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Apaf1 is a key regulator of the mitochondrial intrinsic pathway of apoptosis, as it activates executioner caspases by forming the apoptotic machinery apoptosome. Its genetic regulation and its post-translational modification are crucial under the various conditions where apoptosis occurs. Here we describe Ku70/86, a mediator of non-homologous end-joining pathway of DNA repair, as a novel regulator of Apaf1 transcription. Through analysing different Apaf1 promoter mutants, we identified an element repressing the Apaf1 promoter. We demonstrated that Ku70/86 is a nuclear factor able to bind this repressing element and downregulating Apaf1 transcription. We also found that Ku70/86 interaction with Apaf1 promoter is dynamically modulated upon DNA damage. The effect of this binding is a downregulation of Apaf1 expression immediately following the damage to DNA; conversely, we observed Apaf1 upregulation and apoptosis activation when Ku70/86 unleashes the Apaf1-repressing element. Therefore, besides regulating DNA repair, our results suggest that Ku70/86 binds to the Apaf1 promoter and represses its activity. This may help to inhibit the apoptosome pathway of cell death and contribute to regulate cell survival. [ABSTRACT FROM AUTHOR]

Chromatin three-dimensional (3D) organization inside the cell nucleus determines the separation of euchromatin and heterochromatin domains. Their segregation results in the definition of active and inactive chromatin compartments, whereby the local concentration of associated proteins, RNA and DNA results in the formation of distinct subnuclear structures. Thus, chromatin domains spatially confined in a specific 3D nuclear compartment are expected to share similar epigenetic features and biochemical properties, in terms of accessibility and solubility. Based on this rationale, we developed the 4f-SAMMY-seq to map euchromatin and heterochromatin based on their accessibility and solubility, starting from as little as 10 000 cells. Adopting a tailored bioinformatic data analysis approach we reconstruct also their 3D segregation in active and inactive chromatin compartments and sub-compartments, thus recapitulating the characteristic properties of distinct chromatin states. A key novelty of the new method is the capability to map both the linear segmentation of open and closed chromatin domains, as well as their compartmentalization in one single experiment., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Living organisms can detect and respond to physical forces at the cellular level. The pathways that transmit these forces to the nucleus allow cells to react quickly and consistently to environmental changes. Mechanobiology involves the interaction between physical forces and biological processes and is crucial for driving embryonic development and adapting to environmental cues during adulthood. Molecular studies have shown that cells can sense mechanical signals directly through membrane receptors linked to the cytoskeleton or indirectly through biochemical cascades that can influence gene expression for environmental adaptation. This review will explore the role of epigenetic modifications, emphasizing the 3D genome architecture and nuclear structures as responders to mechanical stimuli, which ensure cellular memory and adaptability. Understanding how mechanical cues are transduced and regulate cell functioning, governing processes such as cell programming and reprogramming, is essential for advancing our knowledge of human diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Enhancers are noncoding regulatory DNA regions that modulate the transcription of target genes, often over large distances along with the genomic sequence. Enhancer alterations have been associated with various pathological conditions, including cancer. However, the identification and characterization of somatic mutations in noncoding regulatory regions with a functional effect on tumorigenesis and prognosis remain a major challenge. Here, we present a strategy for detecting and characterizing enhancer mutations in a genome-wide analysis of patient cohorts, across three lung cancer subtypes. Lung tissue-specific enhancers were defined by integrating experimental data and public epigenomic profiles, and the genome-wide enhancer-target gene regulatory network of lung cells was constructed by integrating chromatin three-dimensional architecture data. Lung cancers possessed a similar mutation burden at tissue-specific enhancers and exons but with differences in their mutation signatures. Functionally relevant alterations were prioritized on the basis of the pathway-level integration of the effect of a mutation and the frequency of mutations on individual enhancers. The genes enriched for mutated enhancers converged on the regulation of key biological processes and pathways relevant to tumor biology. Recurrent mutations in individual enhancers also affected the expression of target genes, with potential relevance for patient prognosis. Together, these findings show that noncoding regulatory mutations have a potential relevance for cancer pathogenesis and can be exploited for patient classification., Significance: Mapping enhancer-target gene regulatory interactions and analyzing enhancer mutations at the level of their target genes and pathways reveal convergence of recurrent enhancer mutations on biological processes involved in tumorigenesis and prognosis., (©2023 The Authors; Published by the American Association for Cancer Research.)

The proper functioning of skeletal muscles is essential throughout life. A crucial crosstalk between the environment and several cellular mechanisms allows striated muscles to perform successfully. Notably, the skeletal muscle tissue reacts to an injury producing a completely functioning tissue. The muscle's robust regenerative capacity relies on the fine coordination between muscle stem cells (MuSCs or "satellite cells") and their specific microenvironment that dictates stem cells' activation, differentiation, and self-renewal. Critical for the muscle stem cell pool is a fine regulation of chromatin organization and gene expression. Acquiring a lineage-specific 3D genome architecture constitutes a crucial modulator of muscle stem cell function during development, in the adult stage, in physiological and pathological conditions. The context-dependent relationship between genome structure, such as accessibility and chromatin compartmentalization, and their functional effects will be analysed considering the improved 3D epigenome knowledge, underlining the intimate liaison between environmental encounters and epigenetics., (Copyright © 2024. Published by Elsevier Inc.)

The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma., (© 2022. The Author(s).)

The regulation of chromatin structure depends on a dynamic, multiple mechanisms that modulate gene expression and constitute the epigenome. The Polycomb group (PcG) of proteins are epigenetic factors involved in the transcriptional repression. Among their multilevel, chromatin-associated functions, PcG proteins mediate the establishment and maintenance of higher-order structures at target genes, allowing the transmission of transcriptional programs throughout the cell cycle.In the nucleus, PcG proteins localize close to the pericentric heterochromatin forming microscopically foci, called Polycomb bodies. Here, to visualize the tissue-specific PcG distribution in the aorta, dorsal skin and hindlimb muscles, we combine a fluorescence-activated cell sorter (FACS)-based method with an immunofluorescence staining., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Polycomb-group (PcG) of proteins are evolutionarily conserved transcription factors necessary for the regulation of gene expression during the development and the safeguard of cell identity in adulthood. In the nucleus, they form aggregates whose positioning and dimension are fundamental for their function. We present an algorithm, and its MATLAB implementation, based on mathematical methods to detect and analyze PcG proteins in fluorescence cell image z-stacks. Our algorithm provides a method to measure the number, the size, and the relative positioning of the PcG bodies in the nucleus for a better understanding of their spatial distribution, and thus of their role for a correct genome conformation and function., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In recent years, there has been an increase in research efforts surrounding RNA modification thanks to key breakthroughs in NGS-based whole transcriptome mapping methods. More than 100 modifications have been reported in RNAs, and some have been mapped at single-nucleotide resolution in the mammalian transcriptome. This has opened new research avenues in fields such as neurobiology, developmental biology, and oncology, among others. To date, we know that the RNA modification machinery finely tunes many diverse mechanisms involved in RNA processing and translation to regulate gene expression. However, it appears obvious to the research community that we have only just begun the process of understanding the several functions of the dynamic web of RNA modification, or the "epitranscriptome". To expand the data generated so far, recently published studies revealed a dual role for N6-methyladenosine (m6A), the most abundant mRNA modification, in driving both chromatin dynamics and transcriptional output. These studies showed that the m6A-modified, chromatin-associated RNAs could act as molecular docks, recruiting histone modification proteins and thus contributing to the regulation of local chromatin structure. Here, we review these latest exciting findings and outline outstanding research questions whose answers will help to elucidate the biological relevance of the m6A modification of chromatin-associated RNAs in mammalian cells.

The role of chromatin-associated RNAi components in the nucleus of mammalian cells and in particular in the context of developmental programs remains to be elucidated. Here, we investigate the function of nuclear Argonaute 1 (Ago1) in gene expression regulation during skeletal muscle differentiation. We show that Ago1 is required for activation of the myogenic program by supporting chromatin modification mediated by developmental enhancer activation. Mechanistically, we demonstrate that Ago1 directly controls global H3K27 acetylation (H3K27ac) by regulating enhancer RNA (eRNA)-CREB-binding protein (CBP) acetyltransferase interaction, a key step in enhancer-driven gene activation. In particular, we show that Ago1 is specifically required for myogenic differentiation 1 (MyoD) and downstream myogenic gene activation, whereas its depletion leads to failure of CBP acetyltransferase activation and blocking of the myogenic program. Our work establishes a role of the mammalian enhancer-associated RNAi component Ago1 in epigenome regulation and activation of developmental programs., Competing Interests: Declaration of interests Authors declare no competing interests., (Copyright © 2021 King Abdullah University of Science and Technology. Published by Elsevier Inc. All rights reserved.)

The Cdkn2a locus is one of the most studied tumor suppressor loci in the context of several cancer types. However, in the last years, its expression has also been linked to terminal differentiation and the activation of the senescence program in different cellular subtypes. Knock-out (KO) of the entire locus enhances the capability of stem cells to proliferate in some tissues and respond to severe physiological and non-physiological damages in different organs, including the heart. Emery-Dreifuss muscular dystrophy (EDMD) is characterized by severe contractures and muscle loss at the level of skeletal muscles of the elbows, ankles and neck, and by dilated cardiomyopathy. We have recently demonstrated, using the LMNA Δ 8-11 murine model of Emery-Dreifuss muscular dystrophy (EDMD), that dystrophic muscle stem cells prematurely express non-lineage-specific genes early on during postnatal growth, leading to rapid exhaustion of the muscle stem cell pool. Knock-out of the Cdkn2a locus in EDMD dystrophic mice partially restores muscle stem cell properties. In the present study, we describe the cardiac phenotype of the LMNA Δ 8-11 mouse model and functionally characterize the effects of KO of the Cdkn2a locus on heart functions and life expectancy.

Background: Chemotherapy is the first line of treatment for cancer patients. However, the side effects cause severe muscle atrophy or chemotherapy-induced cachexia. Previously, the NF-κB/MuRF1-dependent pathway was shown to induce chemotherapy-induced cachexia. We hypothesized that acute collateral toxic effects of chemotherapy on muscles might involve other unknown pathways promoting chemotherapy-induced muscle atrophy. In this study, we investigated differential effects of chemotherapeutic drugs and probed whether alternative molecular mechanisms lead to cachexia., Methods: We employed mouse satellite stem cell-derived primary muscle cells and mouse C2C12 progenitor cell-derived differentiated myotubes as model systems to test the effect of drugs. The widely used chemotherapeutic drugs, such as daunorubicin (Daun), etoposide (Etop), and cytarabine (Ara-C), were tested. Molecular mechanisms by which drug affects the muscle cell organization at epigenetic, transcriptional, and protein levels were measured by employing chromatin immunoprecipitations, endogenous gene expression profiling, co-immunoprecipitation, complementation assays, and confocal microscopy. Myotube function was examined using the electrical stimulation of myotubes to monitor contractile ability (excitation-contraction coupling) post drug treatment., Results: Here, we demonstrate that chemotherapeutic drugs disrupt sarcomere organization and thereby the contractile ability of skeletal muscle cells. The sarcomere disorganization results from severe loss of molecular motor protein MyHC-II upon drug treatment. We identified that drugs impede chromatin targeting of SETD7 histone methyltransferase and disrupt association and synergetic function of SETD7 with p300 histone acetyltransferase. The compromised transcriptional activity of histone methyltransferase and acetyltransferase causes reduced histone acetylation and low occupancy of active RNA polymerase II on MyHC-II, promoting drastic down-regulation of MyHC-II expression (~3.6-fold and ~4.5-fold reduction of MyHC-IId mRNA levels in Daun and Etop treatment, respectively. P < 0.0001). For MyHC-IIa, gene expression was down-regulated by ~2.6-fold and ~4.5-fold in Daun and Etop treatment, respectively (P < 0.0001). Very interestingly, the drugs destabilize SUMO deconjugase SENP3. Reduction in SENP3 protein level leads to deregulation of SETD7-p300 function. Importantly, we identified that SUMO deconjugation independent role of SENP3 regulates SETD7-p300 functional axis., Conclusions: The results show that the drugs critically alter SENP3-dependent synergistic action of histone-modifying enzymes in muscle cells. Collectively, we defined a unique epigenetic mechanism targeted by distinct chemotherapeutic drugs, triggering chemotherapy-induced cachexia., (© 2020 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Genome architecture and function are strictly related to nuclear structures, which contact chromatin at specific regions, regulating its compaction and three-dimensional higher-order structure, therefore contributing to specialized gene expression programs. Recently, growing evidence uncovers a dynamic role of nuclear structures in the plasticity of transcriptional programs. When the cellular microenvironment changes, external cues are transmitted to the nucleus through complex signalling cascades, finally resulting in a genome reorganization that allows the adjustment of the cell to a new condition. This process can be very rapid, especially in cells whose function is to contain sudden threats to the organism. Some examples are stem cells that switch from a quiescent to an activated state to replace damaged tissues or immune cells that, with a similar dynamic, identify and eliminate pathogens.Experimental treatments often require the isolation of cells from their physiological environment, exposing them to possible sudden changes in their nuclear architecture. Here we propose an early cross-linking on primary cells, a fixing method that can help to minimize the risk of nuclear structure alteration during the isolation process. We also bring some examples of downstream studies on early-fixed cells.

Hutchinson-Gilford progeria syndrome is a genetic disease caused by an aberrant form of Lamin A resulting in chromatin structure disruption, in particular by interfering with lamina associated domains. Early molecular alterations involved in chromatin remodeling have not been identified thus far. Here, we present SAMMY-seq, a high-throughput sequencing-based method for genome-wide characterization of heterochromatin dynamics. Using SAMMY-seq, we detect early stage alterations of heterochromatin structure in progeria primary fibroblasts. These structural changes do not disrupt the distribution of H3K9me3 in early passage cells, thus suggesting that chromatin rearrangements precede H3K9me3 alterations described at later passages. On the other hand, we observe an interplay between changes in chromatin accessibility and Polycomb regulation, with site-specific H3K27me3 variations and transcriptional dysregulation of bivalent genes. We conclude that the correct assembly of lamina associated domains is functionally connected to the Polycomb repression and rapidly lost in early molecular events of progeria pathogenesis.

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin Δ8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth. We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin Δ8-11 -/- mutant mice these capabilities are severely impaired. This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

The recent advances, offered by cell therapy in the regenerative medicine field, offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors, such as myoblasts or satellite cells, possess a marked regenerative capacity, but the exploitation of this potential still encounters significant challenges in clinical application, due to low rate of proliferation in vitro, as well as a reduced self-renewal capacity. In this scenario, induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches, but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs, generated from pericytes and fibroblasts, exploiting skeletal muscle-derived extracellular vesicles (EVs), in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological "shuttles" to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches.

Lamin A is a component of the inner nuclear membrane that, together with epigenetic factors, organizes the genome in higher order structures required for transcriptional control. Mutations in the lamin A/C gene cause several diseases belonging to the class of laminopathies, including muscular dystrophies. Nevertheless, molecular mechanisms involved in the pathogenesis of lamin A-dependent dystrophies are still largely unknown. The polycomb group (PcG) of proteins are epigenetic repressors and lamin A interactors, primarily involved in the maintenance of cell identity. Using a murine model of Emery-Dreifuss muscular dystrophy (EDMD), we show here that lamin A loss deregulated PcG positioning in muscle satellite stem cells, leading to derepression of non-muscle-specific genes and p16INK4a, a senescence driver encoded in the Cdkn2a locus. This aberrant transcriptional program caused impairment in self-renewal, loss of cell identity, and premature exhaustion of the quiescent satellite cell pool. Genetic ablation of the Cdkn2a locus restored muscle stem cell properties in lamin A/C-null dystrophic mice. Our findings establish a direct link between lamin A and PcG epigenetic silencing and indicate that lamin A-dependent muscular dystrophy can be ascribed to intrinsic epigenetic dysfunctions of muscle stem cells.

Among rare diseases caused by mutations in LMNA gene, Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B are characterized by muscle weakness and wasting, joint contractures, cardiomyopathy with conduction system disorders. Circulating biomarkers for these pathologies have not been identified. Here, we analyzed the secretome of a cohort of patients affected by these muscular laminopathies in the attempt to identify a common signature. Multiplex cytokine assay showed that transforming growth factor beta 2 (TGF β2) and interleukin 17 serum levels are consistently elevated in the vast majority of examined patients, while interleukin 6 and basic fibroblast growth factor are altered in subgroups of patients. Levels of TGF β2 are also increased in fibroblast and myoblast cultures established from patient biopsies as well as in serum from mice bearing the H222P Lmna mutation causing Emery-Dreifuss Muscular Dystrophy in humans. Both patient serum and fibroblast conditioned media activated a TGF β2-dependent fibrogenic program in normal human myoblasts and tenocytes and inhibited myoblast differentiation. Consistent with these results, a TGF β2 neutralizing antibody avoided fibrogenic marker activation and myogenesis impairment. Cell intrinsic TGF β2-dependent mechanisms were also determined in laminopathic cells, where TGF β2 activated AKT/mTOR phosphorylation. These data show that TGF β2 contributes to the pathogenesis of Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B and can be considered a potential biomarker of those diseases. Further, the evidence of TGF β2 pathogenetic effects in tenocytes provides the first mechanistic insight into occurrence of joint contractures in muscular laminopathies.

The alteration of the several roles that Lamin A/C plays in the mammalian cell leads to a broad spectrum of pathologies that - all together - are named laminopathies. Among those, the Emery Dreifuss Muscular Dystrophy (EDMD) is of particular interest as, despite the several known mutations of Lamin A/C, the genotype-phenotype correlation still remains poorly understood; this suggests that the epigenetic background of patients might play an important role during the time course of the disease. Historically, both a mechanical role of Lamin A/C and a regulative one have been suggested as the driving force of laminopathies; however, those two hypotheses are not mutually exclusive. Recent scientific evidence shows that Lamin A/C sustains the correct gene expression at the epigenetic level thanks to the Lamina Associated Domains (LADs) reorganization and the crosstalk with the Polycomb Group of Proteins (PcG). Furthermore, the PcG-dependent histone mark H3K27me3 increases under mechanical stress, finally pointing out the link between the mechano-properties of the nuclear lamina and epigenetics. Here, we summarize the emerging mechanisms that could explain the high variability seen in Emery Dreifuss muscular dystrophy.

The evolution of chromatin-based epigenetic cell memory may be driven not only by the necessity for cells to stably maintain transcription programs, but also by the need to recognize signals and allow plastic responses to environmental stimuli. The mechanistic role of the epigenome in adult postmitotic tissues, however, remains largely unknown. In vertebrates, two variants of the Polycomb repressive complex (PRC2-Ezh2 and PRC2-Ezh1) control gene silencing via methylation of histone H3 on Lys27 (H3K27me). Here we describe a reversible mechanism that involves a novel isoform of Ezh1 (Ezh1β). Ezh1β lacks the catalytic SET domain and acts in the cytoplasm of skeletal muscle cells to control nuclear PRC2-Ezh1 activity in response to atrophic oxidative stress, by regulating Eed assembly with Suz12 and Ezh1α (the canonical isoform) at their target genes. We report a novel PRC2-Ezh1 function that utilizes Ezh1β as an adaptive stress sensor in the cytoplasm, thus allowing postmitotic cells to maintain tissue integrity in response to environmental changes.

Epigenetic mechanisms modulate and maintain the transcriptional state of the genome acting at various levels on chromatin. Emerging findings suggest that the position in the nuclear space and the cross talk between components of the nuclear architecture play a role in the regulation of epigenetic signatures. We recently described a cross talk between the Polycomb group of proteins (PcG) epigenetic repressors and the nuclear lamina. This interplay is important for the maintenance of transcriptional repression at muscle-specific genes and for the correct timing of muscle differentiation. To investigate the synergism between PcG factors and nuclear architecture we improved a chromatin fractionation protocol with the aim to analyze the PcG nuclear compartmentalization. We thus separated PcG proteins in different fractions depending on their solubility. We surprisingly found a consistent amount of PcG proteins in the matrix-associated fraction. In this chapter we describe the chromatin fractionation procedure, a method that can be used to study the nuclear compartmentalization of Polycomb group of proteins and/or PcG targets in murine and Drosophila cells.

The large amount of data generated in biological experiments that rely on advanced microscopy can be handled only with automated image analysis. Most analyses require a reliable cell image segmentation eventually capable of detecting subcellular structures.We present an automatic segmentation method to detect Polycomb group (PcG) proteins areas isolated from nuclei regions in high-resolution fluorescent cell image stacks. It combines two segmentation algorithms that use an active contour model and a classification technique serving as a tool to better understand the subcellular three-dimensional distribution of PcG proteins in live cell image sequences. We obtained accurate results throughout several cell image datasets, coming from different cell types and corresponding to different fluorescent labels, without requiring elaborate adjustments to each dataset.

Beyond its role in providing structure to the nuclear envelope, lamin A/C is involved in transcriptional regulation. However, its cross talk with epigenetic factors--and how this cross talk influences physiological processes--is still unexplored. Key epigenetic regulators of development and differentiation are the Polycomb group (PcG) of proteins, organized in the nucleus as microscopically visible foci. Here, we show that lamin A/C is evolutionarily required for correct PcG protein nuclear compartmentalization. Confocal microscopy supported by new algorithms for image analysis reveals that lamin A/C knock-down leads to PcG protein foci disassembly and PcG protein dispersion. This causes detachment from chromatin and defects in PcG protein-mediated higher-order structures, thereby leading to impaired PcG protein repressive functions. Using myogenic differentiation as a model, we found that reduced levels of lamin A/C at the onset of differentiation led to an anticipation of the myogenic program because of an alteration of PcG protein-mediated transcriptional repression. Collectively, our results indicate that lamin A/C can modulate transcription through the regulation of PcG protein epigenetic factors., (© 2015 Cesarini et al.)

Background: Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated., Principal Findings: Here we show that the endogenous Dicer-2/Argonaute-2 RNAi pathway is dispensable for the PcG mediated silencing of the homeotic Bithorax Complex (BX-C). Although Dicer-2 depletion triggers mild transcriptional activation at Polycomb Response Elements (PREs), this does not induce transcriptional changes at PcG-repressed genes. Moreover, Dicer-2 is not needed to maintain global levels of methylation of lysine 27 of histone H3 and does not affect PRE-mediated higher order chromatin structures within the BX-C. Finally bioinformatic analysis, comparing published data sets of PcG targets with Argonaute-2-bound small RNAs reveals no enrichment of these small RNAs at promoter regions associated with PcG proteins., Conclusions: We conclude that the Dicer-2/Argonaute-2 RNAi pathway, despite its role in pairing sensitive gene silencing of transgenes, does not have a role in PcG dependent silencing of major homeotic gene cluster loci in Drosophila.