Eric Lieberman Greer

Mu opioids, such as morphine, are effective analgesics, but they produce reward and tolerance that lead to opioid use disorder. A major goal is to achieve analgesia without these harmful effects. Here we show that morphine reward and tolerance are controlled by a reversible RNA modification in small nuclear RNA (snRNA) that is removed by the RNA demethylase FTO. Genetic depletion and pharmacologic inhibition of FTO each reduced morphine reward, measured by conditioned-place preference, and reduced tolerance to morphine and fentanyl, without altering analgesia. Depleting Fto in the nucleus accumbens shell reduced reward but not tolerance, while depleting Fto in the dorsal root ganglion reduced tolerance but not reward, suggesting region-specific effects of FTO. Although FTO is known to erase m ⁶ A on mRNA, we found no effect of FTO depletion on m ⁶ A sites, but a marked increase in m ⁶ Am levels in snRNA. The effects of FTO depletion were suppressed in mice that cannot make m ⁶ Am, supporting the importance of elevated m ⁶ Am in suppressing morphine reward and tolerance. Consistent with altered snRNA function, FTO depletion reshapes gene expression linked to morphine signaling. FTO inhibitors may therefore provide useful adjuvants for mu opioids in pain management and treatment of opioid use disorder.

As organisms age there is a progressive decline in protein homeostasis, yet how mRNAs are selectively translated during aging remains poorly understood. Since cells do not have limitless energy, choices must be made as to which set of proteins are translated at the expense of other proteins which might be less essential. Here, we identify that the Hsp70 chaperone protein HSP-1/HSPA8 binds to the ribosome and helps to direct selective translation in Caenorhabditis elegans. We find that intestine-specific depletion of HSP-1 accelerates ageing and abolishes the longevity conferred by reduced translation. Intestinal translatome profiling revealed that HSP-1 depletion decreases ribosome occupancy on lysosomal transcripts while increasing occupancy on immune- and stress-response mRNAs, including MAPK pathway components. This translational shift activates the p38 TIR-1-MAPK innate immune pathway and impairs autophagy through impaired lysosomal acidification. Similarly, depletion of the human orthologue HSPA8 increases p38 phosphorylation and decreases lysosomal acidification, suggesting that this could be a conserved regulator of selective translation. Although this response enhances resistance to bacterial infection in C. elegans, it comes at the cost of shortened lifespan, revealing a trade-off between host defense and longevity. While decreased lifespan is dependent on autophagy pathways, increased resistance to infection depends on the p38-MAPK pathway. Together, these findings identify HSP-1 as a ribosome-associated chaperone that couples selective translation to autophagy, innate immunity, and ageing.

SUMMARY: Mu opioids, such as morphine, are effective analgesics, but their reward and tolerance drive opioid use disorder. A major goal is to achieve analgesia without these harmful effects. Here we show that morphine reward and tolerance require the RNA demethylase FTO. Genetic depletion and pharmacologic inhibition of FTO each reduced morphine reward, measured by conditioned-place preference, and reduced antinociceptive tolerance to morphine and fentanyl, without altering analgesia. Although FTO is known to erase m ⁶ A on mRNA, we found no effect of FTO depletion on m ⁶ A sites, but markedly increased levels of m ⁶ Am on snRNA. The effects of FTO depletion were suppressed in mice that cannot make m ⁶ Am, supporting the role of m ⁶ Am in morphine reward and tolerance. We show that FTO depletion regulates a gene expression network linked to morphine signaling. FTO inhibitors may therefore provide useful adjuvants to mu opioids in pain management and treatment of opioid use disorder.

Post-translational modifications of histone tails alter chromatin accessibility to regulate gene expression. Some viruses exploit the importance of histone modifications by expressing histone mimetic proteins that contain histone-like sequences to sequester complexes that recognize modified histones. Here we identify an evolutionarily conserved and ubiquitously expressed, endogenous mammalian protein Nucleolar protein 16 (NOP16) that has homology to H3K27. NOP16 binds to EED in the H3K27 trimethylation PRC2 complex and to the H3K27 demethylase JMJD3. NOP16 competitively inhibits the interaction between EED and H3K27me3 and causes EED to translocate from the nucleoplasm to the nucleolus, thereby negatively regulating H3K27me3 modification in the nucleoplasm. NOP16 is overexpressed and linked to poor prognosis in breast cancer. Depletion of NOP16 in breast cancer cell lines causes cell cycle arrest, decreases cell proliferation and selectively decreases expression of E2F target genes and of genes involved in cell cycle, growth and apoptosis. Conversely, ectopic NOP16 expression in triple negative breast cancer cell lines increases cell proliferation, cell migration and invasivity in vitro and tumor growth in vivo, while NOP16 knockout or knockdown has the opposite effect. Thus, NOP16’s histone homology allows it to compete with Histone H3 for H3K27 methylation and demethylation. When it is overexpressed in cancer, it derepresses genes that promote cell cycle progression to augment breast cancer growth.

DNA N6-methyladenine (6mA) has emerged as a significant epigenetic modification across a broad range of eukaryotes, from unicellular protists to metazoa. However, its role in unicellular eukaryotic parasites with highly AT-rich genomes, such as malaria-causing Plasmodium falciparum , remains unclear. Using mass spectrometry, South-western blotting, and Single Molecule Real-Time sequencing (Pacific Biosciences) across four stages of P. falciparum intra-erythrocytic development (IED), we confirmed that 0.02–0.04% of genomic adenines are modified to 6mA, with over 60% of the sites being stably maintained during the IED cycle. Notably, 6mA is enriched at transcription start sites, with genes bearing 6mA marks within their 5’ and 3’ untranslated regions exhibiting significantly elevated steady-state transcript levels. Consistent with this, 6mA loci show a strong positive correlation with activating histone post-translational modifications, while showing no significant association with repressive histone marks. Furthermore, in contrast to unicellular ciliates such as Oxytricha and Tetrahymena – organisms that share ancestry with Plasmodium – 6mA-marked genomic regions do not occlude nucleosomes. Lastly, we identified a putative 6mA methyltransferase belonging to the METTL4 family in P. falciparum, PfN6AMT encoded by the PF3D7_1303100 gene, and demonstrate that recombinant PfN6AMT exhibits robust methyltransferase activity in vitro , with mutation of its active site residues abolishing catalytic activity. Collectively, our findings demonstrate that 6mA is a low-abundance, yet reproducible, feature of the P. falciparum epigenome that is associated with transcriptionally active chromatin, and that the molecular mechanisms governing DNA adenine methylation may have undergone substantial evolutionary divergence, even among closely related eukaryotic lineages.

Type I interferons (IFN-I) are critical for antiviral defense but can drive severe pathology when dysregulated. Excess IFN-I is associated with prominent neutrophil accumulation; however, the contribution of neutrophils to IFN-I overproduction remains underexplored. In all cell types previously studied, IFN-I is synthesized de novo following sensing of microbial or host-derived inflammatory stimuli. Contrary to this paradigm, we find that neutrophils express IFNα during development and store it in the nucleolus, a membrane-less intranuclear condensate classically functioning in ribosome biogenesis. TLR-mediated bacterial sensing induces a nucleolar stress response in neutrophils that triggers rapid release of nucleoli-stored IFNα independent of de novo protein synthesis. These findings reveal roles for the neutrophil nucleolus in IFN-I storage and secretion, identify the first naturally occurring immunoregulatory function of nucleolar stress, and provide insight into the relationship between detrimental IFN-I levels and neutrophil accumulation.

Epigenetic inheritance alerts naïve descendants to prepare for stresses that could still be present, whereas distant descendants return to a basal state after several generations without stress. However, organisms are frequently exposed to stresses successively across generations. We found that parental hypoxia exposure increased P0 longevity, caused intergenerational lipid reduction, and elicited transgenerational fertility reduction that was dependent on generationally transmitted small RNAs. Here, we find that Caenorhabditis elegans adapt to repeated generational stresses. We show that, upon two repeated generational hypoxia exposures, the life-span extension is eliminated, and after four repeated generational hypoxia exposures, the reduced fertility is eliminated. Transgenerational adaptation also occurred in response to changes in glucose availability. Transgenerational hypoxia adaptation is dependent on the H3K27 trimethyltransferase PRC2 complex, and we identified transgenerationally adapted genes. Our findings reveal that transgenerational adaptation occurs and suggest that H3K27me3 is a critical modification for adapting to repeated generational stresses.

Specialized ribosomes help determine which proteins are synthesized, however, the influence of age on ribosome heterogeneity and whether dysregulation of this process drives organismal aging is unknown. Here we examined the role of ribosomal RNA (rRNA) methylation in maintaining appropriate translation as organisms age. In a directed RNAi screen, we identified 18S rRNA N6’-dimethyl adenosine (m^6,2A) methyltransferase, dimt-1, as a regulator of C. elegans lifespan and stress resistance. We demonstrate that DIMT-1 functions in the germline after mid-life to regulate lifespan. Depletion of dimt-1 leads to selective translation of transcripts important for stress resistance and lifespan regulation in the C. elegans germline including the cytochrome P450 daf-9, which synthesizes a steroid that signals from the germline to the soma. dimt-1 induced lifespan extension is dependent on the daf-9 signaling pathway. Our findings highlight ribosome heterogeneity, and specific rRNA modifications, in maintaining appropriate translation later in life to promote healthy aging.

This book chapter will focus on modifications to chromatin itself, how chromatin modifications are regulated, and how these modifications are deciphered by the cell to impact aging. In this chapter, we will review how chromatin modifications change with age, examine how chromatin-modifying enzymes have been shown to regulate aging and healthspan, discuss how some of these epigenetic changes are triggered and how they can regulate the lifespan of the individual and its naïve descendants, and speculate on future directions for the field.

Cancer cells edit gene expression to evade immunosurveillance. However, genome-wide studies of gene editing during early tumorigenesis are lacking. Here we used single-cell RNA sequencing in a breast cancer genetically engineered mouse model (GEMM) to identify edited genes without bias. Late tumors repressed antitumor immunity genes, reducing infiltrating immune cells and tumor–immune cell communications. Innate immune genes, especially interferon-stimulated genes, dominated the list of downregulated tumor genes, while genes that regulate cell-intrinsic malignancy were mostly unedited. Naive and activated CD8⁺ T cells in early tumors were replaced with exhausted or precursor-exhausted cells in late tumors. Repression of immune genes was reversed by inhibiting DNA methylation using low-dose decitabine, which suppressed tumor growth and restored immune control, increasing the number, functionality and memory of tumor-infiltrating lymphocytes and reducing the number of myeloid suppressor cells. Decitabine induced important interferon, pyroptosis and necroptosis genes, inflammatory cell death and immune control in GEMM and implanted breast and melanoma tumors.