Search

DNA is organized into chromatin-like structures that support the maintenance and regulation of genomes. A unique and poorly understood form of DNA organization exists in chloroplasts, which are organelles of endosymbiotic origin responsible for photosynthesis. Chloroplast genomes, together with associated proteins, form membrane-less structures known as nucleoids. The internal arrangement of the nucleoid, molecular mechanisms of DNA organization, and connections between nucleoid structure and gene expression remain mostly unknown. We show that Arabidopsis thaliana chloroplast nucleoids have a unique sequence-specific organization driven by DNA binding to the thylakoid membranes. DNA associated with the membranes has high protein occupancy, has reduced DNA accessibility, and is highly transcribed. In contrast, genes with low levels of transcription are further away from the membranes, have lower protein occupancy, and have higher DNA accessibility. Membrane association of active genes relies on the pattern of transcription and proper chloroplast development. We propose a speculative model that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active periphery., Competing Interests: Competing interests statement:The authors declare no competing interest.

Ribosome heterogeneity has emerged as an important regulatory control feature for determining which proteins are synthesized, however, the influence of age on ribosome heterogeneity is not fully understood. Whether mRNA transcripts are selectively translated in young versus old cells 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 the 18S rRNA N6'-dimethyl adenosine (m 6,2 A) methyltransferase, dimt-1, as a regulator of C. elegans lifespan and stress resistance. Lifespan extension induced by dimt-1 deficiency required a functional germline and was dependent on the known regulator of protein translation, the Rag GTPase, raga-1, which links amino acid sensing to the mechanistic target of rapamycin complex (mTORC)1. Using an auxin-inducible degron tagged version of dimt-1, we demonstrate that DIMT-1 functions in the germline after mid-life to regulate lifespan. We further found that knock-down of dimt-1 leads to selective translation of transcripts important for stress resistance and lifespan regulation in the C. elegans germline in mid-life including the cytochrome P450 daf-9, which synthesizes a steroid that signals from the germline to the soma to regulate lifespan. We found that dimt-1 induced lifespan extension was dependent on the daf-9 signaling pathway. This finding reveals a new layer of proteome dysfunction, beyond protein synthesis and degradation, as an important regulator of aging. Our findings highlight a new role for ribosome heterogeneity, and specific rRNA modifications, in maintaining appropriate translation later in life to promote healthy aging., Competing Interests: Competing interests: Authors declare no competing interests.

Heritable non-genetic information can regulate a variety of complex phenotypes. However, what specific non-genetic cues are transmitted from parents to their descendants are poorly understood. Here, we perform metabolic methyl-labeling experiments to track the heritable transmission of methylation from ancestors to their descendants in the nematode Caenorhabditis elegans (C. elegans). We find heritable methylation in DNA, RNA, proteins, and lipids. We find that parental starvation elicits reduced fertility, increased heat stress resistance, and extended longevity in fed, naïve progeny. This intergenerational hormesis is accompanied by a heritable increase in N6'-dimethyl adenosine (m 6,2 A) on the 18S ribosomal RNA at adenosines 1735 and 1736. We identified DIMT-1/DIMT1 as the m 6,2 A and BUD-23/BUD23 as the m 7 G methyltransferases in C. elegans that are both required for intergenerational hormesis, while other rRNA methyltransferases are dispensable. This study labels and tracks heritable non-genetic material across generations and demonstrates the importance of rRNA methylation for regulating epigenetic inheritance., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 functions as a H3K27 mimic. NOP16 binds to EED in the H3K27 trimethylation PRC2 complex and to the H3K27 demethylase JMJD3. NOP16 knockout selectively globally increases H3K27me3, a heterochromatin mark, without altering methylation of H3K4, H3K9, or H3K36 or acetylation of H3K27. 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 is a histone mimic that competes 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.

While heredity is predominantly controlled by what deoxyribonucleic acid (DNA) sequences are passed from parents to their offspring, a small but growing number of traits have been shown to be regulated in part by the non-genetic inheritance of information. Transgenerational epigenetic inheritance is defined as heritable information passed from parents to their offspring without changing the DNA sequence. Work of the past seven decades has transitioned what was previously viewed as rare phenomenology, into well-established paradigms by which numerous traits can be modulated. For the most part, studies in model organisms have correlated transgenerational epigenetic inheritance phenotypes with changes in epigenetic modifications. The next steps for this field will entail transitioning from correlative studies to causal ones. Here, we delineate the major molecules that have been implicated in transgenerational epigenetic inheritance in both mammalian and non-mammalian models, speculate on additional molecules that could be involved, and highlight some of the tools which might help transition this field from correlation to causation., (© 2022 Wiley Periodicals LLC.)

Non-coding transcription is an important determinant of heterochromatin formation. In Arabidopsis thaliana a specialized RNA polymerase V (Pol V) transcribes pervasively and produces long non-coding RNAs. These transcripts work with small interfering RNA to facilitate locus-specific establishment of RNA-directed DNA methylation (RdDM). Subsequent maintenance of RdDM is associated with elevated levels of Pol V transcription. However, the impact of DNA methylation on Pol V transcription remained unresolved. We found that DNA methylation strongly enhances Pol V transcription. The level of Pol V transcription is reduced in mutants defective in RdDM components working downstream of Pol V, indicating that RdDM is maintained by a mutual reinforcement of DNA methylation and Pol V transcription. Pol V transcription is affected only on loci that lose DNA methylation in all sequence contexts in a particular mutant, including mutants lacking maintenance DNA methyltransferases, which suggests that RdDM works in a complex crosstalk with other silencing pathways., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Eukaryotic genomes are pervasively transcribed, yet most transcribed sequences lack conservation or known biological functions. In Arabidopsis thaliana , RNA polymerase V (Pol V) produces noncoding transcripts, which base pair with small interfering RNA (siRNA) and allow specific establishment of RNA-directed DNA methylation (RdDM) on transposable elements. Here, we show that Pol V transcribes much more broadly than previously expected, including subsets of both heterochromatic and euchromatic regions. At already established RdDM targets, Pol V and siRNA work together to maintain silencing. In contrast, some euchromatic sequences do not give rise to siRNA but are covered by low levels of Pol V transcription, which is needed to establish RdDM de novo if a transposon is reactivated. We propose a model where Pol V surveils the genome to make it competent to silence newly activated or integrated transposons. This indicates that pervasive transcription of nonconserved sequences may serve an essential role in maintenance of genome integrity., Competing Interests: The authors declare no competing interest.

RNA-mediated transcriptional silencing, in plants known as RNA-directed DNA methylation (RdDM), is a conserved process where small interfering RNA (siRNA) and long non-coding RNA (lncRNA) help establish repressive chromatin modifications. This process represses transposons and affects the expression of protein-coding genes. We found that in Arabidopsis thaliana AGO4 binding sites are often located distant from genes differentially expressed in ago4. Using Hi-C to compare interactions between genotypes, we show that RdDM-targeted loci have the potential to engage in chromosomal interactions, but these interactions are inhibited in wild-type conditions. In mutants defective in RdDM, the frequency of chromosomal interactions at RdDM targets is increased. This includes increased frequency of interactions between Pol V methylated sites and distal genes that are repressed by RdDM. We propose a model, where RdDM prevents the formation of chromosomal interactions between genes and their distant regulatory elements.

RNA-mediated transcriptional gene silencing is a conserved process where small RNAs target transposons and other sequences for repression by establishing chromatin modifications. A central element of this process are long non-coding RNAs (lncRNA), which in Arabidopsis thaliana are produced by a specialized RNA polymerase known as Pol V. Here we show that non-coding transcription by Pol V is controlled by preexisting chromatin modifications located within the transcribed regions. Most Pol V transcripts are associated with AGO4 but are not sliced by AGO4. Pol V-dependent DNA methylation is established on both strands of DNA and is tightly restricted to Pol V-transcribed regions. This indicates that chromatin modifications are established in close proximity to Pol V. Finally, Pol V transcription is preferentially enriched on edges of silenced transposable elements, where Pol V transcribes into TEs. We propose that Pol V may play an important role in the determination of heterochromatin boundaries., Competing Interests: The authors declare that no competing interests exist.