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Simple Summary: Acute myeloid leukemia (AML) is characterized by the expansion of clonally derived hematopoietic precursors undergoing a partial or complete maturation block. De novo AML is characterized by recurrent cytogenetic alterations such as chromosomal translocations. However, in the recent past, it was shown that several somatic mutations and epigenetic alterations also contribute to AML onset and progression. Epigenetic mechanisms regulate the equilibrium between self-renewal and differentiation of hematopoietic stem cells and precursors. In this context, Polycomb-group (PcG) proteins regulate the expression of genes involved in cell-cycle regulation and differentiation, and aberrant expression and/or mutations of PcG genes have been shown to occur in hematopoietic neoplasms. In this study we analyzed the expression of Yin and Yang 1 protein (YY1), a member of the noncanonical PcG complexes, in AML patient samples and AML cell lines and the effect of YY1 downregulation on the AML differentiation block. Our results show that YY1 is significantly overexpressed in AML patient samples and AML cell lines and that YY1 knockdown relieves the differentiation block. YY1 downregulation in two AML cell lines (HL-60 and OCI-AML3) and one AML patient sample restored the expression of members of the CEBP protein family, increased the expression of extrinsic growth factors/receptors and surface antigenic markers, induced morphological cell characteristics typical of myeloid differentiation, and sensitized cells to retinoic acid treatment and to apoptosis. Overall, our data show that YY1 is not a secondary regulator of myeloid differentiation but that, if overexpressed, it can play a predominant role in myeloid differentiation block. [ABSTRACT FROM AUTHOR]

Simple Summary: The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50–300 nm spatial scale, is particularly interesting because the tridimensional arrangement of chromatin is implicated in multiple regulatory mechanisms. Indeed, a crucial hallmark of cellular life is the widespread ordering of many biological processes in nano-/mesoscopic domains (10–200 nm), which now may be revealed by an imaging toolbox referred to as super-resolution microscopy. In this context, polycomb proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription. This work reviews the current state-of-the-art super-resolution microscopy applied to polycomb proteins. Of note, super-resolution data have complemented cutting-edge molecular biology methods in providing a rational framework for understanding how polycomb proteins may shape 3D chromatin topologies and functions. The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50–300 nm spatial scale, is particularly interesting, as the 3D arrangement of chromatin is implicated in multiple regulatory mechanisms. In this context, polycomb group (PcG) proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription by combining chemical modifications of target histones with physical crosslinking of distal genomic regions and phase separation. The recent development of super-resolution microscopy (SRM) has strongly contributed to improving our comprehension of several aspects of nano-/mesoscale (10–200 nm) chromatin domains. Here, we review the current state-of-the-art SRM applied to PcG proteins, showing that the application of SRM to PcG activity and organization is still quite limited and mainly focused on the 3D assembly of PcG-controlled genomic loci. In this context, SRM approaches have mostly been applied to multilabel fluorescence in situ hybridization (FISH). However, SRM data have complemented the maps obtained from chromosome capture experiments and have opened a new window to observe how 3D chromatin topology is modulated by PcGs. [ABSTRACT FROM AUTHOR]

Super-resolution microscopy has been recently applied to understand the 3D topology of chromatin at an intermediated genomic scale (kilobases to a few megabases), as this corresponds to a sub-diffraction spatial scale crucial for the regulation of gene transcription. In this context, polycomb proteins are very renowned gene repressors that organize into the multiprotein complexes Polycomb Repressor Complex 1 (PRC1) and 2 (PRC2). PRC1 and PRC2 operate onto the chromatin according to a complex mechanism, which was recently recapitulated into a working model. Here, we present a functional colocalization study at 100–140 nm spatial resolution targeting PRC1 and PRC2 as well as the histone mark H3K27me3 by Image Scanning Microscopy (ISM). ISM offers a more flexible alternative to diffraction-unlimited SRMs such as STORM and STED, and it is perfectly suited to investigate the mesoscale of PRC assembly. Our data suggest a partially simultaneous effort of PRC1 and PRC2 in locally shaping the chromatin topology. [ABSTRACT FROM AUTHOR]

Keratinocyte differentiation is an essential process for epidermal stratification and stratum corneum formation. Keratinocytes proliferate in the basal layer of the epidermis and start their differentiation by changing their functional or phenotypical type; this process is regulated via induction or repression of epidermal differentiation complex (EDC) genes that play a pivotal role in epidermal development. Epidermal development and the keratinocyte differentiation program are orchestrated by several transcription factors, signaling pathways, and epigenetic regulators. The latter exhibits both activating and repressive effects on chromatin in keratinocytes via the ATP-dependent chromatin remodelers, histone demethylases, and genome organizers that promote terminal keratinocyte differentiation, and the DNA methyltransferases, histone deacetylases, and Polycomb components that stimulate proliferation of progenitor cells and inhibit premature activation of terminal differentiation-associated genes. In addition, microRNAs are involved in different processes between proliferation and differentiation during the program of epidermal development. Here, we bring together current knowledge of the mechanisms controlling gene expression during keratinocyte differentiation. An awareness of epigenetic mechanisms and their alterations in health and disease will help to bridge the gap between our current knowledge and potential applications for epigenetic regulators in clinical practice to pave the way for promising target therapies. [ABSTRACT FROM AUTHOR]

The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50–300 nm spatial scale, is particularly interesting, as the 3D arrangement of chromatin is implicated in multiple regulatory mechanisms. In this context, polycomb group (PcG) proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription by combining chemical modifications of target histones with physical crosslinking of distal genomic regions and phase separation. The recent development of super-resolution microscopy (SRM) has strongly contributed to improving our comprehension of several aspects of nano-/mesoscale (10–200 nm) chromatin domains. Here, we review the current state-of-the-art SRM applied to PcG proteins, showing that the application of SRM to PcG activity and organization is still quite limited and mainly focused on the 3D assembly of PcG-controlled genomic loci. In this context, SRM approaches have mostly been applied to multilabel fluorescence in situ hybridization (FISH). However, SRM data have complemented the maps obtained from chromosome capture experiments and have opened a new window to observe how 3D chromatin topology is modulated by PcGs.

Super-resolution microscopy has been recently applied to understand the 3D topology of chromatin at an intermediated genomic scale (kilobases to a few megabases), as this corresponds to a sub-diffraction spatial scale crucial for the regulation of gene transcription. In this context, polycomb proteins are very renowned gene repressors that organize into the multiprotein complexes Polycomb Repressor Complex 1 (PRC1) and 2 (PRC2). PRC1 and PRC2 operate onto the chromatin according to a complex mechanism, which was recently recapitulated into a working model. Here, we present a functional colocalization study at 100–140 nm spatial resolution targeting PRC1 and PRC2 as well as the histone mark H3K27me3 by Image Scanning Microscopy (ISM). ISM offers a more flexible alternative to diffraction-unlimited SRMs such as STORM and STED, and it is perfectly suited to investigate the mesoscale of PRC assembly. Our data suggest a partially simultaneous effort of PRC1 and PRC2 in locally shaping the chromatin topology.

Jasmonate (JA) regulates various aspects of plant growth and development and stress responses, with prominent roles in male reproductive development and defenses against herbivores and necrotrophic pathogens. JASMONATE-ZIM DOMAIN (JAZ) proteins are key regulators in the JA signaling pathway and function to repress the expression of JA-responsive genes. Here, we show that JAZ proteins directly interact with several chromatin-associated Polycomb proteins to mediate repressive chromatin modifications at JA-responsive genes and, thus, their transcriptional repression in Arabidopsis. Genetic analyses revealed that the developmental defects, including anther and pollen abnormalities, resulting from loss or block of JA signaling were partially rescued by loss of Polycomb protein-mediated chromatin silencing (Polycomb repression). We further found that JAZ-mediated transcriptional repression during anther and pollen development requires Polycomb proteins at four key regulatory loci. Analysis of genome-wide occupancy of a Polycomb factor and transcriptome reprogramming in response to JA revealed that Polycomb repression is involved in the repression of various JA-responsive genes. Taken together, our study reveals an important chromatin-based mechanism for JAZ-mediated transcriptional repression and JA signaling in plants. This study reports that JAZ proteins, the negative regulators of the jasmonate (JA) signaling pathway, directly interact and function together with chromatin-associated Polycomb factors to mediate repressive chromatin modifications and transcriptional repression at JA-responsive genes in Arabidopsis. This reveals a chromatin-based mechanism for JAZ-mediated transcriptional repression and JA signaling in plants. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (ncRNAs) are major regulators of gene expression and cell fate. The INK4 locus encodes the tumour suppressor proteins p15INK4b, p16INK4a and p14ARF required for cell cycle arrest and whose expression increases during senescence. ANRIL is a ncRNA antisense to the p15 gene. In proliferative cells, ANRIL prevents senescence by repressing INK4 genes through the recruitment of Polycomb-group proteins. In models of replicative and RASval12 oncogene-induced senescence (OIS), the expression of ANRIL and Polycomb proteins decreases, thus allowing INK4 derepression. Here, we found in a model of RAF1 OIS that ANRIL expression rather increases, due in particular to an increased stability. This led us to search for circular ANRIL isoforms, as circular RNAs are rather stable species. We found that the expression of two circular ANRIL increases in several OIS models (RAF1, MEK1 and BRAF). In proliferative cells, they repress p15 expression, while in RAF1 OIS, they promote full induction of p15, p16 and p14ARF expression. Further analysis of one of these circular ANRIL shows that it interacts with Polycomb proteins and decreases EZH2 Polycomb protein localization and H3K27me3 at the p15 and p16 promoters, respectively. We propose that changes in the ratio between Polycomb proteins and circular ANRIL isoforms allow these isoforms to switch from repressors of p15 gene to activators of all INK4 genes in RAF1 OIS. Our data reveal that regulation of ANRIL expression depends on the senescence inducer and underline the importance of circular ANRIL in the regulation of INK4 gene expression and senescence. [ABSTRACT FROM AUTHOR]

Abstract Background Numerous target genes of the Polycomb group (PcG) are transiently activated by a stimulus and subsequently repressed. However, mechanisms by which PcG proteins regulate such target genes remain elusive. Results We employed the heat shock-responsive hsp70 locus in Drosophila to study the chromatin dynamics of PRC1 and its interplay with known regulators of the locus before, during and after heat shock. We detected mutually exclusive binding patterns for HSF and PRC1 at the hsp70 locus. We found that Pleiohomeotic (Pho), a DNA-binding PcG member, dynamically interacts with Spt5, an elongation factor. The dynamic interaction switch between Pho and Spt5 is triggered by the recruitment of HSF to chromatin. Mutation in the protein–protein interaction domain (REPO domain) of Pho interferes with the dynamics of its interaction with Spt5. The transcriptional kinetics of the heat shock response is negatively affected by a mutation in the REPO domain of Pho. Conclusions We propose that a dynamic interaction switch between PcG proteins and an elongation factor enables stress-inducible genes to efficiently switch between ON/OFF states in the presence/absence of the activating stimulus.

This study reveals that high-copy satellite II (HSATII) sequences in the human genome can bind and impact distribution of chromatin regulatory proteins and that this goes awry in cancer. In many cancers, master regulatory proteins form two types of cancer-specific nuclear bodies, caused by locus-specific deregulation of HSATII. DNA demethylation at the 1q12 mega-satellite, common in cancer, causes PRC1 aggregation into prominent Cancer-Associated Polycomb (CAP) bodies. These loci remain silent, whereas HSATII loci with reduced PRC1 become derepressed, reflecting imbalanced distribution of UbH2A on these and other PcG-regulated loci. Large nuclear foci of HSATII RNA form and sequester copious MeCP2 into Cancer-Associated Satellite Transcript (CAST) bodies. Hence, HSATII DNA and RNA have an exceptional capacity to act as molecular sponges and sequester chromatin regulatory proteins into abnormal nuclear bodies in cancer. The compartmentalization of regulatory proteins within nuclear structure, triggered by demethylation of “junk” repeats, raises the possibility that this contributes to further compromise of the epigenome and neoplastic progression.

The PLZF transcription factor is essential for osteogenic differentiation of hMSCs; however, its regulation and molecular function during this process is not fully understood. Here, we revealed that the ZBTB16 locus encoding PLZF, is repressed by Polycomb (PcG) and H3K27me3 in naive hMSCs. At the pre-osteoblast stage of differentiation, the locus lost PcG binding and H3K27me3, gained JMJD3 recruitment, and H3K27ac resulting in high expression of PLZF. Subsequently, PLZF was recruited to osteogenic enhancers, influencing H3K27 acetylation and expression of nearby genes important for osteogenic function. Furthermore, we identified a latent enhancer within the ZBTB16/PLZF locus itself that became active, gained PLZF, p300 and Mediator binding and looped to the promoter of the nicotinamide N-methyltransferase (NNMT) gene. The increased expression of NNMT correlated with a decline in SAM levels, which is dependent on PLZF and is required for osteogenic differentiation.

Chromatin modifications, including methylation of histone H3 at lysine 27 (H3K27me) by the Polycomb group proteins, play a broadly conserved role in the maintenance of cell fate. Diverse chromatin organization modifier (chromo) domain proteins act as "readers" of histone methylation states. However, understanding the functional relationships among chromo domains and their roles in the inheritance of gene expression patterns remains challenging. Here, we identify two chromo-domain proteins, CEC-1 and CEC-6, as potential readers of H3K27me in Caenorhabditis elegans, where they have divergent expression patterns and contribute to distinct phenotypes. Both cec-1 and cec-6 genetically interact with another chromo-domain gene, cec-3, a reader of H3K9 methylation. Combined loss of cec-1 and cec-3 leads to developmental defects in the adult that result in decreased fitness. Furthermore, loss of cec-6 and cec-3 surprisingly leads to a progressive loss of fertility across generations, a "mortal germline" phenotype. Our results provide evidence of functional compensation between H3K27me and H3K9me heterochromatin pathways, and show that histone methylation readers contribute to both somatic development and transgenerational fitness. [ABSTRACT FROM AUTHOR]

Background: Numerous target genes of the Polycomb group (PcG) are transiently activated by a stimulus and subsequently repressed. However, mechanisms by which PcG proteins regulate such target genes remain elusive. Results: We employed the heat shock-responsive hsp70 locus in Drosophila to study the chromatin dynamics of PRC1 and its interplay with known regulators of the locus before, during and after heat shock. We detected mutually exclusive binding patterns for HSF and PRC1 at the hsp70 locus. We found that Pleiohomeotic (Pho), a DNA-binding PcG member, dynamically interacts with Spt5, an elongation factor. The dynamic interaction switch between Pho and Spt5 is triggered by the recruitment of HSF to chromatin. Mutation in the protein-protein interaction domain (REPO domain) of Pho interferes with the dynamics of its interaction with Spt5. The transcriptional kinetics of the heat shock response is negatively affected by a mutation in the REPO domain of Pho. Conclusions: We propose that a dynamic interaction switch between PcG proteins and an elongation factor enables stress-inducible genes to efficiently switch between ON/OFF states in the presence/absence of the activating stimulus. [ABSTRACT FROM AUTHOR]

Establishment of Kaposi's sarcoma-associated herpesvirus (KSHV) latency following infection is a multistep process, during which polycomb proteins are recruited onto the KSHV genome, which is crucial for the genome-wide repression of lytic genes during latency. Strikingly, only a subset of lytic genes are expressed transiently in the early phase of infection prior to the binding of polycomb proteins onto the KSHV genome, which raises the question what restricts lytic gene expression in the first hours of infection. Here, we demonstrate that both CTCF and cohesin chromatin organizing factors are rapidly recruited to the viral genome prior to the binding of polycombs during de novo infection, but only cohesin is required for the genome-wide inhibition of lytic genes. We propose that cohesin is required for the establishment of KSHV latency by initiating the repression of lytic genes following infection, which is an essential step in persistent infection of humans. [ABSTRACT FROM AUTHOR]

Natural products are considered as promising tools for the prevention and treatment of cancer. The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase unit of polycomb repressor complexes such as PRC2 complex that has oncogenic roles through interference with growth and metastatic potential. Several agents targeting EZH2 has been discovered but they often induce side effects in clinical trials. Recently, EZH2 has emerged as a potential target of natural products with documented anti-cancer effects and this discloses a new scenario for the development of EZH2 inhibitory strategies with agents with low cytotoxic detrimental effects. In fact, several natural products such as curcumin, triptolide, ursolic acid, sulforaphane, davidiin, tanshindiols, gambogic acid, berberine and Alcea rosea have been shown to serve as EZH2 modulators. Mechanisms like inhibition of histone H3K4, H3K27 and H3K36 trimethylation, down-regulation of matrix metalloproteinase expression, competitive binding to the S-adenosylmethionine binding site of EZH2 and modulation of tumor-suppressive microRNAs have been demonstrated to mediate the EZH2-inhibitory activity of the mentioned natural products. This review summarizes the pathways that are regulated by various natural products resulting in the suppression of EZH2, and provides a plausible molecular mechanism for the putative anti-cancer effects of these compounds. [ABSTRACT FROM AUTHOR]

Summary This study reveals that high-copy satellite II (HSATII) sequences in the human genome can bind and impact distribution of chromatin regulatory proteins and that this goes awry in cancer. In many cancers, master regulatory proteins form two types of cancer-specific nuclear bodies, caused by locus-specific deregulation of HSATII. DNA demethylation at the 1q12 mega-satellite, common in cancer, causes PRC1 aggregation into prominent Cancer-Associated Polycomb (CAP) bodies. These loci remain silent, whereas HSATII loci with reduced PRC1 become derepressed, reflecting imbalanced distribution of UbH2A on these and other PcG-regulated loci. Large nuclear foci of HSATII RNA form and sequester copious MeCP2 into Cancer-Associated Satellite Transcript (CAST) bodies. Hence, HSATII DNA and RNA have an exceptional capacity to act as molecular sponges and sequester chromatin regulatory proteins into abnormal nuclear bodies in cancer. The compartmentalization of regulatory proteins within nuclear structure, triggered by demethylation of “junk” repeats, raises the possibility that this contributes to further compromise of the epigenome and neoplastic progression. [ABSTRACT FROM AUTHOR]

ANRIL is a recently discovered long non-coding RNA encoded in the chromosome 9p21 region. This locus is a hotspot for disease-associated polymorphisms, and it has been consistently associated with cardiovascular disease, and more recently with several cancers, diabetes, glaucoma, endometriosis among other conditions. ANRIL has been shown to regulate its neighbor tumor suppressors CDKN2A/B by epigenetic mechanisms and thereby regulate cell proliferation and senescence. However, the clear role of ANRIL in the pathogenesis of these conditions is yet to be understood. Here, we review the recent findings on ANRIL molecular characterization and function, with a particular focus on its implications in human disease.

Gene expression control mediated by microRNAs and epigenetic remodeling of chromatin are interconnected processes often involved in feedback regulatory loops, which strictly guide proper tissue differentiation during embryonal development. Altered expression of microRNAs is one of the mechanisms leading to pathologic conditions, such as cancer. Several lines of evidence pointed to epigenetic alterations as responsible for aberrant microRNA expression in human cancers. Rhabdomyosarcoma and neuroblastoma are pediatric cancers derived from cells presenting features of skeletal muscle and neuronal precursors, respectively, blocked at different stages of differentiation. Consistently, tumor cells express tissue markers of origin but are unable to terminally differentiate. Several microRNAs playing a key role during tissue differentiation are often epigenetically downregulated in rhabdomyosarcoma and neuroblastoma and behave as tumor suppressors when re-expressed. Recently, inhibition of epigenetic modulators in adult tumors has provided encouraging results causing re-expression of anti-tumor master gene pathways. Thus, a similar approach could be used to correct the aberrant epigenetic regulation of microRNAs in rhabdomyosarcoma and neuroblastoma. The present review highlights the current insights on epigenetically deregulated microRNAs in rhabdomyosarcoma and neuroblastoma and their role in tumorigenesis and developmental pathways. The translational clinical implications and challenges regarding modulation of epigenetic chromatin remodeling/microRNAs interconnections are also discussed.

Polycomb Group (PcG) proteins are best-known for maintaining repressive or active chromatin states that are passed on across multiple cell divisions, and thus sustain long-term memory of gene expression. PcG proteins engage different, partly gene- and/or stage-specific, mechanisms to mediate spatiotemporal gene expression during central nervous system development. In the course of this, PcG proteins bind to various cis-regulatory sequences (e.g., promoters, enhancers or silencers) and coordinate, as well the interactions between distantly separated genomic regions to control chromatin function at different scales ranging from compaction of the linear chromatin to the formation of topological hubs. Recent findings show that PcG proteins are involved in switch-like changes in gene expression states of selected neural genes during the transition from multipotent to differentiating cells, and then to mature neurons. Beyond neurodevelopment, PcG proteins sustain mature neuronal function and viability, and prevent progressive neurodegeneration in mice. In support of this view, neuropathological findings from human neurodegenerative diseases point to altered PcG functions. Overall, improved insight into the multiplicity of PcG functions may advance our understanding of human neurodegenerative diseases and ultimately pave the way to new therapies.

Summary The polycomb repressive complex 2 (PRC2) methylates lysine 27 of histone H3 (H3K27) through its catalytic subunit Ezh2. PRC2-mediated di- and tri-methylation (H3K27me2/H3K27me3) have been interchangeably associated with gene repression. However, it remains unclear whether these two degrees of H3K27 methylation have different functions. In this study, we have generated isogenic mouse embryonic stem cells (ESCs) with a modified H3K27me2/H3K27me3 ratio. Our findings document dynamic developmental control in the genomic distribution of H3K27me2 and H3K27me3 at regulatory regions in ESCs. They also reveal that modifying the ratio of H3K27me2 and H3K27me3 is sufficient for the acquisition and repression of defined cell lineage transcriptional programs and phenotypes and influences induction of the ESC ground state. [ABSTRACT FROM AUTHOR]

Background: Enhancer of Zeste Drosophila Homologue 2 (EZH2) is a key regulator of transcription as a member of polycomb repressive complex 2 (PRC2) which exerts repression of downstream genes and is correlated to invasiveness and progression of different tumours. Therefore, we evaluated the expression of PRC2 proteins in pediatric soft tissue sarcoma (rhabdomyosarcoma, RMS and extraosseous Ewing sarcoma, EES) correlating them to the clinical outcome of the patients. Methods: We analyzed PRC2 protein expression by quantitative real time PCR, western blotting and immunohistochemistry in 17 soft tissue sarcomas (11 RMS and 6 EES) enrolled at Paediatric Oncology Units of the Second University of Naples. Expression analysis was performed for EZH2, SUZ12 and EED. Results: Enhancer of Zeste Drosophila Homologue 2 was expressed with a different degree in 60 % of samples. Interestingly, the magnitude of EZH2 up regulation was significantly higher in patients presenting lymph node and/ or distant metastases at the diagnosis. Moreover, patients overexpressing EZH2 had a lower probability of survival compared to patients negative or with low EZH2 expression. Conclusions: Our study suggests that high EZH2 expression is associated to increased aggressiveness of the disease. Therefore, drugs that control its activity could be potentially used in the epigenetic target treatment of tumors with these alterations. [ABSTRACT FROM AUTHOR]

The Polycomb group of proteins (PcGs) are transcriptional repressor complexes that regulate important biological processes and play critical roles in cancer. Mutating or deleting EZH2 can have both oncogenic and tumor suppressive functions by increasing or decreasing H3K27me3. In contrast, mutations of SUZ12 and EED are reported to have tumor suppressive functions. EZH2 is overexpressed in many cancers, including prostate cancer, which can lead to silencing of tumor suppressors, genes regulating epithelial to mesenchymal transition (EMT), and interferon signaling. In some cases, EZH2 overexpression also leads to its use of non-histone substrates. Lastly, PRC2 associated factors can influence the progression of cancer through progressive mutations or by specific binding to certain target genes. Here, we discuss which mutations and deletions of the PRC2 complex have been detected in different cancers, with a specific focus on the overexpression of EZH2 in prostate cancer. [ABSTRACT FROM AUTHOR]

Background: Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma arising from myogenic precursors that have lost their capability to differentiate into skeletal muscle. The polycomb-group protein EZH2 is a Lys27 histone H3 methyltransferase that regulates the balance between cell proliferation and differentiation by epigenetically silencing muscle-specific genes. EZH2 is often over-expressed in several human cancers acting as an oncogene. We previously reported that EZH2 inhibition induces cell cycle arrest followed by myogenic differentiation of RMS cells of the embryonal subtype (eRMS). MiR-101 is a microRNA involved in a negative feedback circuit with EZH2 in different normal and tumor tissues. To that, miR-101 can behave as a tumor suppressor in several cancers by repressing EZH2 expression. We, therefore, evaluated whether miR-101 is de-regulated in eRMS and investigated its interplaying with EZH2 as well as its role in the in vitro tumorigenic potential of these tumor cells. Results: Herein, we report that miR-101 is down-regulated in eRMS patients and in tumor cell lines compared to their controls showing an inverse pattern of expression with EZH2. We also show that miR-101 is up-regulated in eRMS cells following both genetic and pharmacological inhibition of EZH2. In turn, miR-101 forced expression reduces EZH2 levels as well as restrains the migratory potential of eRMS cells and impairs their clonogenic and anchorage-independent growth capabilities. Finally, EZH2 recruitment to regulatory region of miR-101-2 gene decreases in EZH2-silenced eRMS cells. This phenomenon is associated to reduced H3K27me3 levels at the same regulatory locus, indicating that EZH2 directly targets miR-101 for repression in eRMS cells. Conclusions: Altogether, our data show that, in human eRMS, miR-101 is involved in a negative feedback loop with EZH2, whose targeting has been previously shown to halt eRMS tumorigenicity. They also demonstrate that the re-induction of miR-101 hampers the tumor features of eRMS cells. In this scenario, epigenetic dysregulations confirm their crucial role in the pathogenesis of this soft tissue sarcoma. [ABSTRACT FROM AUTHOR]

Floral organ identity in plants is controlled by floral homeotic A/B/C/D/E-class genes. In Arabidopsis thaliana, several epigenetic repressors that regulate these floral organ identity genes have been characterized. However, the roles of epigenetic factors in rice floral development have not been explored in detail. Here, we report the identification and functional characterization of a rice epigenetic repressor, DEFORMED FLORAL ORGAN1 ( DFO1) gene, which causes abnormal floral morphology when mutated., We isolated dfo1 by mapping, and confirmed its function by rescue experiments, combined with genetic, cytological and molecular biological analysis., We showed that DFO1 is constitutively expressed and encodes a nuclear-localized protein. Mutation of DFO1 causes the ectopic expression of C-class genes in the dfo1-1 mutant, and overexpression of Os MADS58, a C-class gene, phenocopies the dfo1 mutants. In vitro and in vivo experiments demonstrated that DFO1 interacts with the rice polycomb group (PcG) proteins (Os MSI1 and Osi EZ1). Remarkably, trimethylation of histone H3 lysine 27, a mark of epigenetic repression, is significantly reduced on Os MADS58 chromatin in the dfo1-1 mutant., Our results suggest that DFO1 functions in maintaining rice floral organ identity by cooperating with PcG proteins to regulate the H3K27me3-mediated epigenetic repression on Os MADS58. [ABSTRACT FROM AUTHOR]

Background Embryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor- supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts. Methods Embryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo. Results Here we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell Cip1 proliferation up-regulating p21 . It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo. Conclusions These results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS. [ABSTRACT FROM AUTHOR]

Polycomb group ( Pc G) proteins are gene repressors that help to maintain cellular identity during development via chromatin remodeling. Fertilization-independent endosperm ( FIE), a member of the Pc G complex, operates extensively in plant development, but its role in rice has not been fully investigated to date., We report the isolation and characterization of a Pc G member in rice, which was designated Os FIE2 for Oryza sativa Fertilization- Independent Endosperm 2. Os FIE2 is a single-copy gene in the rice genome and shows a universal expression pattern., The Os FIE2 RNAi lines displayed pleiotropic phenotypes in vegetative and reproductive organ generation. In unfertilized lines, endosperm formation could be triggered without embryo formation, which indicates that FIE is indeed involved in the suppression of autonomous endosperm development in rice. Furthermore, lateral root generation was promoted early in the roots of Os FIE2 RNAi lines, whereas the primary root was premature and highly differentiated. As the root tip stem cell differentiated, QHB, the gene required for stem cell maintenance in the quiescent center, was down-regulated., Our data suggest that the Os FIE2- Pc G complex is vital for rice reproduction and endosperm formation. Its role in stem cell maintenance suggests that the gene is functionally conserved in plants as well as animals. [ABSTRACT FROM AUTHOR]

Polycomb-repressive complexes (PRCs) ensure the correct spatiotemporal expression of numerous key developmental regulators. Despite their pivotal role, how PRCs are recruited to specific targets remains largely unsolved, particularly in plants. Here we show that the Arabidopsis ASYMMETRIC LEAVES complex physically interacts with PRC2 and recruits this complex to the homeobox genes BREVIPEDICELLUS and KNAT2 to stably silence these stem cell regulators in differentiating leaves. The recruitment mechanism resembles the Polycomb response element-based recruitment of PRC2 originally defined in flies and provides the first such example in plants. Combined with recent studies in mammals, our findings reveal a conserved paradigm to epigenetically regulate homeobox gene expression during development. [ABSTRACT FROM AUTHOR]

ANRIL is a recently discovered long non-coding RNA encoded in the chromosome 9p21 region. This locus is a hotspot for disease-associated polymorphisms, and it has been consistently associated with cardiovascular disease, and more recently with several cancers, diabetes, glaucoma, endometriosis among other conditions. ANRIL has been shown to regulate its neighbor tumor suppressors CDKN2A/B by epigenetic mechanisms and thereby regulate cell proliferation and senescence. However, the clear role of ANRIL in the pathogenesis of these conditions is yet to be understood. Here, we review the recent findings on ANRIL molecular characterization and function, with a particular focus on its implications in human disease. [ABSTRACT FROM AUTHOR]

Abstract: It has been well documented that FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) plays important regulatory roles in diverse developmental processes in model plant Arabidopsis thaliana. However, it is largely unknown how FIE genes function in economically important crops. In this study, MhFIE gene, which was previously isolated from apomictic tea crabapple (Malus hupehensis Redh. var. pingyiensis), was introduced into tomato. The hemizygous transgenic tomato lines produced curly leaves and decreased in seed germination. In addition, the co-suppression of the transgenic MhFIE and endogenous (SlFIE) genes occurred in homozygous transgenic tomatoes. As a result, FIE silencing brought about abnormal phenotypes during reproductive development in tomato, such as increased sepal and petal numbers in flower, a fused ovule and pistil and parthenocarpic fruit formation. A yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) demonstrated that MhFIE interacted with a tomato protein, EZ2 (SlEZ2). Its ectopic expression and SlFIE co-suppression notably influenced the expression of genes associated with leaf, flower, and fruit development. Therefore, together with other PcG proteins, FIE was involved in the regulation of vegetative and reproductive development by modulating the expression of related genes in plants. [Copyright &y& Elsevier]

Gene expression control mediated by microRNAs and epigenetic remodeling of chromatin are interconnected processes often involved in feedback regulatory loops, which strictly guide proper tissue differentiation during embryonal development. Altered expression of microRNAs is one of the mechanisms leading to pathologic conditions, such as cancer. Several lines of evidence pointed to epigenetic alterations as responsible for aberrant microRNA expression in human cancers. Rhabdomyosarcoma and neuroblastoma are pediatric cancers derived from cells presenting features of skeletal muscle and neuronal precursors, respectively, blocked at different stages of differentiation. Consistently, tumor cells express tissue markers of origin but are unable to terminally differentiate. Several microRNAs playing a key role during tissue differentiation are often epigenetically downregulated in rhabdomyosarcoma and neuroblastoma and behave as tumor suppressors when re-expressed. Recently, inhibition of epigenetic modulators in adult tumors has provided encouraging results causing re-expression of anti-tumor master gene pathways. Thus, a similar approach could be used to correct the aberrant epigenetic regulation of microRNAs in rhabdomyosarcoma and neuroblastoma. The present review highlights the current insights on epigenetically deregulated microRNAs in rhabdomyosarcoma and neuroblastoma and their role in tumorigenesis and developmental pathways. The translational clinical implications and challenges regarding modulation of epigenetic chromatin remodeling/microRNAs interconnections are also discussed. [ABSTRACT FROM AUTHOR]

Abstract: The products of the FIS genes play important regulatory roles in diverse developmental processes, especially in seed formation after fertilization. In this study, a FIS-class gene MhFIE was isolated from apple. It encoded a predicted protein highly similar to polycomb group (PcG) protein FERTILIZATION-INDEPENDENT ENDOSPERM (FIE). MhFIE functioned as an Arabidopsis FIE homologue, as indicated by functional complementation experiment using Arabidopsis fie mutant. In addition, BiFC assay showed that MhFIE protein interacted with AtCLF. Furthermore, transgenic Arabidopsis ectopically expressing MhFIE produced less APETALA3 (AtAP3) and AGAMOUS (AtAG) transcripts than WT control, and therefore exhibited abnormal flower, seed development. These results suggested that polycomb complex including FIE and CLF proteins played an important role in reproductive development by regulating the expression of its downstream genes. In addition, it was found that MhFIE constitutively expressed in various tissues tested. Its expression levels were lower in apomictic apple species than the sexual reproductive species, suggested it was possibly involved into apomixis in apple. Furthermore, the hybrids of tea crabapple generated MhFIE transcripts at different levels. The parthenogenesis capacity was negatively correlated with MhFIE expression level in these hybrids. These results suggested that MhFIE was involved into the regulation of flower development and apomixis in apple. [Copyright &y& Elsevier]

Abstract: There is a growing interest for noncoding RNA (ncRNA)-mediated epigenetic regulation of transcription in diverse biological functions. Recent evidence suggests that a subset of long ncRNA epigenetically regulate the transcription of multiple genes in chromosomal domains via interaction with chromatin. Kcnq1ot1 is one such long chromatin-interacting ncRNA that silences multiple genes in the Kcnq1 domain by establishing a repressive higher order chromatin structure. This is done by the recruitment of chromatin and DNA-modifying proteins. This review looks at recent evidence supporting the notion that Kcnq1ot1-mediated silencing is a multilayered pathway. Comparing the mode of action of Kcnq1ot1 with other well-investigated chromatin regulatory long ncRNAs, such as Xist, HOTAIR and Airn, revealed that chromatin regulatory ncRNAs share common epigenetic pathways in the silencing of multiple genes. [Copyright &y& Elsevier]

Cancer evolution at all stages is driven by both epigenetic abnormalities as well as genetic alterations. Dysregulation of epigenetic control events may lead to abnormal patterns of DNA methylation and chromatin configurations, both of which are critical contributors to the pathogenesis of cancer. These epigenetic abnormalities are set and maintained by multiple protein complexes and the interplay between their individual components including DNA methylation machinery, histone modifiers, particularly, polycomb (PcG) proteins, and chromatin remodeling proteins. Recent advances in genome-wide technology have revealed that the involvement of these dysregulated epigenetic components appears to be extensive. Moreover, there is a growing connection between epigenetic abnormalities in cancer and concepts concerning stem-like cell subpopulations as a driving force for cancer. Emerging data suggest that aspects of the epigenetic landscape inherent to normal embryonic and adult stem/progenitor cells may help foster, under the stress of chronic inflammation or accumulating reactive oxygen species, evolution of malignant subpopulations. Finally, understanding molecular mechanisms involved in initiation and maintenance of epigenetic abnormalities in all types of cancer has great potential for translational purposes. This is already evident for epigenetic biomarker development, and for pharmacological targeting aimed at reversing cancer-specific epigenetic alterations. [ABSTRACT FROM AUTHOR]