In Germany and worldwide, the average age of the population is continuously rising. With this general increase in chronological age, the focus on biological age, meaning the actual health and fitness status, is becoming more and more important. The key question is to what extent the age-related decline in fitness is genetically predetermined or malleable by environmental factors and lifestyle. Many epigenetic studies in aging research have provided interesting insights in this nature-versus-nurture debate. In most model organisms, aging is associated with specific epigenetic changes, which can be countered by certain interventions like moderate caloric restriction or increased physical activity. Since these interventions also have positive effects on lifespan and health, epigenetics appears to be the interface between environmental factors and the aging process. This notion is supported by the fact that an epigenetic drift occurs through the life course of identical twins, which is related to the different manifestations of aging symptoms. Furthermore, biological age can be determined with high precision based on DNA methylation patterns, further emphasizing the importance of epigenetics in aging. This article provides an overview of the importance of genetic and epigenetic parameters for life expectancy. A major focus will be on the possibilities of maintaining a young epigenome through lifestyle and environmental factors, thereby slowing down biological aging.

Summary: Extensive remodeling of the female mammary epithelium during development and pregnancy has been linked to cancer susceptibility. The faithful response of mammary epithelial cells (MECs) to hormone signaling is key to avoiding breast cancer development. Here, we show that lactogenic differentiation of murine MECs requires silencing of genes encoding ribosomal RNA (rRNA) by the antisense transcript PAPAS. Accordingly, knockdown of PAPAS derepresses rRNA genes, attenuates the response to lactogenic hormones, and induces malignant transformation. Restoring PAPAS levels in breast cancer cells reduces tumorigenicity and lung invasion and activates many interferon-regulated genes previously linked to metastasis suppression. Mechanistically, PAPAS transcription depends on R-loop formation at the 3′ end of rRNA genes, which is repressed by RNase H1 and replication protein A (RPA) overexpression in breast cancer cells. Depletion of PAPAS and upregulation of RNase H1 and RPA in human breast cancer underpin the clinical relevance of our findings.

Molecular communication between host and microbe is mediated by the transfer of many different classes of macromolecules. Recently, the trafficking of RNA molecules between organisms has gained prominence as an efficient way to manipulate gene expression via RNA interference (RNAi). Here, we posit a new epigenetic control mechanism based on triple helix (triplex) structures comprising nucleic acids from both host and microbe. Indeed, RNA:DNA triplexes are known to regulate gene expression in humans, but it is unknown whether interkingdom triplexes are formed either to manipulate host processes during pathogenesis or as a host defense response. We hypothesize that a fraction of the extracellular RNAs commonly released by microbes (e.g., bacteria, fungi, and protists) and their hosts form triplexes with the genome of the other species, thereby impacting chromatin conformation and gene expression. We invite the field to consider interkingdom triplexes as unexplored weaponry in the arms race between host and microbe.

Ribosome biogenesis is initiated by RNA polymerase I (Pol I)-mediated synthesis of pre-ribosomal RNA (pre-rRNA). Pol I activity was previously linked to longevity, but the underlying mechanisms were not studied beyond effects on nucleolar structure and protein translation. Here we use multi-omics and functional tests to show that curtailment of Pol I activity remodels the lipidome and preserves mitochondrial function to promote longevity in Caenorhabditis elegans . Reduced pre-rRNA synthesis improves energy homeostasis and metabolic plasticity also in human primary cells. Conversely, the enhancement of pre-rRNA synthesis boosts growth and neuromuscular performance of young nematodes at the cost of accelerated metabolic decline, mitochondrial stress and premature aging. Moreover, restriction of Pol I activity extends lifespan more potently than direct repression of protein synthesis, and confers geroprotection even when initiated late in life, showcasing this intervention as an effective longevity and metabolic health treatment not limited by aging.

Zusammenfassung In Deutschland und weltweit steigt das Durchschnittsalter der Bevölkerung immer weiter an. Mit dieser allgemeinen Zunahme des chronologischen Alters wird der Fokus auf das biologische Alter, d. h. den tatsächlichen Gesundheits- und Fitnesszustand, immer wichtiger. Hier stellt sich die zentrale Frage, inwieweit die altersbedingte Abnahme der Fitness genetisch vorbestimmt oder durch Umweltfaktoren und Lebensstil beeinflussbar ist. Bei dieser Nature-versus-Nurture-Debatte haben viele epigenetische Studien in der Alternsforschung interessante Einblicke geliefert. In den meisten Modellorganismen geht das Altern mit bestimmten epigenetischen Veränderungen einher, denen unter anderem durch moderate Kalorienreduzierung oder vermehrte körperliche Aktivität entgegengewirkt werden kann. Da sich diese Interventionen auch positiv auf die Lebensspanne und Gesundheit auswirken, scheint die Epigenetik im Mittelpunkt zwischen Umwelteinflüssen und Alternsprozessen zu stehen. Hierfür spricht auch, dass es im Verlauf des Lebens von eineiigen Zwillingen eine epigenetische Drift gibt, die mit der unterschiedlichen Ausprägung von Alterserscheinungen zusammenhängt. Darüber hinaus lässt sich anhand von DNA-Methylierungsmustern das biologische Alter sehr präzise bestimmen, was die Bedeutung der Epigenetik für das Altern weiter untermauert. Dieser Beitrag gibt eine Übersicht über die Bedeutung von genetischen und epigenetischen Parametern für die Lebenserwartung. Dabei wird ein Augenmerk auf den Möglichkeiten liegen, durch Lebensstil und Umweltfaktoren ein junges Epigenom zu erhalten, um so das biologische Altern zu verlangsamen.

C (PLC) signaling. We identi fi ed PLCgamma 1 (PLCG1) as a speci fi c target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These fi ndings are extended to and con fi rmed by pharmacologic perturbation of Ca 11 -signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO 1 leukemic stem cells.

In an effort to identify novel drugs targeting fusion-oncogene-induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells.

The genomes of complex eukaryotes largely contain non-protein-coding DNA, which is pervasively transcribed into a plethora of non-coding RNAs (ncRNAs). The functional importance of many of these ncRNAs has been investigated in the last two decades, revealing their crucial and multifaceted roles in chromatin regulation. A common mode of action of ncRNAs is the recruitment of chromatin modifiers to specific regions in the genome. Whereas many ncRNA-protein interactions have been characterised in detail, binding of ncRNAs to their DNA target sites is much less understood. Recently developed RNA-centric methods have mapped the genome-wide distribution of ncRNAs, however, how ncRNAs achieve locus-specificity remains mainly unresolved. In terms of direct RNA-DNA interactions, two kinds of triple-stranded structures can be formed: R-loops consisting of an RNA:DNA hybrid and a looped out DNA strand, and RNA:DNA triple helices (triplexes), in which the RNA binds to the major groove of the DNA double helix by sequence-specific Hoogsteen base pairing. In this essay, we will review the current knowledge about RNA:DNA triplexes, summarising triplex formation rules, detection methods, and ncRNAs reported to engage in triplexes. While the functional characterisation of RNA:DNA triplexes is still anecdotal, recent advances in high-throughput and computational analyses indicate their widespread distribution in the genome. Thus, we are witnessing a paradigm shift in the appreciation of RNA:DNA triplexes, away from exotic structures towards a prominent mode of ncRNA-chromatin interactions.

Ribosome biogenesis takes place mainly in the nucleolus, a nuclear, non-membrane bound organelle forming around the gene arrays encoding ribosomal RNA (rRNA). Nucleolar activity comprises synthesis, processing and maturation of rRNAs, followed by their assembly with ribosomal proteins into pre-ribosomal particles. The final formation of translation-competent ribosomes in the cytoplasm is the prerequisite for protein synthesis, which is the most energy-consuming cellular process. In adult stem cells, ribosome biogenesis and protein synthesis determine the switch between the quiescent and the activated state, but also decide whether activated stem cells self-renew or differentiate. Given this major impact on cellular function, it seems likely that perturbations of the circuitry between nucleolar activity and translation lead to ageing-related stem cell deterioration. This review provides an overview of how ribosome biogenesis and translation govern stem cell function and discusses the resultant implication in stem cell ageing.

A major transcriptional output of cells is ribosomal RNA (rRNA), synthesized by RNA polymerase I (Pol I) from multicopy rRNA genes (rDNA). Constitutive silencing of an rDNA fraction by promoter CpG methylation contributes to the stabilization of these otherwise highly active loci. In cancers driven by the oncoprotein Myc, excessive Myc directly stimulates rDNA transcription. However, it is not clear when during carcinogenesis this mechanism emerges, and how Myc-driven rDNA activation affects epigenetic silencing. Here, we have used the Eµ-Myc mouse model to investigate rDNA transcription and epigenetic regulation in Myc-driven B cell lymphomagenesis. We have developed a refined cytometric strategy to isolate B cells from the tumor initiation, promotion, and progression phases, and found a substantial increase of both Myc and rRNA gene expression only in established lymphoma. Surprisingly, promoter CpG methylation and the machinery for rDNA silencing were also strongly up-regulated in the tumor progression state. The data indicate a dichotomous role of oncogenic Myc in rDNA regulation, boosting transcription as well as reinforcing repression of silent repeats, which may provide a novel angle on perturbing Myc function in cancer cells.

Chromosomal translocations found in acute myeloid leukemia (AML) can generate oncogenic fusions with aberrant epigenetic and transcriptional functions. However, direct therapeutic targeting of leukemia fusion proteins has not been accomplished so far. Although high remission rates can be induced in patients diagnosed with AML1-ETO/t(8;21)-positive AML only half of them achieve long-term disease-free survival (Papaemmanuiel et al., NEJM, 2016). In the other half of these patients, the disease maintaining leukemia stem cell (LSC) clone is not eliminated by chemotherapy. A functional characteristic of LSCs is unlimited self-renewal capacity and several signaling pathways have been identified that maintain stem cell self-renewal. Targeting the oncogene induced self-renewal capacity of LSCs has great potential to eliminate the malignant clone and prevent relapse. To identify oncogenic cellular functions with relevance for LSC self-renewal, we performed global proteome profiling in murine AML1-ETO9a (AE) compared to MLL-AF9 (MA9) driven LSCs. Gene set enrichment analyses revealed a significant enrichment of calcium-dependent cellular functions and Phospholipase C (PLC)-signaling in AE LSCs. These data could be confirmed in sorted CD34+ blasts from AE-positive AML when compared to non-AE-AML. All PLC family members are regulators of Ca2+ homeostasis. However, when analyzing published AML gene expression datasets we found exclusively PLCG1 to be highly expressed in t(8;21) AML. Conditional activation of AE in embryonic stem cells resulted in induction of PLCG1 expression and PLCG1 was identified as a direct target of the AE fusion by ChIP-sequencing in AE-positive Kasumi-1 cells.Here, PLCG1 depletion resulted in reduced Ca2+ release, impaired proliferation and reduced colony formation in vitro. In a xenograft model, inactivation of PLCG1 resulted not only in delayed disease development (median survival shNT vs. shPLCG1: 135 days vs. not reached, p=0.02) but also in reduction of disease penetrance by 87%. Consistent with these results, transcriptome analysis revealed strong induction of gene sets related to myeloid differentiation and down-regulation of gene sets linked to proliferation, stemness and c-Myc targets. To confirm the functional role of PLCG1-signaling in AE-driven LSCs, we generated a new conditional knockout mouse model for Plcg1 and induced leukemia using the oncogenes AE and KRAS-G12D (AE/K). Genetic inactivation of Plcg1in vivo after engraftment of leukemic cells resulted in significant reduction of LSC numbers (p=0.04) and a reduction of disease penetrance by 67% in primary recipients. Isolated LSCs revealed induction of differentiation, loss of cell cycle activity and failed to re-establish disease in secondary recipients (Plcg1+/+ vs. Plcg1-/-: median survival 12 days vs. not reached; p=0.0001). In contrast, genetic deletion of Plcg1 appeared to be dispensable for normal murine HSC function during primary and secondary transplantation. Primary human t(8;21) AML cells (derived from 4 different donors) showed impaired colony forming capacity following PLCG1 inactivation in vitro irrespective of co-occurring mutations while colony formation of human CD34+ BM cells was not affected to a major extent. As Ca2+ signaling appeared deregulated in t(8;21) AML, we aimed to investigate the effects of pharmacologic Ca2+ inhibition as a tractable target downstream of PLCG1. To assess specifically for LSC function, we treated primary recipient mice with established AE/K-driven leukemia with the clinically approved calcineurin inhibitor ciclosporin (CsA), a compound that blocks intracellular Ca2+ release. CsA-treated animals showed reduction in total leukemic burden (spleen weight diluent vs. CsA, p=0.01) and LSC numbers (p=0.02). This resulted in increased survival of secondary recipient hosts (diluent vs. CsA: median 15 vs. 29 days, p=0.0002). These effects could not be observed for other oncogenes (e.g. MA9), confirming its specificity for AE-induced disease. Consistently, CsA treated primary human t(8;21)-positive AML blasts failed to form colonies in methylcellulose. In summary, our findings identified PLCG1-dependent Ca2+ signaling as a critical pathway for t(8;21) LSC maintenance and self-renewal. Most importantly, as PLCG1 is dispensable for maintenance of normal HSPCs, PLCG1 could serve as a novel therapeutic target in t(8;21) AML. Disclosures Döhner: Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria; CTI Biopharma: Consultancy, Honoraria. Bullinger:Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Abbvie: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Bayer: Other: Financing of scientific research; Pfizer: Honoraria.

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer glioblastoma. This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in glioblastoma reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of glioblastoma cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for the occurance of aberrant nucleolar function and increased anabolic processes in glioblastoma, which constitutes a primary event in gliomagenesis.

Long noncoding RNAs (lncRNAs) have important roles in shaping chromatin by targeting chromatin-modifying enzymes to distinct genomic sites. This section covers two methods to analyze lncRNA-protein interactions. The RNA-protein pull-down assays use either bead-bound proteins to capture in vitro transcripts, or immobilized synthetic RNAs to bind proteins from cell lysates. In the RNA immunoprecipitation (RIP) assay, endogenous RNAs are co-immunoprecipitated with a protein of interest. Both the methods can be applied to material from proliferating and quiescent cells, thus providing insights into how lncRNA-protein interactions are altered between these two cellular states.

Background Transcriptional and metabolic dysregulation are known to occur in Huntington’s disease (HD). Mutant huntingtin (mHTT) protein affects several cellular functions hindering the identification of the primary pathogenic event. Impaired transcription of ribosomal DNA (rDNA) genes in the nucleolus – a major non-membrane bound sub-nuclear compartment – represents an emerging mechanism underlying progressive neurodegeneration Aims To identify a sensitive transcriptional and metabolic marker associated with mHTT and disease progression, we tested the hypothesis that changes in rDNA transcription in the nucleolus are early signs of transcriptional dysregulation by mHTT in HD models and human tissue biopsies. Methods/techniques We analyzed by real-time quantitative PCR, RNA in situ hybridization, and immunofluorescence the activity and integrity of the nucleolus in the striatum and skeletal muscle of zQ175 knock-in mice at various ages as well as of patient tissue biopsies at early neuropathological stages. Results/outcome Here, we show that rDNA transcription and distribution of the nucleolar chaperone protein nucleophosmin (NPM1) are differentially altered in brain and muscle tissues from HD mouse models and in muscle biopsies from HD patients. Conclusions Our results indicate that mHTT nuclear inclusions interfere with nucleolar function in a stage- and cell-specific fashion by altering NPM1 in striatal cells. Moreover these studies demonstrate that NPM1 in muscle cells constitutes a molecular signature of the initial stages of HD. These findings could help to provide novel tools to test the functional efficacy of ongoing therapeutic strategies aiming at lowering mHTT levels in specific cells. Funding This work was supported by EHDN seed-fund project 0753, DFG PA 1529/2–1, CEMMA Graduate School

Ribosome biogenesis is a complex and highly energy-demanding process that requires the concerted action of all three nuclear RNA polymerases (Pol I-III) in eukaryotes. The three largest ribosomal RNAs (rRNAs) originate from a precursor transcript (pre-rRNA) that is encoded by multicopy genes located in the nucleolus. Transcription of these rRNA genes (rDNA) by Pol I is the key regulation step in ribosome production and is tightly controlled by an intricate network of signaling pathways and epigenetic mechanisms. In this article, we give an overview of the composition of the basal Pol I machinery and rDNA chromatin. We discuss rRNA gene regulation in response to environmental signals and developmental cues and focus on perturbations occurring in diseases linked to either excessive or limited rRNA levels. Finally, we discuss the emerging view that rDNA integrity and activity may be involved in the aging process.

In complex organisms, stem cells are key for tissue maintenance and regeneration. Adult stem cells replenish continuously dividing tissues of the epithelial and connective types, whereas in non-growing muscle and nervous tissues, they are mainly activated upon injury or stress. In addition to replacing deteriorated cells, adult stem cells have to prevent their exhaustion by self-renewal. There is mounting evidence that both differentiation and self-renewal are impaired upon aging, leading to tissue degeneration and functional decline. Understanding the molecular pathways that become deregulate in old stem cells is crucial to counteract aging-associated tissue impairment. In this review, we focus on the epigenetic mechanisms governing the transition between quiescent and active states, as well as the decision between self-renewal and differentiation in three different stem cell types, i.e., spermatogonial stem cells, hematopoietic stem cells, and muscle stem cells. We discuss the epigenetic events that channel stem cell fate decisions, how this epigenetic regulation is altered with age, and how this can lead to tissue dysfunction and disease. Finally, we provide short prospects of strategies to preserve stem cell function and thus promote healthy aging.