM sodium azide and secured with a coverslip. Imaging on the microfluidic device was performed on a Zeiss AXIO Observer.Z7 inverted microscope using a 40X glycerol immersion objective and DIC and GFP filters controlled by ZEN software (version 2.5). Images were captured using a Hamamatsu C11440 digital camera. For scoring plate level phenotypes, images were acquired using a Moticam CMOS (Motic) camera attached to a Zeiss dissecting microscope.

Wide-field Fluorescence microscopy:

Images were acquired with a Zeiss Axio Observer microscope equipped with Nomarski and fluorescence optics as well as a Hamamatsu Orca Flash 4.0 FL Plus camera. An LED lamp emitting at 470 nm was used for fluorophore excitation. For single images, animals were immobilized on 2% agarose pads supplemented with 100mM Levamisole (Sigma). For single images, animals were immobilized on 2% agarose pads supplemented with 100mM Levamisole (Sigma). For long-term imaging methods, see Microfluidics and Long-term Imaging section.

Fluorescent Reporter Quantification. Reporter lines were imaged using wide-field fluorescence or confocal microscopy as described above. The average intensity (arbitrary unites) per seam cell was measured using ImageJ. Each seam cell intensity was determined by the measurement of fluorescent 32 intensity of the nucleus minus the intensity of a background sample. The fluorescent intensity of each animal was determined by the average of three seam cells. Ten animals per developmental stage were imaged unless otherwise noted.

MCP-GFP live imaging.

Long term imaging: For long-term live imaging across several larval stages, animals were mounted in a microfluidic chamber 6h after L1 arrest and grown on NA22 bacteria suspended in S-medium until mid L4 as previously described (22). At each time point, animals were reversibly immobilized using microfluidic pressures and flows. A z-stack of 51 images separated by 0.5um was acquired at four overlapping positions, covering the entire microfluidic chamber. Thereafter, the animal was released from immobilization and left to roam and feed freely until the next time point.

Short term imaging: For short-term live imaging (Figs. 1, 4 & 5) developmentally staged animals were mounted into the microfluidic chamber as previously described ( 22), a few hours before the expected appearance of MS2 spots. Minutes before the appearance of MS2 spots, animals were immobilized using microfluidic pressures and flows and kept immobilized for the entire duration of the experiment.

This allowed to maintain a stable worm position enabling automated analyses (see below). At each time point (every 4min), a stack of 21 images separated by 0.5um was acquired at four overlapping positions, covering the entire microfluidic chamber. Occasionally, animals arrested development upon prolonged immobilizations, as evident by the cessation of germ line divisions (L2-L4 larvae), the cellcycle arrest of vulval precursor cells (VCPs) (L3 larvae) or by failure to advance through vulval morphogenesis (L4 larvae). These animals were excluded from further analysis. As opposed to all other genotypes imaged, nhr-85(0) mutants animals exhibited a pronounced tendency to roll under these imaging conditions, precluding MS2 spot tracking within nuclei of the lateral hypodermis.

MCP-GFP live imaging data analysis.

Short term imaging:

All events (cell divisions, onset and offset of MCP-GFP spots) were scored manually in the time series. For short-term live imaging, all analysis was performed using custom-written FIJI macros, pixel classification with random forest trees in Ilastik and MATLAB © scripts. The main challenge in this analysis is residual animal movement between time points as well as low signal-to-noise ratio of the MCP-GFP signal.

Long term imaging and 3D tracking:

First, the worm backbone was detected in each frame by skeletonization, using a thresholded probability map, obtained by processing a maximum z-projection of the MCP-GFP channel, through a custom-trained Ilastik pixel classifier. Next, computational straightening was performed along this backbone (22) to obtain a time series of straightened worm z-stacks. This straightened time series has the advantage that residual movement is primarily along the anteroposterior animal axis. Next, we divided the straightened worm z-stack along the worm axis into overlapping (20%) segments of 500 pixels (~91um) in length. Each of these segments was then manually registered to obtain zstacks in which tracking of almost all hypodermal nuclei could automatically performed with minimal user corrections. To improve nuclear signal for segmentation, the histone-mCherry signal was augmented, using a custom-trained Ilastik pixel classifier. 3D tracking and manual correction was then performed on the resulting Ilastik probability maps using the FIJI TrackMate plugin (53) with LoG detector and LAP tracker. For each segment, frames with substantial animal movement between zslices were excluded from subsequent analysis. Whenever nuclei were tracked twice (due to overlapping worm segments), the nucleus with the most tracked time points was chosen for subsequent MCP-GFP spot analysis.

MCP-GFP spot tracking and intensity quantification. MS2 spots were tracked in each tracked nucleus, using the FIJI TrackMate plugin with LoG detector and LAP tracker and the Ilastik nucleus probability maps as mask to include only spots inside nuclei. Each MCP-GFP spot track was manually corrected, and spots were added to frames occasionally, in which the TrackMate LoG detector failed to detect them. For quantification of MCP-GFP spot intensities, a 2D Gaussian fit to the maximum zprojection of three z-slices around the peak slice determines the position of the spot. Background was calculated as the average intensity in a ring between 3 and 5 pixels away from the spot position.

The spot intensity was calculated by integrating the fluorescence over a circle with a radius of 2 pixels around the spot position and subtracting the estimated background.

MS2-MCP-GFP trace analysis. MCP-GFP traces were smoothed with a Gaussian of width 1.5 frames (6 minutes). Using this filtered trace, for each tracked locus, duration and maximum intensity 34 during the transcriptional pulse was determined. Loci were considered "ON", if spot intensity was above 150 counts and "OFF" if below. Duration of transcription for each locus was determined as the time interval between the first "ON" time point and the last. Typically, loci stayed "ON" for the entire duration of the transcriptional pulse within a given larval stage.

Yeast two-hybrid. Plasmids containing target proteins fused to GAL4 DNA-binding-domain (pDBD)

and GAL-4 Activation Domain (pAD) were co-transformed into the pJ69-4a Y2H yeast strain (54) as previously described (55, 56). Transformed yeast was plated on SC-TRP-LEU plates for three days.

Three colonies from each transformation plate were streaked onto SC-HIS-TRP-LEU plates containing 3-AT at the indicted concentrations. Protein interactions were determined by visible growth on 3-AT conditions with negative growth in empty vector controls after three days. For the large-scale LIN-42 screen, pDBD containing LIN-42a, LIN-42b, and the empty vector control were individually mated to each pAD construct from the WTF2.2 yeast library (Reece-Hoyes et al., 2011) (57). For visualization of results, individual colonies were grown overnight in YPD in 96-well plates. Overnight cultures were diluted 1/200 in ddH20 and 3μL was pipetted on to selective 3-AT and control plates.

After three days of growth, plates were imaged on a Fotodyne FOTO/Analyst Investigator/FX darkroom imaging station. Plasmids used in 1-and 2-hybrid experiments are outlined in Table S4. analysis and we defined the promoter region as upstream 3kbp to downstream 300bp around the transcription start site (TSS). Peaks located in promoter regions were annotated to their closest TSS sites for both coding and non-coding genes (Table S1). These potential targets were then overlapped to sets of oscillatory genes identified in a previous mRNA-seq-based study (8). Overlapping target gene sets organized in Figure 2D are from postembryonic ChIP-seq samples for each TF. Raw data for each of the 265 ModEncode data sets (annotation numbers listed individually in Table S1) used in Figure 2E were downloaded from modENCODE (48) and processed in the same way as BLMP- The protein was then concentrated and loaded onto a 10/300 Superdex200 Increase gel filtration column (Cytiva Life Sciences), running in lysis/wash buffer, chromatographed for ~30 mL at 0.6 mL min -1 . Protein purity and cleavage efficiency was assessed by SDS-PAGE. Plasmids used to express recombinant proteins are outlined in Table S4.

Bioinformatic analysis of BLMP

Full length C. elegans protein NHR-85 was cloned as a N-terminal Strep-fusion protein in a pFL vector of the MultiBac Baculovirus expression system to create pCMH2206. NHR-85 was purified using the same method as above, with the exception of the overnight N-terminal tag cleavage step.

  Erb and NHR-23ROR for binding lin-4 regulatory elements dictates the timing and duration of lin-4 transcription and that the duration of these interactions is negatively regulated by LIN-42 Period . Specifically, LIN-42 Period binds to NHR-85 Rev-Erb and controls dynamic nature of its expression. Defects in lin-42 function result in an increase in Pol II density at the lin-4 locus and a dramatic increase in the duration of lin-4 transcriptional pulses by expanding the duration NHR-85 Rev-Erb and NHR-23 ROR co-expression. The resulting alterations in lin-4 dosage (and likely the dosage of additional heterochronic miRNAs) cause precocious heterochronic phenotypes in lin-42 mutants. This suggests that physical and functional interactions between Period orthologs and conserved NHRs are maintained in both circadian and developmental clock systems and that other C. elegans NHRs, in addition to NHR-85 Rev-Erb , may function in the heterochronic pathway.While NHR-85 Rev-Erb and NHR-23 ROR generate pulses of lin-4 transcription in the hypodermis, NHR-23 ROR may coordinate cyclical gene expression patterns with overall animal development (Fig.2A). nhr-23 is an essential gene and nhr-23(0) animals arrest during late embryogenesis/hatching at a time when oscillatory transcription begins(8, 9,44). Depletion of NHR-23 ROR during post-embryonic development results in highly penetrant larval arrest phenotypes(45). These arrests prevent somatic cell proliferation/differentiation, occur at the beginning of each larval stage and correlate with the developmental period where NHR-23 ROR expression begins(45). While the general suspension of developmental processes in NHR-23ROR -depleted animals make it unfeasible to measure direct changes in transcription for individual genes, these arrests resemble post-embryonic developmental checkpoints where somatic cell proliferation and cyclical gene expression patterns (including lin-4) are halted during acute food removal/starvation(11,46). Starvation-induced checkpoints are mediated through regulation of the conserved insulin signaling pathway which generate a variety of sterol-derived hormones (47). These hormones may serve as ligands for individual NHRs and provide a mechanism to organize global patterns of gene expression and regulate the pace of organismal development with the molting cycle.

Fig. 1 .

 1 Fig. 1. lin-4 transcription is highly pulsatile at each larval stage. (A) The MS2/MCP system is composed of a MS2 coat protein GFP fusion (MCP-GFP) which can bind to MS2 RNA hairpins encoded within primary miRNA transcripts. (B) Image of mid-L3 staged larva and magnified insets showing MCP-GFP spots in hypodermal cells along the anteroposterior axis. (C) Two examples of L3/L4 hypodermal and vulval precursor cell lineages, with overlayed expression patterns indicating when MCP-GFP foci were visible in each lineage. (D) Snapshots of individual seam cell and hyp7 cell expression trajectories from L3-staged animals. (E) Expression traces in pairs of V6.p seam cells from L2, L3, and L4 staged animals.

Fig. 2 .Fig. 3 .

 23 Fig. 2. NHR-85 Rev-Erb and NHR-23 ROR form a heterodimeric complex that binds cooperatively to regulatory elements controlling pulsatile lin-4 transcription. (A) BLMP-1, NHR-85 and NHR-23 bind to the lin-4 PCE in one-hybrid assays (11). Browser tracks showing BLMP-1, NHR-23 and NHR-85 bindings sites near the lin-4 locus. Also indicated are the major lin-4 pri-miRNAs (21), the computationally defined binding sites for each TF, and the sub-fragments of the lin-4 PCE element used in gel shifts below. Asterisk indicates location of the direct repeats of GGTCA in PCE element. (B) Sequence relationships between NHR-23 and NHR-85 and human Rev-Erb and ROR. (C) NHR-23 ROR and NHR-85 Rev-Erb domain organization highlighting similarity in DNA binding domains. (D) NHR-23 ROR and NHR-85 Rev-Erb interact with each other in two-hybrid assays. (E) Recombinant, purified strep-NHR-85 Rev-Erb and NHR-23 ROR interact in MST binding assays. (F) Sequences of the PCE subfragment iii from A and the 51bp minimal binding element (grey box) derived from lin-4 PCEiii. (G) Conservation of the direct repeats DRs in the PCE elements of different nematode species. (H and I) EMSA experiments of wild-type and mutant target DNAs using recombinant NHR-85 Rev-Erb and NHR-23 ROR .

Fig. 4 .

 4 Fig. 4. lin-4 transcription occurs during periods where NHR-85 Rev-Erb ::GFP and NHR-23 ROR ::mScarlet are co-expressed and LIN-42 Period ::YFP and NHR-85 Rev-Erb ::GFP exhibit antiphasic expression patterns. (A) Time course experiments of MCP-GFP demonstrate that lin-4::24xMS2 expression occurs immediately before the first Pn.p cell divisions and not until NHR-85 Rev- Erb ::GFP are NHR-23 ROR ::mScarlet are co-expressed in the vulval precursor cells. lin-4::24xMS2 expression terminates by the time NHR-85 Rev-Erb ::GFP expression is extinguished in second division VPC. The cessation of lin-4::24xMS2 expression corresponds to time when LIN-42 Period ::YFP accumulates in these cells. (B) The dynamics of lin-4::24xMS2 transcription in L4 staged vulvas. lin-4::24xMS2 expression occurs at the same developmental stage were NHR-85 Rev-Erb ::GFP and NHR-23 ROR ::mScarlet co-expressed. As with L3-stage expression of lin-4::24xMS2, MCP-GFP foci are diminished around the onset time of LIN-42 Period ::YFP expression. (C) Image summarizing the relationship between dynamic expression patterns of NHR-85 Rev-Erb , NHR-23 ROR , and LIN-42 Period with the period of lin-4 transcriptional activation in the L3/L4 stages.

Fig. S2. lin- 4 :

 4 Fig. S2. lin-4::24xMS2 expression is pulsatile in other somatic cells. Timeseries of lin-4::24xMS2 expression in intestine cells and cells of the pharynx in L3-staged animals. Small arrows indicate

  targeted by NHR-85 Rev-Erb or NHR-23 ROR to those targeted by any of the other 170 TFs queried in by the C. elegans ModEncode Project (using 265 publicly available, validated ModEncode ChIP-seq data-sets (tables S1 and S2)(48). (C) Browser tracks overlaying the BLMP-1, NHR-23 ROR , and NHR-85 Rev-Erb ChIP-seq signal near the indicated loci.

Fig. S4 .

 S4 Fig. S4. NHR-23 ROR can bind the lin-4 sub-fragment III at high protein concentrations while NHR-85 Rev-Erb cannot. (A) NHR-23 ROR can bind specifically to PCE sub-fragment III that contains both direct repeats outlined in Fig. 2F and G. (B) NHR-23 ROR binding to the PCE sub-fragment III requires the two direct repeats but does not require the un-conserved single GGTGA element downstream of the direct repeats (see Fig. 2 for details). (C) NHR-85 Rev-Erb does not bind to any of the PCE subelements to any appreciable degree.

- 1 ,

 1 NHR-82, and NHR-23 ChIP-seq data. ChIP-seq short reads were first clipped off adaptor sequences. Reads of minimum 22bp were mapped to the UCSC C. elegans genome (ce10) using bowtie program76 (58) looking for unique alignments with no more than 2 mismatches. MACS program (v1.4)(59) was used for peak calling with significant p-value cutoff equals 1e-5. Target annotations were based on WormBase (version 220) using customized R scripts and Bioconductor packages. ModeEncode data sets (48) (BLMP-1(ENCFF108AEB and ENCFF615AMZ), NHR-23(ENCFF019QMH) and NHR-85(ENCFF018KHA)) were used in the initial

( 20 mM

 20 Tris pH 8.0, 200 mM NaCl, 5 mM BME, 2 mM ATP). The protein was eluted from the affinity column in 2 column volumes of elution buffer (20 mM Tris pH 8.0, 200 mM NaCl, 5 mM BME, 2 mM desthiobiotin). The Strep-SUMO tag was cleaved from NHR23 by ULP1* protease overnight at 4°C.

Microscale thermophoresis analysis .

 analysis Binding assays of purified NHR23 or strep-NHR85 was measured using a Monolith NT.115 Pico running MO Control version 1.6 (NanoTemper Technologies). Assays were performed in 100 mM NaCl, 20 mM Tris pH 8.0, 0.05% Tween-20. AlexaFluor647 NHS Ester (ThermoFisher Scientific) labeled NHR-23 (200 pM) was mixed with 16 serial dilutions of strep-NHR-85 starting at 31.5 uM and loaded into microscale thermophoresis 36premium coated capillaries (NanoTemper Technologies). MST measurements were recorded at 25°C using 30% excitation power and 60% MST power. Measurements were performed in duplicate.Determination of the binding constant was performed using MO Affinity Analysis v.2.3.AlexaFluor647 NHS Ester (ThermoFisher Scientific) labeled strep-NHR-85 (400 pM) was mixed with 16 serial dilutions of NHR-23 starting at 625 nM. MST measurements were recorded at 25°C using 15% excitation power and 40% MST power. Measurements were performed in triplicate.Determination of the binding constant was performed using MO Affinity Analysis v.2.3.Gel Shifts. For gel shifts with free DNA, 5 ' IRDye (IRDye700 or IRDye800)-labeled and unlabeled oligos were obtained from IDT (Coralville, Iowa) and used to amplify DNA probes of the indicated sequences. For wild-type probes, the indicated PCE fragments were amplified pCMH1954. For mutants probes that harbor mutations in either of the GGTCA repeats, synthetic DNAs were obtained from Synbio Technologies (Manmouth Junction, NJ, USA) and used to amplify the corresponding mutant DNA fragments with the following sequences (mutations underlined and italicized): binding reactions, recombinant proteins were incubated with gel purified DNA probes in 10 mM Tris pH 7.5, 50 mM KCl, 1 mM DTT, 0.1mg/mL poly (dIdC) (Sigma-Aldrich), and 0.25% Tween 20 for 30 minutes at 20 °C (in dark chamber). Samples were then run in a 4% native polyacrylamide gel containing 50mM Tris pH 7.5, 0.38 M glycine and 2mM EDTA in 1x TBE buffer. Gels were imaged and quantified using a Li-Cor Odyssey Imager (Lincoln, Nebraska).Graph Plots and Statistical Analysis. Plots and diagrams were generated using GraphPad Prism v9 (GraphPad Software, San Diego, Ca) or custom-written MATLAB ® scripts. Statistical significance was determined using a two-tailed un-paired Student's t-test. P<0.05 was considered statistically significant. **** indicates P < 0.0001.

Rev-Erb , NHR-23 ROR , and LIN-42 Period expression is phased during each larval stage

  identify common sites with at least one base pair overlapping using BEDTools. All ChIPseq mapping graphs and images were produced in R by customized scripts. Cells were harvested by spinning at 2200 rpm for 20 min and resuspended in lysis/wash buffer (20 mM Tris pH 8.0, 200 mM NaCl, 5 mM BME) and a protease inhibitor cocktail before flash freezing in liquid N2. Cell pellets were stored at -80°C. Cell pellets were thawed and sonicated once. Poly-ethylene imine (PEI) was added at 0.2% to the lysate after cell pellets were thawed and sonicated. The lysate was then spun by ultracentrifuge at 38,000 rpm for 45 min, at 4°C. The supernatant of the lysate was then used for batch binding with Strep-Tactin superflow resin (IBA) for 1 hour while on a rolling shaker at 4°C. The affinity beads were harvested by spinning at 1000 rcf for 5 minutes, then resuspended in lysis/wash buffer and applied to a gravity column. The column was washed with 30 column volumes of lysis/wash buffer and 5 column volumes of ATP wash buffer

	Protein preparation. Full-length C. elegans protein NHR-23 was cloned as a N-terminal Strep-
	SUMO fusion protein in a pFL vector of the MultiBac Baculovirus expression system to create
	pCMH1662 (60). This construct was expressed in insect Sf9 cells grown in CCM3 media (HyClone)
	at 27°C for 60 h.

1, NHR-23 and NHR-85 datasets (11). ModEncode-derived peaks from each TF ChIP-seq dataset were 35 compared to

Acknowledgements:

We would like to thank M. Walhout, J. Ward, J. Kimble for strains and reagents and D. Matus and T.

Medwig-Kinney for help with confocal imaging. We would like to also thank G. Sumati for help with recombinant protein expression. L.J. is an investigator of the Howard Hughes Medical Institute. We acknowledge T. Medwig-Kinney, C. Vakoc, R. Martienssen, U. Pedmale, and D. Jackson for suggestions throughout the course of this study.

Funding: National Institutes of Health R01GM117406 (CMH, NS, KH-M) National Science Foundation 2217560 (CMH, JW) CSHL Cancer Training Grant T32CA148056 (BK) The CNRS ATIP/Avenir program Conseil Regional d'Île de France (DIM ELICIT-AAP-2020 KEIL) (20002719) (WK) Fondation pour la Recherche Médicale FDT202204015083 (SS) Howard Hughes Medical Institute (LJ, DA, MJ)

Data and materials availability: Strains and plasmids listed in the Supplementary Materials and

Methods Section are available through the Caenorhabditis Genetics Center or by request to C.M.H.. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.

Supplementary Materials

Materials and Methods

Figs. S1-S6

Tables S1-S4

References (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) Movies S1-S2

Supplemental Information to Accompany

Circadian rhythm orthologs drive pulses of heterochronic miRNA transcription in C. elegans

Brian Kinney 1 *, Shubham Sahu 2 *, Natalia Stec 1 , Kelly Hills-Muckey 1 , Dexter W. Adams 3,4 , Jing Wang 1 , Matt Jaremako 3 , Leemor Joshua-Tor 3 , Wolfgang Keil (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

MATERIALS AND METHODS:

C. elegans maintenance and genetics. C. elegans strains were maintained on standard media at 20°C and fed E. coli OP50 (49). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). A complete list of strains is outlined in Table S3. RNAi Feeding. RNAi by feeding was performed using E. coli HT115 expressing double stranded RNA corresponding to the indicated target gene or containing a control dsRNA expression plasmid that does not contain sequences corresponding to any C. elegans gene (50, 51). To prevent contamination by E. coli OP50, L4-staged animals were added to RNAi plates individually after removing co-transferred bacteria. For RNAi against nhr-23, bacterial cultures were diluted with control RNAi cultures at the indicated levels prior to experimental onset. In experiments in Fig. S6, starved L1 animals of the indicated genotypes were used. Unless otherwise noted, F1 progeny were analyzed for RNAi-induced phenotypes in the following 2-4 days. Plasmids used for RNAi are outlined in Table S4.

CRISPR genome editing. Genome editing and transgene insertion was accomplished using standard CRISPR/Cas9-mediated genomic editing to the ttTi5605 or ttTi4348 landing site following standard protocols (52). For CRISPR/Cas9 editing of the endogenous lin-42 gene, pCMH1434 (expressing Cas9 and a synthetic CRISPR guide RNA targeting a genomic region encoding the LIN-42 C-terminus) and pCMH1439 (encoding a LIN-42::YFP fragment) were injected in to N2 animals and screened by PCR to identify transgene insertion at the lin-42 gene. Plasmids used to generate CRISPR alleles are outlined in Table S4.

Microfluidics and long-term imaging.

For microfluidics experiments, early to mid L1-staged animals were isolated 6h after starvation-induced L1 arrest at 20°C before an experimental time course. Other stages were individually isolated by observing defined cellular and morphological features indicative of animals developmental stage (1). Animals were mounted into the microfluidic device as previously described (22). During the imaging process, animals were constantly fed NA22 E. coli suspended in S medium. The temperature was kept constant at 20˚C both, at the objective and the microfluidic device using a custom-built water-cooled aluminum ring (for objective) and custom-built aluminum stage inset that was directly coupled to a thermal Peltier device. Table S1. Annotation of ChiP-seq Data from ModEncode used in Fig. S3. Table S2. Table depicting the distribution of ChIP-seq target genes for each TF parsed out into expression categories outlined in Fig. S3. Table S3. C. elegans and yeast strains used this work. Table S4. List of plasmids used in this work.

BIBLIOGRAPHY: