Binding Site Analysis of the Caenorhabditis elegans NR 4 ANuclear Receptor NHR-6 During Development

Members of the NR4A subfamily of nuclear receptors make up a highly conserved, functionally diverse group of transcription factors implicated in amultitude of cellular processes such as proliferation, differentiation, apoptosis, metabolism and DNA repair. The gene nhr6, which encodes the sole C. elegans NR4A nuclear receptor homolog, has a critical role in organogenesis and regulates the development of the spermatheca organ system. Our previous work revealed that nhr-6 is required for spermatheca cell divisions in late L3 and early L4 and spermatheca cell differentiation during the mid L4 stage. Here, we utilized chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to identify NHR-6 binding sites during both the late L3/early L4 and mid L4 developmental stages. Our results revealed 30,745 enriched binding sites for NHR-6,∼70% of which were within 3 kb upstream of a gene transcription start site. Binding sites for a cohort of candidate target genes with probable functions in spermatheca organogenesis were validated through qPCR. Reproductive and spermatheca phenotypes were also evaluated for these genes following a loss-of-function RNAi screen which revealed several genes with critical functions during spermatheca organogenesis. Our results uncovered a complex nuclear receptor regulatory network whereby NHR-6 regulates multiple cellular processes during spermatheca organogenesis.


Introduction
Critical aspects of development rely on the proper spatial and temporal expression of genes necessary for a variety of cellular processes.Transcription factors (TFs) possess the ability to rapidly respond to various cell stimuli and regulate gene expression by binding to DNA sequences found in regulatory elements of a specific target gene.A complete understanding of genome-wide TF activities, including identifying both DNA binding regions and target genes, is key to understanding the often complex gene networks they regulate throughout the development of various tissue types.A widely used tool to investigate genome-wide DNA-protein interactions in vivo is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq).Both the ENCODE (Encyclopedia of DNA Elements) [1] and modENCODE (model organisms ENCODE) [2] projects have performed genome-wide ChIP-seq experiments on more than 140 TFs.However, little downstream analysis of target genes has been performed, particularly with respect to the role target genes play in the cellular processes known to be regulated by the TF being examined.
The NR4A subfamily of the nuclear receptor (NR) superfamily functions as TFs that act as early-immediate response genes which respond to a wide array of environmental stimuli and have been implicated in diverse physiological and cellular processes.[3].Unlike liganddependent NRs, members of the NR4A subfamily are orphan receptors that are activated in a ligand-independent manner through posttranslational modifications [4].Following activation, NR4A NRs bind as monomers or homodimers to the NGFI-B response element (NBRE) or Nur response element (NurRE) DNA sequences and both positively and negatively regulate target gene expression [5,6].The mammalian NR4A NR subgroup includes three closely related members-NR4A1 (Nur77), NR4A2 (Nurr1), and NR4A3 (NOR1)-that are highly expressed in energy-dependent tissues including skeletal muscle, heart, brain, and liver where they display tissue and cell-type specific functions.Due in part to their ability to rapidly regulate multiple target genes in response to cellular stimuli including growth factors [7], these receptors have been strongly implicated in developmental processes.Not surprisingly, several studies have confirmed important roles for NR4A NRs during the development of several cell types.Examples include T-cell, monocyte, myeloid and dopaminergic neuron differentiation [8][9][10][11][12][13], smooth muscle cell and hepatocyte proliferation [7,14,15], and mesenchymal stromal cell migration [16].
The sole Caenorhabditis elegans NR4A NR gene, nhr-6 [17,18], is a lineage-specific regulator of the spermatheca, a structurally simple but functionally complex reproductive organ that functions in oocyte fertilization and ovulation [19].NHR-6 is robustly expressed in all developing spermathecal cells from the middle of the third larval stage (L3) into the middle of the fourth larval stage (L4), a key time point in spermatheca development.Previous work in our lab revealed that nhr-6 has a dualistic function during spermatheca organogenesis, regulating both cell proliferation and differentiation [19,20].NHR-6 has also been shown to bind and activate transcription from the canonical NBRE site in mammalian HEK293 cells, while a cysteine to serine mutation in the DNA binding domain abolished its ability to bind DNA at the NBRE site [21], indicating biochemical conservation with mammalian NR4A NRs.The model organism C. elegans provides an excellent model with which to study genome-wide transcriptional networks during development.The lineage of each somatic cell is invariant and traceable, which provides a unique blueprint to map developmental regulatory networks [22].Additionally, it possesses a compact genome that contains short intergenic regions which simplifies the process of assigning a TF binding site to a target gene [23].
Here, we have used ChIP-seq to examine NHR-6-DNA binding activities during spermatheca organogenesis.Following binding site identification in the ChIP-seq data set using a computational pipeline, further analysis was performed to validate binding sites, identify candidate target genes for each stage, and examine up-regulated signaling pathways.Utilizing a functionally biased approach to filter candidate target genes, we were also able to verify multiple target genes with important roles in spermatheca development, indicating that they function within the NHR-6 regulatory network.

Chromatin immunoprecipitation (ChIP)
Egg preparations were produced from ten large (100 mm) agar plates, containing growing cultures of either nhr-6(lg6001); sgIs1 or nhr-6 (lg6001); sgIs2 animals, by alkaline hypochlorite treatment [26].Hatched, synchronized L1 arrested larvae from this procedure were then added to 20 large plates seeded with OP50 E. coli such that ∼4,000 animals were placed on each plate.nhr-6 (lg6001); sgIs1 animals were collected for the late L3/Early L4 and nhr-6 (lg6001); sgIs2 animals were collected for the early-mid L4/mid L4 time points at 34-37 hours and 37-42 hours after plating respectively.A minimum of 20 animals were examined at high magnification (60x) with Nomarski optics to ensure they were at the correct developmental stage before proceeding.Crosslinking was then performed as previously described [27] to obtain multiple 1.5 mL Eppendorf tubes with 0.1 mL of packed animals each.
Fragmented chromatin was obtained by sonicating animal pellets on ice in 0.7 mL HLB buffer [28] using a Misonix Ultrasonic Liquid Sonicator S-4000 with the following settings: 50% amplitude, 5 second pulses with a 1 minute pulse-off period between each set of pulses.Each immunoprecipitation was performed using 2.2 mg of protein equivalent in the lysate as previously described [28] with either rabbit anti-eGFP IgG fraction (ThermoFisher #CAB4211) or control normal rabbit IgG (ThermoFisher #10500C)) antibodies.50 L of total lysate was used as non-treated input control.Purified DNA from each immunoprecipitation was run on a 1% agarose gel with ethidium bromide staining, and fragments of length 100-300 bp were excised and gel purified using the Omega Gel Extraction Kit (Omega BioTek) as per the manufacturer's protocol.Immunoprecipitated DNA samples along with purified input DNA for each developmental time point were sent to the Genomics Core Facility at Louisiana State University Pennington Biomedical Research Center for quality control analysis and next-generation sequencing using the Life Technologies SOLiD 5500xl Genetic Analyzer.Raw and processed data have been deposited into GEO and can be accessed here: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE96908.
For Western analysis of transgenic and non-transgenic animals, chromatin was immunoprecipitated using the Pierce Co-Immunoprecipitation Kit, according to manufacturer's instructions.Immunoprecipitation was performed with covalently immobilized polyclonal antibody to eliminate antibody interference in subsequent Western analysis.Western blots were probed with a monoclonal anti-eGFP (MAB3580, Millipore Sigma).

Mapping, peak-calling, and identification of target genes
ChIP sequence fragments were mapped to the C. elegans genome (version ce6) with the Short Read Mapping Package (SHRiMP) [29] alignment tool using the R/Bioconductor package BSgenome.Celegans.UCSC.ce6[30].Peaks were called using Model-based analysis of ChIP-Seq (MACS) [31] with the band width set at 300 and a p-value cutoff of 1.00 e-05 to generate a list of statistically significant enriched sequences through comparison to input sequences using default parameters.Enriched ChIP peaks were annotated with the R/Bioconductor package ChIPseeker [32] using the annotation package TxDB.Celegans.UCSC.ce6.ensGene[33].

Quantitative PCR
qPCR was performed using the BioRad CFX96 Real-time system and C1000 thermal cycler.All primers were designed using Primer3 [34] to ensure products of ∼150 bp for each primer pair.The SsoFast TM EvaGreen Supermix (Bio-Rad) was utilized for SYBR Green quantification.Each reaction was set up in triplicate for DNA immunoprecipitated with the anti-eGFP antibody, DNA immunoprecipitated with the non-specific IgG antibody, and non-immunoprecipitated input DNA.The average Ct value for three technical repeats was generated and used to calculate ΔCt [(Ct value (sample) − Ct value (input)] and ΔΔCt values [ΔCt (experimental sample) − ΔCt (control)] [27].Melt curve (disassociation plot) and efficiency ranges were used as quality control measures in each reaction.Fold enrichment between experimental sample and negative control was calculated using the formula 2 (−ΔΔ) .

RNAi screen
All RNAi experiments by feeding were performed on hatched L1 larvae [35] using a gene of interest RNAi plasmid-containing HT115 bacterial strain from the Open Biosystems RNAi library or the Source Bioscience RNAi library.Control RNAi experiments utilized HT115 bacteria transformed with a GFP RNAi plasmid (pPD128.110(L447, Addgene)).Bacteria were freshly streaked out onto ampicillin (100 g/mL)/tetracycline (12.5 g/mL) plates and single colonies were cultured in 2 mL of LB broth in a 15 mL conical tube for 8 hours at 37 ∘ C with shaking.75 L of bacteria were spread onto NGM Lite plates containing 200 g/mL ampicillin with 1 mM IPTG and induced overnight at 20 ∘ C. Synchronized L1 larvae from alkaline hypochlorite preparations were added to the freshly induced bacterial lawns and allowed to grow until the desired stage.When animals reached the adult stage they were screened for brood size abnormalities (decrease in number of eggs laid, abnormal egg morphology) for 48 hrs.RNAi experiments to quantify brood size and spermathecal morphology were performed as above with the following additions: ∼50 animals were added to freshly-induced bacterial lawns and allowed to grow until young adult.Twenty animals for each strain were observed by Nomarski optics and scored for spermatheca cell number and morphology.Concurrently, fifteen animals from each experiment were moved to brood count plates and scored for brood size and abnormal egg morphology after 24 hours.Eggs were scored as abnormal when they displayed a small, roundish shape as previously described [19].

Identification of genome-wide NHR-6 binding sites
The spermatheca of adult C. elegans hermaphrodites consists of 24 cells that form a tube-like structure which serves as the site of sperm storage and oocyte fertilization [36].Each adult animal consists of two spermathecae found on each side of the vulva that are characterized by a narrow distal constriction made up of eight cells while the remaining 16 cells form the wider bag-like chamber that connects to the proximal spermatheca-uterine valve.18 of the 24 spermatheca cells are contributed from two large spermatheca precursor cells (SPCs) (9 from each) while the remaining six arise from the dorsal uterine (DU) lineage [37].Each SPC cell is born in mid L3, the same time when NHR-6 expression is first observed in the spermatheca, and undergoes an asymmetric cell division just prior to entry into the fourth larval stage.Upon entry into L4, the larger of the two SPC daughter cells undergoes several rapid divisions while the smaller daughter cell divides once more to give the final total of 9 SPC daughter cells.Each of the six spermatheca cells contributed by the DU lineage arise very early in L4.High NHR-6 expression is observed in all cells that give rise to spermathecal cells beginning in the mid L3 stage and remains high in all 24 daughter cells until the mid L4 stage when it begins to diminish and is lost in the adult animal.
We have previously uncovered a critical role for nhr-6 during this period of rapid SPC daughter cell divisions as these cells in nhr-6(lg6001) loss-of-function mutant animals fail to enter S-phase and undergo the correct number of divisions, resulting in an adult spermatheca with ∼1/2 the number of normal cells [20].Additionally, nhr-6(lg6001) mutants lack a spermathecauterine valve due to differentiation defects in the sujc cells that form the core of the valve [19].When the cell number phenotype of nhr-6(lg6001) animals is suppressed to or near wild type numbers by loss of function of two different negative G1/S regulators [20], the spermatheca still fails to differentiate properly, indicating a dualistic role for nhr-6 in both cell proliferation and cell differentiation.This work provided a detailed understanding of the timing of both spermatheca cell divisions and spermatheca cell differentiation and established an excellent in vivo model to study NR4A functions during these developmental processes.
To identify NR4A transcriptional programs that regulate spermatheca organogensis, we performed independent ChIP-seq experiments during both the late L3/early L4 and mid L4 time points.During this time, NHR-6 is robustly expressed in spermatheca cells (Figure 1A) which are undergoing several rapid divisions followed by differentiation respectively (Figure 1B).ChIP-seq was performed using an anti-eGFP antibody on animals carrying a functional fulllength GFP tagged NHR-6 transgene (Figure 1C).Immunoprecipitated DNA fragments for each developmental stage along with non-treated control (Input) DNA were sequenced and sequencing tags were mapped to the C. elegans genome.Peaks were then called as enriched compared to input for each stage and were found to be evenly distributed across all chromosomes with a total of 5,071 binding sites found specifically in late L3/early L4; 4,457 specifically in mid L4; and 21,127 sites were found in both data sets (co-located) (Table 1).As described in detail previously [20,21], NHR-6::GFP is also expressed at weaker levels in uterine and sheath cell precursor cells as cell divisions occur in the spermathecal lineage.Additionally, NHR-6::GFP is also expressed in two chemosensory neurons throughout larval development.Thus, binding sites identified from the ChIP-seq analysis would potentially also include NHR-6 binding sites from these cell types in addition to NHR-6 binding sites in spermathecal cells.
In order to immediately asses the reliability of the ChIP-seq data sets, 87 enriched binding sites that were found in both data sets were selected at random for quantitative PCR (qPCR) verification of enrichment.Cycle threshold (Ct) values were calculated for DNA sequences extracted with anti-eGFP and DNA extracted with non-specific anti-IgG antibodies.Fourteen sites were omitted due to poor qPCR quality leaving 73 total test sites.Sixty-five (89%) of these sites were verified as enriched with an average fold enrichment increase of 3.02 (data not shown).doi:10.11131/2017/101288

Identification of candidate NHR-6 target genes
All enriched binding site coordinates for each stage were then annotated to determine their locations in relation to gene regulatory regions (Figure 2A and S1).Though C. elegans genes have been shown to have some distant (>2kb) control regions, it is typically found that the minimal promoter required for expression of RNA polymerase II transcripts is found within ∼2 kb upstream of the TSS and that this distance works well as a starting point when looking for cis-acting control elements [38].Large introns may also contain additional regulatory elements.While most C. elegans introns are small (<100 bp), it has been shown that larger introns at the beginning of a coding region often contain regulatory elements [39,40].The majority of NHR-6 binding sites were found within 3 kb upstream (−3 kb) or 3 kb downstream (+3 kb) of a gene TSS when compared to the location of input peaks (Figure 2B).Therefore, we comprised a list of all genes for which an NHR-6 binding site was found either −3 kb or +3 kb of the TSS to represent a putative NHR-6 gene regulatory network.
Protein encoding genes were of particular interest for our functional analysis, and therefore, all genes encoding a small non-coding RNA or pseudogene were omitted.Additionally, we also filtered out sites within High Occupancy Target (HOT) regions that correspond to loci where multiple transcription factors have been shown to bind [2].This comprehensive list was made to capture all or most candidate protein encoding target genes that have an NHR-6 binding site either in an upstream promoter region, possibly in a large first intron, or upstream of a possible transcript variant.These genes can be grouped depending on whether the binding sites associated with a gene is L3 specific (encompassing late L3/early L4 stages), L4 specific (mid-L4 stage), or co-located in both sets.There are seven possible groupings of these genes.There are 861 distinct genes associated only with L3 specific binding sites (L3 specific genes) and 729 distinct genes associated with L4 specific binding sites (L4 specific genes).The majority of genes only have co-located binding sites (4,153 genes) while the remaining have L3 specific and co-located binding sites (2,230 genes), L4 specific and co-located binding sites (1,723 genes), L3 specific and L4 specific binding sites (182 genes), and L3 specific, L4 specific, and co-located binding sites (1,977 genes).In total there are 13,126 distinct genes with TSSs found +/ − 3 kb of the binding site (S2).Among all peaks from both data sets, ∼70% can be found within −3 kb upstream of a gene TSS indicating NHR-6 primarily binds in the upstream regulatory regions of genes.doi:10.11131/2017/101288

Pathway enrichment and gene ontology analysis of candidate NHR-6 target genes
In order to determine the cellular functions of NHR-6 candidate target genes, we examined them for enriched pathways and gene ontology terms.Analysis of enriched pathways was done for all protein encoding genes associated with stage-specific or co-located peaks using the R package PGSEA [41].A total of 12 pathways in late L3/early L4, 4 in mid L4, and 52 in co-located data sets were found to be enriched (Figure 3).Most of the enriched pathways are involved in a variety of metabolic processes which would be active in rapidly proliferating cells and are known to be regulated by NR4A NR's [42,43].Additionally, quite a few signaling pathways were also enriched including the ErbB, MAPK, NOTCH, mTOR, Wnt , and Jak-STAT pathways.These pathways are involved in several critical processes including the propagation of growth signals doi:10.11131/2017/101288 to facilitate cell cycle progression and proliferation as well as cell differentiation processes [44][45][46][47][48][49].
Gene ontology analysis for protein encoding genes using the DAVID bioinformatics resource (version 6.8) [50] uncovered several enriched biological process terms for candidate target genes in each stage (S3).Interestingly, genes involved in lipid storage, which is known to be regulated by NR4A NR's [3], were enriched in the co-located data set while regulation of Rho signaling, which is well studied for its role in C. elegans cellular morphogenesis [51][52][53], was enriched for candidate target genes in the mid L4 data set.Candidate target genes found in the co-located set doi:10.11131/2017/101288 are involved in multiple cellular processes that are occurring in the spermatheca during these stages including the regulation of cell proliferation and spermatogenesis.It is worth noting that neuron fate commitment and specification were enriched in the co-located gene set.This is likely due to the fact that NHR-6 expression has been reported in some chemosensory neurons [18,19], and mammalian NR4A NR's have been shown to regulate dopaminergic neuron development [54,55].Taken together, these results outline a putative NHR-6 regulatory network for further study of genes involved in cellular processes known to be regulated by NR4A NR's and known to be occurring in the spermatheca during these developmental stages.Additionally, the assortment of enriched processes and pathways these candidate target genes are involved in is congruent with much of the work done in mammalian systems, whereby the pleiotropic nature of NR4A receptors allows them to rapidly interconnect multiple signals into varied cellular responses.

qPCR validation of candidate NHR-6 target genes
We have extensively studied the spermatheca phenotypes of nhr-6(lg6001) null mutants as well as the division pattern of spermatheca cells in both wild type and mutant animals.Therefore, we used this information to manually select for a small subset of protein encoding NHR-6 candidate target genes that were likely, based on cellular function, to be involved in spermatheca development.Genes were analyzed with information found on wormbase.orgas well as the gene ontology term with which they were described.All genes that did not fit into a gene ontology category and/or did not have any functional information on wormbase.orgwere omitted.We selected for genes that met some or all of the following criteria: have experimentally been shown to be involved in cell cycle regulation/division, cell differentiation, chromatin modification, cell signaling, or gene regulation, as well as any genes that are expressed in the spermatheca during L3 and L4.Using these criteria, we selected a total of 67 candidate target genes to validate for functions in spermatheca development as well as validate the associated binding sites through qPCR.
Separate ChIP experiments were performed to attain DNA used in each qPCR reaction, and primers were designed to ensure an amplicon size of ∼150 bp for each pair.Of the 67 genes selected, we were unable to design usable primers for 13 of the associated binding sites and one site was upstream of multiple genes; therefore, a total of 53 binding sites were validated using qPCR.DNA immunoprecipated with anti-eGFP antibodies showed at least some enrichment over non-specific IgG DNA for each binding site using the ΔΔCt method of calculating fold enrichment while 27 (∼51%) showed at least two fold enrichment (Table 2).Eight of these genes are known to be expressed in the spermatheca (cye-1, hmg-1.2,tsp-21, riok-1, paa-1, eat-16, uaf-2, bet-1).The majority of binding sites that were enriched at least two fold are associated with candidate target genes which function in cell cycle regulation, various signaling pathways, and chromatin modification.
Interestingly, the binding site with the largest fold increase corresponds to a site in the promoter of the sole C. elegans cyclin E gene, cye-1, which consequently is also found in the modENCODE data set for L4 animals [2].Cyclin E is a key positive regulator of G1/S cell cycle progression, for which we have previously shown a requirement of nhr-6 [20].NR4A NR's have also been shown to regulate cyclin family members in mammalian tissues.For example, NR4A1 and NR4A3 upregulate Cyclin E in islet -cells [56], and NR4A3 upregulates Cyclin D during doi:10.11131/2017/1012883.5.RNAi screen of selected candidate NHR-6 target genes nhr-6 RNAi has been shown to be hypomorphic in a wild-type N2 background [18].However, nhr-6 RNAi phenotypes in the RNAi sensitized eri-1 mutant background are very similar with respect to spermatheca cell number and morphology to nhr-6(lg6001) mutant animals [18].Additionally, nhr-6 hypomorphic RNAi is strongly enhanced in vab-2 and jun-1 mutant backgrounds and NHR-6 has also been shown to interact with the JUN-1 transcription factor in a yeast two-hybrid assay [25].A decrease in the amount of eggs laid and the presence of eggs laid with abnormal morphology is indicative of abnormal somatic gonad development and defective ovulation.Both of these phenotypes are observed in nhr-6 mutant animals and are a direct result of abnormal spermatheca development.Therefore, these four mutant strains were used in an RNAi screen of the 67 candidate target genes previously selected to determine those that displayed reproductive phenotypes similar to nhr-6(lg6001) mutants.It was predicted that a gene with a critical role in spermatheca development would display a reproductive phenotype in either the wild-type N2 or sensitized eri-1 mutant backgrounds, while an observed phenotype in either vab-2 or jun-1 mutant backgrounds would indicate a possible shared role in an nhr-6 pathway, as well as provide a sensitized background to detect reproductive phenotypes.Thirteen of the 67 genes selected did not contain an RNAi clone in any RNAi library; therefore, a total of 54 genes were used for the screen.
An initial screen was performed that allowed young adult animals to lay eggs for 48 hours following RNAi by feeding, at which time plates were qualitatively screened for low brood sizes and/or an increase in abnormal egg morphology compared to control (GFP RNAi) animals.Of the 54 genes tested, 32 displayed a reproductive phenotype in at least one of the strains used, while 22 genes did not display any reproductive phenotypes (data not shown).We further analyzed the 32 genes which scored positive for reproductive defects by performing a detailed brood count analysis 24 hours after the animals reached the young adult stage.A total of 15 animals for each gene in each genetic background were scored.Eleven of the genes displayed a significant decrease in the average number of eggs laid with some also displaying abnormal egg morphology (Table 3).The majority of these genes contained corresponding NHR-6 binding sites found in the co-located data set while only one was found in each of the late L3/early L4 and mid L4 specific data sets.Interestingly, some of the selected genes showed strong reproductive phenotypes in one or more strains that were suppressed in a different genetic background.The high mobility group box gene hmg-1.2, the SWI/SNF complex component let-526, and gei-1 which encodes a RhoGAP protein all displayed a strong phenotype in eri-1 and jun-1 mutant backgrounds that was suppressed in the vab-2 mutant background, while the wingled-helix like transcription factor lin-31 showed a strong reproductive phenotype in the jun-1 mutant background that was suppressed in the other strains (Table 3).
In order to determine the specific cause of the reproductive defects, we analyzed animals from the 11 genes with significant brood count defects by DIC microscopy for spermatheca cell number and morphology defects similar to nhr-6(lg6001) mutants.Every gene scored showed abnormal spermatheca morphology in at least one genetic background compared to wild-type (Figure 4A), most notably a loss of distal constriction and failure of the SP-UT valve to form correctly.These phenotypes are readily detectable and were similar to those observed in nhr-6(lg6001) mutant animals (Figure 4B).The image in Figure 4C shows a representative example doi:10.11131/2017/101288 of the typical phenotypes observed; in the eri-1; dcp-66(RNAi) example there is clear loss of the distal constriction and SP-UT valve as well as overall loss of integrity of the organ.None of the selected genes scored displayed an abnormal spermatheca cell number phenotype.However, let-526 RNAi did show a likely spermatheca cell number phenotype.An accurate quantification of the cell number phenotype of let-526 could not be determined due to other abnormalities in the somatic gonad and germline that obscured spermatheca morphology.Nevertheless, a small number of animals could be observed that showed a marked decrease in cell number; a representative example of the phenotype observed is shown in Figure 4D.
The presence of an NHR-6 binding site in a regulatory region coupled with observed spermatheca phenotypes similar to nhr-6(lg6001) following RNAi of these genes suggests regulation by NHR-6 in a spermathecal development pathway.Further supporting a role for some these genes in a NHR-6 pathway is the genetic interactions observed in jun-1 and vab-2 mutants.It would be expected that at least some target genes of NHR-6 would exhibit genetic interactions similar to nhr-6.These genes, which were selected with a strong functional bias, represent only a very small number of probable NHR-6 target genes.Several selected genes that are likely to be involved in spermatheca development do not show any phenotypes following RNAi by feeding, which could be due to poor RNAi efficacy or redundancy, indicating that additional methods will need to be used to determine their role.RNA-seq studies on both wild-type and nhr-6 loss of function animals will be necessary to confirm which genes associated with NHR-6 binding sites are either positively or negatively regulated by NHR-6.Nevertheless, these findings validate our selection method and outline an initial regulatory network whereby NHR-6 regulates cell cycle processes during the rapid proliferation stage of late L3 and early L4, and doi:10.11131/2017/101288 doi:10.11131/2017/101288

Figure 1 :
Figure 1: NHR-6::eGFP ChIP. A. Fluorescent and DIC images of NHR-6::eGFP localization in the spermathecae of late L3 (sgIs1) and mid L4 animals (sgIs2).White brackets indicate location of spermatheca cells.The surrounding punctate signal is gut autofluorescence, not GFP.B. Spermatheca precursor cell (SPC) lineage during L3 and L4. Green bars indicate time points at which animals were fixed for ChIP.C. Immunoblot of eGFP immunoprecipated from mixed stage sgIs1 (Transgenic) and wildtype (WT) animals with immobilized polyclonal antibody.

Figure 2 :
Figure 2: Genomic location of ChIP-seq peaks.A. Pie chart indicating the location of ChIP-seq peaks relative to gene locations.B. Total number and location of ChIP-seq peaks in relation to gene transcription start sites (TSS).

Figure 3 :
Figure 3: Heat map indicating enriched pathway scores of candidate NHR-6 target genes.

Figure 4 :
Figure 4: Spermatheca RNAi Phenotypes of NHR-6 Target Genes. A. Spermatheca of young adult N2 (WT) spermathecal.The spermathecal-uterine valve is indicated by the arrow, and the distal constriction is indicated by an asterisk.B. Young adult nhr-6(lg6001) mutant showing absence of both the spermathecal-uterine valve and the distal constriction.C. Spermatheca of young adult dcp-66 (RNAi) animal shows loss of normal morphology of the spermathecal-uterine valve and distal constriction, but spermathecal size (and cell number) is normal.This is the typical spermathecal morphology observed in animals displaying reproductive phenotypes in at least one of the mutant backgrounds.D. Spermatheca of young adult let-526 (RNAi) animal shows loss of normal morphology and a decreased organ size that is likely due to decreased cell number.

Table 1 :
Distribution of NHR-6 binding sites compared across each chromosome.

Table 3 :
Average brood size following RNAi of selected genes.