Natalia Stec

Katja Doerfel

Kelly Hills-Muckey

Victoria M Ettorre

Sevinc Ercan

Wolfgang Keil
email: wolfgang.keil@curie.fr

Christopher M Hammell
email: chammell@cshl.edu

An Epigenetic Priming Mechanism Mediated by Nutrient Sensing Regulates Transcriptional Output during C. elegans Development

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

INTRODUCTION

Many developmental systems produce invariable phenotypes despite being confronted with perturbations, such as fluctuations in temperature and nutrient availability. How this robustness emerges from developmental gene regulatory networks (GRNs) and how aspects of precision and flexibility are balanced to generate robust developmental programs is a major open question in developmental biology. C. elegans serves as a unique genetic model for unraveling the mechanisms underlying precision and robustness of gene regulation during development. This is in part because the GRNs that control the essentially invariant cell lineage can generate the identical developmental outcomes in the context of diverse stochastic, environmental or genetic perturbations. A striking example of such a GRN is the so-called heterochronic pathway which controls the timing, sequence and synchrony of stage-specific patterns of cell divisions throughout post-embryonic development. [START_REF] Ambros | Heterochronic mutants of the nematode Caenorhabditis elegans[END_REF] Analysis of this GRN indicates that it generates modular patterns of gene expression that occur in the context of the repetitive post-embryonic molting cycle which, in turn, demarcates the four successive larval stages (L1-L4). Transitions from one temporal program to the next occur through the expression of distinct microRNAs (miRNAs) that down-regulate the expression of their temporal selector gene targets in a strict dosage-sensitive manner. Ectopic or abnormally lower or higher doses of heterochronic genes (e.g. miRNAs) result in the wholesale skipping or reiteration of stage-specific developmental programs. [START_REF] Rougvie | Developmental Transitions in C. elegans Larval Stages[END_REF][START_REF] Feinbaum | The timing of lin-4 RNA accumulation controls the timing of postembryonic developmental events in Caenorhabditis elegans[END_REF][START_REF] Li | Regulatory mutations of mir-48, a C. elegans let-7 family MicroRNA, cause developmental timing defects[END_REF][START_REF] Perales | the Caenorhabditis elegans PERIOD homolog, Negatively Regulates MicroRNA Transcription[END_REF][START_REF] Van Wynsberghe | Period homolog LIN-42 regulates miRNA transcription to impact developmental timing[END_REF] While the regulatory interactions that establish the sequence of stage-specific events are known, we have very little understanding of how the precise timing or levels of gene expression within this GRN are established and how both of these are buffered against perturbations to achieve robust temporal development of the animal. High-temporal resolution transcriptomics studies have outlined a number of remarkable features associated with global gene expression patterns in developing C. elegans larva. [START_REF] Kim | Dampening of expression oscillations by synchronous regulation of a microRNA and its target[END_REF][START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF] These studies indicate that between 10 to 20% of the post-embryonic transcriptome exhibits highly reproducible periodic expression patterns. Periodic transcription occurs in a variety of environmental conditions and is independent of life history, indicating that it is under tight genetic control. Importantly, the transcriptional rhythm follows the cycle of post-embryonic molting, a process that demarcates patterns of stage-specific developmental programs. Under various environmental conditions that modulate overall developmental pace, the timing of transcription onset scales accordingly, such that the phase of transcription onset within the molting cycle is preserved. [START_REF] Kim | Dampening of expression oscillations by synchronous regulation of a microRNA and its target[END_REF][START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF] Currently, it is not known how these transcriptional rhythms are generated, how they are integrated into the execution of stage-specific cellular programs, and how environmental or internal cues modulate features of these transcriptional patterns to achieve robust progression through developmental programs, even after prolonged developmental arrests.

The heterochronic GRN is integrated with global aspects of transcription as each of the miRNAs in this pathway exhibits an oscillatory expression. The most promising candidate gene that integrates the rhythm of C. elegans post-embryonic molting to changes in repetitive transcriptional patterns is lin-42. The lin-42 gene encodes the C. elegans ortholog of PERIOD/Per proteins that are an essential component of the circadian clock in mice, Drosophila and humans. [START_REF] Hurley | Circadian Oscillators: Around the Transcription-Translation Feedback Loop and on to Output[END_REF][START_REF] Jeon | Similarity of the C. elegans developmental timing protein LIN-42 to circadian rhythm proteins[END_REF][START_REF] Monsalve | LIN-42/PERIOD controls cyclical and developmental progression of C. elegans molts[END_REF] Similar to PERIOD, LIN-42 functions as a transcriptional repressor and exhibits a cyclical expression pattern. In contrast to the temperature-invariant approximate 24-hour periodicity of Period, lin-42 transcription is tied to the larval molting cycle, and thus, not temperature compensated. [START_REF] Perales | the Caenorhabditis elegans PERIOD homolog, Negatively Regulates MicroRNA Transcription[END_REF][START_REF] Monsalve | LIN-42/PERIOD controls cyclical and developmental progression of C. elegans molts[END_REF] Hypomorphic mutations in lin-42 cause animals to prematurely execute adult-specific gene expression patterns in the L4 stage of larval development and these phenotypes are caused by the cumulative, premature over-expression of multiple heterochronic miRNAs. [START_REF] Perales | the Caenorhabditis elegans PERIOD homolog, Negatively Regulates MicroRNA Transcription[END_REF][START_REF] Van Wynsberghe | Period homolog LIN-42 regulates miRNA transcription to impact developmental timing[END_REF][START_REF] Mcculloch | Caenorhabditis elegans period homolog lin-42 regulates the timing of heterochronic miRNA expression[END_REF] In addition to these temporal patterning phenotypes, lin-42 mutants also exhibit lengthened and irregular molting cycles. [START_REF] Monsalve | LIN-42/PERIOD controls cyclical and developmental progression of C. elegans molts[END_REF][START_REF] Tennessen | elegans developmental timing protein LIN-42 regulates diapause in response to environmental cues[END_REF] The mechanisms by which LIN-42 negatively regulates heterochronic miRNA transcriptional output remain unknown. Furthermore, it is not known if other regulatory components exist that antagonize LIN-42 functions to ensure normal transcriptional regulation of developmental timing genes.

Here, we leverage the developmental phenotypes of lin-42 mutations to identify BLMP-1 and ELT-3 as conserved transcription factors (TFs) that modulate the transcriptional output of many cyclically expressed genes to provide developmental robustness. We demonstrate that these TFs coordinate a plethora of post-embryonic developmental programs and we present a unique role for BLMP-1 in transcriptional priming that involves chromatin decompaction of target loci. Specifically, we show that BLMP-1 activity modulates the duration and amplitude of transcription of its targets without altering the timing of expression within the repetitive transcriptional cycle. We hypothesize that BLMP-1 functions specifically to assure proper transcriptional output in diverse environmental conditions. Consistent with this model, we demonstrate that the BLMP-1-dependent remodeling of chromatin is regulated by nutritional inputs and becomes essential when animals resume development after nutrient-mediated developmental arrests.

RESULTS

blmp-1 and elt-3 Function in a partially redundant manner to control temporal patterning during larval development.

To identify genes that antagonize the function of LIN-42 to modulate transcriptional output, we took advantage of a highly penetrant phenotype of the lin-42 loss of function allele lin-42(n0189) -the precocious expression of an adult-specific col-19::GFP reporter in both seam and syncytial hyp7 cells of the hypodermis (Figure 1A). [START_REF] Abrahante | Identification of heterochronic mutants in Caenorhabditis elegans. Temporal misexpression of a collagen::green fluorescent protein fusion gene[END_REF] We used bacterial-mediated RNAi to deplete the expression of approximately 20 C. elegans candidate TFs in lin-42(lf) animals and then scored col-19::GFP expression in F1 progeny. These transcription factors were selected based on known strong expression in the hypodermis [START_REF] Cao | Comprehensive single-cell transcriptional profiling of a multicellular organism[END_REF] and/or previous implication in heterochronic developmental defects. The most reproducible suppressors of lin-42 precocious phenotypes were bacterial RNAi clones expressing dsRNAs that target the blmp-1 and elt-3 loci (data not shown). We validated these findings with null alleles of these genes (blmp-1(0) and elt-3(0)) which revealed a redundancy between blmp-1 and elt-3 that is distributed differentially across hypodermal cell types (Figure 1 A and B). Specifically, removal of blmp-1 activity in lin-42(lf) mutants completely suppresses the precocious col-19::GFP expression in the lateral seam cells of L4-staged animals and only partially prevents early expression in hyp7 cells (Figure 1A andB). lin-42(lf); elt-3(0) mutants were only partially suppressed for the precocious col-19::GFP expression phenotypes. In contrast, combining blmp-1(0) and elt-3(0) mutations with lin-42(lf) mutations completely alleviated precocious col-19::GFP expression in both the seam and hyp7 cells of a majority of L4-staged animals (Figure 1A andB). This celltype specific redundancy is likely due to the partially overlapping expression patterns of these TFs in hypodermal cells throughout larval development and indicate that BLMP-1 and ELT-3 are required for lin-42(lf) phenotypes (Figure 1C). [START_REF] Cao | Comprehensive single-cell transcriptional profiling of a multicellular organism[END_REF] Previous analysis of blmp-1(0) mutations indicate that BLMP-1 plays an important role in terminal fate specification of the hypodermis. [START_REF] Horn | DRE-1/FBXO11-Dependent Degradation of BLMP-1/BLIMP-1 Governs C. elegans Developmental Timing and Maturation[END_REF] We sought to determine if blmp-1 functions earlier in temporal cell fate specification when cells are proliferating. To this end, we assayed genetic interactions between blmp-1 and alg-1, the latter encoding one of the two core miRISC argonaute components. alg-1(0) mutants ineffectively process most miRNAs (including heterochronic microRNAs) and exhibit relatively mild temporal patterning defects that resemble those associated with hypomorphic alleles of let-7. [START_REF] Hammell | nhl-2 Modulates microRNA activity in Caenorhabditis elegans[END_REF] To assay interactions, we quantified the number of seam cells at the mid L4 stage in wild-type or alg-1(0) mutant animals in conditions where we reduced blmp-1 and/or elt-3 activity using RNAi (Figure 1D,E). In wild-type animals, lateral seam cells exhibit highly reproducible cell division patterns that occur at specific stages of larval development (Figure 1F). [START_REF] Sulston | Post-embryonic cell lineages of the nematode, Caenorhabditis elegans[END_REF] Defects in this lineage can be monitored by counting seam cell numbers in the L4 larval stage. Depletion of elt-3 or blmp-1 in wild-type animals resulted in the normal number of seam cells in the L4 stage. We observed a significant increase in the number of SCM::GFP(+) seam cells in alg-1(0); blmp-1 RNAi animals (Figure 1E). This expansion of lateral seam cell number could result from inappropriate reiteration of earlier, L2-specific proliferative cell division programs, or conversion of normally asymmetric seam cell divisions in the L3 and L3-stage into symmetric ones (Figure 1F). Depletion of blmp-1 in other mildly reiterative heterochronic mutants also resulted in similar enhancement of cell lineage and col-19::GFP expression phenotypes (Figure S1). elt-3 RNAi did not significantly alter lateral seam cell numbers in alg-1(0) animals or enhance alg-1(0); blmp-1(RNAi) phenotypes.

This indicates that BLMP-1 functions prior to terminal seam cell fate specification in larval development to control temporal cell fates in the hypodermis.

blmp-1 and elt-3 regulate diverse dosage-dependent developmental processes

Development of C. elegans larva results in a fixed number of cells and a stereotypical size under standard growth conditions. Mutations in a number of pathways (including nutrient signaling, TGF-beta and MAP kinase pathways) or in structural components of the cuticle alter organismal size. [START_REF] Tuck | The control of cell growth and body size in Caenorhabditis elegans[END_REF] A shared feature of these regulatory and structural systems is that they function in a dose-sensitive manner wherein changes in the expression levels of individual elements within these pathways or components result in changes in animal morphology. As shown in Figure 2A, blmp-1(0); elt-3(0) animals exhibit a severe dumpy (dpy) phenotype. The dpy phenotype of blmp-1(0); elt-3(0) mutants manifests during post-embryonic development as freshly hatched blmp-1(0); elt-3(0) larvae are indistinguishable from wild-type animals (Figure 2A). blmp-1(0) single mutants exhibit less severe alterations in size and elt-3(0) animals are structurally normal (Figure 2A andB). These results demonstrate that in addition to modulating aspects of temporal cell-fate specification, blmp-1 and elt-3 cooperate to control animal morphology likely through the co-regulation of hypodermally expressed targets.

A majority of blmp-1(0); elt-3(0) animals precociously die two days after they reach adulthood due to the internal hatching of fertilized embryos (Figure 2C). Examination of dying blmp-1(0); elt-3(0) animals revealed that most are trapped in a partially shed cuticle (Figure 2D). Lethality is preceded by a reduction in movement and pharyngeal pumping approximately 8-10 hours after the L4-to-adult transition; a phenotype shared with other mutants that fail to post-transcriptionally down-regulate the adult expression of key genes involved in larval molting. [START_REF] Hayes | The mir-84 and let-7 paralogous microRNA genes of Caenorhabditis elegans direct the cessation of molting via the conserved nuclear hormone receptors NHR-23 and NHR-25[END_REF] To determine if blmp-1(0); elt-3(0) animals inappropriately re-initiate molting programs during adulthood, we directly monitored the expression of a molting-specific GFPpest transcriptional reporter, mlt-10::GFP-pest, in wild-type, single and double mutant animals.

Wild type animals never expressed mlt-10::GFP as adults (n = 100) while a majority of blmp-1(0); elt-3(0) animals re-expressed the mlt-10::GFP-pest reporter (54%; n = 155) as adults (Figure 2E). blmp-1(0) single mutants also express mlt-10::GFP-pest within the same time period but with a much lower penetrance (18%, n = 137), consistent with previous observations using blmp-1(RNAi). [START_REF] Frand | Functional genomic analysis of C. elegans molting[END_REF] These data indicate that blmp-1 and elt-3 function redundantly to halt the behavioral and physiological molting programs once animals transition into adulthood.

Development of the vulval structure in the L3 stage requires the integration of a number of interconnected signaling pathways that establish specific cell fates in vulval precursor cells (Figure 2F). One of the key initiating components in this process is let-60, encoding the C. elegans ortholog of the human Ras oncogene. Continued let-60 activation commits vulval precursor cells (VPCs) to a 1° vulval cell fate specification pattern (Figure 2F). The induction of this fate specification is sensitive to the level of let-60 activity. [START_REF] Beitel | Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction[END_REF][START_REF] Han | let-60, a gene that specifies cell fates during C. elegans vulval induction, encodes a ras protein[END_REF] For instance, the semidominant let-60(n1046gf) allele can induce ectopic vulval protrusions on the ventral surface (multi-vulva; muv) in 18% of animals harboring a single copy of this mutation (Figure 2G andH). We used this sensitized genetic context to determine whether blmp-1(0) and/or elt-3(0) mutations (or the combination of these mutations) leads to an elevation in VPC induction in let-60(n1046)/+ animals. Lack of blmp-1 activity significantly increased the penetrance and expressivity of the muv phenotype in let-60(n1046)/+ animals (Figure G andH). elt-3(0) mutations did not contribute to let-60-dependent phenotypes consistent with a lack of significant ELT-3 expression in these cell types (Figure 3G). [START_REF] Cao | Comprehensive single-cell transcriptional profiling of a multicellular organism[END_REF] Together, these results indicate that blmp-1 and elt-3 function in a partially redundant manner to regulate diverse of developmental aspects, from animal morphology, to behavior to cell fate specification.

BLMP-1 and ELT-3 co-target overlapping gene sets

Our observations that blmp-1 and elt-3 genetically interact with lin-42 to control the transcription of cyclically expressed miRNAs (e.g., let-7, Figure 1D) and that blmp-1(0); elt-3(0) double mutants exhibit pleiotropic defects led us to hypothesize that these TFs may function more broadly to control dynamic gene expression. To define the full repertoire of BLMP-1 and ELT-3 transcriptional targets, we complemented L1-staged BLMP-1 and ELT-3 ChIP-seq data obtained in the modENCODE project with additional ChIP-seq experiments performed in extracts made from L2-, L3-, and L4-staged animals. [START_REF] Araya | Regulatory analysis of the C. elegans genome with spatiotemporal resolution[END_REF][START_REF] Niu | Diverse transcription factor binding features revealed by genomewide ChIP-seq in C. elegans[END_REF] Analysis of these combined data indicates that the regions bound by BLMP-1 in the first larval stage are also bound in subsequent larval stages. Furthermore, the binding sites of BLMP-1 in the L1-L4 stages significantly overlap with regions also bound by ELT-3 in the L1 stage (Figure 3A). A majority of BLMP-1 peaks are enriched within 500bps upstream of the transcriptional start site (TSS) but the enrichment also extends to 5' intergenic regions with a sharp drop-off approximately 3kb upstream of gene bodies of both protein coding and miRNA genes (Figure 3B andC). Peaks from all datasets were assigned to the nearest proximal gene (both coding and non-coding) using these parameters (Data S1A-E).

To characterize the relationship between BLMP-1, ELT-3 and their targets in more detail, we initially focused our analysis on protein coding genes whose expression can be reliably detected in developing larvae. Our analysis employed multiple, high-resolution, and genome-wide time course analyses of gene expression that parsed the larval developmental transcriptome into distinct temporal expression classes. [START_REF] Kim | Dampening of expression oscillations by synchronous regulation of a microRNA and its target[END_REF][START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF] Both studies identified significant, overlapping sets of transcripts (between 12% and 18.9% of the transcriptome) that exhibit cyclical expression patterns. Hendriks et al. classified two types of dynamic expression: a first class of transcripts that exhibit oscillatory or cyclical patterns (osc) that are tied to the molting cycle (osc; 18.9%; 2,718 of 14,378 total genes) or a second class of genes whose expression increases monotonically throughout larval development (rising; 14.2%) (Figure 3D). To compare how BLMP-1 and ELT-3 targets may be distinguished from the targets of other TFs, we analyzed additional publicly available ChIP-seq data sets obtained by the modENCODE project. This data included the targets of 170 additional C. elegans TFs that represent diverse classes of TFs. [START_REF] Araya | Regulatory analysis of the C. elegans genome with spatiotemporal resolution[END_REF][START_REF] Niu | Diverse transcription factor binding features revealed by genomewide ChIP-seq in C. elegans[END_REF] Using the same target assignment criteria as above, we found that most TFs are associated with a limited number of target genes that exhibit a rising expression program (median targets bound = 21 per TF; interquartile range = 9 to 54)(Figure 3E; Data S2A). This relationship was also conserved for the associations between bulk TFs and the oscillatory class of target genes (median of 43 targets per TF; interquartile range = 18 to 97). Surprisingly, we found that a limited subset of 39 TFs is disproportionally associated with a large fraction of the oscillatory class of genes (Figure 3E). Primary amongst this unique group of 39 TFs were BLMP-1 and ELT-3 (with 1124 and 738 of the 2718 cyclically expressed targets bound per TF, respectively) (Figure 3E). Both BLMP-1 and ELT-3 binding sites are statistically enriched in the promoters of cyclically expressed genes (1124 osc BLMP-1 osc targets vs. 3265 non-osc targets and 738 ELT-3 osc targets vs. 2484 non-soc targets; p < 0.0001, Chi square test with Yates correction). We performed a similar analysis of the Kim et al. RNA-seq data to determine if ELT-3 or BLMP-1 binding sites are also enriched in the promoters of cyclically expressed transcripts as defined by their temporal classifications. Our analysis found that BLMP-1 binding sites, but not ELT-3 binding sites, are enriched in the promoters of each cyclically expressed genes class and de-enriched for those that exhibit monotonic expression patterns (Figure S3 and Data S2B).

One robust feature of cyclically expressed mRNAs is that the timing of expression within each larval stage occurs in the same phase of each transcriptional cycle. This expression/phase relationship is preserved even when animals are grown at lower or higher temperatures which lengthens or shortens the periodicity of these patterns, respectively. [START_REF] Kim | Dampening of expression oscillations by synchronous regulation of a microRNA and its target[END_REF][START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF] Because BLMP-1 and ELT-3 are constitutively expressed through each larval stage (Figure 3F), we aimed to determine if the subset of BLMP-1 and ELT-3 targets that exhibit cyclical expression patterns were concentrated in any particular phase of expression during each larval stage. To compare these patterns, we plotted the peak phase (in degrees/radians of a repetitive cycle) and expression level (log2 change in expression as an increasing radius from the center) for each of the cyclically expressed transcripts on a radial chart (Figure 3G). ELT-3 and BLMP-1 targets were distributed across each sector of the repetitive temporal pattern and were not statistically enriched for any phase of expression. The distributed associations of these TFs and their varied targets combined with the constitutive expression of BLMP-1 and ELT-3 throughout each larval stage suggest that BLMP-1 and ELT-3 are associated with their targets throughout the transcriptional cycle regardless of transcriptional class of the target gene or their timing of expression.

Conserved sequences of the upstream regulatory regions of lin-4 are necessary and sufficient for high amplitude expression in hypodermal tissues

Many of the heterochronic phenotypes associated with a null mutant of lin-4, lin-4(e912), can be partially rescued by a high-copy transformation of a 693bp genomic fragment that contains approximately 500bp of upstream putative regulatory sequence. [START_REF] Lee | elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[END_REF] Analysis of the RNAs that are produced from the lin-4 locus indicate transcription of pri-lin-4 is initiated at least five TSS upstream of the lin-4 gene. [START_REF] Bracht | Regulation of lin-4 miRNA expression, organismal growth and development by a conserved RNA binding protein in C. elegans[END_REF] Of these TSSs, three occur upstream of the minimal rescuing fragment, suggesting that additional regulatory information may be required to control normal aspects of lin-4 transcription. These upstream regions are also associated with the active histone modifications H3K4me3 and open chromatin as measured by ATAC-seq assays (Figure 4A). [START_REF] Daugherty | Chromatin accessibility dynamics reveal novel functional enhancers in C. elegans[END_REF][START_REF] Janes | Chromatin accessibility dynamics across C. elegans development and ageing[END_REF] We compared the syntenic sequences of five additional nematode species to determine if regions of the F59G1.4 ninth intron were conserved through evolutionary selection.

This analysis revealed at least three discrete regions upstream of each lin-4 gene that were disproportionately conserved when compared to similarly sized intronic and intragenic regions (Conserved Elements (CE) A, C and D). Examination of our ChIP-seq data indicate that each of these regions is enriched for BLMP-1 binding (Figure 4A, Data S1A-D). We did not detect a strong enrichment of ELT-3 bound to the putative lin-4 regulatory sequences though multiple predicted ELT-3 consensus binding site sequences are found in this region (Figure 4A).

To test the functional relevance of these elements, we constructed a transcriptional GFP-pest reporter that harbors a SL2 splice acceptor sequence directly upstream of the GFP open reading frame (Figure 4A; Full Length (FL)). This feature enables each reporter to generate a single, defined mRNA transcript from each of the various candidate TSSs. This reporter is robustly expressed in a variety of cell types including the hypodermis, pharynx, body wall muscle, and ventral neurons on the surface of larva. Expression of the lin-4::GFP-pest reporter occurs once per larval stage (similar to other characterized transcriptional reporters) in these cell types indicating that this sequence contains the information to drive temporally and spatially restricted expression in multiple cell lineages. To characterize the temporal expression patterns in detail, we measured the levels of lin-4::GFP-pest expression in lateral seam cells at each of the morphologically defined stages of L4 development. [START_REF] Mok | Morphologically defined sub-stages of C. elegans vulval development in the fourth larval stage[END_REF] These experiments revealed that GFP-pest expression is highly pulsatile with expression peaking at the L4.5 and L4.6 stages (Figure 4B andC). Expression in other cell types (neuronal, pharynx, muscle and hyp7 cells) also peak during these larval substages with different expression trajectories (Data not shown). Specifically, muscle cells exhibit a broader (longer) transcriptional pulse compared to those observed in seam cells. Expression in hyp7 and neuronal cell types exhibit a shorter pulse of transcription either muscle or seam cells (Figure 4B).

We then compared the expression patterns we observed using the full-length construct to previously published reporters or variants of the full-length reporter that lack individual or combinations of the conserved sequence elements outlined above. Each of the reporters express pulses of GFP-pest expression in similar cell types as the full-length version. [START_REF] Perales | the Caenorhabditis elegans PERIOD homolog, Negatively Regulates MicroRNA Transcription[END_REF][START_REF] Bracht | Regulation of lin-4 miRNA expression, organismal growth and development by a conserved RNA binding protein in C. elegans[END_REF] However, peak expression in the hypodermal cells was typically 5-20-fold higher from the fulllength reporter compared to the other derivative reporter genes (Figure 4D). Deletion analyses of the full-length construct indicate that CE A, harboring sequences that bind BLMP-1 in vivo, are primarily responsible for this increased transcriptional output as reporters that lack this element exhibit residual expression to a similar level as the minimal rescuing fragment (Figure 4D andE).

Using the ChIP-seq data sets for BLMP-1::GFP, we identified a consensus sequence for BLMP-1 that is enriched in the CE A regulatory sequence (Figure 4A andC). A recombinant GST translational fusion of the five BLMP-1 zinc fingers (ZnFs), GST-BLMP-1(ZnFs), but not GST alone bound several sequences found in CE A element that conform to this consensus. This binding was diminished by the addition of cold competitor DNA harboring consensus BLMP-1 binding sites but not by a similar sized, non-specific target DNA (Figure 4G). To determine if these elements function as a transcriptional enhancer, we cloned a 138bp fragment from the CE A element, called minimal CE (minCE) (Figure 4A andH), into the 5' regulatory sequences of the gst-5 gene encoding the C. elegans ortholog of human HPGDS (hematopoietic prostaglandin D synthetase). RNA-seq experiments demonstrate that gst-5 mRNAs are expressed throughout larval development in a variety of tissues (including the hypodermis) and exhibit a non-cyclical or flat expression pattern. [START_REF] Kim | Dampening of expression oscillations by synchronous regulation of a microRNA and its target[END_REF][START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF][START_REF] Cao | Comprehensive single-cell transcriptional profiling of a multicellular organism[END_REF] A transcriptional reporter bearing 2.9kb of the gst-5 upstream regulatory sequences drives a low level GFP-pest expression in a number of larval cell types including the pharynx, intestine, neurons and hypodermis and this expression pattern remained below the threshold of imaging during normal development (Figure 4H). In contrast, the transcriptional reporter that included two copies of the minCE in the reverse orientation (reversed compared to the orientation within the lin-4 enhancer element) drove high amplitude, periodic expression of GFP-pest exclusively in hypodermal cells (Figure 4H). Expression of the (2xminCE)gst-5::GFP-pest reporter in blmp-1(0) mutant animals resembled the expression of the simple, gst-5::GFP-pest reporter in wildtype animals (Figure S4), consistent with the hypothesis that this minimal sequence from the lin-4 upstream region functions as both a spatial and temporal enhancer.

BLMP-1 controls the transcriptional output of its targets

A large fraction of BLMP-1 and ELT-3 targets are expressed in a cyclical fashion (approximately 26% and 23%, respectively) (Figure 3D and Data S2A). We reasoned that targets that exhibit this highly dynamic form of gene expression could provide an opportunity to determine the effects of BLMP-1 and/or ELT-3 binding on transcriptional dynamics in more detail. To quantify changes in expression dynamics, we obtained or constructed integrated transcriptional GFP-pest reporters that fit three criteria. First, we identified candidate genes that are predicted to be targets of BLMP-1 and/or ELT-3 as measured by ChIP-seq (Figure 3 and Figure S2). Second, we selected genes that are predicted (by RNA-seq experiments) to be dynamically expressed in hypodermal tissues. [START_REF] Cao | Comprehensive single-cell transcriptional profiling of a multicellular organism[END_REF] Third, we selected a set of genes whose expression peaks are predicted (again, by RNA-seq experiments) to occur at a variety of phases within the molting cycle. [START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF] Based on these criteria, we constructed or obtained multicopy integrated transcriptional GFP-pest reporters for (2xminCE)gst-5, moe-3, mlt-10, and ZK180.5 and a single copy version of a transcriptional GFP-pest reporter for C02E7.6 integrated on chromosome II. To characterize the temporal expression patterns of these reporters, we assembled a series of measurements that spanned the morphologically defined stages of L4 development. As shown in Figures 5A-C, each reporter is expressed in a pulsatile expression pattern with (2xminCE)gst-5 and C02E7.6 reporters exhibiting a peak of expression in the mid-L4 stage, and the moe-3 transcriptional reporter reaching maximal amplitude near the end. We next aimed to determine how mutations in blmp-1 and/or elt-3 altered these expression patterns. GFP-pest expression levels in seam cells were then quantified in each genetic context at a developmental stage where reporter expression peaks in wild-type animals. As illustrated in Figure 5A andB, blmp-1(0) mutations dampen the expression of both (2xminCE)gst-5 and C02E7.6 reporters by approximately 1.7-fold. In contrast, elt-3(0) mutations had little effect on the transcriptional output of either of these two reporters.

Furthermore, the decrease in seam cell expression for the (2xminCE)gst-5 and C02E7.6 reporters was not further decreased in blmp-1(0); elt-3(0) double mutants, consistent with a lack of ELT-3 binding sites in these promoters. In contrast, examination of moe-3::GFP-pest expression in various mutant backgrounds indicate that activity of both blmp-1 and elt-3 are required to maintain high transcriptional output for this gene in the hypodermis (Figure 5C).

We next sought to correlate dynamic changes in gene expression to other aspects of larval development in more detail. To accomplish this, we performed long-term, live imaging of developing larvae in a microfluidic device [START_REF] Keil | Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics[END_REF] , enabling us to continuously monitor gene expression dynamics in individual animals and also correlate this dynamics with other cellular and developmental milestones. We focused on two of the most strongly expressed reporter genes, mlt-10pro::GFP-pest and zk180.5pro::GFP-pest, both exclusively expressed in the hypodermis (Figure 5D) and their respective putative regulatory elements bound by BLMP-1 and ELT-3 (Figure 3 and Figure S6). First, we compared overall developmental pace between WT, blmp-1(0), elt-3(0) and blmp-1(0);elt-3(0) animals in the microfluidic chambers by assessing the time between the successive ecdysis (cuticle shedding). While both WT and mutant animals displayed variability of larval stage duration within each genotype, neither blmp-1(0) nor elt-3(0) mutations (or combination of these mutations) affected the average duration to a substantial degree (Figure 5E and Figure S5). However, our live imaging experiments revealed a partially penetrant larval lethality phenotype, in particular in the L3 stage, that was not apparent on standard, solid NGM plates.

The fact that the larval stage durations are largely preserved between WT and blmp-1(0)/elt-3(0) mutants allowed us to directly compare average quantitative temporal features of transgene gene expression dynamics such as the time within each transcriptional cycle in which transcription begins, peak phase (the point within each cycle in which the peak expression is reached), and the cumulative accumulation of GFP-pest expression (output) between genetic backgrounds. To this end, we extracted individual whole-animal fluorescence time course measurements and rescaled them such as to align the timing of molting events of each animal (see Methods) to the average WT molting times (Figure 5F). Consistent with other blmp-1-or blmp-1; elt-3-dependent reporters, cyclical transcription of ZK180.5::GFP-pest and mlt-10::GFP-pest was maintained throughout larval development indicating that blmp-1 (or elt-3) are not essential for generating pulsatile patterns of transcription (Figure 5F-J and Figure S6). The primary difference between the expression trajectories of wild type and mutant animals was the drastic reduction in the amplitude of individual traces in blmp-1(0) mutants. Surprisingly, the reduction of transcriptional output was not accompanied by any substantial change in the timing of transcriptional induction (Figure 5H and J and Figure S6). Ultimately, the reduction of transcriptional output (as measured by total cumulative GFP-pest expression during each pulse) correlates with a reduction in the relative duration of expression in mutant animals (Figure 5H and J and Figure S6). These results indicate that BLMP-1 functions to amplify the baseline pulsatile transcriptional dynamics that are likely generated by other gene regulatory mechanisms. With respect to the (2xminCE)gst-5::GFP-pest reporter (see above), our findings suggest that the 138bp minCE fragment may contain the regulatory sequences required by these mechanisms to generate oscillatory transcription but the resulting oscillation amplitude is strongly dependent on BLMP-1 binding.

The lin-4 locus adopts a BLMP-1-dependent, active chromatin structure in hypodermal cells

Previous studies indicate that BLMP-1 physically and functionally interacts with multiple chromatin remodeling factors (e.g., SWI/SNF subunits) and that BLMP-1 binding sites are enriched in open chromatin regions (Figure S2). [START_REF] Daugherty | Chromatin accessibility dynamics reveal novel functional enhancers in C. elegans[END_REF][START_REF] Janes | Chromatin accessibility dynamics across C. elegans development and ageing[END_REF][START_REF] Fong | LDB1 and the SWI/SNF complex participate in both transcriptional activation and repression by Caenorhabditis elegans BLIMP1/PRDM1[END_REF] We hypothesized that BLMP-1 may function to potentiate transcription through chromatin remodeling near its binding sites which would potentiate the binding of additional TFs. This model would account for the dependency of the diverse transcriptional targets of BLMP-1 that differ in their temporal pattern of expression (phase) during the repetitive transcriptional cycle and would be mechanistically similar to the activities of other TFs (including pioneer factors) that remodel chromatin accessibility prior to the onset of major transcriptional induction in C. elegans. [START_REF] Cochella | Embryonic priming of a miRNA locus predetermines postmitotic neuronal left/right asymmetry in C. elegans[END_REF][START_REF] Hsu | Recruitment of RNA polymerase II by the pioneer transcription factor PHA-4[END_REF][START_REF] Patel | Coordinated control of terminal differentiation and restriction of cellular plasticity[END_REF] We therefore sought to measure how BLMP-1 activity may alter chromatin dynamics at a specific locus with spatiotemporal resolution. We utilized an established chromosome-tagging strategy that has been employed to visualize both the localization of transgenic arrays and dynamics of chromatin compaction in living animals. [START_REF] Cochella | Embryonic priming of a miRNA locus predetermines postmitotic neuronal left/right asymmetry in C. elegans[END_REF][START_REF] Fakhouri | Dynamic chromatin organization during foregut development mediated by the organ selector gene PHA-4/FoxA[END_REF][START_REF] Meister | The spatial dynamics of tissue-specific promoters during C. elegans development[END_REF] Transgenes containing the lin-4(FL)::mCherry-pest ORF flanked by tandem copies of the bacterial lac operator (lacO) sequences were integrated to a single chromosomal site and then crossed into a strain harboring a soluble, ubiquitously expressed GFP-tagged lac repressor (LacI) fusion that recognizes lacO sequences with high affinity. [START_REF] Patel | Coordinated control of terminal differentiation and restriction of cellular plasticity[END_REF] The enabled us to monitor the lin-4 locus in live animals while simultaneously monitoring transcriptional output of the lin-4 enhancer/promoter (Figure 6A). Animals harboring these transgenes exhibit two foci in each somatic nucleus indicating the stable, single integration site of the lin-4::mCherry/LacO transgene. The intranuclear GFP-LacI foci found in freshly hatched L1-staged animals can be grossly categorized into two basic classes: Class 1) a punctate or "ball-like" pair of GFP-LacI foci that are found in a majority of somatic cells and Class 2) pairs of "puffed" foci that are found in the nuclei of hypodermal cells (Figure 6B). This lineage-specific decompaction of the lin-4 locus is surprising for multiple reasons. First, lin-4 loci appear to be decompacted only in hypodermal tissues where BLMP-1 is normally expressed. Second, chromatin decompaction is typically associated with active transcription in a variety of systems [START_REF] Dietzel | Differential large-scale chromatin compaction and intranuclear positioning of transcribed versus nontranscribed transgene arrays containing beta-globin regulatory sequences[END_REF][START_REF] Tumbar | Large-scale chromatin unfolding and remodeling induced by VP16 acidic activation domain[END_REF][START_REF] Yuzyuk | The polycomb complex protein mes-2/E(z) promotes the transition from developmental plasticity to differentiation in C. elegans embryos[END_REF] and the decompaction of the lin-4 loci in freshly hatched L1-stage animals precedes any detectible lin-4::mCherry-pest expression (Figure 6B).

To test whether BLMP-1 expression correlates with target loci decompaction, we performed two types of analyses. First, we characterized when BLMP-1 expression begins in embryos. BLMP-1::GFP can be detected as early as the bean stage (approximately 365 minutes post fertilization) in most developing hypodermal cells (Figure 6C). This tissue-specific expression pattern remains throughout the rest of embryonic development (Figure 6C). We next aimed to determine how the timing of BLMP-1 expression correlates with the decompaction level of the lin-4 loci by examining changes in GFP-LacI foci. From the onset of GFP-LacI expression to the early gastrulation stage of embryogenesis, most somatic lin-4 GFP-LacI foci are decompacted, consistent with a general lack of condensed chromatin and reduced histone H3 lysine 9 (H3K9) methyltransferase activity in early embryos. [START_REF] Yuzyuk | The polycomb complex protein mes-2/E(z) promotes the transition from developmental plasticity to differentiation in C. elegans embryos[END_REF][START_REF] Mutlu | Distinct functions and temporal regulation of methylated histone H3 during early embryogenesis[END_REF][START_REF] Mutlu | Regulated nuclear accumulation of a histone methyltransferase times the onset of heterochromatin formation in C. elegans embryos[END_REF][START_REF] Towbin | Step-wise methylation of histone H3K9 positions heterochromatin at the nuclear periphery[END_REF] By the end of gastrulation (approximately 330 minutes post fertilization) the foci in most somatic cells are equally condensed consistent with the onset of large-scale heterochromatin formation in somatic cells and a reduction in transcriptional plasticity. [START_REF] Yuzyuk | The polycomb complex protein mes-2/E(z) promotes the transition from developmental plasticity to differentiation in C. elegans embryos[END_REF][START_REF] Mutlu | Distinct functions and temporal regulation of methylated histone H3 during early embryogenesis[END_REF][START_REF] Mutlu | Regulated nuclear accumulation of a histone methyltransferase times the onset of heterochromatin formation in C. elegans embryos[END_REF] At roughly the same time as the onset of BLMP-1 expression in embryonic hypodermal cells, the GFP-LacI foci begin to decompact in posterior seam cells (Figure 6D). The cell type-specific decompaction of lin-4 loci in hypodermal tissues is complete for a majority of wild-type animals by the end of embryogenesis while the same foci in other somatic cell types remain tightly compressed (Figure 6D). GFP-LacI foci dynamics were similar in wild-type and blmp-1(0) animals during early embryogenesis. In contrast to the late embryonic decompaction of lin-4 loci in wild-type animals, hypodermal GFP-LacI foci (and foci in other somatic nuclei) in blmp-1(0) mutants remained condensed (Figure 6D andE). We conclude that BLMP-1 is required for decompaction of the lin-4 loci in hypodermal cells during embryogenesis. Furthermore, this decompaction occurs at a point in development where lin-4 transcription is being actively repressed by multiple FLYWCH proteins [START_REF] Ow | The FLYWCH transcription factors FLH-1, FLH-2, and FLH-3 repress embryonic expression of microRNA genes in C. elegans[END_REF] and transcriptional activity is undetectable with our fluorescent transcriptional reporter; though minimal transcription during this period cannot be excluded.

We next aimed to directly monitor any dynamic changes in "puffs" sizes within the transcriptional cycle of lin-4 during larval development and determine if these changes are dependent on BLMP-1 activity. In head ganglion cells, derived from a cell lineage that never express the lin-4pro::mCherry-pest transgene, the two lin-4pro::mCherry-pest loci are constitutively compacted (Figure 6F andG). To measure changes in cells that do express lin-4 miRNAs, we focused our analysis on developing lateral seam cells for multiple reasons. First, seam cells constitutively express BLMP-1 and dynamically express the lin-4pro::mCherry-pest reporter at specific phases of each larval stage (Figure 1B and Figure 4A). Second, in contrast to hyp7 syncytial cells that endoreduplicate their nuclear DNA content at each division resulting in a stable ploidy of 4n, seam cells replicate their nuclear DNA once per division at specific, defined phases of the larval molt cycle. [START_REF] Hedgecock | Polyploid tissues in the nematode Caenorhabditis elegans[END_REF] To ensure accuracy in both the timing of lin-4::mCherry-pest expression and seam cell divisions, we specifically measured the compaction status of the lin-4::mCherry-pest loci in late L3-staged animals where morphological features of the vulval cell lineage, gonad morphology, and mCherry-pest expression could be used to compare animals of both genotypes at similar developmental stages. We found that the GFP-LacI "puffs" size lateral seam cells with undetectable lin-4pro::mCherry-pest expression was typically three times larger than those found in neurons (Figure 6F andG). Later, during the L3 lethargus, when lin-4pro::mCherry-pest is expressed, we observed that the average size of the GFP-LacI "puffs" further increase in size (approximately an additional 2-fold when compared to seam cells lacking lin-4pro::mCherry-pest expression) (Figure 6F andG). This suggests that the lin-4 locus is constitutively decompacted (compared to the same locus in neuronal cells) and the process of transcribing this locus further relaxes chromatin architecture. We then asked if chromatin decompaction at these loci was dependent on BLMP-1 activity by examining the same features in blmp-1(0) animals. A comparison of the GFP-LacI "puff" sizes in head neurons indicates that they are equally compressed in wild-type and blmp-1(0) animals (Figure 6F andG). In contrast, GFP-LacI foci, in both non-transcribing and lin-4 expressing cells, are significantly reduced in size compared to those observed in stage-matched wild-type animals (Figure 6F andG). These experiments demonstrate that the chromatin structure of the lin-4 locus is dynamically remodeled in seam cells and that BLMP-1 is required for decompaction of the lin-4 locus in hypodermal cells prior to high-amplitude transcription in wild-type, larval animals.

BLMP-1 and ELT-3 are required to resume normal temporal patterning after nutrient deprivation

Development of C. elegans larvae in laboratory conditions is both rapid and continuous due to an abundant, controlled food supply and standardized growth conditions. In contrast, the natural environment in which C. elegans larvae normally live is far more diverse and dynamic leading to a more punctuated developmental trajectory. [START_REF] Frezal | elegans outside the Petri dish[END_REF] Acute food removal induces a defined developmental diapause that can occur immediately after each larval molt. [START_REF] Schindler | Identification of late larval stage developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and steroid hormone signaling pathways[END_REF] The diapause is part of a developmental checkpoint that is distinct from dauer development and controlled by insulin signaling. [START_REF] Schindler | Identification of late larval stage developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and steroid hormone signaling pathways[END_REF] When insulin signaling becomes rapidly inactivated (i.e. during acute starvation), animals finish the current larval stage, including somatic cell divisions and the molt, but then arrest all somatic cell movements and divisions, and suspend the subsequent expression of molting-specific genes. [START_REF] Schindler | Identification of late larval stage developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and steroid hormone signaling pathways[END_REF] To determine if global aspects of cyclical transcription are altered during acute starvation, we monitored the expression of four transcriptional reporters that exhibit distinct phases of expression (Figure 5). These reporters include targets of BLMP-1 and ELT-3 ((minCE)gst-5::GFP-pest, ZK180.5::GFP-pest, and mlt-10::GFP-pest) as well as a transcriptional reporter of blmp-1, since ChIP-seq data indicates that it is expressed in a cyclical expression pattern [START_REF] Kim | Dampening of expression oscillations by synchronous regulation of a microRNA and its target[END_REF][START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF] and BLMP-1 activity regulates its own expression through direct positive feedback (Figure S7). We grew populations of L1-staged animals under nutrient-rich conditions for 22 hours and then one half of each sample was allowed to continue development in nutrient replete conditions while the other half was acutely starved (Figure 7A). Acute food removal results in greater than 90% of transgenic animals arresting development with undivided vulval precursor cells after 24 hours indicating that larval development has been paused. [START_REF] Schindler | Identification of late larval stage developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and steroid hormone signaling pathways[END_REF] During the starvation period, we monitored GFP-pest expression in fed and starved populations. In contrast to the cyclical expression of each reporter under nutrient replete conditions, GFP-pest expression for each of the reporters was extinguished during starvation conditions with similar kinetics (Figure 7B). This transcriptionally inactive state was maintained throughout the starvation period. Re-fed animals resumed normal, cyclical expression patterns within a few hours after food exposure (Figure 7B). These experiments demonstrate that oscillatory transcription is inhibited by the starvation-induced developmental diapause and that wild-type animals can re-initiate these transcriptional patterns in a nutrient-dependent manner.

BLMP-1 expression is actively regulated post-translationally by a conserved F-box protein, DRE-1, that mediates the ubiquitination of target proteins which are degraded by the proteasome. [START_REF] Horn | DRE-1/FBXO11-Dependent Degradation of BLMP-1/BLIMP-1 Governs C. elegans Developmental Timing and Maturation[END_REF] Given that blmp-1 is cyclically transcribed and its transcription is inhibited in nutrient depleted conditions (Figure 7B), we hypothesized that the constitutive expression of BLMP-1 during normal development may be altered during starvation. To test this, we repeated the nutrition-mediated arrest experiments outlined above in strains harboring BLMP-1::GFP and ELT-3::GFP translational fusions. The expression patterns of each translational fusion were compared to a constitutively expressed monomeric GFP transgene (sur-5::GFP) to control for potential changes in GFP half-life under starvation conditions. Surprisingly, BLMP-1::GFP expression was diminished during starvation with >50% of animals loosing detectible BLMP-1::GFP expression after 12 hours of starvation. In contrast to the down-regulation of BLMP-1::GFP, most starved animals retained ELT-3::GFP and soluble GFP expression throughout the starvation period with only a mild reduction after 24 hours (Figure 7C). As with the transcriptional reporter for blmp-1 expression, BLMP-1::GFP expression was detectible again in most animals within 2 hours or re-feeding and resumed normal, pre-starved expression levels after 5 hours (Figure 7C). Similar changes in blmp-1 mRNA and BLMP-1 protein expression were seen in freshly hatched and starved L1 (Figure S8). We conclude that BLMP-1 expression, both transcriptionally and post translationally, is regulated by nutrient availability.

To examine if BLMP-1-dependent chromatin remodeling is also modulated by nutrientdependent signaling, we monitored compaction of lin-4 locus in animals that have entered the acute starvation-dependent developmental diapause. At the mid-L3 stage (first VPC division), a developmental time point that precedes lin-4 transcription during the L3 stage the lin-4 locus was decompacted (Figure 7D). We then rapidly removed the bacteria food source to induce the L4-specific developmental diapause. After 22 hours of starvation, >90% of animals arrested at the invagination stage of vulval development (L4.1-L4.2) which precedes fusion between the anchor cell and vulval precursor cells on the ventral surface of the L4 larva. [START_REF] Mok | Morphologically defined sub-stages of C. elegans vulval development in the fourth larval stage[END_REF] Analysis of lateral seam cell GFP-LacI puffs sizes in starved animals demonstrated that the lin-4 loci in seam cells were dramatically compacted when compared to the same loci in continuously developing animals (Figure 7D). Re-plating of these starved animals lead to the rapid decompaction of the lin-4 locus at a timepoint with BLMP-1 expression has resumed (Figure 7D). Interestingly, the level of decompaction is greater in recovering animals that those found in non-starved, continuously developing animals (Figure 7D). We also monitored lin-4::mCherry/LacO loci in the seam cells of starved, L1-stage animals and also found that loci compacted in these starvation conditions (Figure S7). In sum, these experiments indicate that dynamic chromatin rearrangements are modulated by nutrient sensing.

Changes in BLMP-1 expression and the dramatic compaction and decompaction of BLMP-1 target chromatin during starvation and recovery, respectively, suggests that BLMP-1 could play an essential role in modulating transcriptional output of critical developmental genes in dynamic environments. To determine whether animals that lack blmp-1 and/or elt-3 can robustly adapt to starvation, we repeated the food experiments as described in Figure 7A but maintained the animals in starvation conditions for increasing periods of time. At the end of the starvation period, animals were re-plated on normal food and monitored for developmental phenotypes by scoring changes in col-19::GFP expression when the animals had reached adulthood (Figure 7E). In these experiments, wild-type and elt-3(0) larvae re-initiate normal temporal patterning after prolonged bouts of starvation (Figure 7E). In contrast, a minor fraction of blmp-1(0) mutants failed to express col-19::GFP in hyp7 cells, a phenotype associated with weak reiterative heterochronic mutants. [START_REF] Hammell | nhl-2 Modulates microRNA activity in Caenorhabditis elegans[END_REF][START_REF] Abbott | The let-7 MicroRNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans[END_REF] The relatively mild blmp-1(0) phenotypes were dramatically enhanced in blmp-1(0); elt-3(0) double mutants. These results indicate that BLMP-1 and ELT-3 are essential for resuming normal temporal patterning after continuous development has been interrupted by starvation.

Discussion

Features of the oscillatory expression program impose constraints on modulating transcriptional output

Oscillatory patterns of gene expression are a ubiquitous feature of developmental systems where they orchestrate repetitive biological functions and/or anticipate recurring environmental conditions. [START_REF] Hasty | Systems biology of cellular rhythms: from cacophony to symphony[END_REF] While most GRNs that program cyclical patterns of gene expression utilize a basic negative feedback structure, specific features of their architecture define the precision of the timekeeping mechanism and the ability to modulate and/or preserve features of dynamic transcription. For instance, the periodicity of circadian gene expression persists in the absence of external cues over a wide range of temperatures, yet changes in lighting or nutrition can alter the phase of gene expression or the amplitude of clock target gene transcription, respectively. [START_REF] Shigeyoshi | Light-induced resetting of a mammalian circadian clock is associated with rapid induction of the mPer1 transcript[END_REF][START_REF] Trott | Regulation of circadian clock transcriptional output by CLOCK:BMAL1[END_REF] The genetic control of plant root formation also employs a complex feedback loop that generates cyclical transcriptional cycles that induce the formation of lateral roots at periodic positions along the radial surfaces of the developing primary root. The transcriptional output of this GRN is modulated by a hormone, auxin, that amplifies oscillatory transcription in a dose-dependent manner. [START_REF] Moreno-Risueno | Oscillating gene expression determines competence for periodic Arabidopsis root branching[END_REF] Exposure to light is required for maintaining the oscillatory transcriptional patterns at the pre-branch sites. [START_REF] Kircher | Priming and positioning of lateral roots in Arabidopsis. An approach for an integrating concept[END_REF] Water availability can also modulate the ability to the root clock to induce lateral root founder cell development. Uneven water distribution on the surface of the developing primary root leads to a suppression of LR development on the dryer root surface. Importantly, changes in water distribution do not alter the cycling of core root clock components indicating the environment can control clock output at the phenotypic level. [START_REF] Bao | Plant roots use a patterning mechanism to position lateral root branches toward available water[END_REF] While the molecular components of the C. elegans expression oscillator are not fully known, a detailed characterization of the systems-level behavior of this GRN indicates that two features are hard-wired. First, the biological oscillator is directly tied to the molting cycle and gated by genetically-regulated developmental checkpoints. [START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF][START_REF] Schindler | Identification of late larval stage developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and steroid hormone signaling pathways[END_REF][START_REF] Meeuse | Developmental function and state transitions of a gene expression oscillator in Caenorhabditis elegans[END_REF] Under replete nutritional conditions, transcriptional cycling is continuous. [START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF] Removal of food either at hatching or just prior to the termination of the molt results in a regulated arrest of the developmental clock at a precise point within the transcriptional cycle (Figure 7B). [START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF] Re-initiation of the transcriptional clock (either from starved L1-stage animals, dauer arrest, or the nutrition-mediated arrest points at each larval stage) occurs at this same point. [START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF] These features indicate that once initiated, the transcriptional cycle is modular in nature and progresses until the next checkpoint.

Secondly, the periodicity of thousands of cyclically expressed transcripts scales inversely with temperature over a wide dynamic range and the relative timing of expression for individual genes within the transcriptional cycle is phase-locked. [START_REF] Kim | Dampening of expression oscillations by synchronous regulation of a microRNA and its target[END_REF][START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF] Because the accordion-like, modular structure of this GRN constrains the relative timing of transcriptional events within the cycle, other features of cyclically transcribed genes are likely modulated independently of the clock. This adaption of transcriptional output to various environments would be especially important for genes that are expressed at determinant phases within the transcriptional cycle and function in dosage-sensitive manners. This feature is exemplified by the heterochronic miRNAs that exhibit pulsatile transcriptional patterns at each larval stage and also function to control discrete transitions of temporal cell fate at specific developmental milestones (molting). [START_REF]MicroRNAs and developmental timing[END_REF] It had been previously shown that LIN-42, the C. elegans Period ortholog, functions to limit transcriptional output of this class of genes by negatively regulating the overall duration of transcription within each larval stage. [START_REF] Perales | the Caenorhabditis elegans PERIOD homolog, Negatively Regulates MicroRNA Transcription[END_REF][START_REF] Van Wynsberghe | Period homolog LIN-42 regulates miRNA transcription to impact developmental timing[END_REF][START_REF] Mcculloch | Caenorhabditis elegans period homolog lin-42 regulates the timing of heterochronic miRNA expression[END_REF] In this manuscript, we present evidence that BLMP-1 functions to antagonize LIN-42 activities by increasing the duration of transcription for cyclically expressed genes. As with lin-42, blmp-1 is not essential for the generation of cyclical expression patterns but plays a modulatory role in controlling the duration and amplitude of transcription (Figure 7G). How the interplay between these two antagonistic modulators of gene expression achieves precise tuning of gene-dosage to confer robustness and adaptivity to post-embryonic developmental programs is an exciting route for further investigation given that BLMP-1 targets the lin-42 locus (Data S1A-D).

BLMP-1 couples chromatin de-compaction and the regulation of transcriptional output

In C. elegans, BLMP-1 is known to physically interact with chromatin remodeling components (SWSN-1, a core component SWI/SNF of the complex (orthologous to human SMARCC1/BAF155) and HAM-3, an accessory subunit of the SWI/SNF complex) suggesting that BLMP-1 may direct these activities in a sequence-specific manner. [START_REF] Fong | LDB1 and the SWI/SNF complex participate in both transcriptional activation and repression by Caenorhabditis elegans BLIMP1/PRDM1[END_REF] Mouse Blimp-1 has been shown to alter transcriptional activity of target genes by recruiting chromatin remodeling and histone modifying complexes. [START_REF] Minnich | Multifunctional role of the transcription factor Blimp-1 in coordinating plasma cell differentiation[END_REF] The physical associations are conserved between the human ortholog of BLMP-1, hBlimp1, and the hSWI/SNF complex; suggesting additional features of BLMP-1 function may be shared. Here, we characterize a molecular role for BLMP-1 in regulating the transcriptional output of a number of cyclically expressed genes in C. elegans through a mechanism involving the decompaction of chromatin near its target gene loci. In contrast to the chromatin decompaction that occurs during normal transcriptional activation [START_REF] Dietzel | Differential large-scale chromatin compaction and intranuclear positioning of transcribed versus nontranscribed transgene arrays containing beta-globin regulatory sequences[END_REF][START_REF] Tumbar | Large-scale chromatin unfolding and remodeling induced by VP16 acidic activation domain[END_REF][START_REF] Yuzyuk | The polycomb complex protein mes-2/E(z) promotes the transition from developmental plasticity to differentiation in C. elegans embryos[END_REF] , we demonstrate that BLMP-1-dependent decompaction is temporally separated from the activation of the target gene expression and can occur even when transcription is actively repressed by other mechanisms (e.g. for the lin-4 gene during embryogenesis (Figure 6B andD). [START_REF] Ow | The FLYWCH transcription factors FLH-1, FLH-2, and FLH-3 repress embryonic expression of microRNA genes in C. elegans[END_REF] We hypothesize that this anticipatory priming mechanism constitutively opens chromatin loci near BLMP-1 binding sites. These "primed" loci would then be accessible to additional TFs whose binding capacity may be normally restricted by nucleosome complexes. Once bound, these factors would induce the phased patterns of gene expression that define the transcriptional cycle for each target gene (Figure 7 G). Because BLMP-1 binding sites are associated with a multitude of genes that are expressed in diverse phases of the oscillatory transcriptional pattern (Figure 3), we speculate that the BLMP-1-dependent "priming" activity is not limited to a single partner TF but likely facilitates the association of several TFs that function at distinct phases of the transcriptional cycle. In the absence of BLMP-1, many of these factors would exhibit a reduced capacity to bind to their cognate binding sites. As a result, blmp-1(0) mutants exhibit broad changes in transcriptional dynamics. The diversity of these changes would result in pleiotropic phenotypes whose only common feature would be a change in gene dosage across multiple GRNs. Notably, while our analysis focused on genes whose overall expression is upregulated in the presence of BLMP-1, the mechanism we propose by which BLMP-1 modulates target transcription is equally capable of inhibiting transcription of target genes (by increasing chromatin accessibility for transcriptional repressors).

The molecular mechanism that we propose for BLMP-1 in temporal gene regulation is analogous to the role that the TF Zelda (ZLD) plays in establishing normal spatial gene regulation during D. melanogaster embryogenesis. In this system, ZLD is ubiquitously expressed throughout the embryo where it functions as a pioneer factor to rearrange chromatin near its binding sites. [START_REF] Liang | The zinc-finger protein Zelda is a key activator of the early zygotic genome in Drosophila[END_REF][START_REF] Foo | Zelda potentiates morphogen activity by increasing chromatin accessibility[END_REF][START_REF] Mcdaniel | Continued Activity of the Pioneer Factor Zelda Is Required to Drive Zygotic Genome Activation[END_REF] Spatially ubiquitous ZLD activity facilitates the transcriptional activation of genes that are co-targeted by the transcription factor Dorsal (DL). Unlike ZLD, DL is expressed in a dorsal-ventral gradient that extends from the dorsal surface of the embryo to the lateral edge. DL functions in a dosage-dependent manner to generate a graded expression of target genes. [START_REF] Rushlow | The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila[END_REF][START_REF] Roth | A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo[END_REF] Mutation of ZLD binding sites in DL-dependent reporter transgenes results in the dramatic compression of reporter gene expression along the dorsal-ventral axis. [START_REF] Yamada | The Drosophila Pioneer Factor Zelda Modulates the Nuclear Microenvironment of a Dorsal Target Enhancer to Potentiate Transcriptional Output[END_REF] Importantly, zld mutations do not alter DL distribution along the dorsal-ventral axis suggesting that ZLD activity primes DL targets by reorganizing chromatin architecture that would otherwise impair DL binding and function. As with mutations of BLMP-1 in the context of temporal gene expression, zld mutations additionally alter the duration of DL target expression. [START_REF] Yamada | The Drosophila Pioneer Factor Zelda Modulates the Nuclear Microenvironment of a Dorsal Target Enhancer to Potentiate Transcriptional Output[END_REF]

Regulation of BLMP-1 expression in response to nutrient availability coordinates transcriptional output

We hypothesize that the re-initiation of transcription after starvation elicited by food is essential for animals to re-format the chromatin landscape near their target genes after quiescence and to rapidly modulate the transcriptional output of genes that control temporal patterning. In this manuscript we also show that blmp-1 expression is regulated by nutrient availability and is important for the coordination of transcriptional output in a variety of conditions. Under nutritionally replete growth conditions, blmp-1 transcription is both cyclical and auto-regulatory.

While blmp-1 expression at the mRNA level is highly dynamic, BLMP-1 protein expression is highly stable and maintained at relatively constant levels during continuous growth. [START_REF] Kim | Dampening of expression oscillations by synchronous regulation of a microRNA and its target[END_REF][START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF][START_REF] Horn | DRE-1/FBXO11-Dependent Degradation of BLMP-1/BLIMP-1 Governs C. elegans Developmental Timing and Maturation[END_REF] Starvation elicits two main changes in blmp-1 expression that directly lead to a reduction in the priming activity. First, the cyclical transcription of blmp-1 (as with most other cyclically expressed genes) is arrested when animals pause development at a starvation-induced developmental checkpoint. Second, BLMP-1 expression is rapidly curtailed during starvation.

BLMP-1 expression is likely dampened by the combined reduction in transcription and the constitutive activity of DRE-1 that functions independently of starvation. [START_REF] Horn | DRE-1/FBXO11-Dependent Degradation of BLMP-1/BLIMP-1 Governs C. elegans Developmental Timing and Maturation[END_REF] This reduction of BLMP-1 expression is coincident with a re-compaction of chromatin near BLMP-1 target genes.

The establishment of non-permissive chromatin architecture may be a common feature of quiescent cells as diverse cell types (from yeast to human cells) exhibit these changes. It has also been previously suggested to control inappropriate transcription in the context of development. [START_REF] Evertts | H4K20 methylation regulates quiescence and chromatin compaction[END_REF][START_REF] Laporte | Quiescent Saccharomyces cerevisiae forms telomere hyperclusters at the nuclear membrane vicinity through a multifaceted mechanism involving Esc1, the Sir complex, and chromatin condensation[END_REF][START_REF] Pinon | Folded chromosomes in non-cycling yeast cells: evidence for a characteristic g0 form[END_REF][START_REF] Rawlings | Chromatin condensation via the condensin II complex is required for peripheral T-cell quiescence[END_REF][START_REF] Rutledge | The yeast genome undergoes significant topological reorganization in quiescence[END_REF][START_REF] Swygert | Condensin-Dependent Chromatin Compaction Represses Transcription Globally during Quiescence[END_REF] The regulation of BLMP-1 expression through nutrient availability is also important for the coordination of gene expression after starvation as blmp-1(0) and blmp-1(0); elt-3(0) animals fail to resume normal temporal patterning after starvation.

Finally, we hypothesize that the active control of BLMP-1 levels/activities may be important for the adaption of global transcriptional patterns to the environment. The separate genetic control of transcriptional timing and transcriptional output would enable gene expression dynamics to be optimized to specific conditions and ensure developmental robustness. Evidence that the levels of BLMP-1 protein are actively regulated are derived from the observation that dre-1(lf) phenotypes result in an inappropriate over-expression of BLMP-1 and a coincident increase in precocious developmental phenotypes. [START_REF] Horn | DRE-1/FBXO11-Dependent Degradation of BLMP-1/BLIMP-1 Governs C. elegans Developmental Timing and Maturation[END_REF] The active control of BLMP-1 protein expression may play an important role in the adaptation of transcriptional output to different temperatures where the periodicity of the transcriptional cycles and developmental pace changes dramatically. [START_REF] Kim | Dampening of expression oscillations by synchronous regulation of a microRNA and its target[END_REF][START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF] This homeostatic strategy, mediated by controlling the expression of a single pioneer transcription factor, could buffer differences in transcriptional dynamics across the transcriptional cycle by modulating chromatin accessibility. (G) A radar chart plotting oscillation amplitude over the phase of peak expression reveals that BLMP-1 and ELT-3 bind the putative regulatory regions of periodically expressed genes in all phases of the cyclical transcriptional cycle (see Data S1).

See also Figure S2. (I,J) As (F, G) but for mlt-10::GFP-pest (WT, N=7; blmp-1(0), N=5). See also Figure S6. (J) A diagram outlining the changes of blmp-1 mRNA and BLMP-1 protein levels during normal grown and during starvation conditions. marker and unc-119 rescue to select transformants. Gamma ray mutagenesis was used to induce integration of extrachromosomal arrays. [START_REF] Mello | Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences[END_REF]

GFP tagged alleles of blmp-1

We amplified two 0.5kb-long homology arms from N2 genomic DNA flanking the blmp-1 gene and the GFP open reading frame using standard PCR. These fragments were used with Gibson cloning to insert them into a pMCS5 backbone. The resulting plasmid, pCMH1406, was used in combination with pCMH1410 and standard protocols [START_REF] Dickinson | Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination[END_REF] to integrate GFP at the blmp-1 locus. The resulting animals were characterized phenotypically for alae defects that are associated with blmp-1(0). The GFP-tagged allele was wild type.

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 longterm imaging methods, see Microfluidics and Long-term Imaging section.

Chromatin immunoprecipitation

BLMP-1 short reads were first clipped off adapter sequences. Reads of minimum 22bp were mapped to UCSC C. elegans genome (ce10) using bowtie program 76 , looking for unique alignments with no more than 2 mismatches. MACS program (v1.4) [START_REF] Zhang | Model-based analysis of ChIP-Seq (MACS)[END_REF] 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. We defined the promoter region as upstream 3kbp to downstream 300bp around transcription start site (TSS). Peaks located in promoter regions were annotated to their closest TSS sites for both coding and non-coding genes. These potential targets were then overlapped to sets of oscillatory genes identified in previous mRNA-seq-based studies [START_REF] Kim | Dampening of expression oscillations by synchronous regulation of a microRNA and its target[END_REF][START_REF] Hendriks | Extensive Oscillatory Gene Expression during C. elegans Larval Development[END_REF] and the two-tailed Fisher's test was used to calculate the p-values associated with these classifications. ELT-3 (Embryo and L1-staged) raw data were downloaded from modENCODE and processed in the same way as BLMP-1. Peaks from each CHiP-seq dataset were compared to identify common sites with at least one base pair overlapping using BEDTools. [START_REF] Quinlan | BEDTools: a flexible suite of utilities for comparing genomic features[END_REF] All ChIP-seq mapping graphs and images were produced in R by customized scripts.

Microfluidics and long-term imaging

For microfluidics experiments, early to mid L1-staged animals were isolated through a 1-2 hours timed egg-lay approximately 12-14 hours at 20°C before an experimental time course.

Animals were mounted into the microfluidic device as previously described. [START_REF] Keil | Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics[END_REF] During the imaging process, animals were constantly fed NA22 E. coli suspended in S medium. Imaging was done with a 40x, 1.3NA objective using an Andor Zyla 4.2 scMOS camera. 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. [START_REF] Keil | Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics[END_REF]

Native gel shifts

Chromatin decompaction assays

To analyze the compaction state of the lin-4 locus, we exploited the LacI/LacO approach as previously described. [START_REF] Meister | The spatial dynamics of tissue-specific promoters during C. elegans development[END_REF] The arrays containing lin-4 were generated by co-injecting the following components: a) 2ng/µL of linearized pIF9 (unc-119(+)), b) 3ng/µL of linearized pCMH1972 (256x LacO sites), 3ng/µL of pCMH1984 (lin-4pro::SL2::mCherry-pest), and 90ng/µL of sheared salmon sperm DNA (Invitrogen). Transgenic lines produced in this manner (e.g. cshIs116) were integrated using standard procedures and outcrossed into a strain harboring otIs593 (ttx-3::mCherry; let-858pro::GFP::LacI)(Gift of O. Hobert). For imaging nuclear spots assay of either eggs or staged L3/L4 molt animals, Z-stacks (0.3μm spacing) were collected using three channels: DIC, GFP (150ms exposure) and mCherry (60ms exposure). Both acquisition and post-acquisition processing was performed using ZEN Pro software (Zeiss).

Maximum-intensity projections of the planes that span the nuclei of interest were obtained separately for seam and head area. The projections were then exported to Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] and manual traces of areas of the nuclei with GFP intensity above background were made to measure locus compaction/decompaction as described. [START_REF] Cochella | Embryonic priming of a miRNA locus predetermines postmitotic neuronal left/right asymmetry in C. elegans[END_REF]

Food removal assays

Starvation experiments were performed as previously described. [START_REF] Schindler | Identification of late larval stage developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and steroid hormone signaling pathways[END_REF] Briefly, embryos were isolated from gravid adult animals using a standard, hypochlorite treatments and allowed to hatch in M9 buffer. L1 staged/arrested animals were plated onto NGM plates seeded with OP50 E. coli at a concentration lower that 2,500 animals per 60 mm petri dish. Animals were then grown for the indicated amount of time at 20°C unless otherwise noted. Assessment of developmental age was made by direct observation of gonad growth or by specific cell lineage landmarks of lateral seam cells or developing vulval precursor cells. Acute starvation was induced by washing animals off the petri dishes and washing them with M9 for nine times. After the final was animals were resuspended in M9 buffer to a concentration of ~1 animal per µL of sterile M9. Animals were maintained in these starvation conditions at 20°C for the indicated times. Animal development was re-initiated by plating animals onto fresh OP50 E. coli-seeded NGM plates at 20°C. ecdysis event to the next in the L1 to L2 molt cycle. Image analysis was performed as in Keli et al. 2016. [START_REF] Keil | Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics[END_REF] Estimation plots were calculated as previously describted. [START_REF] Ho | Moving beyond P values: data analysis with estimation graphics[END_REF] For Figure 6, panel E, n = number of animals scored in each assay. For Panel G, GFP-LacI areas were quantified from greater that 18 individual nuclei from separate animals for each genotype. GraphPad Prism software was used to plot image data and box plots show data within the 10-90 th percentile with whiskers. For Figure 7, n = number of animals scored in each assay. GraphPad Prism software was used to calculate Mean. For panel D, GFP-LacI areas were quantified from greater than 20 seam cell nuclei from separate animals in each condition.

Figure 1 .

 1 Figure 1. blmp-1 and elt-3 antagonize lin-42 activity to control the expression of heterochronic miRNAs. (A) Representative col-19::GFP expression patterns in wild-type, lin-42(lf), blmp-1(0); lin-42(lf), and blmp-1(0); lin-42(lf); elt-3(0) animals. (B) A quantification of the col-19::GFP expression phenotypes of various mutant combinations in L4-and adult-staged animals. (C) Expression patterns of BLMP-1::GFP and ELT-3::GFP transgenes in hypodermal cells. Sn = Seam cell nuclei. (D) SCM::GFP is expressed in lateral seam cells and can be used to monitor cell division patterns during larval development. Mid-L4-staged alg-1(0); blmp-1(RNAi) animals exhibit a supernumerary number of SCM::GFP cells. (E) Quantification of lateral seam cell numbers of mid-L4-staged animals exposed to bacteria expressing dsRNAs complementary to elt-3, blmp-1 or both blmp-1 and elt-3. Red bars indicate mean and SEM. Asterisks indicate statistically significant differences in phenotype (p = < 0.0001) calculated from a two-tailed chi-square analysis. n = > 40 animals per genotype/RNAi condition. (F) Lateral seam cell lineage of wild-type, blmp-1, and alg-1 mutants and experiments outlined in panel F. See also Figure S1.

Figure 2 .

 2 Figure 2. blmp-1 and elt-3 regulate several additional morphological, behavioral, and cellular phenotypes. (A) blmp-1(0); elt-3(0) animals exhibit a synthetic Dpy phenotype that manifests during larval development. (B) Quantification of animal length-to-width ratio in freshly hatched larvae (left) and young adult animals (right) of the indicated genotypes (n = >20). Red bars indicate the mean and SEM. (C) blmp-1(0) and blmp-1(0); elt-3(0) animals prematurely die after the L4-adult transition. (D) The lethality of blmp-1(0); elt-3(0) adult animals correlates with an inability to shed the cuticle of a supernumerary molt. (E) blmp-1(0); elt-3(0) inappropriately reanimate the expression of a mlt-10::GFP-pest transcriptional reporter during adulthood. Animals were imaged at the L4-adult transition or as gravid adults. (F) Major components of the vulval induction pathway (see text for details). Components that are genetically involved in vulval induction are colored green whereas those involved in

Figure 3 .

 3 Figure 3. BLMP-1 and ELT-3 bind similar sites in the genome and are associated with the putative regulatory regions of gens that exhibit cyclical expression patterns. (A) A matrix of global pairwise factor co-association strengths as defined by promoter interval statistics indicate that BLMP-1 and ELT-3 bind similar genomic regions. (B) The distribution of the peak summit positions of BLMP-1 ChIP-seq data mapped relative to the TSS of protein coding genes or to the +1 nucleotide of the DNA sequence encoding the miRNA precursor RNA. (C) A heat map of ChIP-seq signals +/-3 kb centered on the TSS of each of the 14,252 protein coding genes measured in the developmental RNA-seq data set. 8 Genes are organized for each heat map in a 1:1 fashion according to their density of unique reads present in the BLMP-1 L1-stage ChIP-seq sample (see Data S1). (D) The C. elegans larval transcriptome can be categorized into two dynamic expression classes (rising and oscillating) that are distinct from the predominantly flat expression pattern of most mRNAs (grey region; 66.9%). 8 (E) A graph displaying the number of predicted rising or oscillating class targets for each of the 172 TFs assayed by the ModEncode Projects (data derived from a total of 265 ChIP-seq data sets assayed). Red bars indicate the median (thick) with the interquartile range (thin). Numbers in parenthesis indicate ranks of BLMP-1 or ELT-3 among all TFs (Data S2 and Figure S3).

(F)

  Quantification of GFP intensity for translational reporters of BLMP-1 and ELT-3 during the L4 stage.

Figure 4 .

 4 Figure 4. Conserved elements in the lin-4 promoter are both necessary and sufficient for high-amplitude expression in hypodermal tissues. (A) A browser track overlaying the predicted BLMP-1 consensus binding sites, the BLMP-1::GFP ChIP-seq signal from L1-staged larvae, and conservation of these sequence features found in C. elegans, C. brenneri, C. tropicalis, C. remanei, C. briggsae and C. japonica near the lin-4 locus and promoters used in the expression analysis. Green bars indicate H3K4me3 ChIP-seq data tracks (indicative of active promoters near transcriptional start sites). Black bars indicate the localization of open chromatin as measured from ATAC-seq experiments. 28 (B) Representative measurements of full-length lin-4::GFP-pest expression levels in lateral seam cells. The fluorescent intensity of five seam cells per animal were averaged and 8-15 animals were measured per indicated L4 morphological substage. (C) Representative fluorescence and DIC images of the full-length lin-4::GFP-pest transcriptional reporter in the hypodermal cells of developing L4-staged animals. L4-stage numbers correspond to the defined sub-stages of vulval morphological development (Sn = seam cell nuclei, h = hyp7 cell nuclei, and m = muscle cell nuclei). 30 (D) Measurements of seam cell expression levels for the various lin-4::GFP-pest reporter constructs outlined in panel A at the L4.5 stage of development. (n= 5-10 animals per transgenic strain) (E) Representative images of each transcriptional reporter outlined in D. Individual labels defining cell types are the same as in panel C. (F) Position Weight Matrix of the consensus BLMP-1 binding site calculated from ChIP-seq data outlined in Figure 3A and two sequences located in the minCE element that conform to this consensus. (G) Gel shift experiments demonstrating that a GST fusion protein harboring the 5 ZnF domains of BLMP-1 (but not GST alone) can bind to the two sequences outlined in panel F. (H) Comparison of the GFP-pest expression patterns of gst-5::GFP-pest or (2xminCE)gst-5::GFP-pest in late L1-stage animals. The pulsatile expression of the (2xminCE)gst-5::GFPpest expression correlates with a single pulse in the middle of each larval stage. For the developmental time course, a minimum of forty animals expressing gst-5::GFP-pest or (2xminCE)gst-5::GFP-pest were scored. See also Figure S2 and S4.

Figure 5 .

 5 Figure 5. blmp-1 modulates the amplitude and transcriptional duration of their cyclically expressed targets. (A-C) Quantification of GFP-pest expression from the (2xminCE)gst-5, C02E7.6 and moe-3 transcriptional reporters in L4-staged animals. Top panel for each reporter

Figure 6 .

 6 Figure 6. The lin-4 locus is maintained in an open chromatin state in hypodermal cells in a blmp-1-dependent manner. (A) Schematic of the transgenic array used for visualization of the lin-4 locus in living animals. (B) Representative image of L1-staged animals expressing the GFP-lacO-labeled lin-4 locus. (C) Analysis of the expression pattern of an endogenously GFP-tagged translational fusion of BLMP-1 indicating that expression begins in hypodermal cells around the bean stages and remains stably expressed throughout embryonic development.

For

  recombinant GST-BLMP-1(ZnFs) protein expression, an approximately 400bp PCR fragment (with BamHI ends) was subcloned into the BamHI sites of pGex-3X. The resulting plasmid, pCMH1417, or pGEX-3X alone, was transformed into BLM21(DE3) E. coli. Cultures of these bacteria were grown to an O.D. of 0.5 A600 in LB+amp and induced with 0.2mM IPTG for 2 hours at 37°C. Cell pellets were resuspended in PBS and then sonicated and clarified by centrifugation. Fusion proteins were isolated on glutathione-agarose and eluted with glutathione. The resulting fusion proteins were then dialyzed in 10 mM Tris 7.5, 50 mM NaCl, 0.01% NP-40 1 mM DTT overnight and snap frozen in liquid nitrogen. For gel shifts with free DNA, 5' IRDye (IRDye700 or IRDye800)-labeled and unlabeled oligos were obtained from IDT (Coralville, Iowa). The sequences of these oligos are the following: lin-4 site 1 sequence (+) CTTTCTCCTTCACTTTCTCTCTCTCGGATCACCAG lin-4 site 1 sequence (-) CTGGTGATCCGAGAGAGAGAAAGTGAAGGAGAAAG lin-4 site 2 sequence (+) ATGGGAAAATAGAAAGAGAAAATGAGAGAAAGGAT lin-4 site 2 sequence (-) CTTTCTCTCATTTTCTCTTTCTATTTTCCCAT non-specific sequence (+) GGCTCTTCCGGTCGTGCAAGAGGAAATACAAAAAA non-specific sequence (-) TTTTTTGTATTTCCTCTTGCACGACCGGAAGAGCC Complementary oligos were annealed in TE to a final concentration of 0.1 pmol/µL. 0.1 pmol of these probes were incubated with recombinant GST or GST-BLMP-1(ZnFs) in 10 mM Tris pH 7.5, 50 mM KCl, 1 mM DTT, 0.1µg/µL poly (dIdC) 0.25% Tween 20 for 30 minutes at 20°C (in dark chamber) and 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 using a Licor Odyssey Imager (Lincoln, Nebraska).

Data S1 .Figure 3 .(Continuous

 S13 Figure 3. Tables include gene assignments for L1-L4 BLMP-1 (Data S1A-D) and L1 ELT-3 ChIP-seq data sets (Data S1E).

ACKNOWLEDGEMENTS

We thank A. Zinovyeva, V. Ambros, L. M. Kutscher, and members of the Hammell laboratory for critical review of this manuscript. We received C. elegans strains and recombinant DNAs from S. Gasser, O. Hobert, L. Cochella. The ATIP/Avenir Young Investigator program of the CNRS supported to W.K. W.K. initiated and performed part of this work while being a postdoctoral fellow in the laboratories of Shai Shaham (S.S.) and Eric D. Siggia (E.D.S) at Rockefeller University, supported by NIH grant R35NS105094 to S.S., NSF grant PHY 1502151 to E.D.S. and a postdoctoral fellowship (LT000250/2013-C) from the Human Frontier Science Program (HFSP) to W.K.. Cold Spring Harbor Laboratory, the Rita Allen Foundation, HIH NIGMS R01GM117406 supported C.M.H..

Data S2. Comparison of ChIP-seq data to mRNA expression classes as defined by Hendriks et al. 8 and Kim et al. 7 time course data sets, related to Figure 3. Tables include gene class assignments (flat, oscillating and rising) of ModEncode ChIP-seq data (Data S2A) and assignments of BLMP-1 ChIP-seq data sets to expression cluster data (Data S2B).

DECLARATION OF INTERESTS

The authors declare no competing interests

STAR METHODS

LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Christopher M. Hammell (chammell@cshl.edu). Plasmids generated in this study are available upon request made to the Lead Contact. C. elegans strains generated in this study are available upon request made to the Lead Contact.

Data and Code Availability

A public repository containing MATLAB scripts used for figures and statistical analyses in this paper can be found at https://github.com/wolfgangkeil/Stec_et_al_2020_code.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

C. elegans maintenance and genetics

C. elegans strains were maintained on standard media at 20°C and fed E. coli OP50. [START_REF] Brenner | The genetics of Caenorhabditis elegans[END_REF] A list of strains used in this study is provided in the Key Resources Table. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). blmp-1(tm548) was obtained from Shohei Mitani the National BioResource Project (NBRP) at the Tokyo Women's Medical University.

METHOD DETAILS

RNAi feeding

RNAi by feeding was performed using E. coli HT115 expressing double stranded RNA corresponding to blmp-1 or elt-3, or containing a control dsRNA expression plasmid that does not contain sequence corresponding to any C. elegans gene. [START_REF] Fraser | Functional genomic analysis of C. elegans chromosome I by systematic RNA interference[END_REF][START_REF] Kamath | Systematic functional analysis of the Caenorhabditis elegans genome using RNAi[END_REF] To prevent contamination by E. coli OP50, gravid adult animals were added to RNAi plates individually after removing cotransferred bacteria. Unless otherwise noted, F1 progeny were analyzed for RNAi-induced phenotypes following 2-4 days.

Transgenesis

Cloning was performed using the Gibson Assembly Master Mix (New England Biolabs, #E2611L). Extra-chromosomal arrays were generated using ttx-3::GFP as a co-injection

Quantification Of Gene Expression using GFP reporters

Fluorescence intensities were quantified using ImageJ custom-written MATLAB scripts. software. For measurement of reporter gene expression for time course experiments, we performed dual channel imaging, visualizing gene expression patterns by visualizing GFP-pest expression and overall worm morphology with Nomarski microscopy (DIC). We straightened each three-dimensional image stack using a previously published algorithm [START_REF] Keil | Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics[END_REF] based on a worm backbone which was manually outlined using the DIC channel. The resulting straightened worm z-stacks were then cropped in xy to a region of interest. To deconvolve the straightened, cropped GFP-pest reporter z stacks, we measured the point-spread function (PSF) of our optical setup using red (580/605nm) fluorescent 200nm beads (ThermoFisher Scientific). Using the obtained PSFs, we deconvolved stacks using the classic maximum likelihood estimation (CMLE) algorithm with standard parameters (refractive index of imaging medium: 1.338) in the Huygens Essential software (Huygens Essential 3.7.1) by Scientific Volume Imaging (SVI).

Statistical Analysis

All statistical analyses were performed in MATLAB or GraphPad Prism 9, either using built-in functions (chi squared, Student's t tests) or custom-written scripts (bootstrap permutation test).

Differences between two groups are judged to be statistically significant when p < 0.05 by Student's t test or bootstrap permutation test, where appropriate. Mean and SEM were calculated and plotted using GraphPad Prism or custom scripts. T-tests analyses are based on the normal distribution theory. We did not test the assumption of normality because even when the raw data are not exactly normally distributed, these analysis methods are very robust to departures from normality as long as the sample sizes are larger than approximately 30. For Figure 3, changes in translational reporter expression for BLMP-1::GFP and ELT-3::GFP were quantified as previously described. [START_REF] Keil | Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics[END_REF] Each circle in panel F represents the average GFP fluorescence for 3 hypodermal cells per animal for each translational reporter. For Figure 4,n