Like father, like son: how signatures can be inherited
Figure 1: Epigenetic inheritance of DNA methylation [Image created by BioRender.com]
Abstract
Cytosine-phosphate-Guanine Islands (CGIs) are genomic regions with a high density of CpG sites, typically ranging from 300 to 3000 base pairs in mammalian genomes. The DNA methylation (DNAm) on promoter CGIs correlates with silencing of the associated gene. Aberrant DNAm on CpG islands can lead to epigenetic and hereditary diseases. A previous study showed that connecting a CpG-free DNA sequence to the genome induces methylation of the surrounding CpGs [1]. In this study, Takahashi et al. introduced a CpG-free DNA cassette into the promoter loci of Ankrd26 and Ldlr, which are genes linked to obesity and hypercholesterolemia, to induce DNAm [2]. Then they generated methylated chimera mice to study the transgenerational inheritance of the methylation and the related phenotypes in vivo. The findings powerfully demonstrated the existence of an epigenetic memory. Taken together these data increases our knowledge about inheritance of acquired epigenetic modifications and lay the basis for future potential therapeutic interventions.
Review
Introduction
Epigenetic modifications influence gene activity without altering the DNA sequence. Among these modifications there is DNA methylation, which involves the addition of a methyl group to the nitrogenous base cytosine on both DNA strands. DNAm correlates with gene repression and regulates specific cellular functions. Aberrant newly acquired DNAm can potentially induce heritable diseases in subsequent generations.
Plasmid Integration and Chimera Generation
Researchers have studied how a specific disease in mammals (mice) is transmitted to the progeny by silencing the expression of two responsible genes: Ankrd26 (Ankyrin Repeat Domain 26), which causes obesity, and Ldlr (Low-Density Lipoprotein Receptor), which leads to high cholesterol levels. To induce methylation, a plasmid containing a CpG-free DNA sequence, enclosed between two inverted repeat TTAA sequences (ITRs), was engineered. Additionally, a sequence for the endonuclease enzyme Cas9, guided by a single guide RNA (sgRNA), and a gene conferring resistance to the aminoglycoside antibiotic Geneticin (G418), in order to distinguish plasmid-integrating cells from non-integrating ones, were included. The DNA cassette was integrated into the genome using the PiggyBac transposase [3], an enzyme that catalyzes the movement, integration, and removal of DNA segments on a specific genetic locus. The integration of this CpG-free DNA led to the generation of mouse embryonic stem cells (mESCs), with the promoter of Ankrd26 or Ldlr methylated. mESCs were microinjected into eight-cell-stage male mouse embryos. These embryos were then instilled into female mice for continuing pregnancy and generate chimera mice.
Methylation Levels and Phenotype
Before injecting these cells into embryos, methylation levels were checked during integration of the DNA cassette and after its removal. Using bisulfite sequencing, sporadic CpG methylation in the CGIs of both alleles of the Ankrd26 gene was observed. 8 mESC clones were generated called HR (1-8) clones: clone HR1 showed 65.5% methylation, HR2 showed 56.2%, HR4 showed 17.9%, and HR7 showed 31.2%. In the remaining four clones, methylation was negligible. For the Ldlr gene, among the nine clones, only two showed significant methylation: HR1 with 34.8% and HR2 with 20.2%.
In the methylated clones the DNA cassette was removed and DNAm checked again to assess if it was maintained. The removal was carried out using PiggyBac transposase, which caused the creation of TTAA sequences. It was found that in the clone Ankrd26 HR1 CGI methylation was 85.2%, in Ankrd26 HR7 21.2%, and in Ldlr HR1 27.1%. From these data, especially for HR1 and HR7, a relationship between pre- and post-removal seems to emerge: if the DNAm was high it tended to increase further; if it was low it tended to be reduced.
The percentage of methylation is crucial for phenotype expression, as higher hypermethylation makes the phenotype more evident. Indeed, from the injection of Ankrd26 HR1 cells, 2 chimera mice were generated, which both showed high obesity. The Ankrd26-HR7-derived chimera instead did not show obesity. However, the Ldlr-HR1-derived chimera, showed high cholesterol levels despite a medium-low DNAm level.
Crosses Between Healthy Individuals and Chimeras, and Between Chimeras
By crossing chimeras with healthy (non-methylated) mice and with each other, it was possible to verify how CGI methylation was transmitted to subsequent generations, along with the respective phenotypes of obesity and hypercholesterolemia.
In the cross between chimeras and healthy mice, CGI methylation was stably inherited, regardless of the mouse sex, up to the fourth generation for mice where Ankrd26 is silenced and up to the sixth generation where Ldlr is silencing.
The somatic cells of first-generation methylated mice showed a “mosaic methylation” of the CGIs due to the variable level of methylation (from 20 to 70%) of the mESCs. This can be caused by the chromatin state, which is owed by the initial mESCs that generated the initial chimera.
To explain the inheritance of chromatin state, immunoprecipitation assays showed the enrichment of H3K4me3 (activator) and H3K9me3 (repressor) histones around the Ankrd26 CGI in the second and third generation of heterozygous chimeras (one TTAA allele, which is methylated). A significant increase in H3K9me3 was demonstrated, especially in homozygous mice (two TTAA alleles, both methylated), which reduces the gene-associated protein, increasing body weight. Similar observations were made with the repression of the Ldlr gene, crossing methylated and non-methylated heterozygous mice, resulting in high cholesterol levels and an abnormal phenotype.
During chromatin studies, it was observed that methylation was transmitted during epigenetic reprogramming in the embryonic and postnatal development of mice. Through whole-genome bisulfite sequencing analysis and whole-genome sequencing techniques, a global de novo methylation was observed during embryo implantation, increasing between the blastocyst stage (third day) and the epiblast stage (sixth day). A similar trend is observed in CGI methylation, more or less marked depending on the parent chimera’s methylation. Subsequently, between the eighth and thirteenth day, a global demethylation occurs: it is different between primordial germ cells (PGCs) and somatic cells. Indeed, in PGCs, CGI methylation decreases, while in somatic cells it remains constant. In addition, highly methylated CGIs in the Ankrd26-HR1-chimera are never completely demethylated in PGCs, whereas low-methylation CGIs in the Ankrd26-HR7- and Ldlr-chimeras are completely demethylated.
In conclusion, despite detailed work, it was not possible to establish the main cause of the development of methylation memory.
Conclusions
The study by Takahashi et al. demonstrated that epigenetic signatures are stably transmitted to subsequent generations. Methylation of CpG islands in the Ankrd26 and Ldlr genes not only led to gene repression but also contributed to the emergence of specific phenotypes. Chromatin analysis revealed that the enrichment of H3K4me3 and H3K9me3 histones plays a crucial role in regulating gene expression in these two genes. Researchers hypothesize that methylation memory may be achieved through the combined action of non-coding RNAs and Polycomb group proteins, which are capable of remodeling chromatin and causing epigenetic silencing. These results open new perspectives for the study of epigenetic diseases and related therapies. Moreover, it would be beneficial to explore the therapeutic potential of targeting these epigenetic regulators. For instance, developing specific inhibitors or activators of the identified non-coding RNAs or Polycomb group proteins might offer new strategies for modulating gene expression in diseases characterized by aberrant epigenetic modifications.
Comparative studies in different model organisms could also help to generalize the findings and understand the evolutionary conservation of these mechanisms. In conclusion, while the study from Takahashi et al significantly advances our understanding of transgenerational epigenetic inheritance, further research is needed to fully elucidate the underlying mechanisms and to translate these findings into clinical applications. The integration of multi-omics approaches, including genomics, transcriptomics, and proteomics, could provide a more comprehensive view of the complex regulatory networks involved in epigenetic inheritance and their implications for human health and disease.
References
- Takahashi, Y., Wu, J., et al. (2017). Integration of CpG-free DNA induces de novo methylation of CpG islands in pluripotent stem cells. Science, 356, 503-508. https://doi.org/10.1126/science.aag3260
- Takahashi et al. (2023). Transgenerational inheritance of acquired epigenetic signatures at CpG islands in mice. Cell, 186, 715–731. https://doi.org/10.1016/j.cell.2022.12.047
- Chen, Q., Luo, W., et al. (2020). Structural basis of seamless excision and specific targeting by piggyBac transposase. Nature, 11(1), 3446. https://doi.org/10.1038/s41467-020-17128-1