Complex interaction between genetics, epigenetics, environment, and nutrition affect the physiological activities of adipose tissues and their dysfunctions, which lead to several metabolic diseases including obesity or type 2 diabetes. Here, adipogenesis appears to be a process characterized by an intricate network that involves many transcription factors and long noncoding RNAs (lncRNAs) that regulate gene expression. LncRNAs are being investigated to determine their contribution to adipose tissue development and function. LncRNAs possess multiple cellular functions, and they regulate chromatin remodeling, along with transcriptional and post-transcriptional events; in this way, they affect gene expression. New investigations have demonstrated the pivotal role of these molecules in modulating white and brown/beige adipogenic tissue development and activity. This review aims to provide an update on the role of lncRNAs in adipogenesis and adipose tissue function to promote identification of new drug targets for treating obesity and related metabolic diseases. In most cases, it is caused by an unhealthy lifestyle: food and drink intakes that exceed what is needed for metabolic and physical functions, exacerbated by lack of physical activity and sedentary behavior. Genetic and epigenetic factors can also contribute to obesity. Extra energy is stored in extra fat depots. The term Adipose tissue is no longer considered a static tissue that stores excess energy in fat depots: it is considered a real, metabolically-active endocrine organ that is able to produce several hormones and signaling molecules (adipokines) that play a key role in regulating energy homoeostasis, satiety, blood pressure, and angiogenesis. In humans and other mammals, adipocytes originate in stem cells, which are present in mesenchymal stromal cells (MSCs). Human MSCs are heterogeneous and include: i) multipotent stem cells that can differentiate into osteocytes, chondrocytes, and adipocytes; ii) progenitor cells; iii) stromal cells; and iv) fibroblasts. MSCs were first isolated from bone marrow and then from other tissues with a stromal component, including adipose tissue, endometrium, Wharton’s jelly, dental pulp, and the umbilical cord .Anatomically and physiologically, adipose tissue can be classified into brown adipose tissue (BAT) and white adipose tissue (WAT), with each having different functions. BAT specializes in spreading energy by producing heat. Brown adipocytes present in BAT are characterized by a high number of mitochondria, and they generate heat via the UCP1 protein (uncoupling protein 1) . BAT depWAT occurs due to a low presence of blood vessels and appears as a yellowish tissue. White adipocytes present a single lipid droplet containing triglycerides and vary in the number of mitochondria. In addition to the brown adipocytes present in BAT, human WAT contains adipocytes that possess the ability to transdifferentiate into ‘brown-like’ cells following thermogenic stimuli or pharmacological treatments. This process is called WAT ‘browning’, and these brown adipocytes are known as ‘inducible beige/brite’ (brown-white). Brite adipocytes express UCP1 and may potentially become heat generators that dissipate energy .In terms of body distribution, WAT can be classified into subcutaneous adipose tissue (sWAT)—located under the skin—and visceral adipose tissue (vWAT)—found near the primary organs within the abdominal cavity. Thus, abdominal obesity reflects vWAT growth, which is associated with an increased risk of obesity-related disorders such as insulin resistance, cardiovascular diseases, and hyperlipidemia .Several studies have demonstrated that, in addition the well-known high-caloric intake, genetic and epigenetic factors play a role in obesity . Indeed,Large-scale microarray and comparative genomics analyses suggest that the transcriptome—the collection of all RNA molecules present in one cell—is substantially greater than previously hypothesized . Only a In general, ncRNAs are grouped into two categories, based on transcript length. RNAs shorter than 200 nucleotides are identified as sncRNAs, such as snRNAs, snoRNAs, miRNAs, and piRNAs. LncRNAs represent the other category and include RNA molecules with an average length longer than 200 nucleotides . These RThe functions of lncRNAs are largely dependent on their subcellular localization. LncRNAs are primarily localized in the nucleus and are involved in the regulation of gene expression .In general, nuclear-retained lncRNAs modulate gene expression by acting either close (‘in cis’) or far (‘in trans’) from transcription sites. Several clues suggest that lncRNAs are transcribed from intergenic regions by RNA polymerase II. Indeed, lncRNAs show mRNA features: they are 5’-end capped, spliced, and polyadenylated .Are lncRNAs really noncoding molecules? Very recent studies have indicated the existence of small polypeptides (less than 100 amino acids in length) that, in some cases, can be encoded by lncRNAs . These pEven though the role of many lncRNAs is uncertain, an increasing amount of research is trying to characterize their potential function as transcriptional regulators involved in human diseases, development, and differentiation .Cytoplasmic lncRNAs are less investigated, but new studies have shown that they can form complexes with other biomolecules with structural and regulatory functions. In particular, cytoplasmic lncRNAs can modulate mRNA stability, translation, and protein phosphorylation. Some cytoplasmic lncRNAs hybridize with miRNAs and avoid their binding with mRNA targets (lncRNA sponge activity) .LncRNAs engage in multiple cellular functions and regulate nuclear organization by acting as chromatin remodelers, thus affecting gene expression . Gene exwww.cuilab.cn/lncrnadisease.Many studies have demonstrated that lncRNAs are involved in several physiological and pathological phenomena, including proliferation, apoptosis, and DNA repair and response. A curated list of experimentally-supported associations between lncRNAs and diseases can be found at In this review, attention is focused on the role of lncRNAs in adipogenesis and the regulation of adipose tissue function . FurtherDuring the transcription phase, chromatin remodeling plays a crucial role in generating an open status (euchromatin), which is accessible to polymerases and transcriptional factors. Chromatin status can be modified by chromatin remodeling enzymes, which fall into two classes: ATP-dependent chromatin remodeling complexes and covalent histone modifiers  Panel A.ATP-dependent chromatin remodeling complexes  disrupt histone-DNA interactions and increase nucleosome mobility along chromatin fibers. They may act either as co-activators or co-repressors, depending on the molecular context  .Histone covalent modifiers are enzymes that perform stable chemical modifications on histones. Changes in acetylation status of histones and/or methylation in specific amino acids (primarily lysine) can activate or deactivate the transcription process. Histone deacetylases (HDACs) and histone acetyltransferases (HATs), with their opposing activities, determine the pattern of histone acetylation. HDACs delete the acetyl groups from lysine residues on histone tails, which may play a critical role in gene repression. In contrast, HATs add an acetyl group on the same amino acid residues, promoting chromatin de-condensation and thus activating transcription .Several histone methyltransferases and histone demethylases have been identified. These enzymes can influence transcription by acting as either co-activators or co-repressors, depending on the amino acid’s position. For example, SET1 methylates lysine in position 4 of histone H3 and activates transcription, while its corresponding demethylase LSD represses transcription. In contrast, SUV39H1/2 methylates lysine in position 9 of histone H3 and represses transcription, while its corresponding demethylase JMJD activates transcription .Other enzymes covalently modify nucleotides and contribute to chromatin remodeling. In the mammalian genome, one epigenetic mechanism that influences gene expression is associated with the specific DNA sequences called CpG islands. These are regions with a high frequency of CpG dinucleotides . These sIn this context, lncRNAs support gene regulation at the epigenetic level, acting with enzymes that modify histones and/or DNA, or with ATP-dependent chromatin remodeling complexes .In detail, some studies have demonstrated a functional interaction between DNMTs and lncRNAs  . For exaDifferent lncRNAs are involved in controlling the preliminary transcription stages  Panel B.LncRNAs can also regulate the elongation phase of the transcription process. LncRNAs, transcribed by RNA Pol II, interact with elongation factors, such as P-TEFb (Positive Transcription Elongation Factor b) .The primary transcript of RNA polymerase II in eukaryotic cells undergoes several modifications during the transcriptional process, including 5’-capping, 3’-polyadenylation, and splicing. These events lead to mature mRNA, which is transported from the nucleus into the cytoplasm. LncRNAs are involved in several steps of mRNA maturation.For example, MALAT1 modulates alternative splicing by interacting with splicing factors (SFs) such as SRSF1s, a conserved family of serine/arginine-rich (SR) proteins involved in RNA splicing regulation that is concentration- and phosphorylation-dependent .LncRNAs like ZEB2/SIP1 NAT can bind splicing sites, suppressing them and resulting in intron retention. In the retained intron, the translation apparatus recognizes and binds an internal ribosome entry site (IRES), resulting in a more efficient ZEB2 protein translation .Other functions of lncRNAs have been described by Zhang and colleagues: they have shown a possible role in regulating protein stability through the block of the polyubiquitination that symbolizes the ‘kiss of death’ signal for protein turnover .Finally, lncRNAs can act as sponges, able to sequester miRNAs and thus avoid their binding with specific mRNA targets. In this way, mRNAs show greater stability and higher protein expression. An example is provided by the GUARDIN lncRNA. This molecule controls the expression of telomeric repeat binding factor 2 (TRF2), which is involved in the genomic integrity process under exogenous genotoxic stress. GUARDIN sequesters miR-23a, which has a specific target on TRF2 mRNA GUARDIN, and preserves TRF2 stability . LncRNAsThe lncRNAs have pleiotropic activities: a single lncRNA may regulate different biological functions depending on cellular context. The tissue specific expression of a given lncRNA it is not a pre-requisite for its peculiar activity, rather it depends on target molecules that are present in a given cell in a specific moment. Several lncRNAs, that are describe in the following paragraphs, are not exclusively expressed in adipose tissues, nevertheless they regulate adipose tissues’ specific functions. Brite or beige adipocytes appear in WAT following thermogenic stimuli . They arRecently, several studies have addressed the role of lncRNAs in the network governing adipogenesis and adipocyte biology. Some lncRNAs play a key role in these phenomena by interacting with different transcription factors, such as PGC-1alpha, EBF2, ZBTB7B, ZFP516, and PRDM16 .Alvarez and colleagues used RNA-seq to identify 1500 lncRNAs expressed in brown, inguinal white, and epididymal white fat in mice. A subgroup of 127 lncRNAs was exclusively expressed in BAT; most of these targeted key regulators of adipogenesis, including PPAR-gamma, C/EBP-alpha, and C/EBP-beta . In thisEven though the molecular mechanism remains largely unknown, Blnc1 localizes in the nucleus and stimulates thermogenic gene expression via the Blnc1/hnRNP-U/EBF2 ribonucleoprotein complex. EBF2 (early B-cell factor 2) is a helix-loop-helix member of the EBF transcription factor family, which controls thermogenic brown and beige adipogenesis. Specifically, the interaction of Blnc1 with EBF2 promotes the increase of EBF2 transcriptional activity on the Ucp1 promoter. EBF2 controls PPAR-gamma binding activity on gene promoters to govern brown versus white adipocyte differentiation . Blnc1 iDuring adipogenic differentiation, a feedforward positive regulatory loop is generated: EBF2 is recruited onto the Blnc1 promoter and stimulates its transcription. Subsequently, a heterogeneous nuclear ribonucleoprotein (hnRNP-U) simulates a platform—used for the assembly of a molecular complex—that promotes the aggregation of transcribed Blnc1 and EBF2. This event induces the release of EBF2 from the Blnc1 promoter and the silencing of its transcription. The feedforward loop is relevant to the expression of genes regulating thermogenesis in brown adipogenesis.Li and colleagues recently identified a transcriptional factor, named ZBTB7B (zinc finger and BTB domain-containing 7b), that promotes differentiation into brown and beige adipocytes and thermogenesis . ZBTB7B Functional studies have highlighted that one more lncRNA is necessary for BAT induction, maintenance, and thermogenic activity. This RNA—lnc-BATE1—acts ‘in trans’ and is essential to brown adipogenesis; moreover, it also suppresses the white adipocyte selective gene program . InteresXiong and colleagues have identified a brown adipocyte-enriched lncRNA they named AK079912. AK079912 expression is upregulated during brown adipocyte differentiation and following cold-stimulated browning of white adipocytes; accordingly, AK079912 silencing blocks the brown adipocyte differentiation. The action of this lncRNA has not been elucidated. Still, AK079912 contains an ORF that could be translated into a small peptide. This observation adds further complexity to the lncRNA mechanism of action, suggesting that a lncRNA may act either as noncoding RNA or as coding RNA. It is also possible that a region of an annotated lncRNA could give rise to a peptide and the rest of the molecule might function as a lncRNA .LncBATE10 is another lncRNA involved in brown adipogenesis . LncBATEThe transcription of lncBATE10 is under the control of the cAMP response element-binding protein (CREB) pathway . Indeed,Recently, Chen and colleagues have characterized three lncRNAs—MIAT, LINC01119, and LINC02202—that seem to play a key role during adipogenesis . In partA recent analysis compared adult and newborn BAT in mice. Several morphological and molecular differences were identified, including lncRNA expression . For exaRecent findings have led to the discovery of other lncRNAs that play a key role in BAT biology, such as the lncRNA H19 . This RNIn terms of BAT, lncRNAs take part in multiple gene expression networks that oversee WAT differentiation and its functions, both as energy reserve and as endocrine organ .New investigations have shown a pivotal role for three lncRNAs—ASMER-1, ASMER-2, and Plnc1—during adipogenesis, which regulate the gene expression of key adipogenic transcription factors, such as PPAR-gamma . ASMERs Gao and colleagues identified ASMER-1 and ASMER-2 in subcutaneous WAT obtained from patients with obesity and insulin resistance. They showed that in vitro silencing of ASMERs via antisense oligonucleotides suppressed adipogenesis, adiponectin production, and lipid mobilization .Other attractive lncRNAs include SRA, ADINR, and HOTAIR, which are involved in fat accumulation .Steroid receptor RNA activator (SRA) requires special attention. It was the first reported functional lncRNA present in fat cells, although its role in adipocyte differentiation appears undetermined. In vitro studies have suggested that SRA increases PPAR-gamma transcriptional activity in 3T3-L1 adipocytes . The SRAADINR (adipogenic differentiation-induced noncoding RNA) was identified as a regulator of C/EBP-alpha, a critical transcriptional factor in adipogenesis . In humaHOTAIR (HOX transcript antisense intergenic RNA) is a lncRNA expressed in gluteal adipose depots without any sex distinction and with a possible role in the regulation of human subcutaneous pre-adipocyte differentiation . SpecifiYang and colleagues have identified a lncRNA (UC001KFC.1) involved in glucose homeostasis . In obesLeptin is secreted by white adipocytes and other cell types, and it plays a key role in regulating the body’s intake and energy consumption . FindingThe lncRNA PVT1 could be a potential target for obesity treatment, since its expression is positively associated with full differentiation of 3T3-L1 preadipocytes. Moreover, PVT1 expression in WAT of obese mice is higher than that in WAT of healthy mice. PVT1 appears to be involved in lipid accumulation in white adipocytes by upregulating the expression of peroxisome proliferator activated receptor gamma, CCAAT/enhancer-binding protein α, and adipocyte protein 2 .An interesting study has enabled the identification of Mist, a lncRNA expressed in peritoneal macrophages and adipose tissue macrophages. Mist downregulation was associated with the expression of pro-inflammatory factors, as it occurs in obesity. Mist regulates the expression of the PARP1 gene that is involved in DNA repair mechanisms. Disruption of Mist-PARP1 interaction increases PARP1 recruitment onto the promoters of inflammatory genes, resulting in increased gene expression .The ncRNAs display an amazing variety of sizes, forms and functions. The classification in lncRNAs and sncRNAs is somewhat arbitrary and may hamper the discovery of their mechanism of actions. Indeed, the functions of many sncRNAs are well defined, while further studies are needed to completely dissect the molecular mechanisms of lncRNAs. In this context a further complexity derives from investigations showing that some lncRNAs may be translated and produce short peptides.All together these caveats represent a hurdle for a complete knowledge of lncRNA role in adipose tissue development and activity.Adipogenesis appears to be a process that is characterized by an intricate network in which many transcription factors and lncRNA are involved as regulators of gene expression.In this review, some examples have been provided of a new class of RNA molecules that participate in a wide range of cellular functions during white and brown/beige adipogenic development and activity. The vast majority of clinical drugs target proteins ; howeverCurrently, certain important drugs target nucleic acids in the areas of antibacterial and anticancer therapy. Targeting lnCRNAs for therapy relies on different approaches. For example, deregulated high lncRNA levels may be lowered with antisense oligonucleotides (ASO) that block lncRNA activity and induce their degradation. Alternatively, lncRNA function may be blocked by small molecules that mask the binding site of interacting proteins, or by antisense oligonucleotides that bind to the lncRNAs and inhibit their protein binding capacity .In detail, the lncRNAs regulating brown adipogenesis and BAT functions cannot be target of antisense therapy, since their silencing with ASO will impair BAT activities. On the contrary, there are many lncRNAs  that positively regulate white adipogenesis and are upregulated in obese individuals. These molecules are suitable ASO targets. Preliminary in vitro studies  showed tThe study of lncRNAs in adipogenesis and related diseases, along with the therapeutic approaches described above, may pave the way to developing new strategies for fighting obesity and related metabolic diseases.


Derived from linear  precursor mRNA, circRNA are recycled exons and introns whose ends are ligated. By titrating microRNAs and RNA binding proteins, circRNA interconnect networks of competing endogenous RNAs. Without altering chromosomal DNA, circRNA regulates skeletal muscle development and proliferation, lactation, ovulation, brain development, and responses to infections and metabolic stress. This review integrates emerging knowledge of circRNA activity coming from genome-wide characterizations in many clades of animals. circRNA research addresses one of the main pillars of the One Health vision – to improve the health and productivity of food animals and generate translational knowledge in animal species. Linear polymers of DNA and RNA convey the flow of genetic information, whereas circularized forms of DNA and RNA serve regulatory roles in all domains of life. Given circularized DNA were identifiable before the advent of molecular biology and the current genomic-computational era, decades of study have established their significance.The circularized DNA of mitochondria is necessary for energy generation, thereby governing growth, development and aging . ExtrachCircRNA was first documented in viroids, infectious pathogens of higher plants . The cirSimilar to viroids, the circRNA of hepatitis delta virus (HDV) undergoes recombination and is assisted by hepatitis delta antigen. HDV is a defective human pathogen that is dependent on hepatitis B virus for replication in liver. Because of its circRNA genome, HDV has been considered unique amongst all known animal viruses . In archRecently, two gamma herpesviruses, Epstein-Barr virus  and KapoA growing list of dsRBPs and splicing factors have been shown to mediate exon circularization and potentially may undergo sequestration and sorting by exogenous circRNA  . Mature In canonical splicing of pre-mRNA, spliceosomes catalyze the excision of introns and ligation of consecutive exons . AlternaNon-canonical splicing has been characterized by delayed or aberrant spliceosome activity . LigatioDrosophila cells and may play an important role  . Despiteant role . It was The deposition of nuclear factors to pre-mRNA is necessary for nucleo-cytoplasmic transport and subsequent commitment to decay, storage or efficient translation to protein . circRNAcircRNA serve as microRNA (miR) decoys and harbor copies of miR response elements (MREs). Unlike mRNA, circRNAs bound to miR are completely resistant to miR-mediated target destabilization . One miRMyoD, which has a critical role in muscle differentiation. By sequestering miR-7 and Argonaute, ciRS-7 was shown to derepresss miR-7 target mRNAs and activate embryonic muscle development in goat, e.g., insulin like growth factor 1 receptor , contains 70 MRE, and ciRS-7 binding does not activate miR-mediated destabilization . The expreceptor .CDK2 and p21 , some circRNA exhibit no difference in the abundance in plasma or serum between patients and healthy controls . More reThe expression of circRNAs was an almost completely uncharacterized component of eukaryotic gene expression until sample preparation protocols and deep sequencing were advanced . WhereasProtocols to enrich circRNAs in RNA samples have been established. In transcriptome analysis, depletion of rRNA is desirable since 90% of cellular RNA preparations are ribosomal RNA and only 5% is non-coding RNA or mRNA, respectively . FollowicircRNA isolation methodology with high confidence standards was described by As a group, circRNA generally are less abundant than their linear RNA counterparts. However, with the advancement of RNA library preparation, the reliability of RNA-seq technology and new computational strategies, results of quantitative investigations reveal certain human and chicken circular transcripts are more abundant than their homologous mRNAs .Numerous algorithms have been developed to detect junction-spanning sequences in deep-sequencing data set . Two appThe first group of programs is a candidate-based approach (pseudo-reference), where a circRNA reference set of all possible combinations of scrambled exon–exon junctions is built from a gene annotation repository. The back-splice junction reads are aligned contiguously to the computational reference set by algorithms such as KNIFE and NCLscan  Table 1Table 1. The second group of programs, fragmented-based, is also referred to as segment- or split-alignment-based and relies on the identification of back-splicing junctions from the mapping information provided by aligning reads to the reference genome or transcriptome  Table 1Table 1. Once detected by the algorithm, circRNAs in published data are deposited into one or more databases . Some daComputational reproducibility has become an integral issue in scientific research, particularly due to the rapid advancement of computer environments . SeveralEmerging data indicate circRNA connect feed-back loops in all life processes, from embryonic development to growth, differentiation and response to infection. Aberration of ceRNA networks is consequential to infectious disease, neoplastic transformation, developmental and degenerative disease. In the following sections, perspectives coming from circRNA investigations in livestock, companion animals and wild species are discussed and considered in relation to food production and biomedical science. We emphasize emerging issues and promising avenues to utilize circRNA to improve health of people and non-rodent animal species.Stunning advances have been made in the genetics of food-producing animals. Selection for certain production traits has created a very different livestock compared to the historical primitive breeds. For decades, breeding efforts have been undertaken to select for higher milk yield in dairy cows, faster muscle growth in cattle or larger eggs in poultry. With that in mind, genomic information alone explains only a part of the phenotypic variance in traits .Non-coding RNAs serve epigenetic mechanisms, in addition to DNA methylation, histone tail modification and chromatin remodeling, that have the ability to modify phenotype without altering chromosomal DNA . EpigeneThe genome-wide identification and function analysis of circRNAs in chicken skeletal muscle were characterized over the stages of embryonic development . ThirteeRBFOX mRNA is template for the translation of RBFOX-splicing factors that are essential for the maintenance of skeletal muscle mass and proteostasis  that were annotated by GO enrichment analyses. One circRNA, circCSN1S1_2, specifically stood out and was positively associated with the expression of the CSN1S1, CSN1S2, CSN3, and WAP genes. Furthermore, the MRE identified in circCSN1S1_2 were predicted to competitively bind miR-204 to increase expression of parental gene, CSN1S1. These trends were similar to observations in dairy cows (more below)  .Consistent egg production is a characteristic of healthy hens and depends on hormonal and developmental regulation. Infection is the major concern to avoid in the production of high quality and quantity of poultry meat and eggs. Avian leukosis virus (ALV) infection heightens mortality of hens, reduces egg laying performance and diminishes the quality of eggs by reducing size and increasing fragility of the shell . The retALV strain J (ALV-J) was first isolated from commercial broilers in 1988 in the United Kingdom and has since spread to other countries . ALV-J wChickens vary in susceptibility or resistance to ALV infection through activity of divergent receptors  and celleIF4E and PI3K, are known to positively regulate the mTOR pathway, thus activating protein synthesis. mTOR activity is essential for cell growth and its dysregulation is linked to uncontrolled cell proliferation in cancer. Therefore, circRNA attenuation of mTOR activity by sequestering miRs was posited to be tumor-suppressive  were found to express circRNAs in the mammary gland. Expression levels were up to nine-fold increased on day 90 compared to day 250, correlating with the observed decrease in milk casein production as lactation wanes. Furthermore, cattle casein circRNAs had an abundance of MRE for miR predicted to target CSN1S1 and CSN2 mRNAs. The results support the hypothesis CSN circRNA regulate milk production through competitive binding of miR that upregulates translation of Casein proteins  (The biology of the brain is prominently regulated by non-coding RNAs . In the = 10–12) .Sus scrofa circRNAs was measured in the cortex, hippocampus, cerebellum, brainstem and basal ganglia at several time-points from early embryonic development to the time of birth. The authors reported clear fluctuations in circRNA expression as the gestation progressed. But, in contrast to results in poultry  . The intSry transcript in mouse testis  in dogs is an established model to study atrial fibrillation (AF), a common arrhythmia, which in humans leads to heart failure . RNA seqThe relationship between circRNAs and atherosclerosis was investigated after inducing atherosclerotic plaques on the right carotid arteries in New Zealand white male rabbits by endothelial injury and high fat diet . DiffereNumerous experiments have established that muscle in humans is one of the tissues that is enriched in circRNAs. The dystrophy gene is among the first genes identified to generate circRNAs in skeletal muscle . It is tvastus lateralis) from young, middle-aged and elderly rhesus macaques (age range 0.003–41 years old). The majority of the detected circRNAs were similar among the different age groups, but 19 were significantly downregulated circRNA as individuals aged. The significance of circRNA to muscle development, musculoskeletal pathology and age-related changes in primates is likely to be informed by studies of swine and poultry skeletal muscle with high statistical power.On the other hand, In a landmark publication, One Health is a systems biology vision that encompasses the health of people, animals and global environment under one large umbrella . Under tKB-L outlined the manuscript, tables, and figures. DZ wrote the manuscript and created the figures. DZ and KB-L finalized the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Polycomb repressive complex 2 (PRC2) is a key epigenetic multiprotein complex involved in the regulation of gene expression in metazoans. PRC2 is formed by a tetrameric core that endows the complex with histone methyltransferase activity, allowing it to mono-, di- and tri-methylate histone H3 on lysine 27 (H3K27me1/2/3); H3K27me3 is a hallmark of facultative heterochromatin. The core complex of PRC2 is bound by several associated factors that are responsible for modulating its targeting specificity and enzymatic activity. Depletion and/or mutation of the subunits of this complex can result in severe developmental defects, or even lethality. Furthermore, mutations of these proteins in somatic cells can be drivers of tumorigenesis, by altering the transcriptional regulation of key tumour suppressors or oncogenes. In this review, we present the latest results from structural studies that have characterised PRC2 composition and function. We compare this information with data and literature for both gain-of function and loss-of-function missense mutations in cancers to provide an overview of the impact of these mutations on PRC2 activity. PRC1 catalyses the mono-ubiquitination of lysine 119 on histone H2A (H2AK119ub),6 whereas PRC2 catalyses the mono-, di- and tri-methylation of lysine 27 on the histone H3 tail (H3K27me1/2/3).7 In mice and humans, PRC2 is essential for proper embryonic stem cell (ESC) fate specification, as it regulates the expression of key developmental genes.10 Indeed, depletion of PRC2 subunits leads to severe developmental defects with early embryonic or perinatal lethality.17 Mutations and/or dysregulation of PcG genes are found in several cancer types, especially haematological ones,19 as well as in rare genetic diseases associated with overgrowth, such as Weaver syndrome.20 These alterations can affect PRC2 recruitment and enzymatic activity, leading to changes in the expression of tumour suppressors or oncogenes.Transcriptional diversity is one of the hallmarks of cellular identity. It is largely regulated at the level of chromatin, where different protein complexes act as initiators, enhancers and/or repressors of transcription. Among these complexes, are epigenetic modifiers, which are able to catalyse post-translational modifications (PTMs)—such as methylation, acetylation, phosphorylation or ubiquitination—of histone proteins. These modifications can influence gene expression by modulating chromatin accessibility, its interaction with other proteins and its three-dimensional organisation. The Polycomb group (PcG) proteins form histone-modifying complexes whose activity is associated with transcriptional silencing of facultative heterochromatin.21 suppressor of Zeste (SUZ) 12; and embryonic ectoderm development (EED)  comprises one of three Polycomb-like (PCL) proteins  as well as Elongin BC and Polycomb repressive complex 2-associated protein (EPOP) or PRC2-associated LCOR isoform 1 (PALI1/2), while the other variant (PRC2.2) comprises Jumonji and AT-rich interaction domain 2 (JARID2) and adipocyte enhancer-binding protein 2 (AEBP2),30 in addition to the core components.The PRC2 core comprises three stoichiometric factors: enhancer of Zeste (EZH)1 or EZH2, which has a SET domain and is the catalytic subunit of the complex;Along with the interest in characterising the functional role of accessory factors in regulating PRC2 activity, effort has also been put into trying to gain structural insights into the complexity of PRC2 and its subtypes. In this review, we discuss the latest findings regarding the PRC2 structure, focusing on the aspects that define the formation of different complex subtypes, its chromatin targeting and its enzymatic activity. Finally, building on all the current structural knowledge, we highlight the potential effect of PRC2 mutations on complex integrity and activity, and the role of mutations of PRC2 components in cancer.28 that comes together to mediate HMT activity.The association of the trimeric core EZH2, SUZ12 and EED) with RBBP4 results in a stable, four-lobed structure  and the SET domain, forms the catalytic lobe, in which the HMT activity of PRC2 resides Fig. . The act36 This conformation is necessary to maintain EED in a stable position while leaving the opposite side of the propeller open for stimulatory ligand binding.37 The open end of the propeller faces the rest of the EZH2 N-terminal region. The SET activation loop (SAL) and the stimulation-responsive motif (SRM) of the EZH2 N-terminal region are responsible for the allosteric changes in the catalytic lobe upon trimethyl-peptide binding in the EED pocket. Indeed, although the trimeric core retains basal HMT activity, the recognition of the H3K27me3 mark further activates the complex resulting in an enhanced catalysis (discussed below). Finally, the loop around EED is closed by the association of the SANT1 domain of EZH2 with the distal part of EBD, named SANT1-binding domain (SBD).The catalytic lobe is in close contact with the regulatory lobe, which is formed by the association of EED with the N-terminal domain of EZH2 Fig. . The lon35The central part of EZH2 (comprising the MCSS and SANT2 domains) and the globular VEFS domain of SUZ12 are part of a bridging middle lobe Fig. . In part39 RBBP4/7 is inserted into this lobe, its WD propeller tied by multiple interactions with the SUZ12 C2, WDB1 and WDB2 domains. The ‘cage’ built by SUZ12 around RBBP4/7 prevents the latter from binding many of its known nuclear interactors, including nucleosomes,39 thereby inhibiting its ability to sense active chromatin states. The functional relevance of incorporating an ‘inactive’ WD propeller into the PRC2 complex is still unclear.Finally, the SUZ12 N-terminal region protrudes away from VEFS to form an extended fourth lobe that serves as a docking platform for factors that associate with PRC2 Fig. .38,39 RBOverall, spatial segregation of the docking lobe with respect to the rest of the complex reflects the functional separation between recruitment and catalysis, with the former mediated by accessory proteins, and the latter endowed into the other three lobes.40 These structures show that a non-canonical C2 domain in SUZ12 provides a binding platform for either the C2B domain of AEBP2 or the RC domain of PHF19 . Binding of ELOBC to EPOP  is mediated by the BC box motif, a classical ELOBC binding motif, on the N-terminus of EPOP. Mutation of a crucial leucine residue (L40) in this motif impairs the association of ELOBC with EPOP and PRC2.29 The interaction of EPOP with ELOBC is crucial for proliferation of several human cancer cell lines. Indeed, depletion of EPOP leads to a decrease in their proliferative capacity. This phenotype is dependent on EPOP interaction with ELOBC as only the wild-type version of the EPOP protein (but not the L40 mutant) is able to rescue cancer cells proliferation.29Although these structures provide crucial insight into PRC2 holocomplex formation, many PRC2-associated factors have yet to be characterised in such detail, such as the ELOB and ELOC heterodimer , and the PALI1/2 proteins. However, some biochemical evidence provides preliminary insights into how these factors associate both with the core complex and other accessory proteins. In mouse ESCs, ELOBC associates with PRC2 on chromatin30 Although this biochemical evidence provides crucial information about how these factors associate with PRC2, the exact molecular functions of EPOP, ELOBC or PALI1/2 within PRC2 so far remain unknown.PALI1 and 2 are vertebrate-specific subunits of the PRC2.1 complex, which associate with PRC2 through their highly conserved C-terminal PALI-interaction with PRC2 (PIP) domain , which are cis-regulatory DNA sequences that provide a platform for Polycomb subunit binding and subsequent gene repression.42 Although Polycomb proteins are well conserved between flies and mammals (reviewed in 43), no PRE-like specific DNA sequences have been identified to date in mammals.44In 45 Stable interaction with the chromatin is indeed key for PRC2 stimulation: HMT activity is enhanced on di-/oligonucleosomes as compared with mononucleosomes, especially for linker DNA with lengths of 35–40 bp, allowing for engagement of both the stimulating and the substrate lysine residues.46 Interestingly, this feature seems to be specific for EZH2–PRC2, as EZH1–PRC2 does not display such a preference for its substrate.46Of the core components of PRC2, both EZH2 and SUZ12 possess zinc finger domains, which could potentially mediate an interaction with DNA. However, rather than playing a major role in targeting PRC2 to chromatin, binding of the core components of the complex to DNA seems to stabilise the interaction of PRC2 with the nucleosome template and thereby facilitate catalysis; specifically, the EZH2 CXC Zn-binding clusters contact the DNA exiting from the substrate nucleosome. Additionally, the EZH2 SBD domain contacts the DNA minor groove in the H3K27me3-marked nucleosome, after it has been bound by EED. Concurrently, EED might also contact the DNA of the modified nucleosome, through an unstructured, positively charged N-terminal stretch.48 and were therefore the first candidates to be considered for facilitating DNA-mediated recruitment of PRC2. AEBP2 was shown to have a binding preference for a highly degenerate consensus sequence with a bipartite structure (CTT(N)15–23cagGCC),49 suggesting that the recognition of this motif could provide a targeting mechanism for PRC2. In addition, in vitro biochemical assays showed that the presence of AEBP2 increases the capacity of PRC2 to bind nucleosomes, through its KR motif (a lysine/arginine-rich positively charged patch), resulting in enhanced HMT activity.46 Nonetheless, AEBP2 does not seem to be essential for PRC2 recruitment in vivo, as depletion of AEBP2 in mouse ESCs does not affect PRC2 occupancy on chromatin and even leads to a slight increase in H3K27me3 levels.14 For JARID2, in vitro experiments have demonstrated its ability to bind DNA, with a slight bias for GC-rich elements. This binding is mediated by the C-terminal region of JARID2, potentially through its C5HC2 zinc finger.50 Independent of its DNA-binding ability, JARID2 can also mediate the interaction with nucleosomes, resulting in enhanced PRC2 activity. This interaction is mediated by a region with no annotated structure, which spans residues 349–450.51 In line with these observations, evidence has shown that PRC2.2—but not PRC2.1—is able to bind nucleosomes in vitro.39 In agreement with a role for JARID2 in mediating PRC2 recruitment to chromatin loci, depletion of JARID2 reduces PRC2 levels at specific target genes.54AEBP2 and JARID2 contain three and one zinc finger domains, respectively, with JARID2 also harbouring an AT-rich-interacting domain (ARID). Both proteins display DNA-binding ability in vitro56 This suggests that JARID2-mediated PRC2 recruitment is essential for its tumour-suppressive functions.Notably, JARID2 is mutated and/or deleted in various types of leukaemia where PRC2 plays an onco-suppressor role.59 In 2017, Li and colleagues59 resolved the crystal structure of human N-terminal PHF1, which contains Tudor, PHD1 and PHD2 domains as well as a newly identified extended homologous (EH) domain. The EH domain folds into three α-helices and a curved three-stranded β-sheet. The W1 loop of the winged helix structure within the EH domain (EHWH) binds to unmethylated CpG DNA motifs, but not to GpC or AT-rich elements. Binding specificity is provided by the first two lysine residues of the W1 loop (K323 and K324 in PHF1), which form extensive contacts with both cytosine and guanine nucleotides  are required for MTF2 to bind to unmethylated CpG motifs,62 therefore narrowing down the potential target regions. These observations point to a more degenerate definition of vertebrate PREs that would be defined by DNA helical shape rather than specific primary sequence.PCL proteins are essential for PRC2 recruitment, as their depletion in mouse ESCs leads to a strong reduction in the levels of PRC2 on chromatin.des Fig. , and theAnother crucial aspect of PRC2 regulation and recruitment on chromatin comes from its direct interaction with many post-translation modifications that are present on histone tails. Indeed, PRC2 activity strongly depends on the epigenetic state of its chromatin environment.64 These dynamics result in 4–6-fold enhanced substrate binding35 and 3–7-fold increased catalytic activity.65 Interestingly, H3K27me3 is not the only epigenetic mark that can be recognised by EED, as other repressive chromatin marks —but not active marks —are bound with comparable affinity. However, H3K27me3 is the only mark that is able to elicit PRC2 catalytic stimulation.65 Although it is tempting to speculate that EED-mediated recognition of repressive histone marks could serve as a mechanism for recruitment of PRC2 to silenced loci, a large body of evidence has shown that stimulation of PRC2 catalytic activity and its recruitment on chromatin are completely uncoupled.71 Rather, this mechanism accounts for a positive-feedback loop: upon PRC2 deposition of the H3K27me3 mark through its basal level of activity, methylation of the neighbouring nucleosome is favoured, resulting in spreading of the histone mark along the linear chromatin fibre as well as along the regions that are in spatial proximity.72On the one hand, recognition of PRC2-deposited H3K27me3 products by EED results in catalytic stimulation of the complex: the open end of the EED WD-propeller is oriented towards the side of the catalytic lobe, thereby exposing its aromatic cage in this direction. Following the engagement of H3K27me3 with the aromatic cage  Fig. . This mo74 this inter-complex crosstalk appears to be bidirectional, as PRC2 can bind the H2AK119ub mark deposited by PRC1.75 This binding is specifically mediated by an ubiquitin-interaction motif (UIM) on the N-terminus of JARID2 and is essential for PRC2 recruitment to H2Aub119-marked regions  complex retains both HMT activity and inhibition by H3K4me3/H3K36me2/3.77 Two studies published in 2018 have helped to explain how RBBP4 progressively loses its nucleosome-binding capacity in the context of the human PRC2 complex: distinct residues from SUZ12 (C2 R196) and AEBP2  directly compete with the H3 tail for binding to the RBBP4/7 acidic pocket   wrapping around RBBP4/7 eventually imposes steric hindrance to the interactions of RBBP4/7 with histone H4.39On the other hand, however, histone post-translation modifications associated with an active transcriptional state (e.g. H3K4me3 and H3K36me2/3) have the opposite effect, strongly inhibiting PRC2 di- and tri-methylation activity.19) Fig. . Moreove84 Although the functional relevance of H4 binding is still unclear, Jani and colleagues83 have shown that unmodified H3K36 (but not H3K36me2/3) can increase the catalytic activity of EZH2, providing a rationale for the lower PRC2 activity in active chromatin environments.An alternative hypothesis has therefore been put forward by two reports showing that EZH2 is able to sense the modification state of both H3K36 and H4K16/20 via two independent pockets located in its CXC/SET and SANT1 domains, respectively.87 Interactions with these histone marks are abolished when any of these residues is mutated to an alanine.85 The significance of this interaction for PRC2 recruitment remains unclear, considering the limited overlap between the genomic localisation of H3K36me3 and H3K27me3 on chromatin in mouse ESCs.57 One of the proposed mechanisms is that PCL-containing PRC2 transiently binds to H3K36me3, together with a H3K36me2/3-specific demethylase (such as KDM2B or NO66), in order to establish de novo H3K27me3 domains and to impose gene silencing during differentiation.58 Thus, while histone post-translation modifications associated with active transcription could mediate negative feedback on EZH2 resulting in inhibition of PRC2 activity, an intriguing possibility is that incorporation of PCL proteins into PRC2.1 confers on the complex the ability to initiate the silencing of actively transcribed regions.As well as the PRC2 core components, the PCL family of proteins has long been known to interact directly with the H3K36me2/3 mark. PHF1, PHF19 and MTF2 can all bind the H3K36me2/3 mark through their aromatic cage  located in their highly conserved Tudor domain Fig. .57–59,8588 Notably, H3K36me2/3 decorated regions seem to be devoid of PRC2,89 suggesting that H3K27me2 deposition at these loci is the result of a transient interaction. Whether PCL proteins play a role in this mechanism is not clear yet.However, many genomic regions in mouse ESCs show co-localisation of the H3K36me2 and H3K27me2 marks: in this scenario, the H3K36me2 mark, deposited by NSD1, prevents the spreading of H3K27me3.92 Moreover, direct binding to RNA results in inhibition of PRC2 HMT activity both in vitro93 and in vivo,96 probably due to competition with chromatin binding .Along with the many chromatin features that regulate PRC2 recruitment and activity, other studies suggest that RNA might play a role in both of these two processes: despite the lack of a known RNA-binding domain, PRC2 binds RNA in vivo and in vitro in a non-sequence-specific manner, although it does show a preference for GC-rich sequences and G-quadruplexes.96 used a targeted proteomic identification of RNA-binding regions (RBR-ID) approach to map, at high resolution, the interactions within the nucleus between PRC2 components and RNA. Their results showed that binding involves neither a single subunit of the complex nor discrete RNA-binding domains within them but, rather, dispersed amino acid patches on the exposed surface of the complex;96 this is in line with previous in vitro observations by hydrogen deuterium exchange mass spectrometry.100 Interestingly, RNA interactions were observed for all PRC2 subunits . However, the most consistent points of interaction were at regions of EZH2 at the interface with EED , leading the authors to speculate that inhibition of PRC2 HMT activity by RNA could be, at least partially, due to interference with transduction of the stimulatory signal.96 These observations suggest that the inhibition of PRC2 activity in active chromatin environments is mediated not only at the epigenetic level (through negative regulation by H3K4me3 and H3K36me2/3) but also by RNA itself, as the final output of actively transcribed regions.However, the molecular mechanisms of binding and of the resulting inhibition are not yet clear. To address these questions, Zhang and colleagues101 Since then, many reports have shown that other PRC2 factors are subject to deletions, mutations, translocations and/or dysregulation in different cancer types. In the majority of cancer types, high expression of PRC2 factors correlates with sustained proliferation of cancer cells.103 However, whether this reflects a causative role for PRC2 in tumorigenesis is difficult to assess. In some cases, PRC2 has been shown to repress key tumour-suppressor genes, therefore directly promoting tumour progression.105 Accordingly, a lot of effort is being put in developing targeting strategies to inhibit the activity of PRC2 in order to treat various cancer types (for a comprehensive review on PRC2 inhibition strategies see 107).Early observations demonstrated that EZH2 was overexpressed in prostate tumours and played a role in the progression of this cancer type.19 In haematological malignancies, contrary to most other cancer types, PRC2 can exhibit both oncogenic and tumour-suppressor functions, depending on the cellular context.105PRC2 genes, both core and associated factors, are mostly mutated in haematological malignancies. This is in line with their role in haematopoiesis, where they regulate crucial processes such as hematopoietic stem cells self-renewal and B-cells germinal centre formation.108 108 Fig. , in line108 Fig. .Fig. 3Th116 Disruption of one of these residues could result in impaired substrate recognition and, possibly, loss of methyltransferase activity, as demonstrated for the Y726D mutant.113 The R685 residue, which lies just below the bottom of the hydrophobic pocket, seems to be essential for the correct orientation of F665, and its mutation to cytosine abolishes methyltransferase activity in vitro.117 Indeed, this amino acid is found mutated (R685C/H) in AML, early T-cell precursor acute lymphoblastic leukaemia (ETP-ALL), MDS and myelofibrosis.124As previously discussed, the lysine substrate is accommodated in an aromatic cage Fig. , and mut130 These mutations can either cause steric hindrance to the H3 tail (e.g. G655R) or abolish interactions between the H3 backbone and the iSET . However, to our knowledge, the impact of these mutations on the HMT activity or stimulation of PRC2 has not been assessed.iSET is also responsible for stabilisation of the H3 substrate, as it is sensitive to the activating signal coming from the EED–trimethyl-peptide–SRM axis. Specifically, the α-helix of iSET shapes the channel in which the H3 tail lies and is essential for enhancing PRC2 activity upon stimulus Fig. . Mutatio134  are essential, and their relevance is underscored by their relatively high frequency of mutation in AML, CML, ETP-ALL and MDS134 Fig. . In part149 as well as in those with melanoma152 and Ewing sarcoma.154 Mutations of this residue are thought to be an early driving event in cancer progression, as expression of the EZH2 Y641F mutant in mouse B-cells and melanocytes leads to the formation of lymphoma and melanoma, respectively.155 At the biochemical level, the EZH2 Y641F mutant shows an increased ability to modify dimethyl substrates yet is almost unable to act on nucleosomes with unmethylated or monomethylated H3K27.158 Molecularly, Y641 is positioned at the interface between the substrate lysine and the SAM cofactor, with the potential to form hydrogen bonds with the unmethylated lysine  for the Y641 mutant form, resulting in overall hyper-trimethylation.161 Indeed, mutations in Y641 are exclusively found in heterozygosity. How this excess of H3K27me3 confers tumorigenicity is not well understood, but evidence points to a role for this mark in mediating the silencing of multiple tumour suppressors through changes in promoter-enhancer contacts within chromatin compartments.162Mutations in Y641 of EZH2 have been observed to occur at a high frequency (in around 25% of patients) in patients with follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCLs),ine Fig. . Mutatio163 The A677G mutation enlarges the lysine-binding pocket of EZH2 without affecting the interaction of Y641 with the unmodified substrate, therefore resulting in highly efficient catalysis on all methylation states and eventually leading to aberrantly high levels of H3K27me3 and lower levels of H3K27me2.156 On the other hand, A687V increases the efficiency of EZH2 for monomethyl substrates, by weakening the interaction with a water molecule that needs to be displaced for demethylation to happen. This results in a more balanced substrate preference with respect to wild-type EZH2, thereby increasing overall levels of H3K27me3 without affecting those of H3K27me2.164 Presumably, the resulting aberrantly high levels of H3K27me3 are responsible for the tumorigenicity of these EZH2 mutants.Two other gain-of-function mutations, A677G and A687V, are present in around 1–2% of patients with FL or DLBCL.45 These clusters accumulate a high frequency of mutations, probably leading to loss-of-function mutant isoforms that are not able to stabilise the interaction of PRC2 with the nucleosome. So far, only the C571W mutation has been found in homozygosis in myelodysplasia patients. EZH2 proteins bearing this mutation show complete loss of PRC2 HMT activity and can partially destabilise the integrity of the PRC2 complex.113The two zinc-binding clusters inside the CXC domain (Zn3C9 and Zn3C8H1) are placed right on top of the active site and are involved in contacting DNA in the substrate nucleosome.165  lead to the very same stimulation-deficient phenotype.64 S259F and R302G mutations have been observed in T-ALL and myelofibrosis patients, respectively.132The SAL and SRM domains of EZH2 are responsible for transducing the stimulatory signal from the recognition of the H3K27me3 or JARID2K116me3 mark by EED to the iSET motif Fig. . Almost 165 Fig. . Interes167 In addition to these mutations, other mutations such as W591C/R and N618Y have been observed in osteosarcoma and T-ALL168 and could have deleterious effects: mutations of the homologous residues in D. melanogaster Su(z)12 result in decreased HMT activity and impaired complex assembly.169 Interestingly, N618 forms H bonds with the amine and carboxyl groups of Y292 in the CXC domain, suggesting that mutation to a tyrosine residue would disrupt this interaction and thereby destabilise SUZ12’s association with EZH2.The VEFS domain has a moderately high frequency of mutations, with a diffuse mutational pattern rather than specific hotspots Fig. . Many of171  or stabilising its interaction with nucleosomes through AEBP2 and JARID2.51 By contrast, an active chromatin state is maintained by inhibition of PRC2 activity through the inhibition of the core complex by H3K4me3 and H3K36me3, and by PRC2 binding to RNA, which impedes chromatin binding and/or transduction of the stimulatory signal. In other words, both chromatin states are able to maintain a positive-feedback loop through, in part, inhibition or activation of the PRC2 complex. However, in some cases, maintenance of a chromatin state can be challenged. For instance, both AEBP2 and JARID2 can mimic histone tails through their binding to PRC2. This competition between PRC2 subunits and histone tails could be a mechanism by which PRC2 autoregulates its activity to override the epigenetic context of the targeted chromatin.The PRC2 complex is a tightly regulated machine that governs the correct spatial and temporal expression of key developmental regulators via its function as a transcriptional repressor. Its activity is regulated via interactions with both facultative subunits and its chromatin environment. In general, the interaction of PRC2 with chromatin appears to be a way to safeguard the different chromatin states. Indeed, PRC2 is able to perpetuate a repressive state in a number of ways: first, by binding its own H3K27me3 mark; second, by binding the H2A119ub mark deposited by PRC1; and, third, by trimethylating and binding to JARID2K116. All of these interactions increase PRC2 activity, either by increasing residence time on chromatin  is sufficient to mediate enzymatic activity and modulate it by sensing the chromatin state, as is reflected by the higher incidence of missense mutations in the domains that mediate the catalysis and stimulation of methyltransferase activity.Interestingly, the ability of core components to interact with chromatin features seems to be limited to modulation of the catalysis rather than recruitment to target regions. This observation is in line with the emerging view that differential recruitment of PRC2 during distinct developmental stages is achieved by incorporating accessory factors that are dynamically expressed during development. Cell-type-specific expression and partial redundancy of accessory subunits could therefore account for their lower mutation rates observed in cancer In line with this, it is worth noting that depletion of PRC2 accessory subunits only slightly affects genome-wide levels of H3K27me3. This suggests that, once the pattern is established, the core complex alone (without accessory factors) is able to maintain it, potentially through the positive-feedback mediated by the EED–SRM–iSET axis. Therefore, mutations/dysregulation of core components might be necessary to induce changes in the methylation pattern, as observed during tumorigenesis. While structural studies have greatly deepened our understanding of the function and regulation of PRC2, many unanswered questions relating to PRC2 and its role in controlling gene expression remain to be addressed. For instance, what is the role of the recently characterised associated subunits , for which structures with the complete core complex are still lacking? How much of our knowledge about EZH2 function and structure applies to EZH1, considering their differential response to stimulatory elements as well as their distinct mutational landscape? Furthermore, what is the impact of those mutation hotspots for which we have neither structural nor functional details? Answering these questions will help us to better understand both the physiological and the cancer-associated functions of PRC2.


One major role of the eukaryotic peptidic post-translational modifier SUMO in the cell is transcriptional control. This occurs via modification of virtually all classes of transcriptional actors, which include transcription factors, transcriptional coregulators, diverse chromatin components, as well as Pol I-, Pol II- and Pol III transcriptional machineries and their regulators. For many years, the role of SUMOylation has essentially been studied on individual proteins, or small groups of proteins, principally dealing with Pol II-mediated transcription. This provided only a fragmentary view of how SUMOylation controls transcription. The recent advent of large-scale proteomic, modifomic and genomic studies has however considerably refined our perception of the part played by SUMO in gene expression control. We review here these developments and the new concepts they are at the origin of, together with the limitations of our knowledge. How they illuminate the SUMO-dependent transcriptional mechanisms that have been characterized thus far and how they impact our view of SUMO-dependent chromatin organization are also considered. Since the beginning of the 2000s, there has been accumulating evidence that the eukaryotic peptidic post-translational modifiers of the Small Ubiquitin-like Modifier (SUMO) family (collectively termed SUMO hereafter) plays an important and multifaceted part in the regulation of gene transcription. This is achieved via their conjugation to transcription factors (TFs), positive and negative transcriptional coregulators, histones and other chromatin proteins, chromatin-remodeling complexes, chromatin-modifying enzymatic systems, as well as to the transcriptional machineries themselves. Indeed, one of the first roles that has been characterized for SUMO is regulation of transcription. It is also the most documented one in the literature ,2,3,4,5.Although this review principally deals with RNA polymerase II (Pol II)-mediated transcription regulation by SUMO in mammalian cells, major concepts originating from other organisms will also be addressed together with transcription by RNA Polymerases I (Pol I) and -III (Pol III). A number of preliminary considerations on SUMOylation itself are required before getting into the mechanistic details of gene regulation by SUMO. For more general information on protein SUMOylation and its biological consequences, the reader is referred to general reviews ,9,10,11.The term SUMO defines a group of polypeptides of approximately 100 amino acids belonging to a broader family of eukaryote-specific peptidic post-translational modifiers called the ubiquitin-likes (Ubls). Their founder, ubiquitin, is a major actor in, not only proteasomal degradation of intracellular proteins, but also many non-proteolytic signaling events ,13,14. 7 molecules/cell in the human cervix cancer cell line HeLa, i.e., only slightly less than its kin ubiquitin [SUMO  is among the most abundant cellular proteins with, for example, approximately 10biquitin . Like ubbiquitin ,7,9,10. biquitin ,7,9,10. biquitin ,17. SUMObiquitin . All theSUMO is present in different forms and numbers in the various eukaryotes, ranging from one in an organism such as yeast up to eight in certain plants ,20. In mSeveral sets of enzymes are involved in the SUMOylation cycle . First, The SUMO E3s characterized so far are relatively few in number. The best-studied ones include the members of the PIAS family , several TRIM family members, RANBP2 or the Pc2/CBX4 component of the Polycomb complex ,11,24. CFinally, SUMOylated proteins can be deSUMOylated and SUMO chains can be depolymerized by SUMO isopeptidases  ,30,31. TThere is now ample evidence that protein SUMOylation can no longer be considered in an isolated manner due to multiple cross-talks with protein modification by other Ubls. For example, the FAT10 Ubl can inhibit the activation of the SUMO-1, -2 and -3 via covalent binding to the catalytic cysteine of the SUMO E1 . Yet, itThus far, proteomic approaches have characterized >6700 proteins SUMOylated at >40,000 sites, usually in flexible and disordered peptidic domains ,38. It iMany proteomics studies have now addressed the precise nature of both SUMOylated proteins and SUMOylated sites at the system-wide level in different species , sometimes in pathological situations ,87,88,89In the regard of its predominant conjugation to nuclear proteins, SUMOylation departs significantly from other post-translational modifications (PTMs) such as phosphorylation, acetylation or ubiquitylation, which largely occur throughout the whole cell. This however does not prevent SUMO to crosstalk with other PTMs. Thus, SUMO substrates can also be modified by acetylation, methylation or ubiquitylation or other PTMs occurring on lysines (see below) but also by other PTMs such as phosphorylation occurring on other amino acid residues. Moreover, the SUMO proteins themselves an also be subjected to PTMs . Taken wAnother striking difference with many other PTMs is that the fraction of a given protein that is SUMOylated at a given time in the cell is very low , at the exclusion of a few exceptions such as the RanGAP1 nucleopore-associated protein or the PML component of the PML nuclear bodies ,7,9,10. Another paramount point to consider is that numerous lysines identified as SUMOylated in vivo can also be ubiquitylated, acetylated or methylated as already mentioned . On its The interplay between SUMOylation and other PTMs can also occur on different amino acid residues. This is, for example, the case between SUMOylation and phosphorylation at closely located lysines and threonines residues in the TFs STAT1  and STATImportantly, interactions between PTMs are not necessarily antagonistic but can also be collaborative at the substrate level. For example, certain phosphorylations are known to promote SUMOylation of substrates at so-called phosphorylation-dependent extended consensus SUMOylation motifs  found in many (but not only) TFs ,37,112. SUMO-targeted Ubiquitin E3 ligases (or StUbLs). This is followed by ubiquitylation of SUMO substrates and/or SUMO itself [The diversity of protein modifications by SUMO itself and the crosstalk with ubiquitylation are not necessarily mutually exclusive and may have possible consequences on transcriptional control. Not only proteins can be monoSUMOylated, possibly at multiple sites (multiSUMOylation), but also polySUMOylated , i.e., subjected to modification by SUMO chains that can be of different natures ,31. ThusO itself ,31, whicO itself  and certRNF4 was the first StUbL identified in mammals ,120,121.Arkadia  is the second mammalian StUbL identified so far. It harbors 3 SIMs that preferentially recognize SUMO-2/3 chains capped by SUMO-1 over homotypic SUMO-2/3 chains . If it iAn important lesson of recent functional- and large-scale genomic and proteomic studies is that SUMOylated proteins, at least for the most abundantly modified ones, are often, not only functionally interconnected within the same pathway, but also capable of physical association . Moreover, SUMOylation has most often been associated with transcriptional repression in the literature ,2,3,4,5 The long-standing and dominant assumption of transcriptional repression by SUMO was, in fact, largely the consequence of a historical bias, as most initial studies (strengthened by many later reports) on transcription regulation by SUMO pointed to repression of gene expression ,3,91,146The picture of gene regulation by SUMO, however, progressively appeared more contrasted when data supporting a positive role for SUMOylation in transcription activity/activation begun to accumulate. Thus, and among other examples, SUMOylation was shown to facilitate the recruitment of the basal transcription machinery, to prevent that of transcriptional co-repressors in certain situations and at certain genes ,151,152,limitation rather than just repression of transcription to ensure optimal gene expression, at least under certain conditions and for certain genes. Genome-wide experiments revealing a positive correlation between highly active gene transcription and SUMOylation of promoter-bound proteins also showed that this transcription limitation effect does not only apply to genes induced in response to stimulatory cues but also to numerous cellular genes in cells grown under standard conditions to maintain homeostasis [Other reports have also consolidated the idea that gene regulation by SUMO is not necessarily a matter of mere on/off transcriptional switch. Along this line, a first series of observations showed that SUMOylation of TFs can occur concomitantly to the activation of their target genes to buffer the transcriptional output and, thereby, most probably avoid unphysiological and detrimental overexpression of the activated genes ,173,174.eostasis .maintain maximal expression of certain genes essential for cell survival [Another important concept, applying at least to an acute stress that can entail cell death such as HS, is that SUMOylation of factors binding to gene promoters may be essential to avoid transcriptional down-regulation and, thereby, survival . This efsurvival ,176,177 survival . As a coMaking the picture more complex, it must also be considered that the same transcriptional actor can be SUMOylated/deSUMOylated at several stages of its life. The reasons for this can be different depending on the biological process concerned or, on the contrary, can potentially cooperate towards a common aim. Additionally, SUMOylation pathway components can collaborate with TFs independently of any SUMOylation activity. Elk-1, one of the first TFs whose SUMOylation has been studied, illustrates the latter two points as (i) its SUMOylation can down-regulate its transcriptional activity  and contThat SUMOylation can be associated with transcriptional activity/modulation/limitation should however not hide that SUMO is also crucial for heterochromatin formation, which most often corresponds to a full transcriptional shut-off mechanism due to dense packing of DNA rendering it less, or no longer, accessible to both TFs and the transcriptional machinery. This point has already been mentioned above when addressing the issue of SUMO-2/3 modiform in mouse ESCs ,137 and Pol II transcribes protein-coding genes and endogenous retroviruses together with a number of non-protein-encoding genes such as those for snoRNAs, snRNAs, miRNAs and lncRNAs whereas RNA Polymerase I (Pol I) is responsible for transcription of the multiple genes (rDNA) coding for the 45S precursor of the ribosomal 18S and 28S RNAs ,92. On iSaccharomyces cerevisiae [Trypanosoma brucei where Pol I, not only transcribes rDNA, but also the active gene of the variant surface glycoprotein (VSG), which allows the parasite to escape the immune response of the infected host [Concerning Pol I, it has been reported that SUMOylation limits Pol I-dependent transcription of rDNA in human cells cultured under standard conditions ,180. Recrevisiae . In addirevisiae ,62. In cted host . This SUted host . Moreoveted host .Saccharomyces cerevisiae showing that SUMO may regulate it at multiple steps and in different ways. Thus, SUMO promotes the activity of Pol III under standard growth conditions, as depletion of the SUMO E2 reduces tRNA synthesis [As far as Pol III-mediated transcription is concerned, SUMOylation of tRNA genes has been associated with limitation of transcription in mammalian cells . Moreoveynthesis . Howeverynthesis ,186. Masynthesis ,62. FuncBesides these studies, Wang et al.  have recSUMO can also repress Pol III transcription via other means, in particular via modification of the Maf1 regulator. The latter factor is a master repressor of Pol III-dependent transcription in response to a broad range of intra- and extracellular cues largely controlled by the mTOR pathway . Though Thus, taken together, the above data highlight a role for SUMO in the control of Pol I- and Pol III-mediated transcriptions. Nevertheless, much remains to be studied to fully understand the most probably multiple roles of SUMO in these processes, including in cells subjected to different cues. It is also important to take into account that there exist cross-talks between the three eukaryotic RNA polymerase systems. In particular, they share a number of components and regulators and their respective actions can be coordinated by cell signaling ,92. It wIt is now firmly established that PTMs of chromatin- and chromatin-associating proteins at gene regulatory elements such as transcriptional promoters and enhancers are central for gene expression control ,104,105.Due to the well-established role of SUMO in transcriptional regulation, various laboratories have addressed its distribution, as well as that of SUMOylation enzymes, on chromatin in the recent years. This was achieved using either large-scale standard chromatin immunoprecipitation (ChIP)- or genome-wide ChIP-seq approaches coupled to transcriptomic- and, sometimes, proteomic experiments. These were conducted in budding yeast ,152,173 A first overall conclusion of these studies is that SUMO is widely distributed over the genome with increased concentration at a quite high number of discrete sites. These are found in the range of one to several tens of thousands, depending on the sequencing depth achieved  of SUMO peaks lies just upstream the transcription start site (TSS) in the so-called nucleosome-free region ,175,177 With regard to transcriptional regulation, SUMO enrichment is, however, not limited to gene promoters. For example, putatively active enhancers were found highly SUMOylated in mouse embryonic fibroblasts . SUMO-2 Another important conclusion of these experiments is that the chromatin SUMOylation landscape can be partly or dramatically cell-specific ,137,177 A last important general conclusion of these studies is that the SUMO signals characterized in ChIP and ChIP-seq experiments are likely to be largely accounted for by SUMOylation events occurring within chromatin as (i) they are most often associated with SUMO E2 and E3 enzyme signals in the same ChIP/ChIP-seq experiments and (ii) RNAi-mediated elimination of the latter enzymes entails substantial reduction of SUMO signals in ChIP/ChIP-seq experiments ,196,197.Saccharomyces cerevisiae [Concerning Pol II-dependent transcription, Rosonina  recentlyrevisiae . Strikinrevisiae , led theAdditional studies involving other TFs are still required to confirm on a broader experimental basis the notion of restricting TF binding to appropriate sites by SUMOylation. Moreover, how SUMOylation mechanistically controls TFBS selection constitutes an important ensuing question. In particular, it will be interesting to address whether SUMOylation influences TFBS selection by modulating protein-protein interactions with other nearby-binding TFs and/or with co-factors not binding to DNA and/or via promoting conformational changes within the TFs that alter their DNA-binding specificity and/or affinity. Finally, SUMO-dependent TFBS selection does not exclude other functions for TF SUMOylation (see below).As already illustrated above, the regulation of Pol II-dependent transcription by SUMO can follow a wide variety of mechanisms. However, caution is required when interpreting many past and current functional studies for at least two reasons. First, the experimental methods available to investigate the role of SUMO in gene expression control, although informative, present serious biases. Second, the SUMO field still suffers from the lack of essential tools that would secure entirely unambiguous conclusions. Thus, in in cellulo studies, the functional consequences of SUMOylation were most often addressed via altering the SUMOylation level of the proteins of interest using one, or several, of the following methods: (i) inhibition of protein SUMOylation by mutating either SUMO acceptor lysines (into arginines) or nearby residues, which may alter modification(s) by other PTMs essential for the function of the studied proteins, (ii) N- or C-terminal fusions of SUMO to these proteins, which neither recapitulate natural lysine SUMOylation and the dynamics of SUMOylation/deSUMOylation cycles they can be subjected to, nor reflect that only a minor fraction of the concerned protein is usually SUMOylated at a given time and (iii) altering the overall cellular levels of SUMOylation by either overexpressing SUMO or components of the SUMO pathway  or reducing their expression by RNAi or genetic knock-out of their genes, which also unavoidably affects SUMOylation of other cell components possibly interfering with the process under study.Moreover, there is no easily and efficiently implementable technology currently available to generate antibodies specifically directed to the SUMOylated sites of SUMO substrates similar to those used to generate antibodies recognizing phospho-sites in phosphorylated proteins. The only such antibodies we are aware of are those raised against the SUMOylated c-Fos TF . They haSeveral categories of transcriptional mechanisms regulated by SUMO are (non-exhaustively) considered hereafter. They illustrate that there is no univocal mechanism whereby SUMO controls transcription TFs are central in the nucleation and firing of the transcriptional act or, on the contrary, transcription repression. As illustrated several times above, there is ample evidence that SUMOylation of TFs can alter their intrinsic properties with very different transcriptional outcomes, depending on the TF and on the experimental setting. These properties non-exclusively include subcellular localization, metabolic stability, interference or cooperation with other PTMs, homo- or heterodimerization, binding to- or clearance from DNA and/or association with chromatin and ability to interact with other protein partners (see next section) .As described above, an important lesson of genomic/transcriptomic studies is that SUMOylation of TFs (and other proteins) can largely occur within chromatin ,196,197.If SUMOylation within chromatin may largely be associated with TFBS selection by TFs (see above and ), there On the opposite, SUMOylation can also enhance binding to DNA. This is the case for Oct4, a TF affecting the fate of certain progenitor- or cancer cells . This exSaccharomyces cerevisiae where Gcn4 undergoes SUMOylation after binding to its target promoters and after Pol II has been recruited owing to this TF binding event [Besides this, SUMO-dependent clearance from chromatin can be achieved by simple removal of TFs from the DNA template or protein destruction. This helps adjust transcription to the right level, control the duration of transcriptional responses, reset promoters and/or recycle TFs for other transcriptional actions when the latter are not proteolysed ,174. Thing event . This fang event ,173. Morng event . Along tng event ,130,131.Various studies have indicated that SUMO can facilitate, or, on the contrary, inhibit the recruitment of transcriptional co-regulators by TFs . The finAcetylation of lysines lying in the N-terminal tail of histones is considered as a PTM associated with transcriptional activity/activation. It is deposited by histone acetyltransferases , which also acetylate other proteins, some of them being implicated in transcription . HistoneThe picture of the actions of SUMO on HDACs is however more complex than mere HDAC recruitment by SUMOylated TFs owing to SIM/SUMO interactions. Thus, when they are part of multisubunit transcriptional regulatory complexes, HDACs can be recruited indirectly via other SUMOylated proteins, as discussed later. Moreover, in certain cases, TF SUMOylation limits rather than enhances HDAC recruitment . This isSUMOylation can also regulate the function of certain lysine-specific methyl transferases (KMTs). They transfer methyl groups to lysines of a variety of proteins, including histones ,235. HisOther corepressors can be recruited by SUMOylated TFs. Some well-documented examples include NCoR1, CoREST1 and SMRT. These transcriptional coregulators interact with other nuclear proteins, including various HDACs and KMTs or lysine-specific demethylases (KDMs). They play a key part in modulating the activity of various members of the TF superfamily of nuclear receptors by modulating epigenetic marks on chromatin ,239. ThuH17 cells [Though less frequent, recruitment of coactivators by SUMOylated TFs to promote transcription has also been reported . This is17 cells . Alterna17 cells . In anot17 cells . Finally17 cells .All the above discussed examples highlight the concept that SUMO, not only regulate protein-protein interactions via SIM-bearing protein recognition, but also helps organize multi-subunit complexes involved in transcription regulation. They also pose the question of coordination of multiple SUMOylation events within and between these supramolecular complexes, possibly via group SUMOylation.Pol II-dependent transcription is usually subdivided in four stages: initiation, pausing, elongation and termination. The fact that high levels of SUMOylation are observed in gene promoter regions, especially just upstream of TSSs, suggests that the former three stages may be governed by SUMOylation ,196,197 Xenopus laevis embryo. Its transrepression activity depends on NZFP interaction with TBP (TATA-binding protein), a factor typically binding just upstream of TSSs, which is promoted by NZFP SUMOylation and leads to inhibition of basal transcription complex formation [During the transcription initiation step, the GTF complex TFIID binds to the proximal region of promoters, where it triggers the loading of both other GTFs and Pol II to form the so-called preinitiation complex (PIC). Shortly after initiation, Pol II pauses in a transcription-competent state a few dozens of nucleotides downstream of TSSs in approximately 30% of genes. This pausing is promoted by the multi-subunit complexes NELF and DSIF, which halt RNA polymerization by Pol II. This pause can then be released by the P-TEFb factor made of two subunits, cyclin T and the kinase cdk9. This serves to fine tune gene expression in response to changing cellular environments. Upon appropriate stimuli, P-TEFb phosphorylates NELF, DSIF and the C-terminal domain (CTD) of Pol II on Ser2 of the heptad repeats it is made of. This releases NELF from the PIC, turning DSIF into a positive elongation factor and stimulating the elongation activity of Pol II. Interestingly, TFIID has long been reported as a SUMO-regulated factor via modification of several of its subunits. These include TAF5, which modulates its binding to DNA, TAF1, TAF8, TAF12 and the TATA-binding protein TBP . Of noteormation .Besides this, SUMO might also participate in transcriptional pausing regulation. First, heat-shock increases the SUMOylation of subunits of DSIF (hSpt5), NELF  and P-TEF (cdk9), which is associated with changes in gene expression ,177. SecThe epigenetically-controlled local, long-distance or global 3D organization of chromatin can be dynamically affected in response to numerous cues ,250. TheMuch work has now shown that SUMO can affect chromatin dynamics at different levels, which is not surprising when considering the large number of chromatin proteins found SUMOylated in proteomic/modifomic studies ,38. A fiInterestingly, PML- and PcG bodies are, not only nuclear substructures showing SUMO-dependent nucleation or maintenance and association with SUMOylation pathway components, but also SUMOylation centers impacting chromatin organization and transcriptional activity. For example, the histone H3.3-specific chaperone complex DAXX/ATRX traffics between PML bodies and DNA, with SUMOylation of DAXX being required for localization at PML bodies . MoreoveAt a much lower scale, TFs binding to gene promoters can SUMO-dependently recruit transcriptional coregulators that are chromatin remodelers or post-translational modifiers of chromatin-associated/-constituting proteins affecting nucleosome compaction/decompaction or movements (see above). This also argues for an important role of SUMO in controlling local chromatin configuration in relation with transcriptional competence/incompetence. That many of these coregulators were found themselves regulated by SUMOylation strengthens this idea (see above). In this respect, the case of the poly(ADP-ribose) polymerase PARP1 is worth being noted. Upon heat shock, the SUMO E3 PIASy interacts with PARP-1 and enhances its multi-polySUMOylation at the HSP70.1 gene promoter . This apThere is additional evidence that SUMO plays a part in the control of chromatin 3C organization at different levels. Saccharomyces cerevisiae, also supports the idea that histone SUMOylation may affect chromatin configuration. RSC is an abundant, essential chromatin-remodeling complex of the SWI/SNF family involved in transcription and several other aspects of chromosome biology. It was recently shown that its interaction with nucleosomes is modulated, not only by H3K14 acetylation, but also by SUMOylation of H2B, which regulates genetic information accessibility [Histones are major actors of chromatin organization and their PTMs largely decide the level of nucleosome packaging by favoring or disadvantaging internucleosomal interactions as already discussed. Both mammalian  and yeassibility .Metazoan chromatin is organized into spatially segregated regions called topologically-associating domains (TADs) defined by frequent intra-TAD contacts and infrequent contacts with neighboring regions ,250. MorDrosophila melanogaster gypsy insulator are SUMOylated, which attenuates chromatin insulator activity [Evidence has accumulated for SUMO controlling all these aspects of chromatin organization with impact on transcription ,200. A factivity . SUMOylaactivity , as wellactivity . SUMOylaactivity . Also woactivity . Along tactivity . Finallyactivity ,210. Foractivity , were shactivity . Moreoveactivity . PML proactivity . The rolPartitioning of euchromatin and heterochromatin is an important layer of chromatin organization. Whereas euchromatin comprises transcriptionally active regions of the genome, heterochromatin corresponds to condensed forms of chromatin with poor, or no, transcriptional activity . HeterocSchizosaccharomyces pombe was found less efficiently recruited in heterochromatin, which correlated with decreased HP1-mediated repression of transcription [Over the year, evidence has accumulated for multiple controls of heterochromatin formation by SUMO. As already mentioned, it was initially reported that SUMOylation of core histones can be associated with heterochromatin protein HP1γ recruitment  and thatcription . Moreovecription . It was cription . Moreovecription ,291. Whecription , indicatAnother noteworthy situation in mammalian cells is that of endogenous retroviruses. ERVs are vestiges of the long history of coevolution with retroposons that have shaped the genome. ERV proviruses are associated with H3K9me3 and their transcriptional silencing through heterochromatinization is important to avoid mutagenic events that would result from their retroposition . A genomIn the recent years, large-scale proteomics-, modifomics- and genomics studies have considerably improved our view of the role of SUMO in transcriptional regulation and unveiled novel layers of mechanistical complexity. Coupled to functional studies, they have shown that the three types of transcription  are concerned via SUMOylation of numerous molecular actors. These range from transcription factors and transcription co-regulators up to the transcriptional machineries themselves, as well as to many chromatin components and their organizers/remodelers. These studies also have established the existence of a complex dialog between SUMOylation and other post-translational modifications operating in the cell through molecular mechanisms that can be cooperative or antagonistic, depending on the situation. Numerous challenges however still remain ahead of us before featuring a precise picture of the many roles of SUMO in transcription. In particular, not only the complete repertoire of the post-translational modifications of all transcriptional actors will have to be established in a cell signaling- and/or development-dependent manner, but these data will also have to be integrated in scenarii taking into account the structure of the genome and its diverse transcription regulatory elements without forgetting the dynamics of its tridimensional organization. It will also be necessary to understand how SUMOylation events are coordinated in, and between, the supramolecular complexes involved in transcription and how this molecularly serves transcriptional control. Achieving this goal will undoubtfully require novel technological developments and investigation methodologies.


RB1) is the rate-limiting step in the initiation of both the hereditary and sporadic forms of retinoblastoma tumor. Furthermore, loss of function of the retinoblastoma tumor suppressor protein (pRB) is frequently found in most human cancers. In retinoblastoma, tumor progression is driven by epigenetic changes following pRB loss. This review focuses on the diverse functions of pRB in epigenetic regulation.Loss of function of the retinoblastoma gene (Mutations that result in the loss of function of pRB were first identified in retinoblastoma and since then have been associated with the propagation of various forms of cancer. pRB is best known for its key role as a transcriptional regulator during cell cycle exit. Beyond the ability of pRB to regulate transcription of cell cycle progression genes, pRB can remodel chromatin to exert several of its other biological roles. In this review, we discuss the diverse functions of pRB in epigenetic regulation including nucleosome mobilization, histone modifications, DNA methylation and non-coding RNAs. RB1) or deregulation of its gene product, the retinoblastoma tumor suppressor protein (pRB), are associated with several forms of cancer including retinoblastoma, osteosarcoma, adenocarcinomas, small cell lung cancer, breast cancer, prostate cancer, and others .,11.10,11The three pRB domains are connected by unstructured linker sequences that provide flexibility to the protein. These linker sequences are subjected to post-translational modifications, most notably CDK-dependent phosphorylation, affecting pRB interaction with other proteins and its functional roles ,17. In gGenomic DNA is packaged by histones and non-histone proteins into nucleosomes, the structural units of the eukaryotic chromatin and chromosomes. SWI/SNF complexes are multiprotein chromatin remodeling complexes found in eukaryotes that rearrange nucleosomes in an ATP-dependent manner ,20,21,22BRG1 and BRM binding to pRB is crucial for pRB-mediated repression of E2F1 activity and the resulting cell cycle arrest ,34. BRM Despite being predominantly associated with transcriptional activation, BRG1 forms pRB-SWI/SNF and pRB-SWI/SNF-HDAC repressor complexes, acting as cell cycle checkpoints during G1-S transition by inhibiting CYCLIN A and CYCLIN E, respectively ,33,34. TBrg1 in combination with pRB-loss enhances retinoblastoma tumorigenesis, suggesting that BRG1 acts as a tumor suppressor in the developing retina [It is incontestable that the interplay between pRB and SWI/SNF is critical for cellular homeostasis. SWI/SNF complexes are considered tumor suppressors and are mutated in nearly 20% of human malignancies . Specifig retina . In summHistone acetylation involves the transfer of an acetyl group from acetyl coenzyme A to lysine residues on histone tails by histone acetyltransferases (HATs). Histone acetylation relaxes the chromatin structure, allowing access of transcriptional activators to gene promoter regions to stimulate transcription. Several HATs have been identified, including transcriptional coactivators CBP/p300, PCAF, GCN5L, SRC1, ACTR and TAFII250. Removal of these acetyl groups from the tails of histone octamers is mediated by any of the 18 histone deacetylases (HDACs 1 to 18), ultimately influencing heterochromatinization of the nucleosome . These mThe binding to HDACs and other chromatin remodelers is facilitated by the LxCxE motif within the pocket structure of pRB . Since bAs mentioned previously, E2F1 and pRB also participate in transcription-independent functions in DNA repair ,67. In tAnother important mechanism for chromatin regulation involves histone methylation. Histone methylation is catalyzed by histone methyltransferases (HMTs) and removal of these methyl groups is mediated by histone demethylases (KDMs). In humans, there are over 100 predicted HMTs and more than 20 KDMs. Histone methylation comprises an elaborate network that can activate or repress chromatin depending on the amino acid residue modified and the proteins that bind to these modified tails. Among the most studied histone methylation marks associated with repressed chromatin are histone H3 lysine 9 di- and tri-methylation (H3K9me2/3), histone H4 lysine 20 trimethylation (H4K20me3), and histone H3 lysine 27 trimethylation (H3K27me3). Several studies indicate that pRB can interact with HMTs and KDMs independent of the LxCxE motif to repress transcription as well as to maintain heterochromatin in intergenic zones, centromeres, peri-centromeres, and telomeres ,68,69,70SUV39H1 is an HMT that functionally interacts with the heterochromatin protein 1 (HP1) to repress transcription at heterochromatic and euchromatic sites. SUV39H1 trimethylates H3K9, creating a repressive histone mark that serves as a binding site for HP1. The interaction between H3K9me3 and HP1 changes the nucleosome structure into a packed, transcriptionally inactive conformation. pRB can associate with SUV39H1 and HP1 through the pocket domain to repress transcription, including cell cycle genes cyclin E and cyclin A2 ,69. In tpRB also binds to HMTs SUV4-20H1 and SUV4-20H2, two H4K20 trimethylating enzymes . TrimethThe pRB/E2F complex can recruit enhancer of zeste homolog 2 (EZH2) to mediate H3K27me3 deposition to repress transcription during differentiation and stress . H3K27meThe role of pRB in regulating histone methylation also extends to interactions with KDMs. pRB can bind to KDM1A  and KDM5A , two KDMs that catalyze the removal of methyl groups from histone H3 lysine 4 (H3K4) ,76. Methde novo DNA methylation [DNMT1 and DNMT3A/B [DNA methylation involves the addition of a methyl group to the 5-carbon position of cytosine, forming 5-methylcytosine, at CpG dinucleotides ,79. Methhylation . pRB notDNMT3A/B ,86,87, bDNMT3A/B ,89,90,91As was discussed previously for histone modifiers, pRB transcriptional repression of E2F-regulated genes can also occur through direct recruitment of DNMT1. pRB can form a stable complex with E2F1, HDAC1, and DNMT1 and repress E2F-driven transcription . Even thde novo DNA methylation and stable gene silencing during cellular differentiation. In the HELLS-mediated transcriptional repression, HELLS also acts as a recruiting factor for DNMT1 and HDACs to establish transcriptionally repressive chromatin [The areas of the genome to be silenced by DNA methylation are recognized by chromatin-remodeling complexes that recruit DNMTs to the site. In this context, pRB is known to regulate the expression of chromatin remodeling proteins UHRF1  and HELLS , which recruit DNMT1 and DNMT3, respectively. UHRF1 is a direct downstream target of the pRB/E2F pathway ,89. As ahromatin . In retihromatin . ConsequThe majority of transcriptional outcomes depend on non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). MiRNAs are short nucleotide sequences (~22 nucleotides) known to regulate gene expression post-transcriptionally. MiRNAs are transcribed by RNA polymerase II, cleaved in the nucleus by the endonuclease, Droscha, and then exported to the cytoplasm for further processing by another endonuclease, Dicer, allowing the transcript to become a mature miRNA duplex (reviewed in ). The miCip1 is upregulated; however, the overexpression of miR-17~92 caused by pRB-deficiency counteracts the upregulation of p21Cip1, leading to tumor formation [Loss of pRB function and derepression of E2F-target genes results in deregulation of several ncRNA in RB (reviewed in ). Howeveormation . Severalormation .Several ncRNAs participate in auto-regulatory feedback loops. Therefore, many ncRNAs that regulate pRB may also be regulated by the pRB/E2F pathway. Research revealing ncRNA regulation by pRB should be further investigated for future engineering of potential therapeutic targets. In addition to the uncertainty regarding pRB regulation of ncRNAs, the degree of how much the regulatory networks differ between cancers compared to those in retinoblastoma is still unknown. A ncRNA biomarker for a specific cancer type may be different than those for retinoblastoma, even if that ncRNA is regulated by the pRB-E2F pathway. Further investigations on tissue specific expression patterns should be performed to reveal tissue-specific mechanisms of pRB regulation of ncRNAs.Even though pRB and the pRB/E2F pathway have been extensively studied for decades, the molecular mechanisms behind the wide-ranging roles of this protein and pathway are still yet to be fully elucidated. As reviewed here, several of pRB functions, both within its canonical role in cell cycle control and other non-canonical cellular functions, are exerted through its interactions with epigenetic modifiers (summarized in INK4A, CCND, CDK4/6, RB1, or E2Fs, genetic of functional inactivation of the pRB/E2F pathway seems to be indispensable for deregulated proliferation in most cancer settings [RB1 gene are found in retinoblastoma, osteosarcoma, and small cell lung cancer at a high frequency, and with less frequency in other types of cancer [INK4A are frequently detected in pancreatic cancer and non-small cell lung cancer, and CCND amplification is often found in breast cancer [RB1 gene is not completely synonymous to loss of E2F binding activity due to hyper-phosphorylation of pRB. From the multiple examples presented, it is evident that many of the pRB functions are likely preserved when pRB is hyper-phosphorylated. Thus, not every one of pRB’s function should be considered as integral for its role as a tumor suppressor. Recent studies on the interplay between pRB and chromatin remodelers have been able to explain, in part, the role of pRB in the epigenetic mechanisms that lead to tumorigenesis. Whether these can be harnessed to develop novel cancer therapeutics remains to be determined.As almost all human cancers carry abnormalities in the pRB/E2F pathway components including settings . Mutatiof cancer ,108. Mutt cancer ,109. Whi