The structural proteins of DNA viruses are generally encoded by late genes, whose expression relies on recruitment of the host transcriptional machinery only after the onset of viral genome replication. β and γ-herpesviruses encode a unique six-member viral pre-initiation complex (vPIC) for this purpose, although how the vPIC directs specific activation of late genes remains largely unknown. The specificity underlying late transcription is particularly notable given that late gene promoters are unusually small, with a modified TATA-box being the only recognizable element. Here, we explored the basis for this specificity using an integrative approach to evaluate vPIC-dependent gene expression combined with promoter occupancy during Kaposi’s sarcoma-associated herpesvirus (KSHV) infection. This approach distinguished the direct and indirect targets of the vPIC, ultimately revealing a novel promoter motif critical for KSHV vPIC binding. Additionally, we found that the KSHV vPIC component ORF24 is required for efficient viral DNA replication and identified a ORF24 binding element in the origin of replication that is necessary for late gene promoter activation. Together, these results identify an elusive element that contributes to vPIC specificity and suggest novel links between KSHV DNA replication and late transcription. Gene expression in DNA viruses often occurs in temporal waves, with expression of essential structural proteins occurring late in infection, after viral genome replication has begun. Strategies underlying expression of these viral late genes are often sophisticated; for example, the β- and γ-herpesviruses encode a six-component viral complex that directs late gene transcription, largely by unknown mechanisms. Here, we evaluated how this complex specifically recognizes late promoters during infection with the oncogenic human γ-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). We found that one of the components of the late transcription complex was required for robust viral DNA replication and binds at the origin of replication, suggesting new links between KSHV replication and transcription. Combined measurements of late gene expression and promoter occupancy then revealed which KHSV genes are directly controlled by the late gene transcription complex, leading to identification of a key new regulatory element in KSHV late promoters. Together, these data help explain how the late gene transcription complex is able to bind seemingly minimal promoters with high specificity, ensuring robust expression of viral factors necessary for assembly of progeny virions. A conserved feature of double-stranded (ds) DNA viruses is that they possess a set of genes that become strongly activated only following viral DNA replication . These “Several studies suggest that the expression of late genes is also uniquely regulated in β- and γ-herpesviruses, which include human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and Kaposi’s sarcoma-associated herpesvirus (KSHV) . Unlike The core component of the KSHV vPIC is ORF24 (BcRF1 in EBV and UL87 in HCMV), a viral TBP mimic that displays putative structural similarity to cellular TBP ,15. UnliThe mechanistic basis for promoter selectivity has also been challenging to grasp in the context of the minimal, largely nondescript nature of late gene promoters. While a modified TATA box with a TATT sequence is enriched in several late promoters of KSHV, MHV68, and EBV ,22, the One possible reason that additional DNA elements involved in vPIC promoter recognition have not been identified is that we do not yet know the subset of viral promoters under the direct influence of the vPIC. Techniques such as RNA Sequencing (RNA Seq) and microarrays have helped define the set of genes that are expressed with late kinetics and in a manner dependent on viral DNA replication –26. FurtHere, we integrated RNA Seq with chromatin immunoprecipitation high-throughput sequencing (ChIP Seq) for vPIC components during KSHV infection to distinguish genes whose expression is directly versus indirectly controlled by the vPIC. In doing so we made four key findings that provide insight into late gene promoter activation in KSHV. First, we defined the set of direct vPIC promoter targets within the KSHV genome and showed that other promoters are indeed nonproductively bound or only indirectly regulated by the vPIC. Second, by restricting our analysis to these direct targets, we revealed a novel promoter motif that contributes significantly to vPIC binding and thus provides insight into features governing vPIC specificity. Notably, this motif did not emerge when the indirect vPIC targets were included in the analysis. Third, we found that the KSHV TBP mimic ORF24 is required for efficient viral DNA replication. This was unexpected given that the other studied vPIC components have been shown to be dispensable for this process. Finally, we identified a binding site for ORF24-34 at the lytic origin of replication that is essential for activating late gene expression, providing a new connection between viral DNA replication and late gene transcription.RLLLG->RAAAG mutation (ORF24RAAAG) into KSHV BAC16 to precisely evaluate the impact of the transcriptional function of ORF24 during lytic infection. We also generated the corresponding mutant rescue (ORF24RAAAG-MR) to ensure that any observed phenotypes were not the effect of secondary mutations in the BAC. Identical banding patterns from RsrII digestion of WT and mutant BAC16 DNAs verified that there were no gross rearrangements of the viral genome during mutagenesis  to enable lytic reactivation [RAAAG mutant was selectively unable to express the K8.1 late protein or produce infectious virions  are essential for its interaction with RNA Pol II and its ability to promote late gene transcription [agenesis -lanes1-3tivation . During  virions . These fRAAAG mutant  that has a premature stop codon in the ORF24 coding sequence. Inclusion of the ORF24Stop virus allowed us to evaluate whether the point mutants were functioning in a dominant negative manner, for example through nonproductive DNA binding. Finally, we also N-terminally tagged endogenous WT ORF24 with HA (HA-ORF24) to assess whether any alterations to this locus impacted viral DNA replication. Notably, we observed a similar ~5-6-fold defect in DNA replication in the ORF24RAAAG, ORF24R328A, and ORF24Stop viruses, which was restored to WT levels in the ORF24RAAAG and ORF24R328A MR viruses. Moreover, there was no DNA replication defect in the HA-ORF24 virus  versus both early and late (48 h) viral gene expression  confirmed that the majority of viral genes are upregulated between 24 and 48 h post reactivation  [2 fold change greater than one standard deviation from the mean) showed that TATTWAA is the only motif that is enriched in this set of sequences (We then looked for motifs enriched in the promoters of genes most strongly downregulated in the ORF24n (MEME) , a progrequences .We next monitored for the occurrence of this motif across all promoters in the viral genome using Find Individual Motif Occurrences (FIMO) , which sRAAAG mutation . One explanation for this observation is that these genes could be regulated by cellular transcription factors and other cis elements which are absent in the genes that are downregulated in the ORF24RAAAG mutant. The data also suggest that the motif may not be sufficient to mark regulation by the vPIC, which is consistent with a similar observation in MHV68 [2 fold change greater than one standard deviation from the mean) but lack the TATTWAA motif. The number of genes in this set increases to 26 if we broaden the criteria to include all the genes that show greater than average repression in the presence of the mutation. These genes could either be down regulated by an indirect mechanism in the absence of the ORF24-Pol II interaction or could be bound by the vPIC in a manner independent of the TATT motif.Notably, a subset of the genes containing the TATTWAA motif showed less than average repression by the ORF24Our RNA Seq experiments identified KSHV genes whose expression is influenced by the transcription regulatory function of ORF24. However, RNA Seq does not distinguish genes that are directly bound by the vPIC from those that are indirect transcriptional targets (e.g. controlled by other factors whose expression is induced by ORF24). We therefore sought to identify promoters bound by the KSHV vPIC complex using ChIP Seq. Previous studies suggest that ORF34 acts as a scaffolding factor that brings together all components of the vPIC  includinRsrII digestion  , and genigestion -Lane7. Wigestion  but was igestion . Notablyigestion . Given tigestion . One of igestion . While sTo confirm that the observed binding pattern was representative of vPIC assembly, we independently validated several prominent ChIP Seq peaks by ChIP-qPCR from cells infected with KSHV containing HA-tagged ORF24 (HA-ORF24). Similar to HA-ORF34, the HA-ORF24 virus produced WT levels of infectious virions . Indeed,A MEME analysis of the promoters associated with the peaks again showed that the only motif enriched in these promoters is TATTWAARV . 15 of tRAAAG mutant but lacked ORF34 binding at their promoters are likely to be indirect targets of the vPIC  downstream of the TATTWAA motif was enriched in these promoter sequences .cis significantly boosts late gene promoter activation during infection [RAAAG mutant , suggesting a role for the overall architecture of this motif rather than the primary sequence. In support of this hypothesis, a preliminary analysis of four structural features of the expanded motif in the WT and mutant predict that the average roll of the DNA in the expanded motif is lower in the mutant promoter compared to the WT promoter  or bound by ORF34 (7/23 ORFs). Second, the TATTWAA motif is not a pre-requisite for regulation or binding by the vPIC. For example, although ORF53 expression is dependent on the ORF24-Pol II interaction and its promoter is bound by ORF34, its promoter does not have the conserved TATTWAA sequence but a TATATAA sequence in its place. Third, several genes that exhibit late kinetics escape repression in the mutant, indicating that not all late genes are direct or indirect targets of vPIC. It is notable that several of the kinetic late genes that escape repression are involved in immune regulation, consistent with a similar observation in EBV [Several key points emerge from independent analysis of the RNA Seq and ChIP Seq data. First, the previously identified TATTWAA motif is insufficient to mark binding and regulation by the vPIC, as demonstrated by the multiple ORFs that have the motif in their promoter (23/83 ORFs) but are not repressed in the ORF24n in EBV . In partOur integrative analysis revealed that a distinct 5 bp motif is integral to vPIC promoter recognition. Rather than engaging in base specific contacts (as is the case for the TATTWAA sequence) , we instd) [d element also shows low sequence conservation and does not require an active promoter sequence to conform with the consensus sequence at all positions [d element is thought to stabilize the TFIIB-TBP interaction on the promoter DNA, leading to activation or repression of the promoter in a context-dependent manner. While there is some evidence to suggest that TFIIB is present at late gene promoters [The location of the expanded motif is reminiscent of the downstream recognition element of the general transcription factor TFIIB (BREd) . Interesositions , as seenromoters , whetherNotably, we found that KSHV DNA replication was reduced in the absence of functional ORF24, including with an ORF24 null mutant or ORF24 point mutants that fail to bind either RNA Pol II or the vPIC component ORF34. This was surprising, given that deletion or mutation of several other vPIC components has been shown not to affect DNA replication ,13,17,18One possibility for how KSHV ORF24 might contribute to DNA replication is through its indirect transcriptional targets. We observed that components of the helicase/primase complex , which are involved in DNA replication, show more than average repression in the absence of functional ORF24 . ImportaInterestingly, we also observed that binding of ORF24-34 at the origin is critical for origin dependent late gene expression, as deletion of the ORF24-34 binding element at the origin prevented vPIC assembly and transcription at the distal K8.1 late promoter. We note that although the binding element overlaps the promoter of K4.1/K4.2A, we observe only average or less than average reduction in these transcript levels in the mutant compared to the mutant rescue. We hypothesize that alternate mechanisms of maintaining steady-state K4.1/K4.2A RNA levels are adopted in the absence of a functional vPIC and binding of ORF24-34 at the origin is likely related to the link between DNA replication and late gene activation. The proximity of the ORF24-34 binding element to the other origin elements also raises the possibility of vPIC recruitment by other origin binding proteins. Exploring the mechanism underlying this link between origin binding and late gene activation will be an exciting area for future study.HEK293T cells (ATCC CRL-3216) were maintained in DMEM (Invitrogen) + 10% Fetal Bovine Serum (FBS). iSLK BAC16 cells [A 100 bp fragment of the K8.1 promoter or ORF57 promoter was generated by PCR from the BAC16 genome and cloned into the KpnI and HindIII sites of the pGL4.16 reporter plasmid using Infusion cloning (Clontech). The left origin of replication was similarly generated by PCR from the BAC16 genome and cloned into the NotI and BstXI sites of the K8.1Pr-pGL4.16 plasmid. The promoter mutants were generated by site-directed mutagenesis using KAPA HiFi DNA polymerase using the primers shown in RAAAG, ORF24R328A and ORF24Stop mutants, the corresponding mutant rescues (MR) for ORF24RAAAG, ORF24R328A and N terminal HA tagged ORF24 and ORF34 were engineered using the scarless Red recombination system in BAC16 GS1783 E. coli [The ORF24 E. coli  as descrRAAAG, ORF24R328A, and ORF24Stop (which are defective in infectious virion production), HEK293T cells that stably express ORF24-3xFlag were used for the initial transfection and co-culture.To establish iSLK cell lines latently infected with the modified virus, HEK293T cells were transfected with 5–10 ug of the modified BAC16 using linear polyethylenimine  at a 1:3 DNA:PEI ratio. The following day, the 293T cells were mixed 1:1 with iSLK-puro cells (which contain inducible RTA but lack KSHV) and treated with 30 nM 12-O-Tetradecanoylphorbol-13-acetate (TPA) and 300 nM sodium butyrate for 4 days to induce lytic replication. Cells were then grown in selection media containing 300 ug/ml hygromycin B, 1 ug/ml puromycin and 250 ug/ml G418. The hygromycin concentration was gradually increased to 500 ug/ml and then 1 mg/ ml until all the HEK293Ts cells died. To generate the ORF246 cells in 10 cm dishes and treated with 1 ug/mL doxycycline and 1 mM sodium butyrate the following day. 72 h post induction, the supernatant was filtered through a 0.45 uM syringe filter and 2 ml of the supernatant was mixed with 1x106 freshly trypsinized HEK293T cells in a 6 well plate and centrifuged at 1,500xg for 2 h at 37°C. The following day, cells were trypsinized, fixed in 4% paraformaldehyde and the percentage of cells expressing GFP was determined by flow cytometry .For supernatant transfer assays to assess virion production, BAC16-containing iSLK cells were plated at a density of 1x10To measure viral genome replication, iSLK-BAC16 cells were reactivated for 72 h as described above. The cells were then scraped into the media and the media + cells were digested with proteinase K (80μg/mL) (Promega) in 5x proteinase K digestion buffer  overnight at 55°C. The gDNA was isolated using Zymo Quick gDNA Miniprep Kit according to the manufacturer’s instructions. Quantitative PCR (qPCR) was performed on the isolated DNA using iTaq Universal SYBR Green Supermix on a QuantStudio3 Real-Time PCR machine. DNA levels were quantified using relative standard curves with primers specific for KSHV ORF59 promoter and human CPSF6 promoter . The relTo look at viral transcript levels, total RNA was isolated from reactivated iSLK cells at the indicated time points using the Zymo Direct-Zol RNA kit following the manufacturer’s protocol. The purified RNA was treated with DNase and cDNAs were synthesized using AMV reverse transcriptase (Promega). The cDNAs were directly used for qPCR analysis using the iTaq Universal SYBR Green Supermix. The qPCR signal for each ORF was normalized to 18s rRNA.g for 15 min at 4°C. The supernatant was quantified by Bradford assay, and equivalent amounts of each sample were resolved by SDS-PAGE and western blotted with rabbit polyclonal antibodies against vinculin , ORF59, or K8.1. The ORF59 and K8.1 antibodies were generated by injecting rabbits with purified MBP-ORF59 or MBP-K8.1  by rotating for 30 min at 4°C. The lysate was clarified by centrifugation at 21,000 x e Whitby ) .To compensate for the problem of quantitative assessment of transcripts due to the overlapping transcripts in the KSHV genome, the primary counts of the transcripts were calculated as recommended in Bruce et al  with theHA-ORF34 and KSHVORF24-HA) reactivated for 48 h with 1 ug/ml dox and 1 mM sodium butyrate. Cells were crosslinked in 2% formaldehyde for 15 min at room temperature, quenched in 0.125 M glycine, and washed twice with ice-cold PBS. Crosslinked cell pellets were mixed with 1 ml ice-cold ChIP lysis buffer  and incubated on ice for 10 min, whereupon the lysate was dounce homogenized to release nuclei and spun at 1700 g for 5 min at 4°C. Nuclei were then resuspended in 800 μl of nuclei lysis buffer  and rotated for 10 min at 4°C followed by sonication using a QSonica Ultrasonicator with a cup horn set to 25 amps for 20 min total . Chromatin was spun at 16,000 x g for 10 min at 4°C and the pellet was discarded. The chromatin was precleared with protein A + protein G beads blocked with 5 ug/ml glycogen, 50 ug/ml BSA, 100 ug/ml Ecoli tRNA in 1ml dilution buffer  for 2 hours.ChIP was performed on 3*15-cm plates of iSLK-cells  to 500 μl and incubated with 10 μg anti-HA antibody  overnight, whereupon samples were rotated with 20 μl pre-blocked protein A + G beads (Thermofisher) for 2 h at 4°C. Beads were washed with low salt immune complex , high salt immune complex , lithium chloride immune complex , and Tris-EDTA for 10 min each at 4°C with rotation. DNA was eluted from the beads using 100 μl of elution buffer  and incubated at 50°C for 2 h, then 65°C overnight. DNA was purified using the Zymo PCR purification kit. For ChIP-qPCR, purified DNA was quantified by qPCR using iTaq Universal SYBR Mastermix (BioRad) and the indicated primers . Each saFor ChIP-Seq, the concentration of the eluted DNA was measured using Qubit. 10–15 ng of eluted DNA was used to generate libraries using the Accel-NGS 2S Plus DNA Library Kit (Swift Biosciences). The libraries were sequenced on a Illumina HiSeq 4000 sequencer at the QB3 Berkeley core facility to generate 100 bp single end reads.The quality of the raw reads was checked using FastQC  and adap2 fold change lower than the mean Log2 fold change in the ORF24RAAAG mutant vs MR were considered to be down regulated in the mutant. Although ORF8 and ORF26 were not included in the differential expression analysis, they were considered to be downregulated because of negative values for their levels in the mutant and not in the mutant rescue on calculation of primary transcript levels. Raw and processed data files are available from the GEO database (accession number: GSE126601).To identify the direct targets, transcripts that had a LogWe defined the promoter sequence as 100 base pairs (bp) upstream from the transcription start site (TSS) or 300 bp upstream from the translation start site when the TSS was unknown. The TSS data was obtained from 5 iSLK-BAC16 (WT or ORF24RAAAG) cells were plated in a 6 well plate 24 h before transfection and reactivation. The cells were reactivated with 1 μg/ml doxycycline and 1 mM Sodium Butyrate and simultaneously transfected with 0.95 μg luciferase reporter plasmid and 0.05 μg renilla plasmid using polyjet transfection reagent. 48 h post transfection and reactivation, the cells were rinsed twice with room temperature PBS, lysed by rocking for 15 min at room temperature in 300 μl of Passive Lysis Buffer (Promega), and clarified by centrifuging at 14,000 g for 1 min. 20 μl of the clarified lysate was added in triplicate to a white chimney well microplate (Greiner bio-one) to measure luminescence on a Tecan M1000 using a Dual Luciferase Assay Kit (Promega). The firefly luminescence was normalized to the internal Renilla luciferase control. The induced sample was then normalized to the uninduced (but transfected) control to look at the fold induction during reactivation.5*106 iSLK-BAC16 were plated in a 15 cm dish 24 h before transfection and reactivation. The cells were reactivated with 1 μg/ml doxycycline and 1 mM Sodium Butyrate and simultaneously transfected with 8 μg of reporter plasmid using polyjet transfection reagent. 48 h post reactivation and transfection, cells were crosslinked and lysed as mentioned above. Nuclei were sonicated using a Covaris S220 (Covaris) for 5 min with the recommended sonication settings . The chromatin was pre-cleared as mentioned above and the concentration measured using a qubit. 25 μg of pre-cleared chromatin was used in the IP and the following steps were same as mentioned in the ChIP protocol above.3.5x10S1 FigRsrII digestion.The integrity of the various BAC mutants used in this study was verified by (TIF)Click here for additional data file.S2 FigRAAAG and MR were reactivated with doxycycline and sodium butyrate for the indicated time points, whereupon total RNA was extracted, converted to cDNA, and quantified by qPCR using primers specific to the CDS of K8.1 (late gene) or ORF57 (early gene). The fold induction was measured relative to uninduced sample.iSLK-ORF24(TIF)Click here for additional data file.S3 Fig(A) Western blot showing expression of HA-ORF24 and HA-ORF34. iSLK cells harboring WT BAC16, HA-ORF34 BAC or HA-ORF24 BAC were reactivated with doxycycline and sodium butyrate for 72 hours. Protein lysate was immunoprecipitated with HA beads and visualized by SDS-PAGE-Western blot. (B) iSLK cells harboring WT BAC16, HA-ORF34 BAC or HA-ORF24 BAC were reactivated with doxycycline and sodium butyrate for 72 hours. Progeny virion production was measured by supernatant transfer on to 293T cells and quantified by flow cytometry. (B) IGV image showing ChIP-Seq data from iSLK-HA-ORF34 cells reactivated with doxycycline and sodium butyrate for 48 h and immunoprecipitated with anti-HA antibody. Also shown are plots for control data from untagged iSLK cells and input DNA for two biological replicates.(TIF)Click here for additional data file.S4 FigFour structural parameters for 40 bp of the WT and expanded motif mutants were calculated using the DNAShape program in R . Average(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Further, REIII safeguards epigenetic memory against environmental perturbations. Our results suggest that the most prevalent type of spreading, driven by noncoding RNA-nucleators, is epigenetically unstable and requires collaboration with accessory elements to achieve high fidelity.The heterochromatin spreading reaction is a central contributor to the formation of gene-repressive structures, which are re-established with high positional precision, or fidelity, following replication. How the spreading reaction contributes to this fidelity is not clear. To resolve the origins of stable inheritance of repression, we probed the intrinsic character of spreading events in fission yeast using a system that quantitatively describes the spreading reaction in live single cells. We show that spreading triggered by noncoding RNA-nucleated elements is stochastic, multimodal, and fluctuates dynamically across time. This lack of stability correlates with high histone turnover. At the mating type locus, this unstable behavior is restrained by an accessory  The formation of gene-repressive heterochromatin domains is critical for genome integrity and for the establishment and maintenance of cell identity. Most heterochromatin formation occurs by a sequence-indifferent spreading reaction that propagates heterochromatic marks, structural proteins, and associated effector proteins outwards from nucleation sites. The precise extent of the spreading reaction has critical heritable consequences for cell identity. For example, in early pluripotent precursors, pre-existing heterochromatin domains spread, sometimes over megabases, to repress specifiers of inappropriate fates. Importantly, the final extent of spreading from a locus depends on the lineage pathway, hence it varies across different precursors  and has We have some understanding of how cells inherit silencing at nucleation sites, which constitute the DNA-sequence driven component of heterochromatin. Recent results in heterochromatin systems signaled by Histone 3 Lysine 9 and Lysine 27 methylation (H3K9me and H3K27me) indicate that several mechanisms act together to ensure intergenerational inheritance: continuous DNA-mediated recruitment of the histone methylase , low hisThese insights concerning nucleation sites do not necessarily account for how regions of heterochromatin distal to these sites are maintained. Unlike nucleation, which depends on DNA-based enzyme recruitment , spreadiTo determine how the spreading reaction acts in the maintenance of cell fate, it is central to understand the native behavior of two interconnected but separable phases of spreading: The initial spreading event, and the propagation of the states formed by this initial event through cell divisions. There is evidence that the initial spreading, at least in contexts outside the native chromosomal position, is stochastic, that is only some nucleation events result in a spreading event. This was first demonstrated by observing position effect variegation (PEV) in flies . Such stIntergenerational propagation of spreading is straightforward to conceptualize when epigenetic information is strongly reinforced, but more challenging in situations where modified nucleosomes are less likely to persist. This is the case for H3K9me-signaled heterochromatin in the fission yeast system, which lacks DNA methylation that can reinforce the epigenetic state. Persistence of the modified state is opposed by an anti-silencing protein Epe1 , which acis elements that directly recruit H3K9me. (1) cenH, which is related to the dg and dh repeats at the pericentromere and tlh2 at the subtelomere . This is dependent on REIII-bound transcription factors , which enables quantitative examination of spreading separately from nucleation in single ade6 gene promoter (ade6p) (ade6p-driven recoded super-folder GFP (ade6p-driven triple fusion of E2Crimson (clr4 (Δclr4), encoding the only S. pombe H3K9 methyltransferase. We show that in the absence of heterochromatin, expression of the noise reporter (‘red’) correlates well with that of reporters for both nucleation (‘green’) and spreading (‘orange’) , we employed transcriptionally encoded fluorescent reporters to read silencing by heterochromatin at a given locus, as previously reported. Several critical improvements over prior systems enable documentation of the spreading reaction at high sensitivity . First,  (ade6p)  to drive (ade6p) . Second,lder GFP   , especiaorange’) , which bize gate . Thus, cize gate .dh) is inserted proximal to the endogenous ura4 gene (ura4+ open-reading frame (ORF) with ‘green’ to track nucleation element-proximal events. Then, to track distal events, we inserted ‘orange’ at one of several sites downstream from ‘green’  of the ‘orange’ spreading sensor. The proportion of cells that are fully repressed by spreading declines linearly with distance  and low H3K4me3 ; the intermediate population had intermediate H3K9me2 (0.49 of dh) and H3K4me3 (0.23 of actin), and the high population had low H3K9me2 (0.2 of dh) and higher H3K4me3 (0.44 of actin). Hence, successfully nucleated cells with intermediate fluorescence also exhibit intermediate amounts of the mRNA for ‘orange’ and histone marks reflecting heterochromatin (H3K9me2) and transcriptional activity (H3K4me3). These results support the notion that intermediate states of repression observed by cytometry represent intermediate states of spreading.We next assessed the nature of these intermediate states in the 3 kb distance reporter strain, where ~30% of cells had maximal repression at the ‘orange’ locus and the remainder had intermediate states ranging from strongly to weakly repressed. Using Fluorescence Activated Cell Sorting (FACS), we gated for successful nucleation in the ‘green’ channel and then binned the ‘orange’ channel for fully repressed (low), intermediate and de-repressed (high) populations , cartoonhis1 locus  in the ‘spreading zone’, rather than several transcriptional units. Additionally, switching the nucleation and spreading reporter fluorophores produced similar results  , which is tightly repressed  and a boisrupted . The MATed REIII . At REIIerodimer , recogni (HDACs)  and are promised . We valiof cells , which iof cells . Howeverfindings . We conccenH element. The REIII nucleator was inactivated by deleting the critical cis-acting Atf1/Pcr1-binding sites, to create a strain designated ΔREIIIHSS  or de-repressed at both reporters  or OFF (ΔKHSS-OFF) colonies, consistent with each state being metastable . We found that methylation correlates with the repression state . Our ability to observe cells that were initially fully de-repressed allowed us to trace ‘green’ and ‘orange’ repression kinetically. Consistent with linear heterochromatin spread outward of the dh nucleator, we find that ‘orange’ repression is anticipated by repression at ‘green’ (dh), over time intervals where nucleation does persist, we observed dynamic fluctuations in the distal ‘orange’ color without a fixed temporal pattern , which is difficult with traditional microscopy because of cell crowding effects. To deal with this issue, we used the Fission Yeast Lifespan Micro-dissector (FYLM) microfluidic device , 2014, wto 60 hr . We notestically . Thus, ibehavior . To capt ‘green’ . While n pattern  and 2, w0, which  pattern .HSS cells maintained ‘orange’ repression for the majority of their measured lifespans . ΔKHSS-OFF behaved markedly differently from ΔREIIIHSS: in all the cells analyzed, ‘green’ and ‘orange’ reporters remained OFF throughout the time course . Under this constraint, the three strains exhibit vastly different behaviors . WT MATHifespans . Howeverifespans . In contREIIIHSS  83% of tREIIIHSS . For thiREIIIHSS . In factREIIIHSS . ImportaON state , we anale course , up to 2ed spore . HoweverREIII-dependent heterochromatin. We established two ancestral states (OFF) depends on the ancestral state, which would be indicated by separation of the unperturbed (light orange) and perturbed (red) lines. In contrast, no memory is indicated by convergence of the two lines , we compared cells containing an intact MAT locus to those lacking either ncRNA- or l states ; one witl states . Followil states . Cells eHSS exhibited clear memory at 25 generations (OFF) cells, those that derived from untreated ancestral cells showed a greater fraction of silencing (‘orange'OFF) than those derived from treated cells throughout the entire TSA gradient, with a half-resistance point of ~2 µM , memory of silencing (‘orange'OFF) is significantly weaker. Memory collapsed beyond low TSA concentrations (>0.2 µM TSA), with the red and light orange lines coinciding for much of the gradient. Even at 0 μM TSA, history dependence was erased at 35 generations  is only minimally affected by the perturbation and regains full spreading rapidly (ΔREIIIHSS) strains lose a significant amount of spreading   difference of ~22 hr, or ~9–10 generations between WT MAT and ΔREIIIHSS  . Indeed,REIIIHSS . Therefotrans-acting factors required for the initial recruitment of nucleation factors such as Clr4, Swi6/HP1 and HDACs. This experiment is similar to the induced removal of the cis-acting sites in S. cerevisiae  with established heterochromatin were crossed to mutants disrupting recruitment of nucleation factors at each element  allowedKHSS-OFF∆pcr1 cells remains robustly repressed over 456 hr, around 200 generations  system  system  to assay strains . Tag swi strains . We comp strains  and is hhromatic , and is hromatic . HoweverS. pombe cells, separate from DNA-directed events at nucleation elements, and probe its behaviors and memory characteristics. The central principle that emerges form this work is that heterochromatin spreading in fission yeast, driven predominantly by ncRNA elements, is epigenetically unstable and requires stabilization by accessory elements for high fidelity epigenetic inheritance. At the MAT locus, which carries cell identity information, a separate type of heterochromatin, independent from nc-RNA elements and dependent on the REIII element, safeguards epigenetic propagation by repressing histone turnover.The patterning of the genome into regions of activity and inactivity underlies the formation of cellular identity. In many systems heterochromatin spreading is the dominant contributor to the pattern . MaintaiS. pombe, triggered by ncRNA nucleators, leads to stochastic spreading of both silencing and H3K9 methylation that only occurs in some cells, and forms intermediate states , could e eLife. Your article has been reviewed by a Senior Editor, a Reviewing Editor, and three reviewers.. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "A memory element imposes epigenetic behavior on intrinsically labile RNAi-induced heterochromatin spread" for consideration by eLife. The overall conclusion is that this work is potentially of interest and could be worthy of publication in eLife in the future. However, in its current version it would require too much work and thus it cannot be deemed appropriate for revision within a reasonable time frame.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife. In that case, the resubmitted manuscript would be considered a "new" submission and go through the same 2-stage process. However, unlike a true new submission, it would be typically seen by the editor and if needed the original reviewers.If you can incorporate the important critiques and suggestions made by the reviewers below, you are encouraged to resubmit your manuscript to Summary:REIII, that they term deterministic, and that appears to show greater memory and fidelity in local spreading, being better buffered. This finding is novel. However, in the absence of a mechanism for REIII -mediated spreading, the work remains preliminary and there may be other interpretations of the data presented, as highlighted in the reviewers' comments. In summary, the authors do not really address the questions of heterochromatin spreading fidelity or memory as they set out to. In particular, evidence for actual "spreading" of heterochromatin from the so-called nucleation site and the genes assayed for silencing at some distance from it, is lacking. Based on the reviewers' comments, the overall conclusion is that this work is potentially of great interest and could be worthy of publication in eLife in the future. However, in its current version it would require too much work and thus it cannot be deemed appropriate for revision within a reasonable time frame.In this manuscript the authors set out to explore nature of heterochromatin spreading in fission yeast using a system that quantitatively measures heterochromatic silencing in living cells and follows the spreading reaction using fluorescent reporters. They report that spread of silencing from RNAi-nucleated elements is stochastic and multimodal and fluctuates over time. This is also supported by recent work by Obersriebnig et al. The authors also find another form of spreading, nucleated by the cis-acting element Reviewer #1:S. pombe or its relative S. cerevisiae. The crucial missing datum for all spreading models is some kind of time resolution between events at the cis-regulatory site, called the nucleation site in this study and others, and the genes at a distance that fall under the control of this regulatory site. Moreover, the way the experiments are conducted, for example by starting with cells and colonies that are preselected to be nucleated (green off) prevents the authors from the opportunity to observe the very data that could falsify the hypothesis (i.e. observing cells in which green is on and orange is off.)The fundamental problem conceptual with this paper is that the experiments are designed and interpreted in the context of a model in which spreading is assumed to be a process that emanates from a regulatory site and progressively reaches a detector site or sites. There is no evidence that "spreading" of heterochromatin of this type actually occurs in The second major problem in the paper is the assertion of being able to distinguish between stochastic spreading and deterministic spreading. Let's for the moment imagine that spreading actually occurs. The distinction the authors make between stochastic and deterministic spreading is based upon the range of expression of the detector genes that are positioned to be under the influence of the nucleation site. If that range of expression is narrow, spreading is defined to be deterministic. If it is broad, spreading is defined to be stochastic. The competing model, never mentioned nor tested, is that heterochromatin formed in once case is more stable than heterochromatin formed in the other, but "spreading" is identical in both. Distinguishing between these two models would be easier than some of the experiments in the manuscript, but one needs to acknowledge the competing model before it can be tested.The concept of fidelity of spreading is never defined and is less than obvious, yet experiments are interpreted in the context of this fidelity entity.S. cerevisiae by the Broach and Gartenberg labs in which the nucleation sites is removed and then the persistence of a state is measured over time in the absence of the element that "nucleated" it. Alternatively, the microfluidic technology behind Figure 3 could be used to address this issue.The "memory" experiments are interesting but again memory implies a time over which something is remembered. The experiments in Figure 4 may bear upon memory, but they are done in a rather complicated way show the sensitivity of those cells that have been pregrown for 25 generations to a subsequent challenge of various concentrations of TSA with respect to the stability of their expression state at that one time. A demonstration of memory requires a time component over which the thing of interest is remembered, but this experiments design precludes that. To be sure, there is some interesting biology behind the curves in this figure, but at present the experimental design would allow the authors to make interesting claims about the contribution of the various elements to the efficiency of re-establishing heterochromatin…. interesting in itself, but it isn't memory. It seems to me that the issue of memory would be resolved by the kind of clever experiments performed in Reviewer #2:cenH) is multimodal and occurs stochastically. This finding is consistent when measured at ectopic sites as well as at the endogenous MAT locus. By contrast, they argue that spreading driven solely from the endogenous REIII element at the MAT locus shows no intermediate levels and is complete ("deterministic"). They conclude that the REIII element is required to preserve the epigenetic memory of an ancestral state, as well as to ensure heterochromatin domain maintenance during environmental stress.The assembly of heterochromatin domains involves nucleation and spreading of silencing factors. However, the degree of fidelity and heritable stability among heterochromatin domains throughout the genome can vary. Here, the authors dissect the origins of the intrinsic high-fidelity and memory capacity associated with stable epigenetic heterochromatin states. To do this, they utilize a system that simultaneously monitors nucleation and spreading driven by individual regulatory elements. They find that heterochromatin spreading driven by an RNAi nucleation site  was initially shown to be critical for the stability of heterochromatin at MAT, particularly when heterochromatin nucleation by RNAi is compromised . A recent study confirmed this function of REIII . However, more relevant to this paper are subsequent studies that revealed the mechanism as follows:1) Regarding the mechanism for the role of REIII recruits an HDAC Clr3 , the activity of which is critical for the stability and spread of heterochromatin. Further work showed that Clr3 is a component of the SHREC complex that localizes to REIII . These studies set the stage for a critical finding that explained the role of REIII /Clr3 in heterochromatin stability. Aygun et al., 2013 discovered Clr3 HDAC as a unique factor that promotes heterochromatin stability by suppressing turnover of histones. Indeed, loss of Clr3 is linked to loss of tri-H3K9me , which provides the binding site for the chromodomain of Clr4 to reinforce a feedback loop in which tri-H3K9me recruits Clr4 HMTase to promote heterochromatin spreading/maintenance .Yamada et al., 2005 found that REIII → Atf1-Pcr1 → Clr3/SHREC → Suppresses histone turnover/stabilizes tri-H3K9me → Clr4 chromo binding to tri-H3K9me → Heterochromatin spreading/maintenance via feedback loop.In short: REIII impair the recruitment of Clr3 HDAC , which in turn results in increased in turnover of H3K9 methylated histones that are required for the spreading and maintenance of heterochromatin. The authors shall test whether ∆REIII cells show an elevated level of histone turnover, hence affecting spreading and epigenetic memory.In light of these previous findings, an important possibility is that mutations in REIII cells). This should be tested experimentally by assaying H3K9me levels by ChIP at GREEN and ORANGE loci. Also, GREEN inserted in cenH is likely silenced post transcriptionally (transcripts of markers inserted near centromeric repeats are converted to siRNAs) and its silencing might not provide an accurate measure of silencing caused by heterochromatin modifications. If true, this would complicate the interpretation of the results.2) An important assumption in this study is that cells carrying ORANGEON or ORANGEoff states contain comparable levels of heterochromatin at the nucleation site. However, it is possible that ORANGEON cells contain a relatively lower level of heterochromatin (but still sufficient to repress GREEN with a weak promoter) than ORANGEoff cells . In cells with GREEN located closer to the edge of the heterochromatin domain, the silencing of ORANGE located in the middle of the silenced domain might precede GREEN. In this situation, when silencing of ORANGE is assayed in GREENoff cells it would give the false impression of deterministic spreading. Consistent with this, markers inserted near mat3 are known to be silenced less efficiently. Similarly, if nucleation in ∆K cells occurred on the left side of ORANGE, all cells showing GREENoff will carry the ORANGEoff state. Can they confirm that heterochromatin in ∆K cells is indeed nucleated at REIII, or if REIII simply stabilizes heterochromatin ? Moreover, it is also possible that heterochromatin in ∆K cells is formed in such as a way that it does not involve stepwise mechanism of nucleation and spreading and that the entire domain is silenced simultaneously.3) This reviewer is concerned that the evidence that 4) The authors argue in the abstract that the epigenetic capacity contributing to heterochromatin spreading and maintenance is not encoded in the spreading reaction itself, but rather requires specialized memory elements. This statement is misleading. The factors involved in spreading and maintenance of heterochromatin, such as HDACs, are known to be loaded across silenced domains by HP1, which is also required for heterochromatin spreading. However, these same factors (HDACs) can be also loaded by so called specialized/memory elements, further enhancing the stability of heterochromatin. In this respect, the epigenetic capacity is at least partially encoded in the spreading reaction and can be further strengthened by elements that also load activities, such as HDACs, promoting heterochromatin stability.Reviewer #3:The fidelity and robustness are two essential characteristics to epigenetic inheritance, which is important both for chromatin structure and cell differentiation in many species. Its full establishment comprises of two stages: nucleation and spreading, which can be executed through RNA interference (RNAi)-dependent and RNAi-independent pathways.REIII. Further, they have tested the persistence of "memory" as nucleated by RNAi or REIII with and without perturbations. The biological importance and complicated nature should make it appealing to a broad audience.In this study, Greenstein and colleagues set up an elegant and sensitive single-cell reporter system to dissect these two characteristics. They have used it for population studies and cell-lineage tracking for trans-generation memory. Consistent with previous findings, they showed spreading of heterochromatin nucleated by RNAi alone is robust but low on fidelity, as compared to that nucleated by wild-type locus. Importantly, they have attributed the fidelity in heterochromatin spreading to an RNAi-independent DNA element REIII -alone are tested, the fidelity in spreading and robustness in nucleation segregated clearly in these population studies. However, one concern still remains. As spreading is greatly affected by the distance from the nucleator to the reporter, the actual distance for REIII -nucleated spreading should be measured between REIII and "orange" reporter, rather than between "green" and "orange" reporter as they are on different sides of the REIII nucleator, and to be consistent with Figure 2A and 2B. If the genome feature in Figure 2C is drawn to scale, it is around 1-2 kb in distance. At this distance, even RNAi-nucleated heterochromatin is demonstrating high fidelity in spreading as in the top panel of Figure 1C. At least one testing should be done with the reporter cassette moved further away from REIII nucleator by inserting "inert" DNA sequence (from a euchromatic region), to the similar 3-4 kb distance scale as Figure 2A and 2B. It is necessary to exclude the possibility that higher fidelity as in Figure 2C is due to a shorter loop for spreading. If the experiment is not possible, it shall be clearly explained in the text.In Figure 2B and 2C, where modified nucleators of RNAi-alone or [Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your revised article has been reviewed by Kevin Struhl (Senior Editor), a Reviewing Editor, and two reviewers.Thank you for resubmitting your work entitled "A memory element imposes epigenetic behavior on intrinsically labile ncRNA-induced heterochromatin spread" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:REIII that the authors claim has a deterministic role. However, direct evidence for REIII -mediated heterochromatin spreading was found to be lacking, and there was no mechanism. In their revised manuscript, the authors have provided several lines of new evidence to address some of the reviewers' comments. In particular, by deleting the REIII element, they show that this leads to increased turnover of histones which provides some insight into how REIII may be promoting epigenetic stability of heterochromatin. However, the conclusion that REIII promotes deterministic spreading of heterochromatin is still not really supported, as pointed out by reviewer 2. Furthermore, the argument that ∆K cells have better "memory" than WT cells is not supported by previous findings that ∆K cells contain metastable heterochromatin, with frequent conversion from the OFF to the ON state compared to WT. As both reviewers point out, it remains possible that bias may be introduced in the "memory" determinations, due to preselection for ∆KOFF colonies.In the previous submission of this manuscript, the work presented was found to be potentially of great interest, particularly the discovery of a novel form of putative heterochromatin spreading, nucleated by the cis-acting element REIII -based heterochromatin process, we would be willing to consider a revised text, that focused on the single cell assay showing that loss of REIII affects the stability of heterochromatin. In this revised manuscript, specific references to « memory elements » and « deterministic » spreading should be removed, as suggested by reviewer 2.Despite these limitations, and given the new work included in this manuscript on this Reviewer #2:REIII deletion on histone turnover, which has implications for heterochromatin spreading and epigenetic memory. They do indeed find that deletion of REIII causes increased turnover of histones. Together with previously published findings showing that REIII recruits HDAC Clr3 , which is involved in the suppression of histone turnover , this finding provides a possible mechanistic insight into how REIII promotes epigenetic stability of heterochromatin. With the new additions, this part of the story has improved significantly.The authors performed several experiments that addressed the reviewers' comments and strengthened their conclusions. For example, the extensive chromatin immunoprecipitation analyses performed to assay H3K9me2 and H3K9me3 levels in different strains now correlates changes in gene silencing with heterochromatin levels. Also, this reviewer suggested that they investigate the effects of REIII promotes deterministic spreading of heterochromatin remains ambiguous. In particular, unlike cenH that has been shown to nucleate heterochromatin, there is no clear evidence presented that heterochromatin spreads outward from the REIII element in ∆K cells. Rather, given the bistable nature of heterochromatin at mat in ∆K cells, the assembly of heterochromatin in ∆K cells cells might involve cooperativity between yet unknown elements, as opposed to reliance on the REIII element alone. In presenting their case for deterministic spreading from REIII, the authors mention previous work showing that loss of REIII affects heterochromatin in ∆K cells. However, it is important to note that previous studies  and this article only show that REIII is required for the stability of heterochromatin (most likely via recruitment of HDAC Clr3 that suppresses histone turnover to prevent loss of H3K9me) but is not sufficient to nucleate heterochromatin. Similarly, their argument that ∆K cells have better "memory" than WT cells contrasts with a previous finding that ∆K cells contain metastable heterochromatin, as indicated by the frequent conversion from the OFF to the ON state at a rate many fold higher than in WT. Moreover, it remains possible that bias may be introduced in the "memory" determinations, due to preselection for ∆KOFF colonies , in which entire mat locus is known to be coated with heterochromatin .However, their conclusion that eLife that focuses on the results from their elegant single cell assays showing that the loss of REIII affects the stability of heterochromatin and that this behavior of REIII is mechanistically linked to the suppression of histone turnover at mat. The discussion would be focused on succinctly describing this feature of REIII in the context of previous findings, including the results showing that REIII recruits the HDAC Clr3  that also suppresses histone turnover to promote epigenetic stability of heterochromatin . Indeed, suppression of histone turnover by REIII /HDAC Clr3 may promote retention of methylated histones that are critical for the recruitment of Clr4; thus, promoting the efficient spreading of heterochromatin. Framing the results in this way would avoid the problematic issues raised by applying the terms "memory element" or "deterministic" spreading to REIII.My recommendation is to prepare a revised manuscript for publication in Reviewer #3:REIII -mediated memory. In addition, they have now tested the anti-correlation of histone turnover and memory.The authors have addressed all the critiques raised by this reviewer. In particular, they have now provided more compelling evidence of "spreading" of heterochromatin from corresponding nucleation sites with a more complete testing cassette of different distances, directions, nucleation factors, etc. They have used these testing cassettes to demonstrate the stochasticity of ncRNA-mediated spreading and stability of  [Editors’ note: the author responses to the first round of peer review follow.]Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife. The overall conclusion is that this work is potentially of interest and could be worthy of publication in eLife in the future. However, in its current version it would require too much work and thus it cannot be deemed appropriate for revision within a reasonable time frame.If you can incorporate the important critiques and suggestions made by the reviewers below, you are encouraged to resubmit your manuscript to eLife. In that case, the resubmitted manuscript would be considered a "new" submission and go through the same 2-stage process. However, unlike a true new submission, it would be typically seen by the editor and if needed the original reviewers. Summary: In this manuscript the authors set out to explore nature of heterochromatin spreading in fission yeast using a system that quantitatively measures heterochromatic silencing in living cells and follows the spreading reaction using fluorescent reporters. They report that spread of silencing from RNAi-nucleated elements is stochastic and multimodal and fluctuates over time. This is also supported by recent work by Obersriebnig et al. The authors also find another form of spreading, nucleated by the cis-acting element REIII, that they term deterministic, and that appears to show greater memory and fidelity in local spreading, being better buffered. This finding is novel. However, in the absence of a mechanism for REIII -mediated spreading, the work remains preliminary and there may be other interpretations of the data presented, as highlighted in the reviewers' comments. In summary, the authors do not really address the questions of heterochromatin spreading fidelity or memory as they set out to. In particular, evidence for actual "spreading" of heterochromatin from the so-called nucleation site and the genes assayed for silencing at some distance from it, is lacking. Based on the reviewers' comments, the overall conclusion is that this work is potentially of great interest and could be worthy of publication in eLife in the future. However, in its current version it would require too much work and thus it cannot be deemed appropriate for revision within a reasonable time frame.S. pombe and enable us to make mechanistic inferences about how the divergent spreading types we describeare enacted. Additionally, the data point to mechanisms underlying memory of repression that is required at a cell type specification locus like MAT. Together, they have made this a much stronger manuscript. The most important additions are as follows.We very much like to thank the editors and reviewers for their thoughtful feedback. We have made major additions to this work to address the reviewers’ comments. The experiments we have included here resulted in a more robust view of spreading in 1) Establishing directionality of spreading: We have added an additional single cell tacking experiment in the ectopic strain, which together with our previous data, allows us to address the directionality of spreading using the FYLM device. We are happy to report that our data is consistent with a successive nucleation and spreading model. Additionally, we clarify our analysis method, which does not pre-filter cells of any nucleation proximal (“green”) or distal (“orange”) reporter values. We can now report that our cytometry without proximal or distal gating supports a nucleation seeded spreading model.REIII -dependent heterochromatin formation. We can report that the new data support the conclusion that differences in spreading manifest as weaker heterochromatin but are not rooted in weak nucleation.2) Tracking heterochromatin structure in our MAT locus strains: We have performed a suite of Chromatin Immuno-precipitation (ChIP) experiments to address the following issues: a. correlation of repression and heterochromatin structure, b. the stability of nucleation elements in strong and weak spreading strains and c. 3) REIII mechanism and memory: We added an additional experiment, where we remove nucleation factors post-establishment, which more directly addresses the differences in memory over time in our strains. We provide a mechanistic explanation for increased or decreased memory by tracking histone 3 turnover at euchromatic and MAT locus heterochromatin sites. We can report that REIII confers a high degree of memory that is linked to very low histone turnover compared with strains nucleated by RNA-processes that show more euchromatic levels of histone turnover.We have additionally adopted the reviewers’ suggestions to simplify and clarify the writing. Finally, both reviewer #1 and #2, compared our findings to those of Obersriebnig et al., a previous study of heterochromatin in single fission yeast cells. This was an important study and we would like to clarify the differences in experimental design and conclusions that distinguishes the two reports.cenH, near REII or REIII, or with two colors, one at cenH and one near REIII in wild-type MAT. Since in all cases both REIII and cenH nucleators were intact, silencing of either color cannot be ascribed to spreading. In our work, we both inactivate one of the nucleators at a time and track nucleation behavior with one color and spreading with a second. The use of one color can yield silencing kinetics, but by design, the nucleation and spreading events cannot be untangled. The authors make claims about the stochasticity of heterochromatin establishment, which is novel, but not spreading, as is consistent with their experimental design. Conclusions they draw about establishment kinetics are noteworthy, yet those about spreading kinetics again are complicated by having both nucleators intact. This is important, because we believe the novelty of our work, beyond the novel behavior of REIII, is the ability to isolate any type of spreading event from the DNA/RNA sequence-driven nucleation component and define its characteristics, including in real time.In the Obersriebnig work, heterochromatin formation was tracked either by following one color, at Reviewer #1:The fundamental problem conceptual with this paper is that the experiments are designed and interpreted in the context of a model in which spreading is assumed to be a process that emanates from a regulatory site and progressively reaches a detector site or sites. There is no evidence that "spreading" of heterochromatin of this type actually occurs in S. pombe or its relative S. cerevisiae. The crucial missing datum for all spreading models is some kind of time resolution between events at the cis-regulatory site, called the nucleation site in this study and others, and the genes at a distance that fall under the control of this regulatory site.We thank the reviewer for the comments which helped improve this work.S. pombe , S. cerevisiae ), flies ), plants  and humans . However, we agree that experiments documenting kinetic separation of nucleation and spreading are rare. One example is vernalization of the FLC locus in Arabidopsis, where nucleation and spreading are separated in time . Our single cell tracking data already contained information that addresses this question directly, which we expanded in the revision:Spreading outward from nucleation sites, linearly or via loops, is the paradigm for propagating heterochromatin along the chromosome in Three lines of evidence establish that heterochromatin spreads outward from nucleation sites in our assays. First, we examined the kinetics of proximal and distal silencing in cells where the system switches from full derepression to repression. Our 3kb ectopic strain afforded us an opportunity to do so, , while the MAT locus strains remain near uniformly repressed in the “green” nucleation proximal color. Full silencing of the “green” nucleation-proximal color almost exclusively precedes the distal “orange” spreading color in time . We find only one example  with “orange” repression preceding “green”, with the green closely following orange. This small kinetic gap therefore may suggest a fast spreading event.Second, in the FYLM data, we did not observe any cells (N=0/270) where “green” becomes de-repressed while “orange” stays repressed . In contrast we find many cells in which “orange” fluctuates while “green” is OFF for the entire measured lifespan  and more cells where “orange” is fully de-repressed while “green” is at least repressed partially for the entire measured lifespan . This reinforces the notion that silencing is directional with “green” preceding “orange”.OFF (nucleation site) yields a distribution of orange repression, OFF to ON, while gating in “orange”OFF (spreading site) only yields “green”OFF, supporting the above single-cell dynamics with a broader, population-based snapshot . Therefore, we interpret this to indicate that spreading follows nucleation.Third, using our steady state flow cytometry data for all RNA- nucleated strains, both ectopic and at MAT, gating for “green”Moreover, the way the experiments are conducted, for example by starting with cells and colonies that are preselected to be nucleated (green off) prevents the authors from the opportunity to observe the very data that could falsify the hypothesis (i.e. observing cells in which green is on and orange is off.)OFF cells for analysis: All the 2D histogram and scatter plots in the paper show cells normalized only by “red”. No “green” or “orange” value filter is applied at this stage. Even for the ΔK strain, which can be isolated as ON or OFF alleles , we describe the overall distribution of cells after introduction of clr4+ in Figure 2E. Since we established from our data (cytometry and single cell tracking) that nucleation indeed precedes spreading, we produce 1D “orange” histograms with an additional “green”OFF gate to isolate fully nucleated cells . Importantly, in the cytometry data, we do not observe cell populations that are “orange”OFF and “green”ON  or REIII -disrupted context  and REIII in the cenH deleted context . To do so, we performed Chromatin Immuno-precipitation.We examined the stability of the relevant nucleation sites to test this hypothesis, dh) and nearly invariant between the nucleation sites. Critically, there is no difference in H3K9me2 and me3 methylation at cenH between wildtype MAT and ΔREIII, strainswhich feature very different distributions of distal “orange” repression . This new data is included in Figure 2D and E.(ChIP) experiments for the heterochromatin assembly mark H3K9me2 and the repressive/spreading mark H3K9me3 ). Since factors assembling the heterochromatin structure are directly or indirectly attracted by these marks, we believe that they are the central features to examine. We observe that at all nucleation sites, the degree of both H3K9me2 and H3K9me3 methylation is extremely robust  or stochastically (∆REIIIHSS). To this end we plotted the fraction of “green”OFF cells along the [TSA] gradient . In this analysis, memory is manifested by the system behaving differently depending on its starting state. A stimulant-responsive system exhibits hysteresis (memory) if the behavior along the stimulant gradient is divergent between a fully stimulated or naïve starting condition. The similarity in molecular architectures, cooperativity and positive feedback, which drive both the biochemical systems traditionally subjected to hysteresis analysis and epigenetic systems ), motivated us to perform hysteresis-type experiments. Here, we are probing whether the ancestral state biases the decision of system to undergo a spreading event. We measure the retention of this bias along a gradient of inhibitor, which is conceptually similar to measurements for a stimulant. We chose this course because stimulants are not readily available for heterochromatin. We believe this analysis is both rigorous and valid for probing memory capacity.trans-acting nucleation factors on the retention of the state originally formed by spreading. These experiments are conceptually similar to removal of cis-acting sites but avoid the caveat of low induced Cre-lox recombination frequency in S. pombe. We crossed our reporter strains to mutants required for either the cenH or REIII nucleator. Immediately following detection of cross progeny colonies, we proceeded with flow cytometry analysis. We find indeed that REIII nucleated heterochromatin can maintain itself after removal of a required trans-acting nucleation factor for ~ 200 generations, while cenH-nucleated heterochromatin cannot, and appears to require continual nucleation to maintain the state. These new data support our overall conclusions and are presented in the new Figure 5 and new Figure 5—figure supplement 1.However, inspired by the reviewer’s invocation of the Gartenberg experiments, we probed the effect of removing REIII spreading is remembered, while cenH-spreading is not. Intergenerational tracking of spreading in the cenH, noncoding RNA (ncRNA)*-nucleatedstrains  as well as ectopically ncRNA-nucleated strains  show fluctuations of “orange” not compatible with strong memory, while REIII strains  show no such fluctuations over similar generation times, strongly implying memory.Finally, we also believe that our single cell tracking data in Figure 3 and Figure 3—figure supplement 2 lend independent support for the conclusion that cenH and REIII nucleated spreading.Together, our three independent approaches strongly support a differential in the ability to remember the state formed by spreading between cenH and dh rather than RNAi.We now use this more general descriptor for Reviewer #2: The assembly of heterochromatin domains involves nucleation and spreading of silencing factors. However, the degree of fidelity and heritable stability among heterochromatin domains throughout the genome can vary. Here, the authors dissect the origins of the intrinsic high-fidelity and memory capacity associated with stable epigenetic heterochromatin states. To do this, they utilize a system that simultaneously monitors nucleation and spreading driven by individual regulatory elements. They find that heterochromatin spreading driven by an RNAi nucleation site (cenH) is multimodal and occurs stochastically. This finding is consistent when measured at ectopic sites as well as at the endogenous MAT locus. By contrast, they argue that spreading driven solely from the endogenous REIII element at the MAT locus shows no intermediate levels and is complete ("deterministic"). They conclude that the REIII element is required to preserve the epigenetic memory of an ancestral state, as well as to ensure heterochromatin domain maintenance during environmental stress. A major strength of this article is the use of a system that quantitatively measures heterochromatic silencing in living cells. The development of such tools has allowed them to tackle key questions in the field of heterochromatin assembly. The conclusion that the spreading of heterochromatin from an RNAi nucleated element is stochastic is also supported by recent work by Obersriebnig et al,. 2016, which also used single cell assays. A key new finding here is that a cis-acting element, REIII, promotes deterministic spreading of heterochromatin that has high memory capacity. However, in the absence of a mechanism for the REIII -mediated spreading, this aspect of the work remains preliminary. Moreover, the evidence suggesting deterministic spreading from REIII needs further careful evaluation.We thank the reviewer for this assessment.1) Regarding the mechanism for the role of REIII in epigenetic memory, previous studies have provided important insights that are highly relevant to this story and deserve to be addressed clearly as part of the main discussion. REIII (a binding site for ATF-CREB proteins Atf1-Pcr1) was initially shown to be critical for the stability of heterochromatin at MAT, particularly when heterochromatin nucleation by RNAi is compromised . A recent study confirmed this function of REIII . However, more relevant to this paper are subsequent studies that revealed the mechanism as follows: Yamada et al., 2005 found that REIII recruits an HDAC Clr3 , the activity of which is critical for the stability and spread of heterochromatin. Further work showed that Clr3 is a component of the SHREC complex that localizes to REIII . These studies set the stage for a critical finding that explained the role of REIII /Clr3 in heterochromatin stability. Aygun et al., 2013 discovered Clr3 HDAC as a unique factor that promotes heterochromatin stability by suppressing turnover of histones. Indeed, loss of Clr3 is linked to loss of tri-H3K9me , which provides the binding site for the chromodomain of Clr4 to reinforce a feedback loop in which tri-H3K9me recruits Clr4 HMTase to promote heterochromatin spreading/maintenance . In short: REIII → Atf1-Pcr1 → Clr3/SHREC → Suppresses histone turnover/stabilizes tri-H3K9me → Clr4 chromo binding to tri-H3K9me → Heterochromatin spreading/maintenance via feedback loop In light of these previous findings, an important possibility is that mutations in REIII impair the recruitment of Clr3 HDAC , which in turn results in increased in turnover of H3K9 methylated histones that are required for the spreading and maintenance of heterochromatin. The authors shall test whether ∆REIII cells show an elevated level of histone turnover, hence affecting spreading and epigenetic memory.We thank the reviewer for raising these points. We had included a description of possible mechanisms in our supplemental discussion in the original submission, including a number of the references cited by the reviewer. However, we recognize that the mechanistic arguments should be highlighted more prominently in the paper and have included them now in the main discussion.KHSS-OFF, ∆KHSS-ON and ∆REIIIHSS strains to address the role of REIII, with ∆KHSS-ON serving as an isogenic non-heterochromatic control for ∆KHSS-OFF. We measured H3 turnover at two euchromatic control loci and find no difference between the strains. However, when we examined turnover at MAT using amplicons that are shared by all strains, we found significantly decreased histone turnover in ∆KHSS-OFF, while ∆KHSS-ON and ∆REIIIHSS consistently display elevated turnover, more consistent with euchromatic behavior. These new data are included in the new Figure 6 A,B. The data that histone turnover is elevated in the absence of nucleated REIII thus presents a mechanistic explanation for the strongly reduced memory we see in ∆REIII and stochastic spreading, while the exceedingly low turnover in the presence of REIII accounts for the deterministic spreading and robust memory. The robust memory is underlined by a new experiment we include, where we remove by cross factors required for nucleation following establishment .We have now addressed the role of histone turnover as a possible mechanism for the differences in spreading and epigenetic memory by performing additional experiments. We adopted the published  Recombination Induced Tag Exchange system (RITE) to probe H3 turnover. Given the labile nature of heterochromatin at elevated temperatures , replication stalling was performed with hydroxyurea as published . We address turnover in the ∆2) An important assumption in this study is that cells carrying ORANGEON or ORANGEoff states contain comparable levels of heterochromatin at the nucleation site. However, it is possible that ORANGEON cells contain a relatively lower level of heterochromatin (but still sufficient to repress GREEN with a weak promoter) than ORANGEoff cells (e.g. in WT and ∆REIII cells). This should be tested experimentally by assaying H3K9me levels by ChIP at GREEN and ORANGE loci. Also, GREEN inserted in cenH is likely silenced post transcriptionally (transcripts of markers inserted near centromeric repeats are converted to siRNAs) and its silencing might not provide an accurate measure of silencing caused by heterochromatin modifications. If true, this would complicate the interpretation of the results.We thank the reviewer for the comment. We have now performed extensive Chromatin Immuno-precipitation (ChIP) analysis for the heterochromatin assembly mark H3K9me2 and repressive/spreading mark H3K9me3 across the MAT locus.Specifically, we find:cenH nucleation site (“green” in WT and ∆REIIIHSS), even though this strain, unlike WT, features a high proportion of “orange”ON . We also find very similar proportion of H3K9me2 and me3 at “green” in ∆KHSS-OFF, see the new data in Figure 2E. H3K9me2 and me3 marks accumulate to very high extents at these sites in the three strains, which is not consistent with heterochromatin spreading behaviors being solely the result of different level of heterochromatin assembly at nucleation regions.1) H3K9me2 and H3K9me3 marks accumulate to similar extents at the cenH only in the ∆REIIIHSS, but not wild type stain. This is evident even at the edge of cenH, and more strongly at the more distal “orange” . However, H3K9me2 and me3 do not significantly decline at “orange” in ∆KHSS-OFF versus “green” . We note that for H3K9me3 only there is a small, but not significant, difference between “green” and “orange”.2) H3K9me2 and me3 marks decline rightwards of Together, these data are consistent with differences in heterochromatin spreading, as opposed to weakened nucleation, driving the differences in distal “orange” repression, and further that our single cell silencing data for “orange” accurately reflects differences in the spreading of heterochromatin outwards from nucleation sites.S. pombe, heterochromatin assembled via noncoding RNA (ncRNA)* processes triggers co-transcriptional silencing as the reviewer states. However, transcriptional silencing also plays important roles  at these sites, and silencing broadly correlates well with H3K9me2 accumulation, a read-out of heterochromatin structure. Yet, it remains possible that placement within an RNAi generating element affects the behaviors we track. We believe the reviewer’s concern ought to be mitigated by a difference in the placement of “green” at ectopic loci versus the MAT locus. In ectopic strains, “green”  is placed immediately adjacent but not within the dh element, reporting on silencing at the nucleation locus, while for MAT strains “green” is within cenH. As we find the behavior with respect to spreading to be similar  we conclude that the nature of H3K9me- dependent silencing at the nucleation marker not to interfere with our interpretation.In cenH and dh rather than RNAi*We now use this more general descriptor for 3) This reviewer is concerned that the evidence that REIII causes deterministic spreading is an artifact of the system. Unlike WT or ∆REII cells that contain GREEN at a bona fide nucleation site, from which heterochromatin is known to spread outward, GREEN in ∆K cells is likely not placed at a nucleation site . In cells with GREEN located closer to the edge of the heterochromatin domain, the silencing of ORANGE located in the middle of the silenced domain might precede GREEN. In this situation, when silencing of ORANGE is assayed in GREENoff cells it would give the false impression of deterministic spreading. Consistent with this, markers inserted near mat3 are known to be silenced less efficiently. Similarly, if nucleation in ∆K cells occurred on the left side of ORANGE, all cells showing GREENoff will carry the ORANGEoff state. Can they confirm that heterochromatin in ∆K cells is indeed nucleated at REIII, or if REIII simply stabilizes heterochromatin ? Moreover, it is also possible that heterochromatin in ∆K cells is formed in such as a way that it does not involve stepwise mechanism of nucleation and spreading and that the entire domain is silenced simultaneously.We thank the reviewer for this comment.cenH, Atf1/Pcr1 binding sites at REIII are absolutely required for heterochromatin formation ). Independently of these studies, three lines of evidence from our work suggest that ΔKHSS reports specifically on REIII -nucleated spreading, detected by “green”, towards “orange”:Recent evidence suggests that in the absence of REIII blocks any silencing in ΔKHSS (in 34/34 strains examined) and H3K9 methylation at “green” or “orange”. This new data is presented in Figure 2—figure supplement 2A,B. Because ΔREIII does not abolish heterochromatin at MAT, i.e. it is not required to stabilize heterochromatin formed elsewhere , the above result points to REIII nucleating heterochromatin in ΔKHSS.1) Deletion of both Atf1/Pcr1 binding sites at REII, cenH and REIII have been identified at MAT. Technically, it possible that in ΔKHSS, which lacks cenH, silencing is due to REII. We had addressed this in the original submission by probing ΔKHSSREII::LEU2, in which the REII element is deleted. This strain behaved similarly to ΔKHSS , strongly suggesting that in ΔKHSS, REIII is the only functional nucleator.2) No repressive elements other than OFF. Rather, all cells within the size gate are plotted normalized by “red”. In those 2D plots, no significant de-repression of “green” is evident in ΔKHSS in the repressed population. We only use the “green”OFF gate for 1D histograms, to plot spreading in cells that fully nucleate. To address the point concerning spreading from leftwards of the “orange” nucleator, we moved “green” 2.1 kb towards the right IR-R boundary in ΔKHSS. This did not lead to the appearance of significant de-repression of “green” in “orange”OFF cells, which might be expected if spreading emanated from a site leftwards of “orange” in ΔKHSS. This new data is presented Figure 2—figure supplement 2 C,D.3) We should note that in our 2D histogram and scatter plots  cells are not gated for “green”REIII is indeed the nucleating element in ΔKHSS and that it spreads heterochromatin outward.Together, we believe these lines of evidence and previously published data affirms that ΔKHSS and we had discussed this possibility in the previous supplemental, and now moved it to the main discussion. Heterochromatin spreading can occur by a number of theoretical mechanisms , such as linear “oozing”, skipping or looping. Looping mechanisms have been invoked for the polycomb system . Looping is one possible mechanism to account for deterministic behavior of ΔK, while linear “oozing” is more consistent with our observations for ncRNA-nucleated spreading . In experiments outside the scope of this work, we look forward to testing this hypothesis.As to the comment about a non-stepwise mechanism, this is entirely possible for 4) The authors argue in the Abstract that the epigenetic capacity contributing to heterochromatin spreading and maintenance is not encoded in the spreading reaction itself, but rather requires specialized memory elements. This statement is misleading. The factors involved in spreading and maintenance of heterochromatin, such as HDACs, are known to be loaded across silenced domains by HP1, which is also required for heterochromatin spreading. However, these same factors (HDACs) can be also loaded by so called specialized/memory elements, further enhancing the stability of heterochromatin. In this respect, the epigenetic capacity is at least partially encoded in the spreading reaction and can be further strengthened by elements that also load activities, such as HDACs, promoting heterochromatin stability.We thank the reviewer for raising this point. We have modified the language in the Abstract to reflect that the ncRNA-nucleated spreading reaction specifically does not confer the epigenetic fidelity required for robust and precise epigenetic inheritance, which additionally requires memory elements.Reviewer #3: The fidelity and robustness are two essential characteristics to epigenetic inheritance, which is important both for chromatin structure and cell differentiation in many species. Its full establishment comprises of two stages: nucleation and spreading, which can be executed through RNA interference (RNAi)-dependent and RNAi-independent pathways. In this study, Greenstein and colleagues set up an elegant and sensitive single-cell reporter system to dissect these two characteristics. They have used it for population studies and cell-lineage tracking for trans-generation memory. Consistent with previous findings, they showed spreading of heterochromatin nucleated by RNAi alone is robust but low on fidelity, as compared to that nucleated by wild-type locus. Importantly, they have attributed the fidelity in heterochromatin spreading to an RNAi-independent DNA element REIII. Further, they have tested the persistence of "memory" as nucleated by RNAi or REIII with and without perturbations. The biological importance and complicated nature should make it appealing to a broad audience.We thank the reviewer for the favorable assessment of this work.In Figure 2B and 2C, where modified nucleators of RNAi-alone or REIII -alone are tested, the fidelity in spreading and robustness in nucleation segregated clearly in these population studies. However, one concern still remains. As spreading is greatly affected by the distance from the nucleator to the reporter, the actual distance for REIII -nucleated spreading should be measured between REIII and "orange" reporter, rather than between "green" and "orange" reporter as they are on different sides of the REIII nucleator, and to be consistent with Figure 2A and 2B. If the genome feature in Figure 2C is drawn to scale, it is around 1-2 kb in distance. At this distance, even RNAi-nucleated heterochromatin is demonstrating high fidelity in spreading as in the top panel of Figure 1C. At least one testing should be done with the reporter cassette moved further away from REIII nucleator by inserting "inert" DNA sequence (from a euchromatic region), to the similar 3-4 kb distance scale as Figure 2A and 2B. It is necessary to exclude the possibility that higher fidelity as in Figure 2C is due to a shorter loop for spreading. If the experiment is not possible, it shall be clearly explained in the text.REIII contains two potential nucleation sites (s1 and s2), only the distal (s2) sites has been implicated as the dominant nucleation element . The distance in HSSΔKfrom the end of the spreading sensor ORF to s1 is ~1kb, and to s2 ~2.2kb. In comparison, the comparable distance in ΔREIII HSS is ~3.8kb. To mitigate the concern that the spreading distance in ΔK may be much shorter than in ΔREIII , we deleted s1 in the ΔK strain (ΔKΔs1) and inserted two different ~700bp sections of the sib1 gene ORF between the spreading sensor and the s2 site in the ΔKΔs1 background, increasing the spreading distance to ~3kb. Importantly, in both of these strains, we see the same deterministic behavior as in the ΔK parent. One of the ΔKΔs1 +700bp insertions, representative of two behaviorally indistinguishable insertion strains we constructed, is shown in the new Figure 2—figure supplement 2E. Larger insertions where not compatible with heterochromatin nucleation in ΔK. Given these data, and the observation that significant stochastic spreading and intermediate states are detected for the 3kb, and even 1kb spreading distance with ectopically placed ncRNA nucleators , we conclude that the deterministic behavior is ΔK in intrinsic to nucleation from the REIII element.While [Editors’ note: the author responses to the re-review follow.]The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below: In the previous submission of this manuscript, the work presented was found to be potentially of great interest, particularly the discovery of a novel form of putative heterochromatin spreading, nucleated by the cis-acting element REIII that the authors claim has a deterministic role. However, direct evidence for REIII -mediated heterochromatin spreading was found to be lacking, and there was no mechanism. In their revised manuscript, the authors have provided several lines of new evidence to address some of the reviewers' comments. In particular, by deleting the REIII element, they show that this leads to increased turnover of histones which provides some insight into how REIII may be promoting epigenetic stability of heterochromatin. However, the conclusion that REIII promotes deterministic spreading of heterochromatin is still not really supported, as pointed out by reviewer 2. Furthermore, the argument that ∆K cells have better "memory" than WT cells is not supported by previous findings that ∆K cells contain metastable heterochromatin, with frequent conversion from the OFF to the ON state compared to WT. As both reviewers point out, it remains possible that bias may be introduced in the "memory" determinations, due to preselection for ∆KOFF colonies. Despite these limitations, and given the new work included in this manuscript on this REIII -based heterochromatin process, we would be willing to consider a revised text, that focused on the single cell assay showing that loss of REIII affects the stability of heterochromatin. In this revised manuscript, specific references to « memory elements » and « deterministic » spreading should be removed, as suggested by reviewer 2. Reviewer #2: The authors performed several experiments that addressed the reviewers' comments and strengthened their conclusions. For example, the extensive chromatin immunoprecipitation analyses performed to assay H3K9me2 and H3K9me3 levels in different strains now correlates changes in gene silencing with heterochromatin levels. Also, this reviewer suggested that they investigate the effects of REIII deletion on histone turnover, which has implications for heterochromatin spreading and epigenetic memory. They do indeed find that deletion of REIII causes increased turnover of histones. Together with previously published findings showing that REIII recruits HDAC Clr3 , which is involved in the suppression of histone turnover , this finding provides a possible mechanistic insight into how REIII promotes epigenetic stability of heterochromatin. With the new additions, this part of the story has improved significantly.We thank the reviewer for this positive assessment.However, their conclusion that REIII promotes deterministic spreading of heterochromatin remains ambiguous. In particular, unlike cenH that has been shown to nucleate heterochromatin, there is no clear evidence presented that heterochromatin spreads outward from the REIII element in ∆K cells. Rather, given the bistable nature of heterochromatin at mat in ∆K cells, the assembly of heterochromatin in ∆K cells cells might involve cooperativity between yet unknown elements, as opposed to reliance on the REIII element alone. In presenting their case for deterministic spreading from REIII, the authors mention previous work showing that loss of REIII affects heterochromatin in ∆K cells. However, it is important to note that previous studies  and this article only show that REIII is required for the stability of heterochromatin (most likely via recruitment of HDAC Clr3 that suppresses histone turnover to prevent loss of H3K9me) but is not sufficient to nucleate heterochromatin.REIII Atf1 and Pcr1 binding sites. Since there is no pre-existing H3K9me before introduction of clr4+ in our experiments, heterochromatin nucleation must occur in some fashion in ∆K cells. However, sufficiency of Atf1 and Pcr1 binding sites for nucleation remains to be demonstrated. Specifically, REIII, unlike cenH  cannot effect significant repression when placed ectopically in the genome . This result can be interpreted in at least two ways: REIII is not by itself sufficient to nucleate and requires other, yet to be defined elements, or, the local genomic context/or chromatin organization of REIII within MAT is required for its nucleation activity, hence it cannot function ectopically. Since it cannot be easily resolved which case is correct, we now soften the conclusion that REIII drives spreading in ∆K cells in the text and remove the term “deterministic spreading” in favor of REIII -dependent heterochromatin.The reviewer raises an important point. In ∆K cells, formation of H3K9me- marked heterochromatin is dependent on the action of OFF colonies , in which entire mat locus is known to be coated with heterochromatin .Similarly, their argument that ∆K cells have better "memory" than WT cells contrasts with a previous finding that ∆K cells contain metastable heterochromatin, as indicated by the frequent conversion from the OFF to the ON state at a rate many fold higher than in WT. Moreover, it remains possible that bias may be introduced in the "memory" determinations, due to preselection for ∆KWe thank the reviewer for this point. Here, we (1) Clarify our observations in the context of the literature, (2) Revise our interpretation of Figure 4D, and (3) Clarify our ∆K selection process.-4, . The continuous growth in non-selective media of OFF cells over > 25 generations resulted in the appearance 1-2% ON colonies, consistent with these low transitions . A key difference between our and some previous experiments is observing the entire population without selection (our assays) versus selection assays that permit survival of either the ON or OFF state. Thus, the appearance of very small ON populations may be less obvious in our experiments.Broadly, our results from ∆K cells are consistent with the published literature, namely a very low transition rate form OFF to ON per generation, about 10REIII to resistance and memory of MAT heterochromatin. The comparison of wild-type to ∆REIII cells here is central and valid in our view. However, we recognize that in Figure 4D, the results on its face point to stronger memory in ∆K cells than wild type. The “extreme” aspect of this memory is most likely due to the fact that ∆K cells, once ancestrally TSA treated, cannot efficiently re-nucleate. Given this caveat, we now refrain from interpreting Figure 4D as indicative of the degree of memory in ∆K. Overall, we did not intend to advance the notion that ∆K cells have more/stronger memory than wild-type and wherever this is ambiguous, have clarified the language. We note that unlike for ∆K cells, it is not possible to select for WT cells with active REIII, thus the population may contain some cells with REIII in an inactive state, which likely are more labile than the bulk of cells with active REIII. Hence, ∆KOFF populations can appear somewhat more stable as a result.The memory experiments in Figure 4 reveal the key contribution of OFF selection, our approach is consistent with the literature . We produce ∆KHSS strains in a ∆clr4 background and introduce clr4+ by cross. The population resulting from the germinated spore is often mixed, with both ON and OFF cells as shown in Figure 2E. Simply plating this population on nonselective media predominantly yields colonies that are ON or OFF. This reflects the metastable nature of ∆K cells, with some cells nucleating heterochromatin and some not, but then maintaining the state. In that sense, we do not isolate ∆KOFF cells using a directed selection process, promoting a given state, and do not feel that there should be any bias introduced by this method. Isolation of ON/OFF is required in some cases, for example, due to the insufficient number of OFF cells in the mixed population. However, we now qualify our results pertaining to memory clearly and state that bias may have been introduced by picking OFF colonies.As to ∆KREIII. Together with our reinterpretation of the ∆K result in Figure 4, we believe we addressed remaining concerns.In general, as suggested, we re-focused our conclusions on ∆My recommendation is to prepare a revised manuscript for publication in eLife that focuses on the results from their elegant single cell assays showing that the loss of REIII affects the stability of heterochromatin and that this behavior of REIII is mechanistically linked to the suppression of histone turnover at mat. The discussion would be focused on succinctly describing this feature of REIII in the context of previous findings, including the results showing that REIII recruits the HDAC Clr3  that also suppresses histone turnover to promote epigenetic stability of heterochromatin . Indeed, suppression of histone turnover by REIII /HDAC Clr3 may promote retention of methylated histones that are critical for the recruitment of Clr4; thus, promoting the efficient spreading of heterochromatin. Framing the results in this way would avoid the problematic issues raised by applying the terms "memory element" or "deterministic" spreading to REIII.REIII via suppression of histone turnover. We no longer use the terms “deterministic” or “memory element” with respect to REIII.We thank the reviewer for these suggestions. Indeed, we have re-written the manuscript, most prominently the Abstract and Discussion section, to focus on the overall result that ncRNA spreading is unstable and stabilized by Reviewer #3: The authors have addressed all the critiques raised by this reviewer. In particular, they have now provided more compelling evidence of "spreading" of heterochromatin from corresponding nucleation sites with a more complete testing cassette of different distances, directions, nucleation factors, etc. They have used these testing cassettes to demonstrate the stochasticity of ncRNA-mediated spreading and stability of REIII -mediated memory. In addition, they have now tested the anti-correlation of histone turnover and memory.We thank the reviewer for this positive assessment.


Transcription of eukaryotic protein-coding genes by RNA polymerase II (pol II) is highly regulated at initiation, elongation and termination. Transcription is also coordinated with co-transcriptional processing of the emerging pre-mRNA by capping, splicing, and cleavage and polyadenylation. Polyadenylation (poly(A)) site recognition, which defines the end of the mRNA, relies on the cleavage and polyadenylation (CPA) complex. It was previously observed that knocking-down proteins of the CPA complex affects not only recognition of the poly(A) site but also results in increased pausing of pol II at the beginning of genes. This finding suggests that the CPA complex plays a role in regulating pol II turnover after transcription initiation.To explore this possibility, we knocked-down a subunit of the cleavage factor I (CFIm), CFIm68, which is part of the CPA complex and involved in alternative polyadenylation, and performed pol II ChIP-seq in absence or presence of a transcription elongation inhibitor. In addition, we performed pol II ChIP-qPCR on a subset of protein coding genes after knocking down CFIm68. Transcription of a eukaryotic protein-coding gene by pol II requires several steps, including transcription initiation, elongation, and termination. During transcription, co-transcriptional processes such as mRNA capping, splicing, and cleavage and polyadenylation also occur and are required for the production of a mature mRNA. The end of a protein-coding gene is defined by one or more poly(A) sites and recognition of a poly(A) site is essential for the cleavage and polyadenylation of the mRNA , 2. ApprSome proteins of the CPA complex, including CstF64, CPSF73, and the CPA-associated termination factor Xrn2 have been shown to regulate pol II activity at the beginning and end of the transcription cycle , 12. To d-ribofuranoside  for 30 min  supplemented with 10% fetal bovine serum  and 100 units/ml penicillin + 100 µg/ml streptomycin (Gibco). The CFIm68KD cell lines, and its respective control HEK293 Flp, were previously described [t test, *p < 0.05, **p < 0.01, ***p < 0.001.ChIP was performed as previously described . BrieflyAdapters were trimmed with Cutadapt v. 1.9.1  with theThe knockdown of CFIm68 was not complete and may therefore may not be sufficient to completely abrogate the role of CFIm68 in pol II pausing and transcription regulation. The ChIP-seq were performed only once and in only one cell line; HEK293. We also performed pol II ChIP-qPCR on a limited number of protein-coding genes.Additional file 1. List of primers, List of primers used for the ChIP-qPCR presented in additional file.Additional file 2. Pol II ChIP-qPCR results, results of the pol II ChIP-qPCR on three genes in the HEK293 Flp-In and CFIm68KD cell lines.