Specification of the germline by Nanos-dependent down-regulation of the somatic synMuvB transcription factor LIN-15B

The Nanos RNA-binding protein has been implicated in the specification of primordial germ cells (PGCs) in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of PGCs lacking the nanos homologues nos-1 and nos-2 iC. elegans. nos-1nos-2 PGCs fail to silence hundreds of genes normally expressed in oocytes and somatic cells, a phenotype reminiscent of PGCs lacking the repressive PRC2 complex. The nos-1nos-2 phenotype depends on LIN-15B, a broadly expressed synMuvB class transcription factor known to antagonize PRC2 activity in somatic cells. LIN-15B is maternally-inherited by all embryonic cells and is down-regulated specifically in PGCs in a nos-1nos-2-dependent manner. Consistent with LIN-15B being a critical target of Nanos regulation, inactivation of maternal LIN-15B restores fertility to nos-1nos-2 mutants. These studies demonstrate a central role for Nanos in reprogramming the transcriptome of PGCs away from an oocyte/somatic fate by down-regulating an antagonist of PRC2 activity.


Introduction
In animals, formation of the germline begins during embryogenesis when a few cells (~30 in mice, 2 in C. elegans) become fated as primordial germ cells (PGCs) -the founder cells of the germline. PGC specification requires the activity of chromatin regulators that induce genome-wide changes in gene expression. For example, in mice, the transcriptional repressor BLIMP1 initiates PGC specification by blocking the expression of a mesodermal program active in neighboring somatic cells Saitou et al., 2005). In C. elegans, the NSD methyltransferase MES-4 and the PRC2 complex (MES-2, 3 and 6) cooperate to place active and repressive histone marks on germline and somatic genes, respectively (Gaydos et al., 2012). Despite their critical roles during germ cell development, the MES and BLIMP1 regulators are not germline-specific factors and also function during the differentiation of somatic lineages (Cui et al., 2006;Gaydos et al., 2012;Seydoux and Braun, 2006). How the activities of these global regulators are modulated in germ cells to promote a germline-specific program is not well understood.
In C. elegans, genetic analyses have shown that MES activity is antagonized in somatic lineages by the synMuvB group of transcriptional regulators (Curran et al., 2009;Petrella et al., 2011;Unhavaithaya et al., 2002). Loss-of-function mutations in synMuvB genes cause ectopic activation of germline genes in intestinal cells and result in larval growth arrest at elevated temperatures (26 o C). Inactivation of MES proteins suppresses the ectopic germline gene expression and restores viability to synMuvB mutants (Petrella et al., 2011). A similar antagonism has been uncovered in the adult germline between mes-4 and the synMuvB gene lin-54 (Tabuchi et al., 2013). The X chromosome is a major focus of MES repression in C. elegans germline. The X chromosome is silenced throughout germ cell development except in oocytes, which activate the transcription of many X-linked genes in preparation for embryogenesis (Kelly et al., 2002). mes mutants prematurely activate the transcription of somatic and X-linked genes in pre-gametic germ cells leading to germ cell death (Bender et al., 2006;Gaydos et al., 2012;Seelk et al., 2016). Reducing the function of the synMuvB transcription factor lin-54 in mes-4 mutant restores the expression of X-linked genes closer to wild-type levels (Tabuchi et al., 2013).
Together, these genetic studies suggest that competition between the MES chromatin modifiers and the synMuvB class of transcription factors tunes X chromosome silencing and the ratio of soma/germline gene expression in somatic and germline tissues. How the balance of synMuvB/MES activities is initially set for each tissue, however, is not known.
The C. elegans PGCs arise early in embryogenesis from pluripotent progenitors (P blastomeres) that also generate somatic lineages. RNA polymerase II activity is repressed in the P lineage until the 100-cell stage when the last P blastomere P 4 divides to generate Z2 and Z3, the two PGCs (Seydoux et al., 1996). RNA polymerase II becomes active in PGCs, but these cells remain relatively transcriptionally quiescent, and exhibit reduced levels of active chromatin marks compared to somatic cells throughout the remainder of embryogenesis (Kelly, 2014). Active marks and robust transcription return after hatching when the L1 larva begins to feed and the PGCs resume proliferation in the somatic gonad (Fukuyama et al., 2006;Kelly, 2014). The mechanisms that maintain PGC chromatin in a silenced state during embryogenesis are not known, but embryos lacking the nanos homologs nos-1 and nos-2 have been reported to display abnormally high levels of the H3meK4 mark in PGCs (Schaner et al., 2003). nos-1nos-2 PGCs initiate proliferation prematurely during embryogenesis and die during the second larval stage (Subramaniam and Seydoux, 1999). Nanos proteins are broadly conserved across metazoans and have been shown to be required for PGC survival in several phyla, from insects to mammals (Asaoka-Taguchi et al., 1999;Beer and Draper, 2013;Deshpande et al., 1999;Lai et al., 2012;Tsuda et al., 2003). Nanos proteins are cytoplasmic and regulate gene expression post-transcriptionally by recruiting effector complexes that silence and degrade mRNAs in the cytoplasm. Six direct Nanos mRNA targets have been identified to date [Drosophila hunchback, cyclin B and hid(Asaoka-Taguchi et al., 1999;Dalby and Glover, 1993;Kadyrova et al., 2007;Murata and Wharton, 1995;Sato et al., 2007;Wreden et al., 1997), Xenopus VegT (Lai et al., 2012), and sea urchin CNOT6 and eEF1A (Oulhen et al., 2017;Swartz et al., 2014)], but none of these targets are sufficient to explain how Nanos activity might affect PGC chromatin. In this study, we characterize the gene expression defects of PGCs lacking nanos activity in C. elegans. Our findings indicate that nanos activity is required to silence a maternal program active in oocytes and somatic embryonic cells. We identify the synMuvB transcription factor lin-15B as a critical target of Nanos regulation and demonstrate that down-regulation of maternal LIN-15B is essential to establish PRC2 dominance in PGCs.
These analyses identified 461 under-expressed transcripts and 871 over-expressed transcripts in nos-1(gv5)nos-2(RNAi) L1 PGCs compared to wild-type (q >0.05, Figure 1A and Table S5 for list of miss-regulated genes). qRT-PCR of 11 genes confirmed the result of the RNA-seq analysis ( Figure S1A).
To determine the types of genes affected, we used published gene expression data (Gaydos et al., 2012;Meissner et al., 2009;Ortiz et al., 2014;Reinke et al., 2004;Wang et al., 2009) to generate non-overlapping lists of genes with preferential expression in pregametic germ cells, oocytes, sperm, or somatic cells (described and listed in Table S1).

Turnover of maternal transcripts is delayed in PGCs lacking nos-1 and nos-2
Oogenic transcripts in nos-1(gv5)nos-2(RNAi) L1 PGCs could correspond to maternal transcripts that failed to turnover during embryogenesis or to zygotic transcripts synthesized de novo in nos-1(gv5)nos-2(RNAi) PGCs. To distinguish between these possibilities, we isolated PGCs from embryos with fewer than 200 cells, at a time when PGCs are still mostly transcriptionally silent (EMB PGCs) (Schaner et al., 2003;Seydoux and Dunn, 1997). By comparing the EMB PGC transcriptome to a published oocyte transcriptome (Stoeckius et al., 2014), we observed an excellent correlation in relative transcript abundance between oocytes and EMB PGCs ( Figure S2A). This observation suggests that many maternal mRNAs are maintained in the nascent germ lineage up to the 200-cell stage, as suggested earlier by in situ hybridization experiments (Seydoux and Fire, 1994). Next, we compared the transcriptome of EMB PGCs to that of L1 PGCs to identify PGC transcripts whose abundance decline during embryogenesis. We identified 411 down-regulated transcripts, including 197 oocyte transcripts (Figure 2A, 2B and Table S5), consistent with turnover of many maternal mRNAs in PGCs after the 200-cell stage.
Transcription of the X chromosome is silenced in all germ cells except in oocytes, which activate X-linked gene expression in preparation for embryogenesis (Kelly et al., 2002). As expected, we found that transcripts from X-linked genes are rare in wild-type L1 PGCs, with an average 4.7 FPKM per X-linked genes compared to 50.9 for autosomal genes. X-linked transcripts were more abundant in nos-1(gv5)nos-2(RNAi) L1 PGCs (9.6 FPKM for X-linked genes compared to 43.8 for autosomal genes) (Table S3), and strikingly 44% of the "open" genes with ATAC-seq peaks in nos-1(gv5)nos-2(RNAi) PGCs were Xlinked ( Figure 3E). We conclude that silencing of the X chromosome is defective in nos-1(gv5) nos-2(RNAi) PGCs.

MES-2 and MES-4 activities are compromised in nos-1nos-2 PGCs.
Failure to silence X-linked genes has been reported for germ cells lacking the chromatin regulators mes-2 and mes-4 (Bender et al., 2006;Gaydos et al., 2012). To directly compare the effect of nos and mes activities in PGCs, we purified PGCs from L1 larvae derived from hermaphrodites where mes-2 or mes-4 was inactivated by RNAi (Methods). As expected, loss of mes-2 and mes-4 led to a significant upregulation of Xlinked genes in L1 PGCs ( Figure 4A-B, Figure S4A-B and Table S5 for lists of missregulated genes). To directly compare these changes to those observed in nos-1(gv5)nos-2(RNAi) PGCs, we compared, for each genotype, the log2 fold change over wild-type for X-linked genes and for autosomal oocyte genes. As expected, we observed a strong positive correlation between mes-2 and mes-4 in both gene categories (R=0.91 and R=0.76, X-linked and autosomal oogenic genes, respectively) ( Figure 4C and 4D). We also observed a strong correlation between mes-4(RNAi) and nos-1(gv5)nos-2(RNAi) (R=0.75, Figure 4E) and mes-2(RNAi) and nos-1(gv5)nos-2(RNAi) (R=0.73, not shown) for X-linked genes. Interestingly, the correlations were weaker for autosomal oocyte genes (R=0.35, Figure 4F), which tended to be more over-expressed in nos-1(gv5)nos-2(RNAi) L1 PGCs. This finding is consistent with the notion that, while nos-1nos-2 and mes PGCs share a defect in X-linked silencing, nos-1nos-2 PGCs also have an additional defect in maternal mRNA turn over.

Maternal LIN-15B is inherited by all embryonic blastomeres and downregulated specifically in PGCs
A LIN-15B::GFP transgene was reported to be broadly expressed (Sarov et al., 2012). To examine the expression of endogenous LIN-15B, we used a polyclonal antibody generated against LIN-15B protein (modencode project, personal communication with Dr. Susan Strome). We confirmed the specificity of this antibody by staining lin-15B(n744) mutant, which showed no nuclear staining ( Figure S6A). We first detected LIN-15B expression in the germline in the L4 stage in nuclei near the end of the pachytene region where germ cells initiate oogenesis ( Figure 6A). Nuclear LIN-15B was present in all oocytes and inherited by all embryonic blastomeres, including the germline P blastomeres

Downregulation of maternal LIN-15B in PGCs requires nos-1 nos-2 activity
lin-15B transcripts were modestly elevated in nos-1(gv5)nos-2(RNAi) EMB PGCs compared to wild-type EMB PGCs, suggesting that lin-15B may be one of the maternal RNAs that requires Nanos activity for rapid turnover in PGCs (Table S4). lin-15B transcripts rose significantly by ~2-fold when comparing EMB versus L1 stage nos-1(gv5)nos-2(RNAi) PGCs. This increase was not observed in wild-type PGCs, suggesting that lin-15B is also inappropriately transcribed in nos-1(gv5)nos-2(RNAi) L1 PGCs. Unfortunately, we were not able to confirm these RNA-seq observations by in situ hybridization due to the low abundance of lin-15B RNA and its presence in all somatic cells.
To distinguish between these possibilities, we created a lin-15B transcriptional reporter by inserting a GFP::H2B fusion at the 5' end of lin-15B locus in an operon configuration to preserve endogenous lin-15B expression ( Figure S6B and Table S6). We crossed nos-1(gv5) males carrying the lin-15B transcriptional reporter to wild-type or nos-1(gv5)nos-2(ax3103) hermaphrodites and examined crossed progenies for GFP expression. In both cases, we observed strong GFP expression in somatic cells, but no expression in PGCs during embryogenesis (data not shown). In wild-type animals, we first observed zygotic expression of the lin-15B transcriptional reporter in the germline of L4 stage animals ( Figure S6C), in germ cells that have initiated oogenesis. In contrast, in animals derived from nos-1(gv5)nos-2(ax3103) mothers, zygotic expression of the lin-15B transcriptional reporter could be detected as early as the L1 stage in PGCs and their descendants ( Figure   6C). This expression was maintained until the L2 stage when nos-1nos-2 PGC descendants undergo cell death. We conclude that nos-1nos-2 activity is required both to promote the turnover of maternal LIN-15B in EMB PGCs and to prevent premature zygotic transcription of lin-15B in L1 PGCs.

Maternal lin-15B is responsible for nos-1 nos-2 sterility
To determine whether miss-regulation of maternal or zygotic LIN-15B is responsible for nos-1nos-2 sterility, we compared the sterility of nos-1(gv5)nos-2(ax3103) animals that lack either maternal or zygotic lin-15B ( Figure 6D and Figure S7). We found that loss of maternal lin-15B was sufficient to fully suppress nos-1(gv5)nos-2(ax3103) sterility, even in the presence of one zygotic copy of lin-15B ( Figure 6D). The penetrance of the suppression was dependent on the dosage of maternal lin-15B. nos-1(gv5)nos-2(ax3103) animals with only one copy of maternal lin-15B were only 32% sterile compared to 70% sterility for animals with two copies of maternal lin-15B and 0% with animals with zero copies of maternal lin-15B ( Figure 6D). Interestingly animals with only one copy of maternal LIN-15B appeared sensitive to the zygotic dosage of lin-15B ( Figure 6D, compare the sterility M1Z2, M1Z1 and M1Z0). We conclude that maternal lin-15B is primarily responsible for the sterility of nos-1nos-2 animals, although zygotic LIN-15B activity may also contribute.

Discussion
In this study, we have examined the transcriptome of PGCs lacking Nanos function in C. elegans. We have found that nos-1(gv5)nos-2(RNAi) PGCs activate 100s of genes normally expressed in oocytes and somatic cells. Our observations suggest that Nanos activity is required to erase a maternally-inherited somatic program. Importantly, Nanos activity promotes the turnover of LIN-15B, a maternally-inherited transcription factor known to antagonize PRC2 activity in somatic cells. Down-regulation of LIN-15B frees PRC2/MES-4 to silence oocyte and somatic genes and activate germline genes in PGCs.

Nanos activity is required for the timely turnover of maternal mRNAs in PGCs.
During oogenesis, oocytes stockpile mRNAs and proteins in preparation for embryogenesis. These include mRNAs and proteins with housekeeping functions as well as factors required to specify somatic and germ cell fates. During the maternal-to-zygotic transition, these maternal products are turned over to make way for zygotic factors. We have found that Nanos activity is required for the timely turnover of maternal mRNAs in PGCs. RNA-seq analyses comparing embryonic and first stage larval PGCs identified 411 maternal mRNAs whose abundance decrease sharply during embryogenesis in wild-type PGCs, but not in nos-1(gv5)nos-2(RNAi) PGCs ( Figure 2E). We also found that maternal LIN-15B protein levels decline rapidly at the time of gastrulation in wild-type PGCs, but not in nos-1(gv5)nos-2(ax3103) PGCs ( Figure 6B). Nanos is thought to silence mRNAs by interacting with the sequence-specific RNA-binding protein Pumilio and with the CCR4-NOT deadenylase complex which interferes with translation and can also destabilize RNAs. (Lai et al., 2012;Suzuki et al., 2012;Swartz et al., 2014;Wharton et al., 1998). In the C. elegans genome, there are eight genes related to Drosophila pumilio. Depletion of five of these (fbf-1, fbf-2, puf-6, puf-7 and puf-8) phenocopies the nos-1nos-2 PGC phenotypes, including failure to incorporate in the somatic gonad, premature proliferation, and eventually cell death (Subramaniam and Seydoux, 1999). These observations suggest that NOS-1 and NOS-2 function with Pumilio-like proteins to target specific maternal RNAs for degradation. Paradoxically, in sea urchins, Nanos targets the mRNA coding for the CNOT6 deadenylase for degradation in PGCs, which indirectly stabilizes other maternal mRNAs (Swartz et al., 2014). In that system, Nanos was also found to silence eEF1A expression, leading to a transient period of translational quiescence in PGCs (Oulhen et al., 2017).
One possibility is that, at the earliest stages of the maternal-to-zygotic transition in PGCs, Nanos generally silences maternal mRNA translation and targets specific mRNAs for degradation while stabilizing others. In combination, these effects could lead to loss of somatic mRNAs and proteins (e.g. LIN-15B) and maintenance of germline mRNAs (e.g. MES) whose translation could be reactivated at a later time. In C. elegans, the redundant nanos homologs nos-1 and nos-2 are expressed sequentially in PGCs during the maternal-to-zygotic transition and may have overlapping yet distinct effects on mRNAs stability and translation. Genetic analyses already have suggested that nos-1 and nos-2 have both unique and shared functions (Kapelle and Reinke, 2011;Mainpal et al., 2015). It will be important to determine whether nos-1 and nos-2 are both required to keep LIN-15B levels low throughout embryogenesis, and whether they act directly on the lin-15B RNA or indirectly, by silencing other factors required for LIN-15B protein translation and/or stability.

In PGCs lacking nos-1 and nos-2, maternal LIN-15B interferes with MES-dependent silencing of oocyte and somatic genes
Several lines of evidence indicate that nos1nos-2 sterility is caused by a failure to turn over maternally-inherited LIN-15B in embryonic PGCs. First, loss of one maternal copy of the lin-15B locus is sufficient to partially suppress nos-1(gv5)nos-2(ax3103) sterility and loss of both maternal copies maximally suppresses nos-1(gv5)nos-2(ax3103) sterility even in the presence of a zygotic copy of lin-15B ( Figure 6D). These genetic results demonstrate that maternal lin-15B is required for nos-1(gv5)nos-2(ax3103) sterility, and suggest that abnormal perdurance of LIN-15B in PGCs interferes with their reprogramming to become pre-gametic germ cells. Consistent with the genetic findings, we have found that nos-1(gv5)nos-2(RNAi) PGCs activate by the L1 stage the transcription of 100s of somatic and oocyte genes and this ectopic expression is reduced in PGCs also lacking lin-15B activity (Figures 1 and 6). How does ectopic LIN-15B activate oocyte and somatic gene expression in nos-1nos-2 PGCs? LIN-15B activity antagonizes MES-dependent repression of somatic genes and activation of germline genes in somatic cells (Petrella et al., 2011;Wang et al., 2005). Consistent with LIN-15B playing a similar role in PGCs, nos-1(gv5)nos-2(RNAi) PGCs activate the transcription of many of the same genes activated in PGCs lacking mes activity. The strongest correlation is seen for genes on the X chromosome ( Figure 4E), a well-documented focus of MES transcriptional repression (Bender et al., 2006;Garvin et al., 1998;Gaydos et al., 2012). Interestingly, the lin-15B locus itself is on the X chromosome and is ectopically transcribed in nos-1(gv5)nos-2(RNAi) PGCs at hatching. These observations raise the possibility that maternal LIN-15B potentiates zygotic lin-15B expression as MES-dependent silencing of the X-chromosome becomes compromised. How does maternal LIN-15B initially opposes MES activity is not known, but another X-linked gene and potential LIN-15B target is utx-1, a de-methylase that removes the silencing mark deposited by the PRC2 complex. Upregulation of utx-1 was shown recently to promote reprogramming of adult germline stem cells into neurons (Seelk et al., 2016). utx-1 is up-regulated in a lin-15B-dependent manner in nos-1(gv5)nos-2(RNAi) PGCs, and RNAi of utx-1 partially suppresses nos-1(gv5)nos-2(ax3103) sterility ( Figure S5A). Suppression by loss of utx-1 is weaker than that observed when inactivating lin-15B, suggesting that utx-1 is not the only lin-15B target that opposes PRC2. We have found that loss of two other synMuvB genes lin-35/Rb and dpl-1 also suppresses nos-1(gv5)nos-2(ax3103) sterility ( Figure 5A), albeit again less stringently than loss of lin-15B.
It will be interesting to determine whether these genes function with, or in parallel to, LIN-15B to oppose PRC2 activity in PGCs.
Inhibition of LIN-15B by Nanos is unlikely to be the only mechanism that promotes PCR-2 function in PGCs. XND-1 is a chromatin-associated protein that is expressed in PGCs throughout embryogenesis. XND-1 is required redundantly with NOS-2 to maintain low levels of active histone marks in PGCs (Mainpal et al., 2015). An exciting possibility is that XND-1 is a chromatin factor that promotes/maintains PRC2 activity in PGCs, in parallel to NOS-2.

An ancient regulatory switch balances somatic and germline fates throughout the germline cycle
Competition between synMuvB and PRC2 activities has already been implicated in balancing somatic and germline gene expression during larval development in somatic lineages and in the adult germline (Petrella et al., 2011;Tabuchi et al., 2013). Our findings demonstrate that such a competition also occurs in PGCs, where Nanos biases the competition in favor of PRC2 by lowering maternal LIN-15B levels ( Figure 7A). We propose that the ratio of synMuvB and PRC2 activity changes at two key developmental stages during the germline cycle ( Figure 7B). First, during oogenesis, an unknown activity promotes the transcriptional activation of LIN-15B, which allows the demethylase UTX-1 and other LIN-15B targets to begin erasing PRC2 marks. Erasure of PRC2 marks activates the transcription of X-linked genes and other somatic genes in oocytes in preparation for embryogenesis. This oogenic/maternal program is inherited by all embryonic blastomeres. In somatic lineages, which activate transcription first, maternallyinherited LIN-15B continues to oppose PRC2 activity, which permits zygotic activation of the lin-15B and utx-1 loci and eventual complete erasure of the PRC2 program. In the nascent germline, transcription is kept off until gastrulation when Nanos expression is activated in PGCs by unknown mechanisms that both promote the translation of maternal nos-2 RNA and later the zygotic transcription of nos-1. Nanos activity in PGCs promotes the turnover of maternal LIN-15B, freeing PRC2 to re-establish silencing of somatic and Xlinked genes, including the lin-15B and utx-1 loci, until the next round of oogenesis.
What prevents expression of Nanos in somatic cells? Interestingly, evidence in Drosophila and mammals suggest that Nanos is among the germline genes inhibited by synMuvB activity in somatic cells. Loss of the dREAM complex component lethal (3) malignant brain tumor [ l(3)mbt] leads to tumorous growth in Drosophila imaginal disks and ectopic expression of germline genes, including nanos (Janic et al., 2010). Similarly, loss of the synMuv B class transcription factor retinoblastoma protein (RB) leads to activation of nanos transcription in mammalian tissue culture cells and in Drosophila wings . A complex regulatory feedback loop has also been reported between the LSD1 demethylase and the Nanos partner Pumilio in Drosophila and human bladder carcinoma cells (Miles et al., 2015). Taken together, these observations suggest that mutual antagonism between transcriptional regulators and the Nanos/Pumilio C. elegans was cultured according to standard methods (Brenner, 1974).
RNAi knock-down experiments were performed by feeding on HT115 bacteria (Timmons and Fire, 1998). Feeding constructs were obtained from Ahringer or OpenBiosystem libraries or PCR fragments cloned into pL4440. The empty pL4440 vector was used as negative control. Bacteria were grown at 37°C in LB + ampicillin (100 µg/mL) media for 5-6 hr, induced with 5 mM IPTG for 30 min, plated on NNGM (nematode nutritional growth media) + ampicillin (100 µg/mL) + IPTG (1 mM) plates, and grown overnight at room temperature. Embryos isolated by bleaching gravid hermaphrodites, or synchronized L1s hatched in M9, were put onto RNAi plates. For sterility counts, the progeny of at least six gravid adult hermaphrodites were tested. Adult progenies were scored for empty uteri ('white sterile' phenotype) on a dissecting microscope. For all Immunostaining and smFISH experiments shown in Figure 2D, 6A, 6B, S4C and S6A, worms were grown at 25°C. For live embryo imaging and synMuvB related experiments shown in Figure 5, Figure 6C, 6D and Figure S4C, worms were grown at 20°C.

Immunostaining
Adult worms were placed on 3-wells painted slides in M9 solution (Erie Scientific co.) and squashed under a coverslip to extrude embryos. Slides were frozen by laying on prechilled aluminum blocks for >10 min. Embryos were permeabilized by freeze-cracking Sci.) were applied for 1~2 hr at room temperature. MES-3 was tagged with the OLLAS epitope at the C-terminus using CRISPR genome editing (Paix et al., 2015).

Confocal microscopy
Fluorescence microscopy was performed using a Zeiss Axio Imager with a Yokogawa spinning-disc confocal scanner. Images were taken and stored using Slidebook v6.0 software (Intelligent Imaging Innovations) using a 40x or 63x objective. Embryos were staged by DAPI-stained nuclei in optical Z-sections and multiple Z-sections were taken to include germ cells marked by anti-PGL-1 (K76) staining. For images of embryonic PGCs, a single Z-section was extracted at a plane with the widest area of DAPI staining for nuclear signal of LIN-15B, MES-3, and MES-4. For MES-2-GFP, the Z-section was determined based on widest area of GFP signal. Equally normalized images were first taken by Slidebook v6.0, and contrasts of images were equally adjusted between control and experimental sets using Image J.

Germ cell isolation and sorting:
RNAi treatments for sorting experiments were done by seeding synchronized L1 (hatched from embryos incubated in M9 overnight) onto RNAi plates and growing them to gravid adults. Additional RNAi or control bacteria were added once to ensure enough food to support development. Early embryos were harvested from gravid adults. These embryos were either used directly to isolate embryonic PGCs or incubated for 12~16 hours in M9 solution until reaching the L1 stage for PGCs isolation. To isolate L1 PGCs from fed animals, the L1s were plated onto RNAi plates for additional 5 hours before processing for PGC isolation. For RNA-seq experiments described in Figure 1 and  Table S8 for sequencing library information.
To isolate PGCs from embryos, cell dissociation was performed as described in Strange et al. 2007(Strange et al., 2007 with the following modifications: 1x10 6 embryos were treated in 500ul chitinase solution (4.2 unit of chitinase (Sigma # C6137) in 1ml of conditioned egg buffer). After chitinase treatment, embryos were collected by centrifugation at ~900g for 4 mins at 4°C and resuspended in 500µl accumix-egg buffer solution for dissociation (Innovative Cell Techologies, AM105, 1:3 dilution ratio in egg buffer). In the final step, cells were resuspended in chilled egg buffer before sorting using BD FACSAriaII. 65,000~120,000 PGL-1::GFP PGCs were used for RNA isolation.
To isolate PGCs from L1 larvae, 400,000 to 500,000 packed L1s were used for cell dissociation as described in Zhang and Kuhn (Zhang and Kuhn, 2013)(www.wormbook.org/chapters/www_cellculture/cellculture.html#sec6-2) with the following modifications: starved and fed (for 5 hours) L1 were incubated with freshly thawed SDS-DTT solution for 2 min and 3min, respectively, with gentle agitation using a 1000µl pipette tip. Pronase treatment was performed using 150 µl of 15mg/ml pronase (Sigma P6911). Pronase treatment was stopped by adding 1000µl conditioned L-15 medium and spin at 1600g for 6 min. Cells were resuspended in chilled egg buffer and washed three times to remove debris before sorting using BD FACSAriaII or Beckman Coulter MoFlo sorter. ~75,000 sorted cells were pelleted at 1600g for 5 mins, snap freezed and saved in -80°C for later RNAseq analysis.

RNA extraction.
RNA was extracted from sorted cells using TRIZOL. The aqueous phase was transferred to Zymo-SpinTM IC Column (Zymo research R1013) for concentration and DNase I treatment as described in manual. RNA quality was assayed by Agilent Bioanalyzer using Agilent RNA 6000 Pico Chip. All RNAs used for library preparation had RIN (RNA integrity number) >8.

RNAseq library preparation and analysis.
Three different RNA-seq library preparation methods were used for this study: SMART-seq, which uses poly-A selection (Figure 1 and 2), Nugen Ovation, which uses random priming ( Figure S2), and Truseq combined with Ribozero to remove ribosomal RNAs (all other figures). The first two methods allow library construction from <10ng of total RNA, whereas the latter method requires >50ng total RNA. We compared SMART-seq and Truseq-Ribo zero performance on L1 PGCs isolated from wild-type and nos-1(gv5)nos-2(RNAi) and observed identical trends, with an overall higher number of miss-regulated genes identified with Truseq-Ribozero (Compare Figure 1 (SMART-seq) and Figure S1B-D (Truseq/Ribozero). For the experiment shown in Figure S2 where we compared RNA levels between embryonic PGCs and an oocyte library reference, we used Nugen Ovation libraries which avoids any bias due to poly-A selection while allowing library construction from < 3ng of RNA. For all experiments, control and experimental libraries were made using the same method. Table S5 contains lists of miss-regulated genes from analyses. All cDNA libraries were sequenced using the Illumina Hiseq2000/2500 platform.
In Figure 6F, the area-proportional Venn diagram was created using the VennDiagram R package. For comparisons shown in Figure S2A, oocyte transcriptome data was extracted from Stoeckius et al. 2014, and embryonic soma and germ cells expression profiles were from this study (Supplemental Table S8). Expression for each genes were log10 transformed, ranked and ordered. Correlations were plotted using custom R codes.
In this function -c, mapped reads were used as a reference to identify nos-1/2 dependent chromatin features. PAVIS (https://manticore.niehs.nih.gov/pavis2/) uses the output file NAME_summits.bed from MACS2 for peak annotation. Identification of genes with nos-1/2-dependent peaks at their upstream was extracted and gene IDs were cross-referenced with RNA-seq analysis in this study.

Quantitative RT-PCR assay
To verify our analysis pipeline for RNAseq data, quantitative RT-PCR (qRT-PCR) reactions using sequencing libraries as templates were performed. The cDNA libraries were diluted to 1nM before performing qRT-PCR. Primers for qRT-PCR were listed in Supplemental   Table S7. Enrichment of target mRNAs between wild type and nos-1/2 was calculated using ΔΔCt with tbb-2 expression then normalized to wild type control. Fold change were plotted and significance was calculated by paired t-test.

Technical v biological replicates
Biological replicates refer to experiments performed on independently treated hermaphrodites (in the case of RNA-seq libraries, this refers to worms exposed to independent RNAi treatments followed by cell sorting and RNA extraction). All in vivo technical replicates refer to observations in the same strain from separate zygotes.

Datasets
Datasets generated in this paper are available at GEO accession GSE100651 for ATACseq and GSE100652 for RNA-seq. based on their preferential expression patterns as determined in (Gaydos et al., 2012;Ortiz et al., 2014) (Table S1). The lists are non-overlapping and include 2064 pregamete genes, 1688 oocyte genes, 2748 sperm genes, and 3239 somatic genes. Because genes were categorized based on their preferential gene expression pattern, genes on one list may also be expressed in other tissues. See Table S1 for complete gene lists. Pre-gamete genes are overrepresented among down-regulated genes and oocyte genes are overrepresented among up-regulated genes.  (Table S1). Oocyte genes are overrepresented among down-regulated genes and pre-gamete genes are overrepresented among up-regulated genes.

Figure Legends
(C) Box and whisker plot showing the expression levels (log10) of 411 genes that are down-regulated during embryogenesis in wild-type PGCs. Expression of these genes remains high on average in nos-1(gv5)nos-2(RNAi) PGCs. Each box extends from the 25th to the 75th percentile, with the median indicated by the horizontal line; whiskers extend from the 2.5th to the 97.5th percentiles.
(D) Photomicrograph of embryos hybridized with single molecule fluorescence probes (red) against mex-5, C01G8.1 and Y51F10.2. Wild-type and nos-1(gv5)nos-2(ax3103) embryos were raised at 25°C and are compared here at the same stage (as determined by the number of DAPI-stained nuclei shown in blue). By the stages shown, all three transcripts have turned over in wild-type, but are still present (red signal) in PGCs in nos-1(gv5)nos-2(ax3103) embryos. At least ten embryos were examined per probe set in different genotypes shown.
(E) Volcano plot showing log2 fold-change in transcript abundance for each gene. The numbers of genes whose expression were up-or down-regulated in L1 PGCs compared to embryonic PGCs are indicated. Dashed lines mark the significance cutoff of q = 0.05 above which genes were counted as miss-expressed.
(F) Bar graphs showing expected and observed number of genes (Y axis) in the different expression categories (X axis).  Table S5 for lists of genes with differential expression in PGCs.
(B) Working model: Mutual antagonism between LIN-15B and PRC2 balances somatic and germline fates during development. In oocytes, LIN-15B transcription is activated by an unknown mechanism, leading to co-expression of . Competition between LIN-15B and PRC2 begins to erase PRC2 silencing marks, allowing the activation of somatic and X-linked genes in oocytes. In embryos, maternal LIN-15B and PRC2 are co-inherited (purple) by all nuclei. Somatic blastomeres activate zygotic transcription early when maternal LIN-15B levels are still high, causing the complete erasure of PRC2 marks and zygotic activation of somatic and X-linked genes, including lin-15B. In germline blastomeres, the onset of zygotic transcription is delayed until gastrulation by maternal proteins (light blue) that segregate with the nascent germline and also stabilize and promote the translation of maternal RNAs such as nos-2 (Seydoux and Braun, 2006;Tenenhaus et al., 2001). Nanos activity promotes the turnover of maternal LIN-15B, leaving PRC2 (blue nuclei) free to silence somatic and X-linked genes,  Table S2 for a list of 584 genes with more closed chromatin structure in nos-1(gv5)nos-2(RNAi) PGCs compared to wild-type PGCs.
(C) Bar graph showing expected and observed number of genes acquired more repressive chromatin structure in nos-1(gv5)nos-2(RNAi) compared to wild-type L1 in four different expression categories. Consistent with previous RNA-seq analysis, nos-1(gv5)nos-2(RNAi) PGCs failed to activate pre-gamete genes.
(D) Bar graph showing the chromosomal distribution of 584 genes that acquired more repressive chromatin structure in nos-1(gv5)nos-2(RNAi) compared to wild-type L1.
(A) Volcano plot showing log2 fold change of gene expression between mes-2(RNAi) and wild-type L1 PGCs. The numbers of genes whose expression were up-or down-regulated in mes-2(RNAi) compared to wild-type L1 PGCs are indicated. Dashed lines mark the significance cutoff of q = 0.05 above which genes were counted as miss-expressed.  Table S7 for PCR/sequencing oligos).