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Title: Dynamics of RNA m5C modification during brain development
Post-transcriptional RNA modifications have been recognized as key regulators of neuronal differentiation and synapse development in the mammalian brain. While distinct sets of 5-methylcytosine (m5C) modified mRNAs have been detected in neuronal cells and brain tissues, no study has been performed to characterize methylated mRNA profiles in the developing brain. Here, together with regular RNA-seq, we performed transcriptome-wide bisulfite sequencing to compare RNA cytosine methylation patterns in neural stem cells (NSCs), cortical neuronal cultures, and brain tissues at three postnatal stages. Among 501 m5C sites identified, approximately 6% are consistently methylated across all five conditions. Compared to m5C sites identified in NSCs, 96% of them were hypermethylated in neurons and enriched for genes involved in positive transcriptional regulation and axon extension. In addition, brains at the early postnatal stage demonstrated substantial changes in both RNA cytosine methylation and gene expression of RNA cytosine methylation readers, writers, and erasers. Furthermore, differentially methylated transcripts were significantly enriched for genes regulating synaptic plasticity. Altogether, this study provides a brain epitranscriptomic dataset as a new resource and lays the foundation for further investigations into the role of RNA cytosine methylation during brain development. more »« less
Folate is an essential B-group vitamin and a key methyl donor with important biological functions including DNA methylation regulation. Normal neurodevelopment and physiology are sensitive to the cellular folate levels. Either deficiency or excess of folate may lead to neurological disorders. Recently, folate has been linked to tRNA cytosine-5 methylation (m5C) and translation in mammalian mitochondria. However, the influence of folate intake on neuronal mRNA m5C modification and translation remains largely unknown. Here, we provide transcriptome-wide landscapes of m5C modification in poly(A)-enriched RNAs together with mRNA transcription and translation profiles for mouse neural stem cells (NSCs) cultured in three different concentrations of folate.
Results
NSCs cultured in three different concentrations of folate showed distinct mRNA methylation profiles. Despite uncovering only a few differentially expressed genes, hundreds of differentially translated genes were identified in NSCs with folate deficiency or supplementation. The differentially translated genes induced by low folate are associated with cytoplasmic translation and mitochondrial function, while the differentially translated genes induced by high folate are associated with increased neural stem cell proliferation. Interestingly, compared to total mRNAs, polysome mRNAs contained high levels of m5C. Furthermore, an integrative analysis indicated a transcript-specific relationship between RNA m5C methylation and mRNA translation efficiency.
Conclusions
Altogether, our study reports a transcriptome-wide influence of folate on mRNA m5C methylation and translation in NSCs and reveals a potential link between mRNA m5C methylation and mRNA translation.
Rizzardi, Lindsay F.; Hickey, Peter F.; Idrizi, Adrian; Tryggvadóttir, Rakel; Callahan, Colin M.; Stephens, Kimberly E.; Taverna, Sean D.; Zhang, Hao; Ramazanoglu, Sinan; Hansen, Kasper D.; et al(
, Genome Biology)
Abstract Background DNA methylation dynamics in the brain are associated with normal development and neuropsychiatric disease and differ across functionally distinct brain regions. Previous studies of genome-wide methylation differences among human brain regions focus on limited numbers of individuals and one to two brain regions. Results Using GTEx samples, we generate a resource of DNA methylation in purified neuronal nuclei from 8 brain regions as well as lung and thyroid tissues from 12 to 23 donors. We identify differentially methylated regions between brain regions among neuronal nuclei in both CpG (181,146) and non-CpG (264,868) contexts, few of which were unique to a single pairwise comparison. This significantly expands the knowledge of differential methylation across the brain by 10-fold. In addition, we present the first differential methylation analysis among neuronal nuclei from basal ganglia tissues and identify unique CpG differentially methylated regions, many associated with ion transport. We also identify 81,130 regions of variably CpG methylated regions, i.e., variable methylation among individuals in the same brain region, which are enriched in regulatory regions and in CpG differentially methylated regions. Many variably methylated regions are unique to a specific brain region, with only 202 common across all brain regions, as well as lung and thyroid. Variably methylated regions identified in the amygdala, anterior cingulate cortex, and hippocampus are enriched for heritability of schizophrenia. Conclusions These data suggest that epigenetic variation in these particular human brain regions could be associated with the risk for this neuropsychiatric disorder.
Nandety, Raja Sekhar; Oh, Sunhee; Lee, Hee‐Kyung; Krom, Nick; Gupta, Rajeev; Mysore, Kirankumar S.(
, The Plant Journal)
SUMMARY
Medicago truncatulais a model legume for fundamental research on legume biology and symbiotic nitrogen fixation.Tnt1, a retrotransposon from tobacco, was used to generate insertion mutants inM. truncatulaR108. Approximately 21 000 insertion lines have been generated and publicly available.Tnt1retro‐transposition event occurs during somatic embryogenesis (SE), a pivotal process that triggers massive methylation changes. We studied the SE ofM. truncatulaR108 using leaf explants and explored the dynamic shifts in the methylation landscape from leaf explants to callus formation and finally embryogenesis. Higher cytosine methylation in all three contexts of CG, CHG, and CHH patterns was observed during SE compared to the controls. Higher methylation patterns were observed in assumed promoter regions (~2‐kb upstream regions of transcription start site) of the genes, while lowest was recorded in the untranslated regions. Differentially methylated promoter region analysis showed a higher CHH methylation in embryogenesis tissue samples when compared to CG and CHG methylation. Strong correlation (89.71%) was identified between the differentially methylated regions (DMRs) and the site ofTnt1insertions inM. truncatulaR108 and stronger hypermethylation of genes correlated with higher number ofTnt1insertions in all contexts of CG, CHG, and CHH methylation. Gene ontology enrichment and KEGG pathway enrichment analysis identified genes and pathways enriched in the signal peptide processing, ATP hydrolysis, RNA polymerase activity, transport, secondary metabolites, and nitrogen metabolism pathways. Combined gene expression analysis and methylation profiling showed an inverse relationship between methylation in the DMRs (regions spanning genes) and the expression of genes. Our results show that a dynamic shift in methylation happens during the SE process in the context of CG, CHH and CHG methylation, and theTnt1retrotransposition correlates with the hyperactive methylation regions.
Abstract Background Mutations in LMNA , encoding lamin A/C, lead to a variety of diseases known as laminopathies including dilated cardiomyopathy (DCM) and skeletal abnormalities. Though previous studies have investigated the dysregulation of gene expression in cells from patients with DCM, the role of epigenetic (gene regulatory) mechanisms, such as DNA methylation, has not been thoroughly investigated. Furthermore, the impact of family-specific LMNA mutations on DNA methylation is unknown. Here, we performed reduced representation bisulfite sequencing on ten pairs of fibroblasts and their induced pluripotent stem cell (iPSC) derivatives from two families with DCM due to distinct LMNA mutations, one of which also induces brachydactyly. Results Family-specific differentially methylated regions (DMRs) were identified by comparing the DNA methylation landscape of patient and control samples. Fibroblast DMRs were found to enrich for distal regulatory features and transcriptionally repressed chromatin and to associate with genes related to phenotypes found in tissues affected by laminopathies. These DMRs, in combination with transcriptome-wide expression data and lamina-associated domain (LAD) organization, revealed the presence of inter-family epimutation hotspots near differentially expressed genes, most of which were located outside LADs redistributed in LMNA -related DCM. Comparison of DMRs found in fibroblasts and iPSCs identified regions where epimutations were persistent across both cell types. Finally, a network of aberrantly methylated disease-associated genes revealed a potential molecular link between pathways involved in bone and heart development. Conclusions Our results identified both shared and mutation-specific laminopathy epimutation landscapes that were consistent with lamin A/C mutation-mediated epigenetic aberrancies that arose in somatic and early developmental cell stages.
Hoyt, Savannah J.; Storer, Jessica M.; Hartley, Gabrielle A.; Grady, Patrick G.; Gershman, Ariel; de Lima, Leonardo G.; Limouse, Charles; Halabian, Reza; Wojenski, Luke; Rodriguez, Matias; et al(
, Science)
INTRODUCTION Transposable elements (TEs), repeat expansions, and repeat-mediated structural rearrangements play key roles in chromosome structure and species evolution, contribute to human genetic variation, and substantially influence human health through copy number variants, structural variants, insertions, deletions, and alterations to gene transcription and splicing. Despite their formative role in genome stability, repetitive regions have been relegated to gaps and collapsed regions in human genome reference GRCh38 owing to the technological limitations during its development. The lack of linear sequence in these regions, particularly in centromeres, resulted in the inability to fully explore the repeat content of the human genome in the context of both local and regional chromosomal environments. RATIONALE Long-read sequencing supported the complete, telomere-to-telomere (T2T) assembly of the pseudo-haploid human cell line CHM13. This resource affords a genome-scale assessment of all human repetitive sequences, including TEs and previously unknown repeats and satellites, both within and outside of gaps and collapsed regions. Additionally, a complete genome enables the opportunity to explore the epigenetic and transcriptional profiles of these elements that are fundamental to our understanding of chromosome structure, function, and evolution. Comparative analyses reveal modes of repeat divergence, evolution, and expansion or contraction with locus-level resolution. RESULTS We implemented a comprehensive repeat annotation workflow using previously known human repeats and de novo repeat modeling followed by manual curation, including assessing overlaps with gene annotations, segmental duplications, tandem repeats, and annotated repeats. Using this method, we developed an updated catalog of human repetitive sequences and refined previous repeat annotations. We discovered 43 previously unknown repeats and repeat variants and characterized 19 complex, composite repetitive structures, which often carry genes, across T2T-CHM13. Using precision nuclear run-on sequencing (PRO-seq) and CpG methylated sites generated from Oxford Nanopore Technologies long-read sequencing data, we assessed RNA polymerase engagement across retroelements genome-wide, revealing correlations between nascent transcription, sequence divergence, CpG density, and methylation. These analyses were extended to evaluate RNA polymerase occupancy for all repeats, including high-density satellite repeats that reside in previously inaccessible centromeric regions of all human chromosomes. Moreover, using both mapping-dependent and mapping-independent approaches across early developmental stages and a complete cell cycle time series, we found that engaged RNA polymerase across satellites is low; in contrast, TE transcription is abundant and serves as a boundary for changes in CpG methylation and centromere substructure. Together, these data reveal the dynamic relationship between transcriptionally active retroelement subclasses and DNA methylation, as well as potential mechanisms for the derivation and evolution of new repeat families and composite elements. Focusing on the emerging T2T-level assembly of the HG002 X chromosome, we reveal that a high level of repeat variation likely exists across the human population, including composite element copy numbers that affect gene copy number. Additionally, we highlight the impact of repeats on the structural diversity of the genome, revealing repeat expansions with extreme copy number differences between humans and primates while also providing high-confidence annotations of retroelement transduction events. CONCLUSION The comprehensive repeat annotations and updated repeat models described herein serve as a resource for expanding the compendium of human genome sequences and reveal the impact of specific repeats on the human genome. In developing this resource, we provide a methodological framework for assessing repeat variation within and between human genomes. The exhaustive assessment of the transcriptional landscape of repeats, at both the genome scale and locally, such as within centromeres, sets the stage for functional studies to disentangle the role transcription plays in the mechanisms essential for genome stability and chromosome segregation. Finally, our work demonstrates the need to increase efforts toward achieving T2T-level assemblies for nonhuman primates and other species to fully understand the complexity and impact of repeat-derived genomic innovations that define primate lineages, including humans. Telomere-to-telomere assembly of CHM13 supports repeat annotations and discoveries. The human reference T2T-CHM13 filled gaps and corrected collapsed regions (triangles) in GRCh38. Combining long read–based methylation calls, PRO-seq, and multilevel computational methods, we provide a compendium of human repeats, define retroelement expression and methylation profiles, and delineate locus-specific sites of nascent transcription genome-wide, including previously inaccessible centromeres. SINE, short interspersed element; SVA, SINE–variable number tandem repeat– Alu ; LINE, long interspersed element; LTR, long terminal repeat; TSS, transcription start site; pA, xxxxxxxxxxxxxxxx.
@article{osti_10476250,
place = {Country unknown/Code not available},
title = {Dynamics of RNA m5C modification during brain development},
url = {https://par.nsf.gov/biblio/10476250},
DOI = {10.1016/j.ygeno.2023.110604},
abstractNote = {Post-transcriptional RNA modifications have been recognized as key regulators of neuronal differentiation and synapse development in the mammalian brain. While distinct sets of 5-methylcytosine (m5C) modified mRNAs have been detected in neuronal cells and brain tissues, no study has been performed to characterize methylated mRNA profiles in the developing brain. Here, together with regular RNA-seq, we performed transcriptome-wide bisulfite sequencing to compare RNA cytosine methylation patterns in neural stem cells (NSCs), cortical neuronal cultures, and brain tissues at three postnatal stages. Among 501 m5C sites identified, approximately 6% are consistently methylated across all five conditions. Compared to m5C sites identified in NSCs, 96% of them were hypermethylated in neurons and enriched for genes involved in positive transcriptional regulation and axon extension. In addition, brains at the early postnatal stage demonstrated substantial changes in both RNA cytosine methylation and gene expression of RNA cytosine methylation readers, writers, and erasers. Furthermore, differentially methylated transcripts were significantly enriched for genes regulating synaptic plasticity. Altogether, this study provides a brain epitranscriptomic dataset as a new resource and lays the foundation for further investigations into the role of RNA cytosine methylation during brain development.},
journal = {Genomics},
volume = {115},
number = {3},
publisher = {Elsevier},
author = {Johnson, Zachary and Xu, Xiguang and Lin, Yu and Xie, Hehuang},
}
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