Title: Whole-genome DNA methylomes of Tree shrew brains reveal conserved and divergent roles of DNA methylation on sex chromosome regulation
Abstract The tree shrew (Tupaia belangeri) is a promising emerging model organism in biomedical studies, notably due to their evolutionary proximity to primates. To enhance our understanding of how DNA methylation is implicated in regulation of gene expression and the X chromosome inactivation (XCI) in tree shrew brains, here we present their first genome-wide, single-base-resolution methylomes integrated with transcriptomes from prefrontal cortices. We discovered both divergent and conserved features of tree shrew DNA methylation compared to that of other mammals. DNA methylation levels of promoter and gene body regions are negatively correlated with gene expression, consistent with patterns in other mammalian brains studied. Comparing DNA methylation patterns of the female and male X chromosomes, we observed a clear and significant global reduction (hypomethylation) of DNA methylation across the entire X chromosome in females. Our data suggests that the female X hypomethylation does not directly contribute to the gene silencing of the inactivated X chromosome nor does it significantly drive sex-specific gene expression of tree shrews. However, we identified a putative regulatory region in the 5’ end of the X inactive specific transcript (Xist)gene, a key gene for XCI, whose pattern of differential DNA methylation strongly relate to its differential expression between male and female tree shrews. We show that differential methylation of this region is conserved across different species. Moreover, we provide evidence suggesting that the observed difference between human and tree shrew X-linked promoter methylation is associated with the difference in genomic CpG contents. Our study offers novel information on genomic DNA methylation of tree shrews, as well as insights into the evolution of X chromosome regulation in mammals. more »« less
Singh, Devika; Sun, Dan; King, Andrew G.; Alquezar-Planas, David E.; Johnson, Rebecca N.; Alvarez-Ponce, David; Yi, Soojin V.
(, Proceedings of the Royal Society B: Biological Sciences)
null
(Ed.)
X chromosome inactivation (XCI) mediated by differential DNA methylation between sexes is an iconic example of epigenetic regulation. Although XCI is shared between eutherians and marsupials, the role of DNA methylation in marsupial XCI remains contested. Here, we examine genome-wide signatures of DNA methylation across fives tissues from a male and female koala ( Phascolarctos cinereus ), and present the first whole-genome, multi-tissue marsupial ‘methylome atlas’. Using these novel data, we elucidate divergent versus common features of representative marsupial and eutherian DNA methylation. First, tissue-specific differential DNA methylation in koalas primarily occurs in gene bodies. Second, females show significant global reduction (hypomethylation) of X chromosome DNA methylation compared to males. We show that this pattern is also observed in eutherians. Third, on average, promoter DNA methylation shows little difference between male and female koala X chromosomes, a pattern distinct from that of eutherians. Fourth, the sex-specific DNA methylation landscape upstream of Rsx , the primary lnc RNA associated with marsupial XCI, is consistent with the epigenetic regulation of female-specific (and presumably inactive X chromosome-specific) expression. Finally, we use the prominent female X chromosome hypomethylation and classify 98 previously unplaced scaffolds as X-linked, contributing an additional 14.6 Mb (21.5%) to genomic data annotated as the koala X chromosome. Our work demonstrates evolutionarily divergent pathways leading to functionally conserved patterns of XCI in two deep branches of mammals.
Abstract DNA methylation is a critical regulatory mechanism implicated in development, learning, memory, and disease in the human brain. Here we have elucidated DNA methylation changes during recent human brain evolution. We demonstrate dynamic evolutionary trajectories of DNA methylation in cell-type and cytosine-context specific manner. Specifically, DNA methylation in non-CG context, namely CH methylation, has increased (hypermethylation) in neuronal gene bodies during human brain evolution, contributing to human-specific down-regulation of genes and co-expression modules. The effects of CH hypermethylation is particularly pronounced in early development and neuronal subtypes. In contrast, DNA methylation in CG context shows pronounced reduction (hypomethylation) in human brains, notably in cis-regulatory regions, leading to upregulation of downstream genes. We show that the majority of differential CG methylation between neurons and oligodendrocytes originated before the divergence of hominoids and catarrhine monkeys, and harbors strong signal for genetic risk for schizophrenia. Remarkably, a substantial portion of differential CG methylation between neurons and oligodendrocytes emerged in the human lineage since the divergence from the chimpanzee lineage and carries significant genetic risk for schizophrenia. Therefore, recent epigenetic evolution of human cortex has shaped the cellular regulatory landscape and contributed to the increased vulnerability to neuropsychiatric diseases.
Abstract BackgroundMorphologic sex differences between males and females typically emerge after the primordial germ cell migration and gonad formation, although sex is determined at fertilization based on chromosome composition. A key debated sexual difference is the embryonic developmental rate, within vitroproduced male embryos often developing faster. However, the molecular mechanisms driving early embryonic sex differences remain unclear. ResultsTo investigate the transcriptional sex difference during early development,in vitroproduced bovine blastocysts were collected and sexed by PCR. A significant male-biased development was observed in expanded blastocysts. Ultra-low input RNA-seq analysis identified 837 DEGs, with 231 upregulated and 606 downregulated in males. Functional enrichment analysis revealed male-biased DEGs were associated with metabolic regulation, whereas female-biased DEGs were related to female gonad development, sex differentiation, inflammatory pathways, and TGF-beta signaling. Comparing X chromosome and autosome expression ratio, we found that female-biased DEGs contributed to the higher X-linked gene dosage, a phenomenon not observed in male embryos. Moreover, we identified the sex-biased transcription factors and RNA-bind proteins, including pluripotent factors such asSOX21andPRDM14, and splicing factorsFMR1andHNRNPH2. Additionally, we revealed 1,555 significantly sex-biased differential alternative splicing (AS), predominantly skipped exons, mapped to 906 genes, with 59 overlapping with DEGs enriched in metabolic and autophagy pathways. By incorporating novel isoforms from long reads sequencing, we identified 1,151 sex-biased differentially expressed isoforms (DEIs) associated with 1,017 genes. Functional analysis showed that female-biased DEIs were involved in the negative regulation of transcriptional activity, while male-biased DEIs were related to energy metabolism. Furthermore, we identified sex-biased differential exon usage inDENND1B, DIS3L2, DOCK11, IL1RAPL2,andZRSR2Y,indicating their sex-specific regulation in early embryo development. ConclusionThis study provided a comprehensive analysis of transcriptome differences between male and female bovine blastocysts, integrating sex-biased gene expression, alternative splicing, and isoform dynamics. Our findings indicate that enriched metabolism processes in male embryos may contribute to the faster developmental pace, providing insights into sex-specific regulatory mechanisms during early embryogenesis. Plain English summaryMale and female early embryos develop at different speeds, with male embryos often developing faster than female embryos. However, the reasons behind these early differences remain unclear. In this study, we examined gene activity in bovine embryos to uncover the biological factors regulating these early sex differences. We collected in vitro-produced bovine blastocysts, examined their sex, and confirmed that male embryos develop faster. By analyzing global gene activity, including alternative splicing, which allows one gene to code for multiple RNA isoforms and proteins, we found distinct gene expression profiles between male and female embryos. Male embryos showed higher activity in genes related to metabolism and cellular functions, while female embryos had increased activity in genes associated with female-specific gonad development and gene expression regulation. We also examined differences in how genes on the X chromosome were expressed. Female embryos had higher X-linked gene expression, which may contribute to sex-specific developmental regulation. Additionally, we identified sex-specific transcription factors and RNA-binding proteins that regulate early embryo development, some of which are known to control pluripotency and gene splicing. Overall, our study provides new insights into how gene activity shapes early sex differences, suggesting that enhanced metabolism in male embryos may be a key driver of their faster developmental rate. HighlightsMale embryos develop faster due to increased gene expression in metabolism pathwaysFemale embryos exhibit higher X-linked gene expression, suggesting X-dosage compensation plays a role in early developmentSex-biased alternative splicing events contribute to embryonic metabolism, autophagy, and transcriptional regulation in embryosSex-biased isoform diversity contributes to distinct developmental regulation in male and female embryosKey pluripotency factors (SOX21, PRDM14) and splicing regulators (FMR1, HNRNPH2) drive sex-specific gene expression
Bogan, Samuel N.; Strader, Marie E.; Hofmann, Gretchen E.
(, BMC Biology)
Abstract BackgroundEpigenetic processes are proposed to be a mechanism regulating gene expression during phenotypic plasticity. However, environmentally induced changes in DNA methylation exhibit little-to-no association with differential gene expression in metazoans at a transcriptome-wide level. It remains unexplored whether associations between environmentally induced differential methylation and expression are contingent upon other epigenomic processes such as chromatin accessibility. We quantified methylation and gene expression in larvae of the purple sea urchinStrongylocentrotus purpuratusexposed to different ecologically relevant conditions during gametogenesis (maternal conditioning) and modeled changes in gene expression and splicing resulting from maternal conditioning as functions of differential methylation, incorporating covariates for genomic features and chromatin accessibility. We detected significant interactions between differential methylation, chromatin accessibility, and genic feature type associated with differential expression and splicing. ResultsDifferential gene body methylation had significantly stronger effects on expression among genes with poorly accessible transcriptional start sites while baseline transcript abundance influenced the direction of this effect. Transcriptional responses to maternal conditioning were 4–13 × more likely when accounting for interactions between methylation and chromatin accessibility, demonstrating that the relationship between differential methylation and gene regulation is partially explained by chromatin state. ConclusionsDNA methylation likely possesses multiple associations with gene regulation during transgenerational plasticity inS. purpuratusand potentially other metazoans,but its effects are dependent on chromatin accessibility and underlying genic features.
Ruhr, Ilan; Bierstedt, Jacob; Rhen, Turk; Das, Debojyoti; Singh, Sunil Kumar; Miller, Soleille; Crossley, II, Dane A.; Galli, Gina L. J.
(, Epigenetics & Chromatin)
Abstract BackgroundEnvironmental fluctuation during embryonic and fetal development can permanently alter an organism’s morphology, physiology, and behaviour. This phenomenon, known as developmental plasticity, is particularly relevant to reptiles that develop in subterranean nests with variable oxygen tensions. Previous work has shown hypoxia permanently alters the cardiovascular system of snapping turtles and may improve cardiac anoxia tolerance later in life. The mechanisms driving this process are unknown but may involve epigenetic regulation of gene expression via DNA methylation. To test this hypothesis, we assessed in situ cardiac performance during 2 h of acute anoxia in juvenile turtles previously exposed to normoxia (21% oxygen) or hypoxia (10% oxygen) during embryogenesis. Next, we analysed DNA methylation and gene expression patterns in turtles from the same cohorts using whole genome bisulfite sequencing, which represents the first high-resolution investigation of DNA methylation patterns in any reptilian species. ResultsGenome-wide correlations between CpG and CpG island methylation and gene expression patterns in the snapping turtle were consistent with patterns observed in mammals. As hypothesized, developmental hypoxia increased juvenile turtle cardiac anoxia tolerance and programmed DNA methylation and gene expression patterns. Programmed differences in expression of genes such asSCN5Amay account for differences in heart rate, while genes such asTNNT2andTPM3may underlie differences in calcium sensitivity and contractility of cardiomyocytes and cardiac inotropy. Finally, we identified putative transcription factor-binding sites in promoters and in differentially methylated CpG islands that suggest a model linking programming of DNA methylation during embryogenesis to differential gene expression and cardiovascular physiology later in life. Binding sites for hypoxia inducible factors (HIF1A, ARNT, and EPAS1) and key transcription factors activated by MAPK and BMP signaling (RREB1 and SMAD4) are implicated. ConclusionsOur data strongly suggests that DNA methylation plays a conserved role in the regulation of gene expression in reptiles. We also show that embryonic hypoxia programs DNA methylation and gene expression patterns and that these changes are associated with enhanced cardiac anoxia tolerance later in life. Programming of cardiac anoxia tolerance has major ecological implications for snapping turtles, because these animals regularly exploit anoxic environments throughout their lifespan.
Son, Dongmin, Kong, Yifan, Tan, Yulian, Hu, Ting, Shi, Lei, and Yi, Soojin V. Whole-genome DNA methylomes of Tree shrew brains reveal conserved and divergent roles of DNA methylation on sex chromosome regulation. Retrieved from https://par.nsf.gov/biblio/10554737. Web. doi:10.1101/2024.06.05.597676.
Son, Dongmin, Kong, Yifan, Tan, Yulian, Hu, Ting, Shi, Lei, & Yi, Soojin V. Whole-genome DNA methylomes of Tree shrew brains reveal conserved and divergent roles of DNA methylation on sex chromosome regulation. Retrieved from https://par.nsf.gov/biblio/10554737. https://doi.org/10.1101/2024.06.05.597676
Son, Dongmin, Kong, Yifan, Tan, Yulian, Hu, Ting, Shi, Lei, and Yi, Soojin V.
"Whole-genome DNA methylomes of Tree shrew brains reveal conserved and divergent roles of DNA methylation on sex chromosome regulation". Country unknown/Code not available: bioRxiv. https://doi.org/10.1101/2024.06.05.597676.https://par.nsf.gov/biblio/10554737.
@article{osti_10554737,
place = {Country unknown/Code not available},
title = {Whole-genome DNA methylomes of Tree shrew brains reveal conserved and divergent roles of DNA methylation on sex chromosome regulation},
url = {https://par.nsf.gov/biblio/10554737},
DOI = {10.1101/2024.06.05.597676},
abstractNote = {Abstract The tree shrew (Tupaia belangeri) is a promising emerging model organism in biomedical studies, notably due to their evolutionary proximity to primates. To enhance our understanding of how DNA methylation is implicated in regulation of gene expression and the X chromosome inactivation (XCI) in tree shrew brains, here we present their first genome-wide, single-base-resolution methylomes integrated with transcriptomes from prefrontal cortices. We discovered both divergent and conserved features of tree shrew DNA methylation compared to that of other mammals. DNA methylation levels of promoter and gene body regions are negatively correlated with gene expression, consistent with patterns in other mammalian brains studied. Comparing DNA methylation patterns of the female and male X chromosomes, we observed a clear and significant global reduction (hypomethylation) of DNA methylation across the entire X chromosome in females. Our data suggests that the female X hypomethylation does not directly contribute to the gene silencing of the inactivated X chromosome nor does it significantly drive sex-specific gene expression of tree shrews. However, we identified a putative regulatory region in the 5’ end of the X inactive specific transcript (Xist)gene, a key gene for XCI, whose pattern of differential DNA methylation strongly relate to its differential expression between male and female tree shrews. We show that differential methylation of this region is conserved across different species. Moreover, we provide evidence suggesting that the observed difference between human and tree shrew X-linked promoter methylation is associated with the difference in genomic CpG contents. Our study offers novel information on genomic DNA methylation of tree shrews, as well as insights into the evolution of X chromosome regulation in mammals.},
journal = {},
publisher = {bioRxiv},
author = {Son, Dongmin and Kong, Yifan and Tan, Yulian and Hu, Ting and Shi, Lei and Yi, Soojin V},
}
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