Behind the scenes: How RNA orchestrates the epigenetic regulation of gene expression.

Non-coding DNA accounts for approximately 98.5% of the human genome. Once labeled as "junk DNA", this portion of the genome has undergone a progressive re-evaluation and it is now clear that some of its transcriptional products, belonging to the non-coding RNAs (ncRNAs), are key players in cell regulatory networks. A growing body of evidence demonstrates the crucial impact of regulatory ncRNAs on mammalian gene expression. Here, we focus on the defined relationship between chromatin-interacting RNAs, particularly long non-coding RNA (lncRNA), enhancer RNA (eRNA), non-coding natural antisense transcript (ncNAT), and circular RNA (circRNA) and epigenome, a common ground where both protein and RNA species converge to regulate cellular functions. Through several examples, this review provides an overview of the variety of targets, interactors, and mechanisms involved in the RNA-mediated modulation of loci-specific epigenetic states, a fundamental evolutive strategy to orchestrate mammalian gene expression in a timely and reversible manner. We will discuss how RNA-mediated epigenetic regulation impacts development and tissue homeostasis and how its alteration contributes to the onset and progression of many different human diseases, particularly cancer.

LINE-1 RNA causes heterochromatin erosion and is a target for amelioration of senescent phenotypes in progeroid syndromes.

Constitutive heterochromatin is responsible for genome repression of DNA enriched in repetitive sequences, telomeres, and centromeres. During physiological and pathological premature aging, heterochromatin homeostasis is profoundly compromised. Here, we showed that LINE-1 (Long Interspersed Nuclear Element-1; L1) RNA accumulation was an early event in both typical and atypical human progeroid syndromes. L1 RNA negatively regulated the enzymatic activity of the histone-lysine N-methyltransferase SUV39H1 (suppression of variegation 3-9 homolog 1), resulting in heterochromatin loss and onset of senescent phenotypes in vitro. Depletion of L1 RNA in dermal fibroblast cells from patients with different progeroid syndromes using specific antisense oligonucleotides (ASOs) restored heterochromatin histone 3 lysine 9 and histone 3 lysine 27 trimethylation marks, reversed DNA methylation age, and counteracted the expression of senescence-associated secretory phenotype genes such as p16, p21, activating transcription factor 3 (ATF3), matrix metallopeptidase 13 (MMP13), interleukin 1a (IL1a), BTG anti-proliferation factor 2 (BTG2), and growth arrest and DNA damage inducible beta (GADD45b). Moreover, systemic delivery of ASOs rescued the histophysiology of tissues and increased the life span of a Hutchinson-Gilford progeria syndrome mouse model. Transcriptional profiling of human and mouse samples after L1 RNA depletion demonstrated that pathways associated with nuclear chromatin organization, cell proliferation, and transcription regulation were enriched. Similarly, pathways associated with aging, inflammatory response, innate immune response, and DNA damage were down-regulated. Our results highlight the role of L1 RNA in heterochromatin homeostasis in progeroid syndromes and identify a possible therapeutic approach to treat premature aging and related syndromes.

Ubiquitin ligases HUWE1 and NEDD4 cooperatively control signal-dependent PRC2-Ezh1α/ß-mediated adaptive stress response pathway in skeletal muscle cells.

BACKGROUND: While the role of Polycomb group protein-mediated "cell memory" is well established in developmental contexts, little is known about their role in adult tissues and in particular in post-mitotic cells. Emerging evidence assigns a pivotal role in cell plasticity and adaptation. PRC2-Ezh1α/ß signaling pathway from cytoplasm to chromatin protects skeletal muscle cells from oxidative stress. However, detailed mechanisms controlling degradation of cytoplasmic Ezh1ß and assembly of canonical PRC2-Ezh1α repressive complex remain to be clarified. RESULTS: Here, we report NEDD4 ubiquitin E3 ligase, as key regulator of Ezh1ß. In addition, we report that ubiquitination and degradation of Ezh1ß is controlled by another layer of regulation, that is, one specific phosphorylation of serine 560 located at Ezh1ß-specific C terminal. Finally, we demonstrate that also Ezh1α needs to be stabilized under stress condition and this stabilization process requires decreased association pattern between another E3 ubiquitin ligase HUWE1. CONCLUSIONS: Together, these results shed light on key components that regulate PRC2-Ezh1α/ß pathway to direct modulation of epigenome plasticity and transcriptional output in skeletal muscle cells.

Nuclear AGO1 Regulates Gene Expression by Affecting Chromatin Architecture in Human Cells.

The impact of mammalian RNA interference components, particularly, Argonaute proteins, on chromatin organization is unexplored. Recent reports indicate that AGO1 association with chromatin appears to influence gene expression. To uncover the role of AGO1 in the nucleus, we used a combination of genome-wide approaches in control and AGO1-depleted HepG2 cells. We found that AGO1 strongly associates with active enhancers and RNA being produced at those sites. Hi-C analysis revealed AGO1 enrichment at the boundaries of topologically associated domains (TADs). By Hi-C in AGO1 knockdown cells, we observed changes in chromatin organization, including TADs and A/B compartment mixing, specifically in AGO1-bound regions. Distinct groups of genes and especially eRNA transcripts located within differentially interacting loci showed altered expression upon AGO1 depletion. Moreover, AGO1 association with enhancers is dependent on eRNA transcription. Collectively, our data suggest that enhancer-associated AGO1 contributes to the fine-tuning of chromatin architecture and gene expression in human cells.

Memories from the polycomb group proteins.

The first genes composing the Polycomb group (PcG) were identified 50 years ago in Drosophila melanogaster as essential developmental functions that regulate the correct segmental expression of homeotic selector genes. In the past two decades, what was initially described as a large family of chromatin-associated proteins involved in the maintenance of transcriptional repression to maintain cellular memory of homeotic genes turned out to be a highly conserved and sophisticated network of epigenetic regulators that play key roles in multiple aspects of cell physiology and identity, including regulation of all developmental genes, cell differentiation, stem and somatic cell reprogramming and response to environmental stimuli. These myriad phenotypes further spread interest for the contribution that PcG proteins revealed in the pathogenesis and progression of cancer and other complex diseases. Recent novel insights have increasingly clarified the molecular regulatory mechanisms at the basis of PcG-mediated epigenetic silencing and opened new visions about PcG functions in cells. In this review, we focus on the multiple modes of action of the PcG complexes and describe their biological roles.

Enhancer of zeste homolog 2 overexpression has a role in the development of anaplastic thyroid carcinomas.

CONTEXT: Enhancer of zeste homolog 2 (EZH2) is a histone lysine methyltransferase belonging to the polycomb group protein family. Overexpression of EZH2 has been found in several human malignancies including hematological and solid tumors. OBJECTIVES: In this study we investigated the expression levels of EZH2 and its polycomb group protein partners in thyroid carcinoma tissues with different degrees of malignancy to identify potential new therapeutic targets for anaplastic thyroid carcinoma (ATC). RESULTS: We show that high EZH2 expression levels are characteristic of undifferentiated ATC, whereas no significant changes were observed in well-differentiated papillary and follicular thyroid carcinomas as compared with normal thyroid. Knockdown of EZH2 in ATC cell lines results in cell growth inhibition, loss of anchorage-independent growth, migration, and invasion properties. Moreover, we demonstrate that EZH2 directly controls differentiation of ATC cells by silencing the thyroid specific transcription factor paired-box gene 8 (PAX8). CONCLUSIONS: EZH2 is specifically overexpressed in ATC, and it directly contributes to transcriptional silencing of PAX8 gene and ATC differentiation.

Noncoding RNA synthesis and loss of Polycomb group repression accompanies the colinear activation of the human HOXA cluster.

The ratio of noncoding to protein coding DNA rises with the complexity of the organism, culminating in nearly 99% of nonprotein coding DNA in humans. Nevertheless, a large portion of these regions is transcribed, creating the alleged paradox that noncoding RNA (ncRNA) represents the largest output of the human genome. Such a complex scenario may include epigenetic mechanisms where ncRNAs would be involved in chromatin regulation. We have investigated the intergenic, noncoding transcriptomes of mammalian HOX clusters. We show that "opposite strand transcription" from the intergenic spacer regions in the human HOXA cluster correlates with the activity state of adjacent HOXA genes. This noncoding transcription is regulated by the retinoic acid morphogen and follows the colinear activation pattern of the cluster. Opening of the cluster at sites of activation of intergenic transcripts is accompanied by changes in histone modifications and a loss of interaction with Polycomb group (PcG) repressive complexes. We propose that noncoding transcription is of fundamental importance for the opening and maintenance of the active state of HOX clusters.