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Germ Granules Govern Small RNA Inheritance

Summary In C. elegans nematodes, components of liquid-like germ granules were shown to be required for transgenerational small RNA inheritance. Surprisingly, we show here that mutants with defective germ granules can nevertheless inherit potent small RNA-based silencing responses, but some of the mutants lose this ability after many generations of homozygosity. Animals mutated in pptr-1, which is required for stabilization of P granules in the early embryo, display extraordinarily strong heritable RNAi responses, lasting for tens of generations. Intriguingly, the RNAi capacity of descendants derived from mutants defective in the core germ granule proteins MEG-3 and MEG-4 is determined by the genotype of the ancestors and changes transgenerationally. Further, whether the meg-3/4 mutant alleles were present in the paternal or maternal lineages leads to different transgenerational consequences. Small RNA inheritance, rather than maternal contribution of the germ granules themselves, mediates the transgenerational defects in RNAi of meg-3/4 mutants and their progeny. Accordingly, germ granule defects lead to heritable genome-wide mis-expression of endogenous small RNAs. Upon disruption of germ granules, hrde-1 mutants can inherit RNAi, although HRDE-1 was previously thought to be absolutely required for RNAi inheritance. We propose that germ granules sort and shape the RNA pool, and that small RNA inheritance maintains this activity for multiple generations.

In Brief
RNA-rich germ granules are highly conserved and required for germline functions. Lev et al. show that ancestral germ granules shape the gene silencing capacity and small RNA pools of future generations. Altering germ granules in paternal and maternal lineages generates distinct transgenerational dynamics that deviate from Mendelian inheritance.
Recently, P granules were found to be part of a tri-condensate assemblage that also includes the Mutator granule (or M granule) and a newly discovered Z granule [20]. The Z granules colocalize with P granules, but during the mitotic stage in the adult germline the two granules separate, while remaining adjacent to each other [20]. Z granules contain the WAGO-4 argonaute [20,21] and the ZNFX-1 protein [20,22] needed for proper small RNA amplification on targeted mRNAs and for balanced epigenetic inheritance [22]. Both proteins were shown to be required for RNAi inheritance [20][21][22].
The formation and stabilization of germ granules is a tightly regulated process [24]. PPTR-1 is a regulatory subunit of the conserved PP2A phosphatase holoenzyme [36] that stabilizes maternally deposited P granules in the early embryo, regulating the correct segregation of the granules to the germline blastomere across cell divisions [37]. In pptr-1 mutants, P granules are evenly distributed throughout the embryo, and are reduced drastically in number and size [37,38]. PPTR-1 interacts with MEG-3 and MEG-4, which are intrinsically disordered serine-rich proteins that function redundantly to stabilize the condensed phase of P granules via binding of RNA and PGL-1 [39,40]. In comparison to wild-type animals, meg-3/4 zygotes display an 89% loss of P granules [39].
In this manuscript, we examined the contribution of P and Z granules to RNAi inheritance. Surprisingly, we observed enhanced RNAi inheritance in pptr-1 and meg-3/4 germ granule-defective mutants. Our experiments revealed that pptr-1 mutants consistently display enhanced RNAi inheritance, while the RNAi inheritance capacity of meg-3/4 mutants is affected by history, as it is determined by the ancestral, rather than the current, meg-3/4 genotype. While P granules are known to be maternally contributed, we report that germ granule morphological changes are not inherited transgenerationally and therefore these entities do not carry the heritable memory. Instead, we propose that germ granules generate transgenerational changes via shaping of the heritable pool of small RNAs.

Germ Granule Integrity Is Not Required for RNAi Inheritance
To study the role of germ granules in small RNA inheritance, we crossed different germ granule mutants to worms that contain a single-copy gfp driven by the mex-5 promoter in the germline. This gfp transgene is commonly used as a target for RNAi inheritance experiments [5,7,14,35]. Surprisingly, when we tested pptr-1 and meg-3/4 mutants, we found that both strains exhibit enhanced RNAi inheritance. pptr-1 mutants displayed extremely strong heritable responses, persisting for more than 70 generations ( Figures 1A-1C). Stronger RNAi inheritance in pptr-1 and meg-3/4 mutants was observed also when we used the endogenous oma-1 gene as a target ( Figure S1). This was tested by crossing the mutants with the redundant and temperature-sensitive dominant lethal oma-1(zu405) allele. Heritable silencing of this allele is necessary for survival of the oma-1 mutant at the restrictive temperature [6]. After validating previous reports showing that pptr-1 and meg-3/4 mutants indeed display highly defective embryonic germ granules (Figures 1D and S1; Videos S1, S2, and S3), we conclude that germ granule integrity is not necessary for RNAi inheritance in the germline.
To strengthen this conclusion, we examined the ability of pgl-1 mutants that we also crossed to the gfp transgene to inherit RNAi. pgl-1 mutants are also defective in P granule assembly [41]. Although these worms were reported multiple times to be resistant to germline RNAi [33,34], we found that pgl-1 mutants are competent in RNAi inheritance ( Figure S2; see more below). The Z granule argonaute WAGO-4 was also shown to be required for inheritance of dsRNA-derived exogenous siRNAs [20,21]. Our results show that WAGO-4 is indeed required for RNAi inheritance, but only for initiation of the heritable RNAi response, in parents that are directly exposed to dsRNA. Specifically, wago-4(À/À) mutant progeny that derive from RNAiexposed wago-4(+/À) parents can efficiently inherit RNAi for multiple generations ( Figure S2). This last result shows that the consequences of the activity of WAGO-4 are preserved in further generations even in WAGO-4's absence. In contrast, as was reported by others in the past [9][10][11]14], we found that the nuclear argonaute HRDE-1 is required for maintenance of the heritable RNAi response ( Figure S2).

PPTR-1 and MEG-3/4 Are Required for Maintaining
Balanced Expression of Different Small RNA Pathways in the Germline To understand how germ granules regulate small RNA levels, we sequenced small RNAs from dissected germlines of germ granule-defective meg-3/4 and pptr-1 mutants. We found that a large number of protein-coding genes (1,777 in meg-3/4 and 1,059 in pptr-1) were targeted by differential levels of cognate endo-siRNAs in the mutants compared to wild-type (DESeq2 adjusted p < 0.1; Figure 1E; Data S1). To focus our analysis on the specific effects of disruption of germ granules, we analyzed the set of genes that were targeted by differentially expressed small RNAs in both pptr-1 and meg-3/4 mutants ( Figure S3A). We found a disruption in endo-siRNAs that bind the nuclear argonaute HRDE-1 (fold enrichment of 9.23 for downregulated small RNAs and 6.63 for upregulated small RNAs, p < 10 À4 ; Figure 1F). Small RNAs that depend on or bind to proteins localized to germ granules were also substantially disturbed, such as endo-siRNAs associated with P granule proteins (the WAGO-1 [19] and the PRG-1 [42] argonautes), the M granule protein MUT-16 [43], and the Z granule argonaute WAGO-4 [21] ( Figure 1F). Similar results were obtained when examining pptr-1 and meg-3/4 separately ( Figure S3B). Previous studies have shown that different small RNA pathways compete over limiting biogenesis factors [7,8,[44][45][46]. To understand why germ granule mutants display enhanced dsRNA-induced RNAi inheritance, we sequenced small RNAs from gonads of wild-type and pptr-1 mutants, six generations after exposure to anti-gfp dsRNA. We found more than 3-fold increase in the levels of exogenousderived small RNAs targeting the gfp transgene in pptr-1 mutants ( Figure S3C), while the global levels of endogenous small RNAs targeting protein-coding genes were decreased (Figure 1G). Altogether our data suggest that proteins that affect germ granule integrity maintain the balance between different endogenous and exogenous small RNA pathways.
The Function of meg-3/4 in the Ancestors Determines the Ability of the Progeny to Inherit RNAi in a Transgenerational Manner We noticed that the ability of meg-3/4 double mutants to inherit RNAi was gradually lost after multiple generations, in (F) X-fold enrichment and depletion values (log2, bar graphs and color-coded) for protein coding genes that displayed upregulated (left) or downregulated (right) expression levels of small RNA targeting them. Shown are results for genes with differential levels in both pptr-1 and meg-3/4 mutants compared to wild-type (see also Figure S3). We tested the enrichment for the list of genes differentially targeted by small RNAs against lists of genes known to be targeted by endogenous small RNAs of specific pathways (y axis; see STAR Methods for details). p values for enrichment were calculated using 10,000 random gene sets identical in size to the tested group (see STAR Methods for details). Enrichments were considered significant if p < 0.05. ****p < 10 À4 , **p < 0.01, *p < 0.05; ns, p > 0.05. (G) Global analysis of the various small RNA species in dissected germlines of pptr-1 and meg-3/4 mutants. Shown are the mean normalized total number of reads (RPM) aligned to the denoted small RNA species. See also Figures S1-S3, Data S1, and Videos S1, S2, and S3. homozygous mutants that derived from heterozygous parents ( Figure S2B). Eventually, these mutants lost the ability to respond to RNAi. We observed the same gradual decline in RNAi responsiveness in pgl-1 mutants ( Figure S2A), but not in pptr-1 mutants ( Figure 1A). We reasoned that in previous studies researchers have found that meg-3/4 and pgl-1 mutants are refractory to RNAi [20,33,34] because mutants that have been homozygous for many generations have been examined.
To study this intriguing transgenerational dynamic, we attempted to restore the capacity of meg-3/4 mutants to perform RNAi in two different ways: we crossed wild-type worms with RNAidefective meg-3/4 mutants (homozygous for multiple generations), and since germ granules are maternally deposited, we introduced the mutated alleles via either the maternal or paternal germline (Figure 2A). We isolated F2 meg-3/4(À/À) homozygous mutants and F2 meg-3/4(+/+) wild-type animals and examined the capacity of their progeny to initiate RNAi inheritance responses ( Figure 2B). As will be elaborated below, our results clearly demonstrate that (1) the ability of the progeny to inherit RNAi is determined by the genotype of the ancestors, (2) the maternal and paternal lineages have different contributions, and (3) the potency of the phenotypes transmitted from meg-3/4 mutants changes over generations. Unlike meg-3/4 mutants, pptr-1 mutants consistently showed enhanced inheritance of RNAi in all the experiments we performed, independently of the parent's genotype ( Figure S4).
We found that when the great grandmother was meg-3/4 mutant, the mutant progeny did not respond to RNAi at all (Figure 2B, right). Moreover, even the wild-type progeny that derived from such crosses lost their RNAi inheritance ability and regained their RNAi responsiveness only after multiple generations ( Figure 2B, right). While the RNAi defect was stable in the mutants, we found that repetitive crossing of meg-3/4 mutant mothers to wild-type males improves the capacity of meg-3/4 mutants to respond to RNAi ( Figure S5). This suggested that the male germline contributes to the heritable phenotype.
When the great grandfather was meg-3/4 mutant, we found that both mutants and wild-type great grandchildren were always responsive to RNAi, and this phenotype lasted many generations (>8) in all experiments ( Figure 2B, left; Figure S2B).
In some experiments, however, the descendants of meg-3/4 fathers remained responsive to RNAi for more generations (Figure 2B, left panel as described above). We hypothesized that even these descendants gradually lose their RNAi responsiveness, but that the assay is not sensitive enough to detect it. It is known that transgenes can be very sensitive to transgenerational RNAi [15,47,48] and therefore to study these dynamics further we tested RNAi responses against two endogenous germline-expressed target genes (pos-1 and mel-26). These experiments revealed that these meg-3/4 mutants are indeed RNAi competent for a few generations but gradually lose the ability to respond to RNAi ( Figure 3A). Moreover, this transgenerational deterioration is reflected also in the small RNA pools of these animals: we dissected gonads of meg-3/4 mutants (that inherited the mutations from the paternal lineage) and sequenced small RNAs at three time points: 2, 15, and >80 generations after homozygosity. These data show dramatic genome-wide accumulative mis-expression of small RNAs that target protein-coding genes ( Figure 3B). Specifically, we found transgenerational accumulation of mis-expressed small RNAs that are associated with the HRDE-1 ( Figure 3B) and WAGO-4 pathways ( Figure S6). We also found that the levels of poly-uridylation of small RNAs that target protein-coding genes increased over generations ( Figures 3C and S6). Moreover, even HRDE-1-small RNAs, which are typically not poly-uridylated [49], exhibited accumulative poly-uridylation ( Figure 3C). Previous studies suggested that poly-uridylation is involved in sorting of small RNAs to different argonautes [21,49] and also in degradation [50] of small RNAs.
The Integrity of the Progeny's Germ Granules Is Not Affected by the Ancestral Genotype As described above, we found differences in the RNAi responsiveness of animals derived from paternal and maternal meg-3/ 4 mutant lineages. Germ granules are maternally deposited [25,26], and therefore we tested if inheritance of the germ granules themselves could explain these differences. We performed similar crosses (see scheme in Figure 4A) and examined if the morphology, abundance, and distribution of the germ granules changed in the different isolated progeny. In contrast to the RNAi inheritance phenotype of the progeny, which depended on the ancestors' meg-3/4 genotype, wild-type progeny had normal germ granules and meg-3/4 progeny had defective germ granules ( Figures 4B-4E). Moreover, we found that the P granule characteristics are similar in both RNAi responsive and non-responsive progeny of the same lineage, and the levels of silencing did not correlate with the P granules' morphology (Figure S7). In summary, we could not detect significant differences in the characteristics of the germ granules of G2-G5 isogenic animals that derived from wild-type or mutant ancestries.
Endogenous Small RNAs Mediate the Transgenerational Defects of meg-3/4 Mutants Germ granules are not deposited in the sperm; however, we observed that RNAi inheritance defects can transmit via meg-3/4(À/Ø) males to wild-type progeny. The defects that were transmitted via the paternal lineage lasted for multiple generations, but were weaker in comparison to defects transmitted via the maternal lineage ( Figure 2B, left). Unlike germ granules, small RNAs are inherited from both the sperm and the oocyte, and therefore we examined whether small RNAs could mediate the transgenerational inheritance of the RNAi defects. The nuclear germline argonaute HRDE-1 is necessary for the inheritance of many endo-siRNAs and dsRNA-derived small RNAs. Small RNA sequencing data from pptr-1 and meg-3/4 mutants indicated that HRDE-1-associated small RNAs were disturbed ( Figures 1F, 3B, and S3B). We reasoned that inheritance of endogenous small RNAs via the HRDE-1 pathway may transmit the defective RNAi phenotype to the descendants of meg-3/4 hermaphrodites. To test this possibility, we crossed meg-3/4 mutant hermaphrodites with hrde-1 mutant males, isolated meg-3/4;hrde-1 triple mutant progeny, and 3 generations later re-introduced the functional hrde-1 allele by crossing with wildtype males (see scheme in Figure 5A). We found that removing and restoring the functional hrde-1 allele erased the defects inherited from meg-3/4 mutant hermaphrodites, rescuing the ability of the progeny to inherit RNAi ( Figure 5B). Multiple crosses with wild-type males also affect RNAi resistance, albeit to a lesser extent, independently of HRDE-1 function ( Figure S5). In summary, we posit that transgenerational effects that result from defective ancestral germ granules are inherited by HRDE-1dependent endogenous small RNAs.
Next, we sequenced small RNAs from the germline of wildtype and meg-3/4 mutants derived from either paternal or maternal lineages (two generations after homozygosity; see scheme in Figure 5C) and compared their small RNAs to wildtype and to meg-3/4 mutants that had been homozygous for many generations (>80G). Principle component analysis (PCA) of the global small RNA expression levels shows that wild-type A B Figure 2. The Ancestral Germ Granule Function Determines the Ability of the Progeny to Inherit RNAi (A) Schematic diagram depicting the crosses performed to determine the ancestral contribution to RNAi inheritance in the descendants. Long-term meg-3/4 mutants (hermaphrodites and males) were outcrossed using wild-type worms of the opposite sex. All worms contained a transgene expressing GFP in the germline (Pmex-5::gfp). (B) Homozygote descendants of the crosses depicted in (A) were exposed to dsRNA complementary to gfp, to initiate gene silencing via RNAi. To test the transgenerational dynamics of the ability to respond to RNAi, naive descendants were exposed to dsRNA at different time points, 2, 8, 16, and 32 generations following homozygosity. The proportion of GFP-expressing worms (y axis, out of 40$100 worms for each group and time point) was determined over ten generations (x axis) following exposure to RNAi. The colors depict the genotypes and lineages according to the scheme in (A). Shown are mean ± SD from two independent experiments (two independent ancestral crosses). In each row (initiation at different generations following homozygosity), all groups were tested side by side, and therefore the same values for the wild-type control groups are displayed on both left and right panels. Wild-type and meg-3/4(À/À) in the maternal wild-type lineage for the G2 generation (top left panel) are replicates from Figure 1B  The transgenerational dynamics of the ability of meg-3/4 mutants to respond to RNAi targeting endogenous germline genes. Wild-type (gray) and meg-3/4 (red) worm homozygotes for the indicated amount of generations (x axis) were allowed to lay eggs on plates containing dsRNA-producing bacteria targeting the germline-expressed genes pos-1 (left) and mel-26 (right). Shown are the percentages of eggs that did not hatch into larvae (y axis), indicating the penetrance of the RNAi effect. Each dot represents one tested plate (biological replicate) and bars represent the mean. Each group was tested in two independent experiments. (B) Two-way hierarchical clustering of the different examined biological groups and genes targeted by significantly differential levels of small RNAs (compared to wild-type). Small RNA samples were obtained from dissected germlines of meg-3/4 mutants that were homozygote for 2, 15, or >80 generations (x axis). Clustering of small RNAs targeting all protein-coding genes (left panel) and small RNAs targeting genes known to be targeted by small RNAs associated with the HRDE-1 argonaute (right panel [9]) is presented. Each row represents one gene. Only genes targeted by significantly differential levels of small RNAs in at least one sample were included in the analysis (analyzed with DEseq2, adjusted p < 0.1). Each line is color-coded according to the fold change in expression. Lines depicting no significant differential expression appear in gray.
(C) An analysis of poly-uridylation of small RNAs in meg-3/4 mutants across generations. The distribution of the average fractions of poly-uridylated small RNAs against individual genes is shown for each of the examined samples. Data for small RNA samples from extracted germlines of wild-type and meg-3/4 mutants that were homozygote for 2, 15, or >80 generations (x axis) are shown. The fraction of poly-uridylated small RNAs was calculated as the number of reads with untemplated 3 0 Us out of the total aligning reads against a specific gene (only genes with R5 RPM in at least one sample were included). ***p < 10 À3 , ***p < 10 À4 , Wilcoxon rank-sum test.
For the >80 generations group in (B) and (C), data from two replicates were analyzed and a third replicate was left out since it displayed substantially less depth (3 3 10 5 reads compared to >10 7 reads in all other samples). See also Figure S6. and >80G meg-3/4 mutant worms exhibit profoundly distinct small RNA pools. Strikingly, the small RNA expression patterns of the recently isolated wild-type and meg-3/4 worms grouped together according to their maternal ancestry (the meg-3/4 genotype of the great grandmother; Figures 5D-5F). The progeny's own meg-3/4 genotype had only minor contribution to the samples' grouping ( Figure 5E). Hierarchical clustering analysis of the differentially expressed small RNAs also showed that the recently isolated wild-type and mutant worms cluster according to their maternal ancestry ( Figure 5F). These results further suggest that inheritance of mis-expressed endogenous small RNAs, rather than maternal contribution of defective granules, transmits across generations the abnormal RNAi phenotype via the meg-3/4 mutant maternal lineage.

Disrupting the Germ Granules Allows HRDE-1-Independent Inheritance of RNAi
The HRDE-1 argonaute was shown in numerous studies to be absolutely essential for dsRNA-induced RNAi inheritance [9-11, 14, 51, 52]. As we found strong RNAi inheritance in germ granule mutants despite severe defects in HRDE-1-associated small RNAs, we hypothesized that when the germ granules are defective, RNAi might be inherited via alternative routes. To test this, we performed RNAi inheritance experiments in hrde-1;pptr-1 double mutants and hrde-1;meg-3/4 triple mutants.
To enhance the sensitivity of the anti-gfp RNAi silencing assay, we quantified the exact levels of GFP expression (as was conducted in [8,15,21,53]). We found that hrde-1;meg-3/4 triple mutants, and to a lesser extent also hrde-1;pptr-1 double mutants, can weakly inherit RNAi ( Figure 6). Together, our results suggest the intriguing possibility that germ granules function in sorting of heritable small RNAs, and that changes in small RNA assortment can persist for multiple generations with extensive consequences to gene regulation in the progeny.

DISCUSSION
Close to a century ago it was observed that the cytoplasm contains droplets [54]. These droplets were later found to be RNArich granules, and characterized in the germline of more than 80 species [55]. Ingenious studies conducted over the years have elucidated the many crucial roles that germ granules play, and especially the intriguing ways by which these granules shape the transcriptome. Here we show that the influence of germ granule proteins on RNAi and small RNA inheritance changes dynamically over the course of multiple generations, shedding new light on germ cell biology.
Our study revises the current model according to which intact germ granule composition is essential for RNAi and small RNA inheritance. We demonstrate that mutants in which the germ granules are massively disrupted can exhibit potent RNAi inheritance ( Figure 1). We assume that the transgenerationally dynamic nature of RNAi in germ granule mutants has not been noted before, since in the past researchers have assayed mutants that have been homozygous for many generations. We found that the ability of the progeny to inherit RNAi is determined by the genotype of the ancestors, and that the maternal and paternal lineages have different contributions (Figure 2). We discovered that the heritable effects that accumulate due to disruption of germ granule genes can last for multiple generations (Figures 2 and 3). . Both groups as well as uncrossed wild-type controls (gray) were tested side-by-side, and therefore the same wild-type data appear in both panels. Shown are mean ± SD from two independent experiments. Data for the groups with no hrde-1 history (dark yellow and dark red) are replicates from Figure 2B, for convenience of visualization purposes only. Reset versus non-reset groups in both panels display statistically significant differences in silencing (p < 10 À4 ) based on two-way ANOVA with Bonferroni post hoc correction for multiple analysis.
(C) Crosses were performed similarly to the scheme in Figure 2A. (F) Two-way hierarchical clustering of the different biological groups and genes targeted by significantly differential levels of small RNAs. The clustering is based on the differentially expressed small RNAs compared to wild-type control samples. Each row represents one gene. Only genes targeted by significantly differential levels of small RNAs in at least one sample were included in the analysis (analyzed with DEseq2, adjusted p < 0.1). Each line is color-coded by the fold change in expression, where cells depicting no significant differential expression appear in gray. Biological groups are color-coded according to the scheme in (C) and appear above the columns.
Both heritable small RNAs and maternally contributed germ granules could potentially mediate the inheritance of the RNAi defects to the progeny of meg-3/4 mutants. Several lines of evidence suggest that transgenerational inheritance of small RNAs mediates the heritable defects: (1) the integrity of germ granules in the progeny is unaffected by changes to the ancestors' germ granules ( Figure 4); (2) the P granule's morphology and distribution does not correlate with RNAi capacity ( Figure S7); (3) some of the effects can transmit through the paternal lineage, although germ granules are only inherited from the mother (Figure 2, left); (4) the small RNA pools of the progeny are more closely related to the small RNA pools of the great grandmothers regardless of the progeny's genotype ( Figure 5); and (5) removing and then restoring the HRDE-1 argonaute, important for small RNA inheritance, re-sets the heritable effects of germ granule mutants (Figure 5). The dynamic interactions between the germ granules and heritable small RNAs determine eventually how gene regulation would be inherited across generations [2,56].
More generally, our results show that genetically identical animals can have strikingly different phenotypes because of their ancestry. First, we provide evidence that recently outcrossed animals differ from animals that were outcrossed many generations ago due to epigenetic effects. Second, outcrossing using wildtype males or hermaphrodites generates different long-term phenotypes.
Overall, pptr-1 and meg-3/4 mutants had similar effects on RNAi inheritance, small RNA expression, and germ granule morphology. However, the mutants differed in several aspects: while meg-3/4 mutants eventually become refractory to RNAi inheritance (after many generations), pptr-1 mutants are consistently responsive to RNAi, and exhibit enhanced RNAi inheritance. In contrast to meg-3/4 mutants, the RNAi capacity of the pptr-1 mutants is not affected by the maternal ancestry (Figure S4). In addition, pptr-1 mutants exhibited greater reduction in the levels of small RNAs that target protein coding genes (Figure 1G). These differences can stem from the fact that PPTR-1, a subunit of the PP2A phosphatase holoenzyme, affects many other targets (PP2A has $460 putative targets in C. elegans [57]). Additionally, we noticed that the Z granules of pptr-1 and meg-3/4 mutants differ. Whereas in meg-3/4 mutants the Z granules fail to segregate toward the P2 cell, these granules segregate normally in pptr-1 mutants (although the Z granules are smaller in both cases; Figures 1, 4, and S1B).
It is very challenging to account for non-germ-granule-related or indirect functions of the proteins examined in this work. For example, it is possible that these proteins indirectly regulate other genes that function in RNAi inheritance, even genes that encode for proteins that reside outside of granules. Interestingly, we found that genes that affect different epigenetic processes [7] are targeted by small RNAs in meg-3/4 mutants (38/285 epigenetic genes, 3.83 fold enrichment, p < 0.00001). Similarly, we cannot rule out the possibility that in the above-mentioned mutants, the typical germ granule proteins that we assayed, PGL-1 and WAGO-4 (used also as germ granule markers in [20,37,39]), are replaced by other unknown components that have different functions. Nevertheless, our results show that the integrity of the germ granules is not required for RNAi inheritance, and that mutating different genes required for the integrity of the germ granules leads to similar effects on endogenous small RNA expression.
In summary, we suggest a new heritable small RNA-based model to account for the function of germ granules in maintaining the balanced expression of different endogenous small RNA species. The imbalance in endogenous small RNAs in germ granule mutants can result in abnormal exogenous RNAi responses, characterized by unusual intensity, duration, and inheritance of small RNAs via alternative routes (independently of HRDE-1). In meg-3/4 mutants the imbalance exacerbates across generations and eventually hinders novel exogenous dsRNAinduced RNAi responses (see Figure 3 and model in Figure 7). Poly-uridylation of small RNAs by the germ granule localized protein CDE-1 was previously suggested to regulate the stability of small RNAs [50] or their sorting [21] to different argonautes. Interestingly, we detected an aggravating ectopic poly-uridylation of small RNAs in meg-3/4 mutants ( Figure 3C) that correlates with their inability to respond to dsRNA. Previous studies have GFP fluorescence (y axis) of adult worms with the indicated genotype over generations (x axis), following exposure to gfp dsRNA. Fluorescence in each group was normalized to the mean fluorescence value of the corresponding isogenic control worms originally exposed to empty vector. Shown are mean ± SD of two independent pptr-1 experiments (left) and three independent meg-3/4 experiments (right), in which 25$90 animals were analyzed for each group and generation. Tested strains derived from crosses of hrde-1 hermaphrodites with pptr-1 or meg-3/4 males (all with a Pmex-5::gfp transgene in the background) and the experiments were performed up to 6 generations since homozygosity. p values were determined via two-way ANOVA with Bonferroni post hoc correction for multiple comparisons to hrde-1. ***p < 0.001, *p < 0.05.
shown that the morphology of the germ granules is affected by environmental changes (for instance, temperature [58,59]), and also that temperature shifts trigger changes in heritable small RNAs [3,60]. Therefore, germ granules could be important mediators of transgenerationally small RNA-based heritable stress responses.
Finally, prior to Mendel's realization that traits are transmitted as discrete units of information, it was thought that parents' heritable materials mix, as liquids do. According to the now obsolete theory known as ''Blending Inheritance,'' the heritable factors ''dilute'' in the progeny [61]. We find that disruption of maternally deposited liquid condensates [24] leads to heritable changes that dilute eventually. While the modern mechanistic explanation is obviously very different from that used before the re-discovery of Mendel, we propose that the properties of RNA-carrying liquid-like granules lead to deviations from Mendelian inheritance.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Figure 7. A Schematic Model Describing the Roles of Germ Granules in Small RNA Inheritance
Our results suggest that germ granules shape the heritable small RNA pool. Mutants in the meg-3/4 core germ granule proteins accumulate small RNA defects over generations, resulting in abnormal processing of exogenous RNAi responses. The RNAi defects are inherited from meg-3/4 (À/À) maternal lineages to the progeny via endogenous HRDE-1-dependent small RNAs, and not by contribution of the germ granules themselves. In addition, exogenous RNAi-derived small RNAs can be inherited transgenerationally through alternative routes in meg-3/4 mutants (independent of HRDE-1). The scale represents the balance between different small RNA populations. The circle in the middle of the scales represents intact germ granules (the P, Z, and M granules) that ''keep the scale balanced.'' The genotype of the animal is shown below each corresponding scale. The presence of a non-interrupted black arrow originating from dsRNA (represented in red) signifies that the animal can respond to exogenous RNAi. Dotted lines that crossed out by an ''X'' symbol indicate that this animal lost its ability to respond to exogenous RNAi. The arrows between scales represent transitions between generations. Red and blue single-stranded RNA molecules depict exogenous RNAiderived siRNAs and endogenous siRNAs, respectively.

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, Oded Rechavi (odedrechavi@gmail.com). Worm strains generated in this study are available upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cultivation of worms All strains were maintained according to standard methods [67]. Unless noted otherwise, we performed all experiments at 20 degrees. Strains with the oma-1(zu405) temperature-sensitive allele were maintained at 15 degrees, and transferred to 20 degrees (restrictive temperature) for the relevant RNAi experiments. All the strains we used are listed in the Key Resources Table. METHOD DETAILS RNAi experiments HT115 E. coli bacteria bearing the dsRNA-expression bacteria and empty-vector control bacteria were grown overnight in LB medium supplemented with 0.25 mg/mL of Carbenicillin. Bacteria were seeded onto RNAi plates: NGM plates supplemented with isopropyl b-D-1thiogalactopyranoside (IPTG, 1 mM) and Carbenicillin (0.25 mg/mL). A day later, $10 adult hermaphrodites were placed on the plates, and were allowed to lay eggs for $4 hours. The progeny laid on this occasion was considered the P0 treated generation. For anti-gfp RNAi inheritance experiments, adult P0 young worms (40$120 per group) were collected for imaging, and for production of the next generation via synchronized egg-laying. For all generations (other than the P0 treated generation), synchronization was performed on the same day of imaging (4 days after egg-laying), by allowing 20 adult worms of each tested group (x2 plates) to lay eggs for $2 hours on NGM plates seeded with standard OP50 bacteria. For anti-oma-1 RNAi inheritance experiments, in each generation twelve individual larval (L4) staged worms were placed in individual wells of a 12-well plate. Four days later the number of fertile worms was assessed (at least one progeny) and twelve individual L4 progeny worms were chosen from the most fertile well to continue to the next generation. For anti-pos-1 and anti-mel-26 RNAi experiments, larval (L4) staged worms were placed on RNAi plates. From the resulting adult progeny, individual adults were isolated to fresh RNAi plates, one adult per plate. These plates were assayed two days later.
Images of the worms were collected using a BX63 Olympus microscope with x10 magnification, and were used to evaluate GFP expression.

Granules imaging and analysis
Embryos were extracted from young (day 1) adult hermaphrodites. Embryos at the 2-cell stage were chosen and immediately mounted on freshly made 3% agarose pads for imaging. All imaging was carried at 20 C on a Nikon Ti-2 eclipse microscope equipped with a 100X CFI Plan-Apo 1.45 NA objective (Nikon, Tokyo, Japan) and CSU-W1 spinning-disk confocal head (Yokogawa Corporation, Tokyo, Japan). Embryo samples were excited using 488 and 561 nm DPSS-Laser (Gataca, France) and images acquired using Prime95B sCMOS camera (Photometrics, Tucson, AZ). Image acquisition was controlled by MetaMorph software (Molecular Devices, Sunnyvale, CA). We photographed 4 cell stage embryos, using $30 z stacks per embryo with three channels: Bright field, 488 and 561.
Analysis of granules was carried out using the imageJ2 open source software. First, we split the channels and then merged the z stakes using the ''Maximal intensity z projection'' function. Next, we binarized the fluorescent signal using automated threshold using the ''Maximal entropy'' method. In the next step we used the ''Analyze particles'' function to automatically locate the granule foci. Finally, we measured the number and size of the located granule foci. To calculate the percentage of granules in the P2 cell, we manually defined the P2 and embryo regions of interest. Next, we re-applied the ''Analyze particles'' function to count number of foci in the P2 cell and the whole embryo regions. Foci < 0.1 mm 2 were excluded from the analysis of P granules.

Small RNA sequencing
Hermaphrodites were collected on the first day of adulthood, washed 4 times in M9 buffer, and transferred in a microscope slide with egg buffer (1M HEPES, 5M NaCl, 1M MgCl2, 1M CaCl2, 1M KCl and 20% tween-20) containing 2mM levamisole. Worms were cut right under the pharynx with a gauge needle, and the evading gonads separated from the body. Gonads were collected from the slide into an Eppendorf tube on ice prior the addition of 300 mL Trizol (Life Technologies). For RNA extraction from gonads of meg-3/4 and corresponding wild-type samples the miRNeasy Mini kit (QIAGEN) kit was used. To extract RNA from gonads of pptr-1 mutants and corresponding wild-type samples the following phenol-choloroform protocol was used: 60 mL of chloroform was added to the samples, which were then transferred to a pre-spun Heavy Phase Lock tube and centrifuged at 16,000 g for 12 min at 4C. We transferred the aqueous phase to a new Heavy Phase Lock tube, and added 1:1 Phenol:Chloroform:Isoamyl Alcohol, before a centrifugation round at 16,000 g for 15 min at RT. The aqueous phase was transferred to an eppendorf tube, to which we added 1:1 pure ethanol and 20 mg Glycogen (Thermo Fisher), and let incubate overnight in À20C. Samples were then centrifuged at 16,000 g for 30 min at 4C, then washed twice by removing the supernatant and adding ice-cold 70% ethanol. After the last wash and removal of ethanol, the samples were air-dried for 6 min, and resuspended in 10 mL RNase-free water. 150 ng of each sample was treated with RNA 5 0 polyphosphatase (Epicenter) to ensure small RNA capture independently of 5 0 phosphorylation status. Libraries were prepared using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) according to the manufacturer's protocol. We measured the samples concentration via Qubit, and tested their quality on an Agilent 2200 TapeStation. Samples were then pooled together and run on a 4% agarose E-Gel (Life Technologies), and the 140-160 nt length bands were excised and purified using MiniElute Gel Extraction Kit.Samples were tested for their quality and concertation on an Agilent 2200 TapeStation and then pooled together. Pools were separated on a 4% agarose E-Gel (Life Technologies) and the 140-160 nt length bands were excised and purified using MiniElute Gel Extraction kit (QIAGEN). Libraries were run on an Illumina NextSeq500 sequencer.

QUANTIFICATION AND STATISTICAL ANALYSIS
Analysis of GFP silencing For photographing of the worms $60 animals were mounted on a 2% agarose slides and paralyzed in a drop of M9 with 0.05% tetramisole. The worms were photographed with 10x objective using a BX63 Olympus microscope (exposure time of 200 ms and gain of 2). Analysis of the photographed worms was done using Image2 software.
For binary GFP silencing analysis, we manually scored the photographs for the numbers of worms having or lacking any observable germline GFP signal.
For quantification of GFP fluorescence levels, we manually defined in each worm the area of the oocyte and three background regions. Next we calculated a CTCF value as follows: CTCF = Integrated density values of the oocyte X -(area of measured oocyte * mean fluorescence of background regions). We normalized the CTCF value to the average CTCF value obtained from photographs of control animals of the same genotype, generation and age which were fed on control plates.

Quantification of anti-oma-1 RNAi experiments
Each generation we counted the fraction of worms (out of 12) that had progeny.

Quantification of anti-pos-1 and anti-mel-26 experiments
The number of hatched larva and eggs were counted per assayed RNAi-treated worm, and the percentage of hatched larva was calculated.
Reads were filtered to keep reads of 20 to 23 in length. Next, we counted reads aligning in an anti-sense orientation to genes based on the corresponding Ensembl .gff file, via the HTseq [65]: ''-m HTSeq.scripts.count-stranded=reverse-mode=intersectionnonempty input.sam GENES.gff.'' We then used the HTseq output file as an input for DESeq2 [66]. We defined differentially expressed genes using cutoff of adjusted p value < 0.1. Full DESeq2 results comparing pptr-1 and meg-3/4 mutants to wild-type are available in Data S1.
Enrichment of a given gene set i in pptr-1-dependent endo-siRNAs was calculated according to the formula: fraction of genes belong to the i À th set among differentially exp ressed STGs fraction of genes belong to the i À th set among all the genes We calculated p values using 10,000 random gene groups identical in size to that of genes targeted by pptr-1-dependent endo-siRNAs.

Bioinformatics analysis of different small RNA species
Briefly, reads were filtered to sizes of 20-23 in length. Next, we counted reads using HTseq in the sense, and again in the anti-sense orientations based on ce11 Ensembl .gff file. Reads were normalized to the total amounts of aligned reads, and the sum of reads aligning to each small RNA specie was calculated: for small RNAs targeting protein-coding and pseudogenes, reads aligned in the anti-sense orientation were used. For miRNAs and piRNAs, read aligning in the sense orientation were used. and heterozygote (right) worms mutated in the argonautes genes hrde-1 (blue) and wago-4 (red) were exposed to gfp dsRNA for RNAi initiation, and GFP fluorescence (y-axis) was measured over generations (xaxis). To enhance the sensitivity of the assay, the exact GFP expression levels were quantified. The heterozygote worms (right) were heterozygote only at the P0 generation, and in the next generations we measured GFP fluorescence of the homozygote mutant progeny. Fluorescence in each group was Figure S3. pgl-1 and meg-3/4 can inherit RNAi, but lose this ability after multiple generations.
Worms of the indicated genotype containing a transgene expressing g f p in the germline (P m e x -5 : : g f p ) were exposed to g f p dsRNA, to initate an RNAi response. The proportion of GFP-expressing worms (y-axis) was measured over generations (x-axis).
The tested homozygous mutant strains descend from heterozygotes parents, and "G#" indicates the number of generations that have passed since homozygosity at the time of RNAi initiation. Figure S3. pgl-1 and meg-3/4 can inherit RNAi, but lose this ability after multiple generations.
Worms of the indicated genotype containing a transgene expressing g f p in the germline (P m e x -5 : : g f p ) were exposed to g f p dsRNA, to initate an RNAi response. The proportion of GFP-expressing worms (y-axis) was measured over generations (x-axis).
The tested homozygous mutant strains descend from heterozygotes parents, and "G#" normalized to the mean fluorescence value of the corresponding isogenic control worms originally exposed to empty vector. Shown are mean ± SD of three independent experiments, in which 25~80 animals were analyzed for each group and generation. P values were determined via Two-way ANOVA with Bonferroni post-hoc correction for multiple comparisons to hrde-1. ****-p<10 -4 , ns-p>0.05. All groups were tested side by side, therefore the same wild-type group data appears on both panels. are results for genes with differential levels separately in pptr-1 and in meg-3/4 compared to wild type (See also Figure 1). We tested the enrichment for the list of genes differentially targeted by small RNAs against lists of genes known to be targeted by endogenous small RNAs of specific pathways (y-axis, see STAR methods for more details and a description of the statistical analysis). ****-p<10 -4 , **-p<0.01, *-p<0.05. (A) Schematic diagram depicting the crosses performed to determine the ancestral contribution to RNAi capacity in the descendants. Long-term pptr-1 mutants (hermaphrodites and males) were outcrossed using wild-type worms of the opposite sex. All worms contained a transgene expressing GFP in the germline (Pmex-5::gfp).
(B) Homozygote descendants of the crosses depicted in (A) were exposed to dsRNA complementary to gfp, to initiate gene silencing via RNAi. The percentage of GFP-expressing worms (y-axis, out of 40~100 worms for each group and time point) was determined over ten generations (x-axis) following exposure to RNAi. The colors depict the genotypes and lineages according to the scheme in (A). Shown are mean ± SD from two independent experiments (two independent ancestral crosses). All groups were tested side by side, and therefore the same values for the wild-type control groups are displayed on both left and right panels. P values were determined via Two-way ANOVA with Bonferroni post-hoc correction for multiple comparisons to wild type control. ****-p<10 -4 , ***-p<0.001, ns-p>0.05. Figure S5. Repetitive crossing of meg-3/4 mutants to wild type males leads to improvement in the capacity of the mutants to generate an RNAi response. Related to Figure 2. Worms with the indicated genotype were exposed to gfp dsRNA to generate RNAi two generations after homozygosity. The proportion of GFP-expressing worms (y-axis) was measured over generations (x-axis). meg-3/4(-/-) were outcrossed twice (purple), and tested for RNAi inheritance. Shown are mean ± SD of two independent experiments. (A) Two-way hierarchical clustering of the different examined biological groups and genes targeted by significantly differential levels of small RNAs (compared to wild type). Small RNAs samples were obtained from extracted germlines of meg-3/4 mutants that were homozygote for 2, 15 or >80 generations (x-axis) and derived from a cross between mutant males and wild-type hermaphrodites. Clustering diagrams of small RNAs targeting genes known to be targeted by the argonautes WAGO-4 [1] (top) and CSR-1 [2] (bottom) are presented. Each row represents one gene. Only genes targeted by significantly differential levels of small RNAs in at least one sample were included in the analysis (analyzed with DEseq2, adjusted p-value<0.1).
Each line is color-coded by the fold-change in expression, where lines depicting no significant differential expression appear in grey. For the >80 generations group, data from two replicates were analyzed and a third replicate was left out since it displayed substantially less read depth. for small RNAs samples from extracted germlines of wild type and meg-3/4 mutants that were homozygote for 2, 15 or >80 generations (x-axis) are shown. The fraction of poly-uridylated small RNAs was calculated as the number of reads with untemplated 3'Us out of the total aligning reads against a specific gene (only genes with >= 5 RPM in at least one sample were included). ****-p<10 -4 , ***-p<10 -3 Wilcoxon rank sum test.
For the >80 generations group, data from two replicates were analyzed and a third replicate was left out since it displayed substantially less depth.  Transgenic worms (Pmex-5::gfp::h2b, pgl-1::rfp) of the indicated genotype and lineage were exposed to antigfp dsRNA. The embryos extracted from the RNAi exposed animals were analyzed by microscopy. P granule characteristics (y-axis) are plotted against the GFP fluorescence levels (x-axis). Each dot represents one analyzed embryo. The number of analyzed embryos (N) is indicted next to the genotype. Pearson correlation scores and their P values are indicated above the panels.


extfieldsofstudy: Chemistry Medicine

text:
TLP-mediated global transcriptional repression after double-strand DNA breaks slows down DNA repair and induces apoptosis

Transcription and DNA damage repair act in a coordinated manner. Recent studies have shown that double-strand DNA breaks (DSBs) are repaired in a transcription-coupled manner. Active transcription results in a faster recruitment of DSB repair factors and expedites DNA repair. On the other hand, transcription is repressed by DNA damage through multiple mechanisms. We previously reported that TLP, a TATA box-binding protein (TBP) family member that functions as a transcriptional regulator, is also involved in DNA damage-induced apoptosis. However, the mechanism by which TLP affects DNA damage response was largely unknown. Here we show that TLP-mediated global transcriptional repression after DSBs is crucial for apoptosis induction by DNA-damaging agents such as etoposide and doxorubicin. Compared to control cells, TLP-knockdown cells were resistant to etoposide-induced apoptosis and exhibited an elevated level of global transcription after etoposide exposure. DSBs were efficiently removed in transcriptionally hyperactive TLP-knockdown cells. However, forced transcriptional shutdown using transcriptional inhibitors α-amanitin and 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) slowed down DSB repair and resensitized TLP-knockdown cells to etoposide. Taken together, these results indicate that TLP is a critical determinant as to how cells respond to DSBs and triggers apoptosis to cells that have sustained DNA damage.

Results tLp is required for etoposide-induced apoptosis induction. Exposure of cells to genotoxic agents
such as ionizing radiation and the Topo II inhibitor etoposide results in cell growth arrest and apoptosis. We previously reported that siRNA-mediated TLP knockdown confers resistance to etoposide [30][31][32][33] . To confirm this, we examined etoposide sensitivity of cells in which TLP expression was stably knocked down. As expected, stable TLP knockdown conferred etoposide resistance. After continuous etoposide treatment, TLP-knockdown cells exhibited a significantly higher viability than control cells (Fig. 1a). Etoposide-induced cleavage of Caspase 3, a marker of apoptosis induction, was markedly suppressed in TLP-knockdown cells (Fig. 1b).
Cell cycle analysis showed that etoposide treatment increased sub-G1, S, and G2/M population in control cells ( Supplementary Fig. S1), in agreement with previous findings 34,35 . Moreover, TLP knockdown partially suppressed the increase in sub-G1 and S population by etoposide treatment for 24 h, suggesting that TLP is involved in etoposide-induced apoptosis and S-phase arrest. On the contrary, G2/M population was slightly increased rather than decreased by TLP knockdown, suggesting that TLP may contribute to mitotic progression. Incidentally, TLP knockdown cells have a slightly slower growth rate than control cells, with doubling times of approximately 21.6 hours and 18.5 hours, respectively (Supplementary Fig. S2a), which may be due to a slower mitotic progression of TLP knockdown cells.
We also examined the effect of a brief treatment with etoposide and found that etoposide treatment for as short as 30 min was sufficient to induce significant growth inhibition ( Supplementary Fig. S2b). Moreover, TLP knockdown attenuated etoposide-induced growth inhibition in all the cases we examined ( Supplementary  Fig. S2b), further substantiating its critical role in etoposide-induced apoptosis. These results prompted us to examine whether TLP-knockdown cells are also resistant to various other stresses, such as γ irradiation, doxorubicin (another Topo II inhibitor), the alkylating agent methyl methane-sulfonate (MMS), and the proteasome inhibitor MG132. As a result, TLP-knockdown cells were found to be resistant to γ irradiation and doxorubicin, but not to MMS and MG132 ( Supplementary Fig. S2c,d), suggesting that TLP knockdown confers resistance to genotoxic stresses that induce DSBs. To validate the above findings, TLP expression was induced using the Tet-On system. Re-expression of TLP in knockdown cells increased sensitivity to etoposide and doxorubicin ( Fig. 1c and Supplementary Fig. S1e), suggesting that TLP is a critical determinant as to how cells respond to these DNA-damaging agents.
Consistent with our previous study 30,32,33 , the TLP protein level was dramatically increased by etoposide treatment (Fig. 1d). Meanwhile, DNA DSBs caused by Topo II inhibition are known to repress transcription 36 . To investigate a possible role of TLP in DNA damage-induced transcriptional repression, we examined the phosphorylation status of the RNAPII C-terminal domain (CTD). Ser-5 and Ser-2 phosphorylation of the RNAPII CTD is a hallmark of active transcription initiation and elongation, respectively. In control cells, etoposide treatment caused a decrease in the phosphorylation levels of both Ser-2 and Ser-5 (Fig. 1e). In TLP-knockdown cells, however, etoposide had only a modest effect on their phosphorylation levels, suggesting that TLP contributes to etoposide-induced transcriptional repression. TLP expression is induced by 24 h after etoposide addition (Fig. 1d), implying that TLP-dependent transcriptional repression is stronger in cells that have sustained DNA damage.
www.nature.com/scientificreports www.nature.com/scientificreports/ tLp is required for etoposide-induced transcriptional repression. To understand the precise role of TLP in etoposide-induced transcriptional repression, we next investigated etoposide-induced transcriptional changes in control and TLP-knockdown cells. Etoposide treatment strongly repressed expression of a subset of genes at the RNA level (Fig. 2a). On the other hand, TLP knockdown significantly attenuated etoposide-induced transcriptional repression of these genes (Fig. 2a). Consistent results were obtained by mRNA-seq analysis, which showed that 2,758 genes and 2,055 genes were significantly downregulated by etoposide in control cells and in TLP-knockdown cells, respectively (Fig. 2b, left panel). Etoposide-downregulated genes were largely overlapped between control and TLP-knockdown conditions, but the number of affected genes was substantially smaller in the latter case, suggesting that etoposide-induced transcriptional repression was impaired by TLP knockdown. Similarly, 443 genes and 348 genes were significantly upregulated by etoposide in control cells and in TLP-knockdown cells, respectively (Fig. 2b, right panel), suggesting that etoposide-induced transcriptional activation was also attenuated by TLP knockdown. GO analysis showed that genes involved in nucleosome assembly and cell-cell adhesion were particularly enriched in etoposide-affected genes; although there was no big difference in the GO terms identified between control and TLP-knockdown conditions ( Supplementary Fig. S3a). Taken together, these results suggest that TLP influences global gene expression after etoposide exposure.
Since TATA-containing promoters are thought to be a major target of TLP-mediated transcriptional repression, we investigated the proportion of TATA-containing genes in etoposide-affected genes. Enrichment analysis for transcription factor-binding sites using DAVID showed that 60.2% of top 1,000 etoposide-downregulated genes contain TATA, while only 37.1% of top 1,000 etoposide-upregulated genes contain TATA ( Supplementary  Fig. S3b). These findings suggest that TATA-containing genes are more prone to downregulation by etoposide, and that TLP is involved in etoposide-induced downregulation of TATA-containing genes.
In principle, RNA expression levels are determined by the equilibrium between RNA synthesis and degradation. To determine whether the etoposide-induced transcriptomic changes were indeed caused by changes in the  www.nature.com/scientificreports www.nature.com/scientificreports/ rate of mRNA synthesis, we investigated the effect of TLP knockdown on ongoing transcription by labeling nascent transcripts using 4-sU. In control cells, the total quantity of RNA newly synthesized in 45 min was decreased to ~4% by 24 h of etoposide treatment (Fig. 3a). When TLP was knocked down, the quantity of newly synthesized RNA was increased 1.28-fold under DMSO-treated conditions and up to 2.85-fold under etoposide-treated conditions. Concordantly, the quantities of newly synthesized mRNAs for multiple genes were significantly increased The rate of ongoing transcription was analyzed by metabolic labeling of newly transcribed RNA using 4sU. 4sU-RNA labeling was performed using control (ctrl) and TLP-knockdown (shTLP) HeLa cells that were treated with 50 μM etoposide for indicated times, and the total quantity of 4sU-labeled RNA was measured with a Quantus fluorometer (a). qRT-PCR was performed using 4sU-labeled RNA as a template (b). Data were normalized to the level of nontreated cells (0 h) and represent the average and S.D. of three independent experiments. *p < 0.05; **p < 0.01; n.s, non-significant. (c) Control and TLP-knockdown HeLa cells were treated with DMSO or 50 μM etoposide for 24 h, and ChIP was performed using anti-RNAPII CTD. Data were normalized to the level of DMSO-treated conditions and represent the average and S.D. of three independent experiments. *p < 0.05, n.s., non-significant.
www.nature.com/scientificreports www.nature.com/scientificreports/ by TLP knockdown before and after etoposide treatment while rRNA synthesis was unaffected (Fig. 3b), suggesting that the level of ongoing transcription is generally higher in TLP-knockdown cells.
We previously demonstrated that TLP inhibits the initiation step of transcription 24 . To examine whether the etoposide-TLP pathway involves the inhibition of transcription initiation, we indirectly measured promoter activity by ChIP analysis of promoter-bound RNAPII. RNAPII occupancy at the promoter region of GAPDH, EPAS1, and ACTB was significantly reduced by etoposide treatment in control cells, but not in TLP-knockdown cells (Fig. 3c), suggesting that TLP inhibits the recruitment of RNAPII in etoposide-treated cells. Taken together, our observations suggest that TLP mediates etoposide-induced transcriptional shutdown by inhibiting preinitiation complex assembly.
TLP-mediated transcriptional shutdown after etoposide exposure is crucial for efficient apoptosis induction. To investigate the possible impact of TLP-mediated transcriptional shutdown on the chromatin structure of etoposide-treated cells, we analyzed the level of H2B mono-ubiquitination (H2Bub). H2Bub is a hallmark of relaxed and transcriptionally active states [37][38][39][40] . In control cells, etoposide treatment almost eliminated H2Bub. In TLP-knockdown cells, however, etoposide treatment had only a modest effect on the H2Bub level (Fig. 4a). These results suggest that TLP-knockdown cells have relaxed chromatin structure even after etoposide exposure.
H2Bub is also known as a hallmark of active DNA repair, as H2Bub-containing chromatin is more accessible to DNA repair factors 39,40 . Moreover, active transcription is sometimes critical for efficient DNA damage repair [2][3][4] . Based on these reports, we hypothesized that an elevated level of transcription in TLP-knockdown cells leads to more efficient DNA repair and thereby results in resistance to etoposide. To test this idea, we examined the effect of the transcriptional inhibitor α-amanitin 41 . Under the conditions employed, α-amanitin alone had little effect on cell viability ( Fig. 4d and Supplementary Fig. S4a). Coadministration of α-amanitin with etoposide, however, diminished the level of residual H2Bub in TLP-knockdown cells (Fig. 4b), implying that transcriptional de-repression by TLP knockdown was reversed by α-amanitin. Concordantly, α-amanitin treatment resensitized TLP-knockdown cells to etoposide, whereas increasing concentrations of α-amanitin had a less pronounced effect on the viability of etoposide-treated control cells (Fig. 4c). Moreover, coadministration of α-amanitin with etoposide accelerated Caspase 3 cleavage in TLP-knockdown cells, but not in control cells (Fig. 4d), suggesting that transcriptional de-repression in TLP-knockdown cells is critical for etoposide resistance and for the prevention of etoposide-induced apoptosis. Lack of effect of α-amanitin in control cells is probably because transcription is efficiently repressed by etoposide treatment in control cells.
Next, we examined whether the etoposide resistance of TLP-knockdown cells is due to increased efficiency of DNA repair. To this end, we used B02, the RAD51 inhibitor that blocks DSB-induced HR 42 . B02 alone is toxic to cells and induced apoptosis in a concentration-dependent manner, with no apparent difference in sensitivity between control and TLP-knockdown cells ( Supplementary Fig. S4b). Therefore, 10 µM B02, which had little effect on cell viability alone, was used in combination with various concentrations of etoposide. While B02 had only a weak, if any, effect on etoposide sensitivity of control cells, it significantly enhanced etoposide sensitivity of TLP-knockdown cells, making TLP-knockdown cells as sensitive as control cells to etoposide (Fig. 4e). These results suggest that HR repair is more active in TLP-knockdown cells and that this makes TLP-knockdown cells more resistant to etoposide.

DSB is efficiently repaired in transcriptionally active TLP-knockdown cells.
To obtain direct evidence that HR repair is accelerated in TLP-knockdown cells, we quantified γH2AX, a sensitive indicator of DSB 43 , after etoposide treatment of control and TLP-knockdown cells (Fig. 5). Gamma-H2AX foci were formed immediately after exposure to etoposide, and signal intensity gradually diminished after etoposide removal. Five hours after etoposide removal, signal intensity decreased by ~67% in control cells and by ~86% in TLP-knockdown cells from the respective peak levels (Fig. 5, Eto v.s. r5hr). The number of γH2AX foci per cell was also counted. Five hours after etoposide removal, ~80% of control cells still had more than 20 foci per cell, whereas ~55% of TLP-knockdown cells had more than 20 foci per cell (Supplementary Fig. S5a). These results suggest that etoposide-induced DSBs are repaired in TLP-knockdown cells more rapidly. While expression levels of nearly 300 genes were significantly up-or down-regulated by TLP knockdown (Supplementary Fig. S6a), none of the 54 genes involved in HR or NHEJ was significantly affected by TLP knockdown (Supplementary Fig. S6b), suggesting that rapid DSB repair in TLP-knockdown cells does not result from change in expression levels of DSB repair genes.
Finally, we examined the effects of B02 and the transcriptional inhibitors α-amanitin and DRB on the γH2AX level. In control cells, the γH2AX signal that remained 5 h after etoposide removal was not significantly affected by B02, α-amanitin, and DRB ( Fig. 5 and Supplementary Fig. S5b). This implies that transcription is not required for DSB repair in control cells and that HR is not a major DNA repair pathway in etoposide-treated control cells. By contrast, B02, α-amanitin, and DRB all resulted in an increase of the γH2AX level in TLP-knockdown cells, suggesting that transcription is critical for DSB repair in TLP-knockdown cells, and that HR is a major DNA repair pathway in etoposide-treated TLP-knockdown cells.

Discussion
Here we revealed the mechanism by which TLP induces apoptosis through transcriptional repression after DNA damage (Fig. 6). TLP contributes to global transcriptional repression after etoposide exposure, thereby slowing down DSB repair and inducing apoptosis. Transcription is repressed by multiple mechanisms after DNA damage. For example, ATM-dependent ubiquitination of H2A causes transcriptional repression through chromatin remodeling 6,8 . Moreover, it is shown that the recruitment of Polycomb repressive complex 1 to DSB sites contributes to DNA damage-dependent transcriptional repression 9 . While most of the mechanisms described so far (2019) 9:4868 | https://doi.org/10.1038/s41598-019-41057-9 www.nature.com/scientificreports www.nature.com/scientificreports/ involve the inhibition of transcription elongation, the mechanism uncovered in this study involves the inhibition of transcription initiation, implying that DNA damage causes efficient transcriptional inhibition through multiple mechanisms. It is therefore not surprising that etoposide-induced transcriptional repression was only partially reversed by TLP knockdown (Fig. 3). While the TLP protein level is usually maintained at a low level, its expression is significantly induced by etoposide exposure by an as yet unknown mechanism (Fig. 1). On the other hand, our knockdown study showed that TLP represses the transcription of target genes before and after etoposide treatment (Fig. 3). It is therefore plausible that TLP serves as a transcriptional repressor not only in the late stage but also in the early stage of etoposide-induced cellular responses and regulates the processes. More specifically, we speculate that the basal level of TLP slows down DSB repair in the early stage of etoposide treatment and that, when induced by sustained DNA damage, TLP potentiates its function as a transcriptional repressor and accelerates apoptosis to cells that have sustained DNA damage (Fig. 6).
Despite attention, the interplay between transcription and DSB repair is not fully understood. It is considered that transcriptional repression is beneficial for efficient DSB repair, as actively elongating RNAPII could be an impediment to DSB repair machinery 44 . Concordantly, it has been reported that inhibition of transcription elongation around damaged sites results in efficient DSB repair 8 . Other studies have shown, however, that sustained transcription is critical for the recruitment of DNA repair factors to damaged sites, and that nascent RNA around   www.nature.com/scientificreports www.nature.com/scientificreports/ damaged sites facilitates HR repair 2,4 . Taking these findings into account, it is plausible that, while DSB results in the inhibition of transcription elongation, open chromatin structure allows faster access of DNA repair factors to damaged sites, and that nascent RNA associated with arrested RNAPII is used as a template for HR repair, all leading to efficient DNA repair. Given this assumption, TLP-mediated transcriptional repression seems to inhibit DSB repair through global changes to histone modifications and/or through a reduction of chromatin-associated nascent RNA. In support, the total H2Bub level and the level of ongoing transcription after etoposide exposure were higher in TLP-knockdown cells than in control cells (Figs 3 and 4). TLP-knockdown cells showed resistance to etoposide, doxorubicin, and γ irradiation, but not to MMS and MG132, suggesting that there is some specificity in the action of TLP. Unlike MMS, which is mainly repaired by the base excision repair pathway, DSBs induced by etoposide, doxorubicin, and γ irradiation are preferentially repaired by the HR pathway. It is therefore likely that TLP is particularly important in the regulation of transcription-coupled HR repair.
What is the physiological significance of TLP-mediated inhibition of DSB repair? Its possible role includes cancer prevention through elimination of DNA-damaged cells. According to this scenario, TLP expression is increased by sustained DNA damage and triggers the shift from the DNA repair mode to apoptosis in severely damaged cells. A prediction from this study is that cells expressing TLP at a low level are resistant to DSB-induced apoptosis and therefore tumorigenic. Intriguingly, previous microarray analyses of clinical samples indicate that, in testis, in which TLP is normally expressed at a high level, TLP expression is significantly reduced in cancerous tissues such as yolk sac tumor, teratoma, and embryonal carcinoma 45 . Here we demonstrated that a reduced expression of TLP confers resistance to Topo II poisons. Since etoposide is used for the treatment of various types of cancer, including testicular cancer, our findings may have clinical relevance. TLP might be useful as a prognostic marker or as a predictor of chemotherapy efficacy.

Cell culture and drug treatment. Control HeLa cells and TLP-knockdown HeLa cells 31 were maintained
in Dulbecco's modified Eagle's medium containing glucose and 10% fetal calf serum at 37 °C. shTLP-TetOn cells, which harbor the Tet-On expression system for FLAG/polyhistidine-tagged TLP (FH-TLP), were maintained in medium containing glucose, 10% fetal calf serum, and puromycin. Etoposide, doxorubicin, MG132, and B02 were dissolved in dimethyl sulfoxide (DMSO) and used at indicated concentrations. Cell viability was assayed using Cell Count Reagent SF (Nacalai Tesque).

Quantitative reverse-transcription pCR (qRt-pCR).
Total cellular RNAs were prepared from HeLa cells using RNeasy Mini Kit (Qiagen) as described by the manufacturer, quickly frozen, and stored at −80 °C until use. Quantification of purified RNA was performed using NanoDrop One (Thermo Fisher Scientific). Reverse transcription was performed using 1 µg of total RNA, SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and Oligo(dT) 20 (Thermo Fisher Scientific) as described by the manufacturer, without prior DNase treatment. It was confirmed that little or no signal was detected by qPCR without reverse transcriptase. qPCR analysis was performed by using StepOnePlus Real-Time PCR System (Thermo Fisher Scientific), KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems), and 1 µl of RT products as described by the manufacturer. Sequences and information of the primers used for qPCR are listed in Supplementary Table S1. Linear amplification of PCR products was confirmed by serial dilution of template DNA, and quantification was made within the linear range of amplification. Most of the primer sets used in this study have nearly 2.0-per-cyce amplification efficiency with negligible y-intercept. Most of the coefficients of determination derived from standard curves were greater than 0.98. Specific amplification was confirmed by melting curve analysis and agarose gel electrophoresis. qPCR data were analyzed by StepOne Software v2.2.2, and relative quantification was made by the ∆Ct method. Immunofluorescence microscopy. One day before immunofluorescence experiments, cells were seeded on cover slips. Cells were fixed with 4% paraformaldehyde for 5 min, permeabilized with 0.5% Triton X-100 for 10 min, and blocked with Blocking One (Nacalai Tesque) for 1 h at room temperature. Cells were then incubated with primary and Alexa Fluor 488-conjugated secondary antibodies for 1 h each. After counterstained with DAPI, cover slips were mounted on glass slides. Fluorescent images were obtained using a fluorescence microscope CKX53 (Olympus) and analyzed using the CellProfiler software. www.nature.com/scientificreports www.nature.com/scientificreports/ of Protein G Sepharose beads were added to each sample and further incubated for 3 h at 4 °C with rotation. Beads were washed once with Low Salt Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris-HCl (pH 8.0)), once with High Salt Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, and 20 mM Tris-HCl (pH 8.0)), once with LiCl Buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl (pH 8.0)), and twice with TE Buffer (1 mM EDTA and 10 mM Tris-HCl (pH 8.0)). DNA-protein complexes were eluted with 250 μl of Elution Buffer (1% SDS, 0.1 M NaHCO 3 , and 10 mM DTT). Cross-links were reversed by heating at 65 °C in the presence of 0.2 M NaCl at least for 8 h, followed by RNase A treatment at 37 °C for 0.5 h and Proteinase K treatment at 55 °C for 2 h. DNA was purified by phenol-chloroform extraction and ethanol precipitation and dissolved in water. Quantification of purified DNA was performed using NanoDrop One (Thermo Fisher Scientific). ChIP signals were detected by qPCR using StepOnePlus Real-Time PCR System, KAPA SYBR FAST qPCR Master Mix, and the primer sets listed in Supplementary Table S1. Linear amplification of PCR products was confirmed by serial dilution of template DNA, and quantification was made within the linear range of amplification. Specific amplification was confirmed by melting curve analysis and agarose gel electrophoresis. qPCR data were analyzed by StepOne Software v2.2.2, and relative quantification was made by using the ∆Ct method. 4-thiouridine (4sU)-RNA labeling. RNA labeling with 4sU and its purification was performed essentially as described previously 47 . Briefly, nascent RNAs were labelled by incubating cells with 500 μM 4sU (Sigma) for 45 min, immediately extracted with Sepasol-RNA I Super G (Nacalai Tesque), quickly frozen, and stored at −80 °C until use. Seventy micrograms of purified RNA was incubated with EZ-Link Biotin-HPDP (Thermo Fisher Scientific) in Biotinylation Buffer (10 mM Tris (pH 7.4) and 1 mM EDTA) for 1.5 h at room temperature. Following phenol-chloroform extraction and ethanol precipitation, biotinylated RNAs were isolated by µMACS Streptavidin beads (Miltenyi Biotec), eluted with 100 mM DTT, and further purified using RNeasy MinElute Cleanup Kit (Qiagen). The quantity of recovered RNA was measured using a Quantus fluorometer (Promega). qRT-PCR analysis was performed by using StepOnePlus Real-Time PCR System (Thermo Fisher Scientific), One

Chromatin immunoprecipitation (ChIp). Cross
Step TB Green Prime Script RT-PCR (TaKaRa), and the primer sets listed in Supplementary Table S1. mRNA-sequencing (mRNA-seq) and data analysis. Ribosomal RNA was removed from samples using RiboMinus Eukaryote Kit (Thermo Fisher Scientific), and libraries for mRNA-seq were prepared using Ion Total RNA-seq Kit v2 (Thermo Fisher Scientific). The sizes of fragmented RNA and library cDNA were determined using the MultiNA microchip electrophoresis system (Shimadzu), and the quantity of library cDNA was determined by qPCR using KAPA Library Quantification Kit (KAPA Biosystems). mRNA-seq was performed using the Ion Proton sequencer and Ion PI Chip v3 (Thermo Fisher Scientific). Mapping of sequenced reads to the human genome reference hg19 and calculation of fragments per kilobase of transcript per million mapped reads (FPKM) were performed using the software CLC Genomic Workbench (Qiagen). All data were deposited in NCBI's Gene Expression Omnibus and are publicly accessible through GEO accession number GSE120734. Fold change of gene expression was calculated using the following equation. DAVID 6.8 was used for gene ontology (GO) analysis. Flow cytometry. Trypsinized and PBS-washed cells were fixed with ethanol, treated with 0.5 mg/ml RNase A, and stained with 50 μg/mL propidium iodide as described previously 32 . Cell cycle was analyzed by using a FACSCalibur flow cytometer and the ModFit LT software (BD Biosciences).
60 Co γ irradiation. Exponentially growing cells were irradiated with cumulative doses of 25, 50, and 100 Gy using a 60 Co source at a dose rate of 2 kGy/h. After irradiation, cells were and maintained at 37 °C for 72 h, and then cell number was determined by using a Countess automated cell counter (Life Technologies). statistical analysis. Data obtained in this study are shown as means ± standard deviation (S.D.) from at least three independent experiments. Statistical significance was determined by Student's t-test or Bonferroni's method using the R Console version 3.0.3. p < 0.05 was regarded as statistically significant. Statistical significance is indicated in the figures with asterisks as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Data Availability
The datasets generated during and/or analyzed during the current study are available in the NCBI Gene Expression Omnibus, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE120734.


extfieldsofstudy: Medicine Biology

text:
Role of telomerase in the tumour microenvironment

Telomeres are specialized genomic structures that protect chromosomal ends to maintain genomic stability. Telomeric length is primarily regulated by the telomerase complex, essentially consisting of an RNA template (TERC), an enzymatic subunit (telomerase reverse transcriptase, TERT). In humans, telomerase activity is repressed during embryonic differentiation and is absent in most somatic cells. However, it is upregulated or reactivated in 80%–90% of the primary tumours in humans. The human TERT (hTERT) plays a pivotal role in cellular immortality and tumourigenesis. However, the molecular mechanisms of telomerase functioning in cancer have not been fully understood beyond the telomere maintenance. Several research groups, including ours, have demonstrated that hTERT possesses vital functions independent of its telomere maintenance, including angiogenesis, inflammation, cancer cell stemness, and epithelial–mesenchymal transformation (EMT). All these telomere‐independent activities of hTERT may contribute to the regulation of the dynamics and homeostasis of the tumour microenvironment (TME), thereby promoting tumour growth and development. Cancer progression and metastasis largely depend upon the interactions between cancer cells and their microenvironment. In this review, the involvement of TERT in the tumour microenvironment and the underlying implications in cancer therapeutics have been summarized.

consists of a catalytic component TERT, telomerase template RNA component (TERC). 29 Regardless of the telomerase activity, TERC is universally expressed in cells. In contrast, the expression of TERT is the primary determinant of telomerase activity. While this activity is repressed in somatic cells, it is reactivated in 90% of human cancer cells. 30,31 It is well documented that TERT expression or telomerase activity confers the cells with unlimited proliferation capability by elongating telomere length and bypassing the senescence checkpoint. 32,33 However, accumulating evidence also reveals that TERT exhibits multiple oncogenic activities that are independent of the reverse transcriptase activity. A substantial number of clinical studies have shown a strong correlation among levels of TERT expression and tumour malignancy. 34 Consistently, it has been described that TERT can function as a transcriptional co-activator by interacting with several other important transcription factors. This enables TERT to modulate the specific gene expression profiles in favour of epithelial-mesenchymal transformation (EMT), invasion and metastasis, angiogenesis, inflammation and immunosuppression, activation of fibroblasts, and the pluripotency of CSCs. These telomere-independent activities of hTERT could greatly contribute to the dynamics and homeostasis of the TME. [35][36][37][38][39][40][41] In this review, we will focus on the role of TERT in TME and elucidate the underlying implications in the cancer therapeutic intervention.

| TERT PROMOTE S TUMOUR ANG IOG ENE S IS
Angiogenesis is an extremely complex process involving the proliferation, migration, and lumen formation of endothelial cells. In most malignant tumours, angiogenesis is dense and occurs rapidly. New blood vessels not only provide nutrients and oxygen for tumour cell growth but also furnishes a metastatic environment that supports tumourigenesis. 42 Pallini et al revealed a direct correlation between the mRNA expression of TERT in neovascular endothelial cells and the histological grade of human tumours. 43 In a mouse model of angiogenesis, the TERT knockdown in endothelial cells reduced the formation of microvessels. On the contrary, the microvessel formation was highly enhanced upon upregulation of TERT expression. 44 Ectopic TERT expression has specifically been shown to improve the formation of persistent microvascular structures in human dermal microvascular endothelial cells, in addition to the proliferation, migration, and survival of endothelial progenitor cells in immunodeficient mice. 45 Conversely, inhibition of telomerase activity in the human umbilical vein endothelial cells (HUVECs) of glioblastoma xenografts, either by siRNA or by a dominant-negative (DN) allele of hTERT, has been reported to disrupt tumour angiogenesis significantly in the tumour environment. 44 Furthermore, treatment with IFN-γ and knockdown of the TERT gene in human glioblastoma cells can reduce the production of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Subsequently, there was a reduction in the extent of microvascular formation, thereby effectively inhibiting angiogenesis and tumorigenesis. 46 A previous report by our group demonstrated that hTERT could facilitate tumour angiogenesis by upregulating VEGF expression through direct interactions with the Vegf gene and the Sp1 transcription factor. Conversely, deletion of the mTert gene in the first generation of Tert null mice compromised tumour growth, with lowered VEGF expression and CD31-positive tumour-associated microvessel formation. 38 Intriguingly, the angiogenic factors such as VEGF and bFGF have been identified to upregulate TERT expression by activating its promoter via PI3K/Akt, nitric oxide, and ERK 1/2 pathways. 47,48 Thus, this kind of autocrine feedback loop may possibly enable TERT to contribute to angiogenesis and tumour progression. Nonetheless, tumour angiogenesis is a complex process regulated by multiple factors, and the specific molecular mechanism beneath TERT-mediated regulation of tumour angiogenesis needs further investigation.

| TERT CONTRIBUTE S TO S HAPING OF THE INFL AMMATI ON AND IMMUNOSUPPRE SS IVE ENVIRONMENT
Inflammation can upregulate growth factors, survival factors, promote angiogenesis, and induce EMT, all of which contribute to the tumour progression. 24,[49][50][51][52][53][54][55] The key inflammatory cytokines, such as IL-6, IL-8, and TNF-α, that activate the NF-κB pathway, have also been shown to be positively regulated by TERT through the NF-κB pathway. 36 It has been proven that IL-6 is essential in creating a "chemo-resistant niche" and in promoting cancer progression. 56 Besides, IL-8 and TNF-α were also revealed to be involved in the proliferation, invasion, metastasis, and inhibition of apoptosis in cervical and breast cancers. [57][58][59] A few studies, including ours, found that TERT could interact with RelA/p65 as a transcriptional co-activator to activate the NF-κB target genes such as interleukins IL-6, IL-8, and TNF-α, which are critical for inflammation and cancer progression. 36,37 Interestingly, NF-κB has been experimentally demonstrated as an activator of TERT expression, potentially amplifying the TERT-dependent carcinogenic effects. 36,60 These findings suggest that the feedback loop between the NF-κB pathway and TERT may reinforce the inflammatory signalling, leading to the development of inflammation in the TME.
The TME usually showcases its immunosuppressive features in the presence of the tumour and immune cells, allowing immune evasion and tumour progression. 61,62 Chronic inflammation and myeloid-derived suppressor cells (MDSCs) together conduce to constitute an immunosuppressive environment in the TME. 63 In fact, IL-6 has been confirmed as a key inflammatory cytokine that creates an immunosuppressive TME. 63,64 In breast cancer, IL-6 has been demonstrated to contribute the immunosuppressive TME by activating the STAT3/NF-κB/IDO pathways in MDSCs. 65,66 Additional studies reveal that IL-6 inclines the TME towards immunosuppression largely by stimulating the infiltrations of MDSCs. Clearly, an interaction between IL-6 and MDSCs contributes to the formation of the immunosuppressive TME. It has been noted that MDSCs negatively affect immune responses via inhibiting T-cell activity and upregulating immunosuppressive cells and cytokines. 67 Apart from exerting an immunosuppressive effect, MDSCs also promote tumour angiogenesis, invasion, and metastasis via production of matrix metalloproteinase-9 (MMP9), VEGF, and bFGF proteins, all of which are direct targets of TERT. [68][69][70][71] Schupp et al found that VEGF, released by cancer cells induces the production of MDSCs in the bone marrow. 72 All the studies suggest that TERT might directly and indirectly regulate the inflammation and immunosuppression within the TME.

| ROLE OF TERT IN AC TIVATI ON OF FIBROBL A S TS
Telomerase reverse transcriptase promotes cell proliferation by modulating the expression of growth-controlling genes. 16 Human immortalized fibroblasts can be generated by overexpression of hTERT. Compared with normal fibroblasts, the expressions of 172 genes were found to be altered, including that of epiregulin, a growth factor belonging to the epidermal growth factor (EGF) family. 39 Gorbunova et al found that hTERT-expressing fibroblasts were able to avoid apoptosis and necrosis induced by physicochemical factors. 73 It is also shown that TGF-β plays a chief role in the transformation of fibroblasts into cancer-associated fibroblasts (CAFs). 74,75 In mouse embryonic fibroblasts (MEFs), ectopic expression of TERT could antagonize the TGF-β-dependent growth inhibition, elevating the proliferative potential of these cells. 76 The CAFs are activated fibroblasts found in numerous types of solid cancers and are characterized by expression of activation markers such as FAP, α-SMA, and multiple secretory factors. 77 Being highly abundant in the TME, CAFs not only promote tumour initiation, progression, and recurrence, but also participate in the microenvironment remodelling processes such as angiogenesis, ECM degradation, cancer-associated inflammation, and metabolic reprogramming. [78][79][80][81][82][83] Studies show that there is a cross-talk between TLR4/NF-kB signalling and fibroblast activation. The LPS-stimulated fibroblast activation is executed cumulatively by induced expression of FAP, secretion of collagen I and TGFβ, and activation of TLR4/NF-κB signalling of the uterine fibroid fibroblasts. 84 Furthermore, Dickkopf1 (Dkk1), an antagonist of Wnt signalling that reduces the accumulation of β-catenin, was injected into the mouse model of renal fibrosis. Consequently, a decrease in the α-SMA protein levels was observed, indicating that Wnt/β-catenin pathway influences α-SMA expression levels. 85 It TA B L E 1 The role of telomerase reverse transcriptase (TERT) in the tumour microenvironment (TME)

| ROLE OF TERT IN C AN CER S TEM CELL S
Cancer stem cells are a subset of tumour cells that are able to self-renew, proliferate, and differentiate similar to the normal stem cells. 86 CSCs have been shown to possess a tumour-initiating capacity, closely interacting with the TME. Both these elements rely on each other, subsequently favouring tumour promotion.
Increasing evidence reveals that CSCs can not only adapt to alterations in the TME but also affect the TME. Moreover, various factors derived from the tumour microenvironment work in synergistically to promote self-renewal and prevent differentiation of CSCs. Recently, studies demonstrated that CSCs express higher levels of TERT, which can regulate various CSCs-associated cellular behaviours such as pluripotency, proliferation, gene expression, and therapeutic resistance. 87,88 For instance, while hTERT overexpression can promote stemness of gastric CSCs, its inhibition can suppress the stemness by regulating the expression of the CSC marker CD44. 35 The siRNA-mediated silencing of hTERT in gastric CSCs can also inhibit the production of a well-known stem cell marker OCT-4, suggesting that TERT has the potential to maintain the pluripotency in CSCs. 35 Zhang et al further demonstrated that hTERT-mediated CSC properties were at least partly dependent on β-catenin signalling in prostate CSCs. 89 It is also known that most CSCs require EGFR signalling to maintain their stemness. Reports have revealed that TERT promotes stem cell-like features in glioma cells and maintains glioma stem cell properties by inducing EGFR expression, which is independent of its telomerase activity. Conversely, lower TERT expression in turn leads to a reduced expression of EGFR and bFGF, promoting loss of glioma stem cell properties. 40 However, the specific molecular mechanistic details of TERT regulation in maintaining the stemness of CSCs remain unclear and prompt for further investigations.

| TERT PROMOTE S TUMOUR INVA S I ON A N D M E TA S TA S I S
Invasion and metastasis are complex processes that are regulated by several components of the TME. 90 The ECM is a key non-cellular component of TME and acts as a barrier to tumourigenesis. 91 The EMT is an indispensable biological process in cancer progression by which the epithelial-derived malignant cells acquire the ability to migrate and invade. 92 The degradation of ECM and induction of EMT are necessary for tumour invasion and metastasis. The tumour invasion and metastasis in the TME have also been demonstrated to be facilitated by MMPs, which are zinc-containing endopeptidases that cleave the ECM and destruct the basement membrane structure. 90 Ding et al showed that TERT could increase the expressions of MMP1, MMP3, MMP9, and MMP10 proteins of the MMP family to promote tumour invasion independent of telomerase activity. This was executed by TERT-mediated enhancement of the NF-κB pathway, which is a critical pathway for EMT. 37 Overexpressed TERT also activates the Wnt/β-catenin signalling pathway by binding to β-catenin directly as a transcriptional coactivator and upregulating the downstream target genes such as Snail-1 and vimentin, along with facilitating EMT. 35 Similarly, TERT can also synergize with transforming growth factor β (TGFβ)1 and β-catenin to promote EMT in gastric cancer cells. Additional studies have shown that hTERT could facilitate cellular metastasis in HCT116 and SW480 cells by forming a complex with ZEB1 that directly binds to the E-cadherin promoter and inhibits the expression of E-cadherin. 93 Besides, other components of the TME such as angiogenesis, inflammatory environment, and CAFs provide a metastatic environment for tumours and promote tumour invasion and metastasis. 42,58,[80][81][82][83]94 As mentioned previously, TERT can contribute to angiogenesis, inflammation, and the activation of CAFs by regulating several cytokines and growth factors.
Collectively, these findings indicate that TERT directly regulates tumour invasion and metastasis during cancer development and/ or progression.

| CON CLUS I ON S AND PER S PEC TIVE S
Credible evidence indicates that hTERT plays a pleiotropic role independent of telomere maintenance in many aspects of the TME. We speculate that TERT can act as a co-activator to excite multiple signalling pathways for regulating various features of the TME such as angiogenesis, inflammation and immunosuppression, fibroblast activation, and maintenance of CSCs pluripotency.
This contributes to the TME for promoting tumour invasion and metastasis (Table 1). Interestingly, most of the genes regulated by TERT are known activators of TERT expression, that can upregulate transcription of TERT and enhance the telomerase activity ( Figure 1). Therefore, it is debatable whether TERT may be involved in the dynamics and homeostasis of the TME by amplifying transcriptional outputs through such feedback loops. It is well recognized that NF-κB, Wnt/β-catenin, and PI3K/Akt pathways can also cross-talk with other oncogenic pathways such as the Notch, STAT3, MEK/ERK, and MAPK pathways. Thus, the effects of TERT on these pathways may potentially have multiple indirect oncogenic effects. [95][96][97][98][99] However, there are many other unresolved questions regarding the effects of TERT on TME, and the genetic mechanisms involved in its regulation, which demand further evaluation. In addition, recent study demonstrates that telomerase can contribute to telomere shortening by stabilizing stalled replication forks at telomeres in certain genetic context, which compromises replication and leads to telomere dysfunction.
The detailed mechanistic action of telomerase within and beyond telomeres clearly merits future investigation. 100 Since tumour development is highly dependent on the specific TME, a huge effort has been made to develop new therapeutic strategies targeting TME components such as blood, ECM, immune cells, and fibroblasts, to enable a more efficient targeting of TME. However, the TME of solid tumour is highly heterogeneous and complex. The development of effective anti-cancer therapies has been challenged by the overall complexity of tumours. Due to the roles of TERT in regulating the components of TME, developing the therapeutic approaches targeting of TERT in TME may increase the effectiveness of cancer therapy. While targeting TERT in the TME, multiple features of the TME may be targeted simultaneously, avoiding the menace of multidrug resistance and presenting lower toxicity. Thus, a precise understanding of the contribution of TERT in the different components of the TME in specific cancer types is a prerequisite to designing better therapeutic agents. Since TERT is also a determinant of telomerase activity, targeting TERT can simultaneously limit the proliferation potential of cancer cells by hindering the telomerase activity. It would also restrict invasion and metastasis by interfering with the TME.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflict of interests.

AUTH O R S CO NTR I B UTI O N S
All authors were participated in manuscript writing. All authors read and approved the final manuscript.