G-quadruplex folding in Xist RNA antagonizes PRC2 activity for stepwise regulation of X chromosome inactivation.

How Polycomb repressive complex 2 (PRC2) is regulated by RNA remains an unsolved problem. Although PRC2 binds G-tracts with the potential to form RNA G-quadruplexes (rG4s), whether rG4s fold extensively in vivo and whether PRC2 binds folded or unfolded rG4 are unknown. Using the X-inactivation model in mouse embryonic stem cells, here we identify multiple folded rG4s in Xist RNA and demonstrate that PRC2 preferentially binds folded rG4s. High-affinity rG4 binding inhibits PRC2's histone methyltransferase activity, and stabilizing rG4 in vivo antagonizes H3 at lysine 27 (H3K27me3) enrichment on the inactive X chromosome. Surprisingly, mutagenizing the rG4 does not affect PRC2 recruitment but promotes its release and catalytic activation on chromatin. H3K27me3 marks are misplaced, however, and gene silencing is compromised. Xist-PRC2 complexes become entrapped in the S1 chromosome compartment, precluding the required translocation into the S2 compartment. Thus, Xist rG4 folding controls PRC2 activity, H3K27me3 enrichment, and the stepwise regulation of chromosome-wide gene silencing.

Genomic context- and H2AK119 ubiquitination-dependent inheritance of human Polycomb silencing.

Polycomb repressive complexes 1 and 2 (PRC1 and 2) are required for heritable repression of developmental genes. The cis- and trans-acting factors that contribute to epigenetic inheritance of mammalian Polycomb repression are not fully understood. Here, we show that, in human cells, ectopically induced Polycomb silencing at initially active developmental genes, but not near ubiquitously expressed housekeeping genes, is inherited for many cell divisions. Unexpectedly, silencing is heritable in cells with mutations in the H3K27me3 binding pocket of the Embryonic Ectoderm Development (EED) subunit of PRC2, which are known to disrupt H3K27me3 recognition and lead to loss of H3K27me3. This mode of inheritance is less stable and requires intact PRC2 and recognition of H2AK119ub1 by PRC1. Our findings suggest that maintenance of Polycomb silencing is sensitive to local genomic context and can be mediated by PRC1-dependent H2AK119ub1 and PRC2 independently of H3K27me3 recognition.

Unlocking adult T-cell leukemia/lymphoma's epigenetic secrets: delving into the mechanism and impact of EZH1/2 inhibition.

Epigenetic modifications, particularly through methylation of DNA packaging histones, play a pivotal role in controlling gene expression. Aberrant patterns of histone methylation have been associated with the development and progression of hematological malignancies. Unraveling the impact of aberrant histone marks on gene expression and leukemogenesis has spurred a concerted effort to develop clinically effective epigenetic therapies. In malignancies associated with the accumulation of histone H3 lysine trimethylation (H3K27me3), one such intervention involves preventing the deposition of this repressive histone mark by inhibiting the histone-modifying enzymes EZH1 and EZH2. While inhibition of EZH1/2 has demonstrated efficacy in both preclinical studies and clinical trials in various cancers, studies delineating the dynamic effect of EZH1/2 inhibition on H3K27me3 and disease relapse in clinical samples are lacking. In a recent publication, Yamagishi et al. explore how responses of a patient with adult T-cell leukemia/lymphoma to valemetostat, an EZH1/2 inhibitor, are associated with changes in H3K27me3, chromatin accessibility and gene expression, and how these changes can be circumvented in relapsed disease.

BRG1 programs PRC2-complex repression and controls oligodendrocyte differentiation and remyelination.

Chromatin-remodeling protein BRG1/SMARCA4 is pivotal for establishing oligodendrocyte (OL) lineage identity. However, its functions for oligodendrocyte-precursor cell (OPC) differentiation within the postnatal brain and during remyelination remain elusive. Here, we demonstrate that Brg1 loss profoundly impairs OPC differentiation in the brain with a comparatively lesser effect in the spinal cord. Moreover, BRG1 is critical for OPC remyelination after injury. Integrative transcriptomic/genomic profiling reveals that BRG1 exhibits a dual role by promoting OPC differentiation networks while repressing OL-inhibitory cues and proneuronal programs. Furthermore, we find that BRG1 interacts with EED/PRC2 polycomb-repressive-complexes to enhance H3K27me3-mediated repression at gene loci associated with OL-differentiation inhibition and neurogenesis. Notably, BRG1 depletion decreases H3K27me3 deposition, leading to the upregulation of BMP/WNT signaling and proneurogenic genes, which suppresses OL programs. Thus, our findings reveal a hitherto unexplored spatiotemporal-specific role of BRG1 for OPC differentiation in the developing CNS and underscore a new insight into BRG1/PRC2-mediated epigenetic regulation that promotes and safeguards OL lineage commitment and differentiation.

Discovery of a polymorphic gene fusion via bottom-up chimeric RNA prediction.

Gene fusions and their chimeric products are commonly linked with cancer. However, recent studies have found chimeric transcripts in non-cancer tissues and cell lines. Large-scale efforts to annotate structural variations have identified gene fusions capable of generating chimeric transcripts even in normal tissues. In this study, we present a bottom-up approach targeting population-specific chimeric RNAs, identifying 58 such instances in the GTEx cohort, including notable cases such as SUZ12P1-CRLF3, TFG-ADGRG7 and TRPM4-PPFIA3, which possess distinct patterns across different ancestry groups. We provide direct evidence for an additional 29 polymorphic chimeric RNAs with associated structural variants, revealing 13 novel rare structural variants. Additionally, we utilize the All of Us dataset and a large cohort of clinical samples to characterize the association of the SUZ12P1-CRLF3-causing variant with patient phenotypes. Our study showcases SUZ12P1-CRLF3 as a representative example, illustrating the identification of elusive structural variants by focusing on those producing population-specific fusion transcripts.

EZH2 mutations in follicular lymphoma distort H3K27me3 profiles and alter transcriptional responses to PRC2 inhibition.

Mutations in chromatin regulators are widespread in cancer. Among them, the histone H3 lysine 27 methyltransferase Polycomb Repressive Complex 2 (PRC2) shows distinct alterations according to tumor type. This specificity is poorly understood. Here, we model several PRC2 alterations in one isogenic system to reveal their comparative effects. Focusing then on lymphoma-associated EZH2 mutations, we show that Ezh2Y641F induces aberrant H3K27 methylation patterns even without wild-type Ezh2, which are alleviated by partial PRC2 inhibition. Remarkably, Ezh2Y641F rewires the response to PRC2 inhibition, leading to induction of antigen presentation genes. Using a unique longitudinal follicular lymphoma cohort, we further link EZH2 status to abnormal H3K27 methylation. We also uncover unexpected variability in the mutational landscape of successive biopsies, pointing to frequent co-existence of different clones and cautioning against stratifying patients based on single sampling. Our results clarify how oncogenic PRC2 mutations disrupt chromatin and transcription, and the therapeutic vulnerabilities this creates.

Isothiocyanates Potentiate Tazemetostat-Induced Apoptosis by Modulating the Expression of Apoptotic Genes, Members of Polycomb Repressive Complex 2, and Levels of Tri-Methylating Lysine 27 at Histone 3 in Human Malignant Melanoma Cells.

In this study, we utilized an in vitro model consisting of human malignant melanoma as well as non-tumorigenic immortalized keratinocyte cells with the aim of characterizing the therapeutic effectiveness of the clinical epigenetic drug Tazemetostat alone or in combination with various isothiocyanates. In doing so, we assessed markers of cell viability, apoptotic induction, and expression levels of key proteins capable of mediating the therapeutic response. Our data indicated, for the first time, that Tazemetostat caused a significant decrease in viability levels of malignant melanoma cells in a dose- and time-dependent manner via the induction of apoptosis, while non-malignant keratinocytes were more resistant. Moreover, combinatorial treatment protocols caused a further decrease in cell viability, together with higher apoptotic rates. In addition, a significant reduction in the Polycomb Repressive Complex 2 (PRC2) members [e.g., Enhancer of Zeste Homologue 2 (EZH2), Embryonic Ectoderm Development (EED), and suppressor of zeste 12 (SUZ12)] and tri-methylating lysine 27 at Histone 3 (H3K27me3) protein expression levels was observed, at least partially, under specific combinatorial exposure conditions. Reactivation of major apoptotic gene targets was determined at much higher levels in combinatorial treatment protocols than Tazemetostat alone, known to be involved in the induction of intrinsic and extrinsic apoptosis. Overall, we developed an optimized experimental therapeutic platform aiming to ensure the therapeutic effectiveness of Tazemetostat in malignant melanoma while at the same time minimizing toxicity against neighboring non-tumorigenic keratinocyte cells.

Clinical Significance of Upregulation of EZH1 Expression in Hepatocellular Carcinoma Tissues.

BACKGROUND AND AIMS: The incidence and mortality of hepatocellular carcinoma (HCC) are increasing. It is urgent to develop more effective HCC biomarkers for diagnosis and treatment. This project intends to verify the expression of enhancer of zeste 1 polycomb repressive complex 2 subunit (EZH1) and its mechanism in HCC. METHODS: This study integrates global microarray and high-throughput sequencing datasets, combined with internal immunohistochemistry, to analyze the expression and prognostic value of EZH1 in HCC. Functional enrichment analysis was conducted to investigate transcriptional targets, which were achieved by intersecting HCC over-expressed genes, EZH1 co-expressed genes and putative transcriptional targets. The relationship between EZH1 and anticancer drugs was detected by drug sensitivity analysis. RESULTS: In this study, 84 datasets from 40 platforms (3,926 HCC samples and 3,428 non-cancerous liver tissues) were included to show the high expression of EZH1 in HCC. Immunohistochemistry with 159 HCC samples and 62 non-HCC samples confirmed the high expression level. HCC patients with high EZH1 expression had worse survival prognoses. Gene ontology and Reactome analysis revealed that metabolism-related pathways, including autophagy, are critical for HCC. Interestingly, as one of the EZH1 potential transcriptional targets, autophagy-related 7 (ATG7) appeared in the above pathways. ATG7 was positively correlated with EZH1, upregulated in HCC, and mediated poor prognosis. Upregulation of EZH1 was found to be in contact with HCC anti-tumor drug resistance. CONCLUSIONS: The upregulation of EZH1 expression can promote the occurrence of HCC and lead to poor clinical progression and drug resistance; these effects may be mediated by regulating ATG7.

Polycomb-mediated silencing of miR-8 is required for maintenance of intestinal stemness in Drosophila melanogaster.

Balancing maintenance of self-renewal and differentiation is a key property of adult stem cells. The epigenetic mechanisms controlling this balance remain largely unknown. Herein, we report that the Polycomb Repressive Complex 2 (PRC2) is required for maintenance of the intestinal stem cell (ISC) pool in the adult female Drosophila melanogaster. We show that loss of PRC2 activity in ISCs by RNAi-mediated knockdown or genetic ablation of the enzymatic subunit Enhancer of zeste, E(z), results in loss of stemness and precocious differentiation of enteroblasts to enterocytes. Mechanistically, we have identified the microRNA miR-8 as a critical target of E(z)/PRC2-mediated tri-methylation of histone H3 at Lys27 (H3K27me3) and uncovered a dynamic relationship between E(z), miR-8 and Notch signaling in controlling stemness versus differentiation of ISCs. Collectively, these findings uncover a hitherto unrecognized epigenetic layer in the regulation of stem cell specification that safeguards intestinal homeostasis.

Sustained activation of NF-κB through constitutively active IKKß leads to senescence bypass in murine dermal fibroblasts.

Although the transcription factor nuclear factor κB (NF-κB) plays a central role in the regulation of senescence-associated secretory phenotype (SASP) acquisition, our understanding of the involvement of NF-κB in the induction of cellular senescence is limited. Here, we show that activation of the canonical NF-κB pathway suppresses senescence in murine dermal fibroblasts. IκB kinase ß (IKKß)-depleted dermal fibroblasts showed ineffective NF-κB activation and underwent senescence more rapidly than control cells when cultured under 20% oxygen conditions, as indicated by senescence-associated ß-galactosidase (SA-ß-gal) staining and p16INK4a mRNA levels. Conversely, the expression of constitutively active IKKß (IKKß-CA) was sufficient to drive senescence bypass. Notably, the expression of a degradation-resistant form of inhibitor of κB (IκB), which inhibits NF-κB nuclear translocation, abolished senescence bypass, suggesting that the inhibitory effect of IKKß-CA on senescence is largely mediated by NF-κB. We also found that IKKß-CA expression suppressed the derepression of INK4/Arf genes and counteracted the senescence-associated loss of Ezh2, a catalytic subunit of the Polycomb repressive complex 2 (PRC2). Moreover, pharmacological inhibition of Ezh2 abolished IKKß-CA-induced senescence bypass. We propose that NF-κB plays a suppressive role in the induction of stress-induced senescence through sustaining Ezh2 expression.

Characterization of the Pristionchus pacificus "epigenetic toolkit" reveals the evolutionary loss of the histone methyltransferase complex PRC2.

Comparative approaches have revealed both divergent and convergent paths to achieving shared developmental outcomes. Thus, only through assembling multiple case studies can we understand biological principles. Yet, despite appreciating the conservation-or lack thereof-of developmental networks, the conservation of epigenetic mechanisms regulating these networks is poorly understood. The nematode Pristionchus pacificus has emerged as a model system of plasticity and epigenetic regulation as it exhibits a bacterivorous or omnivorous morph depending on its environment. Here, we determined the "epigenetic toolkit" available to P. pacificus as a resource for future functional work on plasticity, and as a comparison with Caenorhabditis elegans to investigate the conservation of epigenetic mechanisms. Broadly, we observed a similar cast of genes with putative epigenetic function between C. elegans and P. pacificus. However, we also found striking differences. Most notably, the histone methyltransferase complex PRC2 appears to be missing in P. pacificus. We described the deletion/pseudogenization of the PRC2 genes mes-2 and mes-6 and concluded that both were lost in the last common ancestor of P. pacificus and a related species P. arcanus. Interestingly, we observed the enzymatic product of PRC2 (H3K27me3) by mass spectrometry and immunofluorescence, suggesting that a currently unknown methyltransferase has been co-opted for heterochromatin silencing. Altogether, we have provided an inventory of epigenetic genes in P. pacificus to compare with C. elegans. This inventory will enable reverse-genetic experiments related to plasticity and has revealed the first loss of PRC2 in a multicellular organism.

Alternative splicing decouples local from global PRC2 activity.

The Polycomb repressive complex 2 (PRC2) mediates epigenetic maintenance of gene silencing in eukaryotes via methylation of histone H3 at lysine 27 (H3K27). Accessory factors define two distinct subtypes, PRC2.1 and PRC2.2, with different actions and chromatin-targeting mechanisms. The mechanisms orchestrating PRC2 assembly are not fully understood. Here, we report that alternative splicing (AS) of PRC2 core component SUZ12 generates an uncharacterized isoform SUZ12-S, which co-exists with the canonical SUZ12-L isoform in virtually all tissues and developmental stages. SUZ12-S drives PRC2.1 formation and favors PRC2 dimerization. While SUZ12-S is necessary and sufficient for the repression of target genes via promoter-proximal H3K27me3 deposition, SUZ12-L maintains global H3K27 methylation levels. Mouse embryonic stem cells (ESCs) lacking either isoform exit pluripotency more slowly and fail to acquire neuronal cell identity. Our findings reveal a physiological mechanism regulating PRC2 assembly and higher-order interactions in eutherians, with impacts on H3K27 methylation and gene repression.

Mechanisms of action and resistance in histone methylation-targeted therapy.

Epigenomes enable the rectification of disordered cancer gene expression, thereby providing new targets for pharmacological interventions. The clinical utility of targeting histone H3 lysine trimethylation (H3K27me3) as an epigenetic hallmark has been demonstrated1-7. However, in actual therapeutic settings, the mechanism by which H3K27me3-targeting therapies exert their effects and the response of tumour cells remain unclear. Here we show the potency and mechanisms of action and resistance of the EZH1-EZH2 dual inhibitor valemetostat in clinical trials of patients with adult T cell leukaemia/lymphoma. Administration of valemetostat reduced tumour size and demonstrated durable clinical response in aggressive lymphomas with multiple genetic mutations. Integrative single-cell analyses showed that valemetostat abolishes the highly condensed chromatin structure formed by the plastic H3K27me3 and neutralizes multiple gene loci, including tumour suppressor genes. Nevertheless, subsequent long-term treatment encounters the emergence of resistant clones with reconstructed aggregate chromatin that closely resemble the pre-dose state. Acquired mutations at the PRC2-compound interface result in the propagation of clones with increased H3K27me3 expression. In patients free of PRC2 mutations, TET2 mutation or elevated DNMT3A expression causes similar chromatin recondensation through de novo DNA methylation in the H3K27me3-associated regions. We identified subpopulations with distinct metabolic and gene translation characteristics implicated in primary susceptibility until the acquisition of the heritable (epi)mutations. Targeting epigenetic drivers and chromatin homeostasis may provide opportunities for further sustained epigenetic cancer therapies.

The competitive mechanism of EZH1 and EZH2 in promoting oral squamous cell carcinoma.

Enhancer of Zeste Homolog 1 (EZH1) and Enhancer of Zeste Homolog 2 (EZH2) are the key components of polycomb repressive complex 2 (PRC2); however, the roles of these proteins in oral squamous cell carcinoma (OSCC) have yet to be elucidated. In this study, we aimed to determine the respective roles of these proteins in OSCC by investigating the expression levels of EZH1 and EZH2 in OSCC tissues (N = 63) by immunohistochemistry. In addition, we used lentiviruses to construct stable OSCC cell lines that overexpressed EZH1 and EZH2. Then, we investigated these cell lines for cell viability, colony formation capacity, stemness, and epithelial-mesenchymal transition (EMT). Binding competition between EZH1 and EZH2 with PRC2 was further evaluated using Co-immunoprecipitation (Co-IP). Compared with normal tissues, the expression levels of EZH2 in OSCC tissues was up-regulated, while the expression of EZH1 was down-regulated. EZH2 enhanced cell viability, colony formation capacity, stemness, and EMT, while EZH1 did not. Furthermore, analysis indicated that EZH1 and EZH2 bound competitively to PRC2 and influenced the methylation status of H3K27. In conclusion, our findings verified that EZH1 and EZH2 play opposing roles in OSCC and that EZH1 and EZH2 compete as the key component of PRC2, thus affecting the characteristics of OSCC via the methylation of H3K27.

A methylation-phosphorylation switch controls EZH2 stability and hematopoiesis.

The Polycomb Repressive Complex 2 (PRC2) methylates H3K27 to regulate development and cell fate by transcriptional silencing. Alteration of PRC2 is associated with various cancers. Here, we show that mouse Kdm1a deletion causes a dramatic reduction of PRC2 proteins, whereas mouse null mutation of L3mbtl3 or Dcaf5 results in PRC2 accumulation and increased H3K27 trimethylation. The catalytic subunit of PRC2, EZH2, is methylated at lysine 20 (K20), promoting EZH2 proteolysis by L3MBTL3 and the CLR4DCAF5 ubiquitin ligase. KDM1A (LSD1) demethylates the methylated K20 to stabilize EZH2. K20 methylation is inhibited by AKT-mediated phosphorylation of serine 21 in EZH2. Mouse Ezh2K20R/K20R mutants develop hepatosplenomegaly associated with high GFI1B expression, and Ezh2K20R/K20R mutant bone marrows expand hematopoietic stem cells and downstream hematopoietic populations. Our studies reveal that EZH2 is regulated by methylation-dependent proteolysis, which is negatively controlled by AKT-mediated S21 phosphorylation to establish a methylation-phosphorylation switch to regulate the PRC2 activity and hematopoiesis.

Roles of HOTAIR Long Non-coding RNA in Gliomas and Other CNS Disorders.

HOX transcript antisense intergenic RNA (HOTAIR) is a long non-coding RNA (lncRNA) which is increasingly being perceived as a tremendous molecular mediator of brain pathophysiology at multiple levels. Epigenetic regulation of target gene expression carried out by HOTAIR is thorough modulation of chromatin modifiers; histone methyltransferase polycomb repressive complex 2 (PRC2) and histone demethylase lysine-specific demethylase 1 (LSD1). Incidentally, HOTAIR was the first lncRNA shown to elicit sponging of specific microRNA (miRNA or miR) species in a trans-acting manner. It has been extensively studied in various cancers, including gliomas and is regarded as a prominent pro-tumorigenic and pro-oncogenic lncRNA. Indeed, the expression of HOTAIR may serve as glioma grade predictor and prognostic biomarker. The objective of this timely review is not only to outline the multifaceted pathogenic roles of HOTAIR in the development and pathophysiology of gliomas and brain cancers, but also to delineate the research findings implicating it as a critical regulator of overall brain pathophysiology. While the major focus is on neuro-oncology, wherein HOTAIR represents a particularly potent underlying pathogenic player and a suitable therapeutic target, mechanisms underlying the regulatory actions of HOTAIR in neurodegeneration, traumatic, hypoxic and ischemic brain injuries, and neuropsychiatric disorders are also presented.

Denaturing purifications demonstrate that PRC2 and other widely reported chromatin proteins do not appear to bind directly to RNA in vivo.

Polycomb repressive complex 2 (PRC2) is reported to bind to many RNAs and has become a central player in reports of how long non-coding RNAs (lncRNAs) regulate gene expression. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here, we revisit the evidence supporting RNA binding by PRC2 and show that many reported interactions may not occur in vivo. Using denaturing purification of in vivo crosslinked RNA-protein complexes in human and mouse cell lines, we observe a loss of detectable RNA binding to PRC2 and chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, and others), despite accurately mapping bona fide RNA-binding sites across others (SPEN, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.

The apparent loss of PRC2 chromatin occupancy as an artifact of RNA depletion.

RNA has been implicated in the recruitment of chromatin modifiers, and previous studies have provided evidence in favor and against this idea. RNase treatment of chromatin is commonly used to study RNA-mediated regulation of chromatin modifiers, but the limitations of this approach remain unclear. RNase A treatment during chromatin immunoprecipitation (ChIP) reduces chromatin occupancy of the H3K27me3 methyltransferase Polycomb repressive complex 2 (PRC2). This led to suggestions of an "RNA bridge" between PRC2 and chromatin. Here, we show that RNase A treatment during ChIP causes the apparent loss of all facultative heterochromatin, including both PRC2 and H3K27me3 genome-wide. We track this observation to a gain of DNA from non-targeted chromatin, sequenced at the expense of DNA from facultative heterochromatin, which reduces ChIP signals. Our results emphasize substantial limitations in using RNase A treatment for mapping RNA-dependent chromatin occupancy and invalidate conclusions that were previously established for PRC2 based on this assay.

BMP-ACVR1 Axis is Critical for Efficacy of PRC2 Inhibitors in B-Cell Lymphoma.

EZH2 is the catalytic subunit of the histone methyltransferase Polycomb Repressive Complex 2 (PRC2), and its somatic activating mutations drive lymphoma, particularly the germinal center B-cell type. Although PRC2 inhibitors, such as tazemetostat, have demonstrated anti-lymphoma activity in patients, the clinical efficacy is not limited to EZH2-mutant lymphoma. In this study, Activin A Receptor Type 1 (ACVR1), a type I Bone Morphogenetic Protein (BMP) receptor, is identified as critical for the anti-lymphoma efficacy of PRC2 inhibitors through a whole-genome CRISPR screen. BMP6, BMP7, and ACVR1 are repressed by PRC2-mediated H3K27me3, and PRC2 inhibition upregulates their expression and signaling in cell and patient-derived xenograft models. Through BMP-ACVR1 signaling, PRC2 inhibitors robustly induced cell cycle arrest and B cell lineage differentiation in vivo. Remarkably, blocking ACVR1 signaling using an inhibitor or genetic depletion significantly compromised the in vitro and in vivo efficacy of PRC2 inhibitors. Furthermore, high levels of BMP6 and BMP7, along with ACVR1, are associated with longer survival in lymphoma patients, underscoring the clinical relevance of this study. Altogether, BMP-ACVR1 exhibits anti-lymphoma function and represents a critical PRC2-repressed pathway contributing to the efficacy of PRC2 inhibitors.

COOLAIR and PRC2 function in parallel to silence FLC during vernalization.

Noncoding transcription induces chromatin changes that can mediate environmental responsiveness, but the causes and consequences of these mechanisms are still unclear. Here, we investigate how antisense transcription (termed COOLAIR) interfaces with Polycomb Repressive Complex 2 (PRC2) silencing during winter-induced epigenetic regulation of Arabidopsis FLOWERING LOCUS C (FLC). We use genetic and chromatin analyses on lines ineffective or hyperactive for the antisense pathway in combination with computational modeling to define the mechanisms underlying FLC repression. Our results show that FLC is silenced through pathways that function with different dynamics: a COOLAIR transcription-mediated pathway capable of fast response and in parallel a slow PRC2 switching mechanism that maintains each allele in an epigenetically silenced state. Components of both the COOLAIR and PRC2 pathways are regulated by a common transcriptional regulator (NTL8), which accumulates by reduced dilution due to slow growth at low temperature. The parallel activities of the regulatory steps, and their control by temperature-dependent growth dynamics, create a flexible system for registering widely fluctuating natural temperature conditions that change year on year, and yet ensure robust epigenetic silencing of FLC.