The RNA-protein-DNA Axis: A new layer of Regulation of LTR7/HERV-H in hESC
Figure 1. Crosstalk between RNA m6A and DNA methylation at LTR7/HERV-H loci. In hESCs, m6A-modified HERV-H RNA recruits YTHDC2 and TET1 to LTR7 loci, promoting DNA demethylation (5hmC) and maintaining open chromatin to preserve pluripotency (left). Depletion of m6A or YTHDC2 leads to DNA hypermethylation (5mC), LTR7 silencing, and induction of neuronal differentiation (right)
Abstract
This study examines the role of m6A RNA modification in regulating transposable elements within human pluripotent stem cells (hESCs). It highlights the crucial function of YTHDC2, an YTH-domain-containing m6A reader protein, in maintaining HERV-H activity by preventing its silencing. The loss of YTHDC2 or reduction of m6A on HERV-H RNA leads to increased repressive chromatin marks and enhances neural differentiation. This provides insights on the mechanisms that maintain pluripotency and influence cell fate decisions in stem cells.
Review
Transposable elements (TEs) are mobile sequences that can move within the genome. Their displacement can cause genomic instability, for this reason their activity must be finely regulated. A recent study by Sun et al. has identified a mechanism that directly connects m6A-modified transcripts of HERV-H, the m6A-reader YTHDC2 and the DNA demethylase TET1 in human embryonic stem cells (hESCs)1. Their work reveals that m6A-modified TE-derived RNAs can guide chromatin regulation by recruiting epigenetic enzymes to their genomic loci, preventing silencing and cell-fate transitions. These discoveries can be used for the study of regenerative medicine, cancer and neurological diseases.
Introduction
Transposable elements (TEs) are an important but often underrated component of the human genome2. These DNA sequences have the unique ability to move within the genome, potentially disrupting gene function and causing genomic instability. To prevent such dangerous effects, the genome employs multiple regulatory mechanisms and the DNA methylation is one of the most significant. The addition of a methyl group to cytosine residues in CpG dinucleotides serves as a key mechanism to silence TEs and maintain genomic stability3. However, RNA modifications, such as m6A, have recently gained attention for their role in regulating gene expression at the post-transcriptional level4,5. These modifications influence RNA stability, splicing, and translation, providing an additional layer of control over TE activity.
The interaction between DNA and RNA modifications is now recognized as a crucial regulatory axis that maintains genomic integrity, but also influences cell fate, differentiation and pluripotency. Understanding how these two epigenetic modifications work together to regulate TEs is essential for advancing our knowledge of gene regulation and its implications in diseases, such as cancer and neurological disorders, as well as in regenerative medicine.
Results
Molecular Mechanism of the YTHDC2–m6A–TET1 Axis in Transposable Element Regulation
One of the achievements of this study is the development of the CARGO-BioID method, a CRISPR-based targeted proteomics strategy designed to map protein complexes associated with repetitive genomic elements, such as HERVH. The technique uses multiplexed guide RNAs (gRNAs) to direct a dCas9-TurboID fusion protein to over 1,800 copies of LTR7 sequences. This approach is particularly innovative because it overcomes a significant challenge in the study of transposable elements. By using CARGO-BioID, the authors were able to identify the most enriched proteins in these loci, providing an overall map of proteins that interact with HERVH regions.
One of the most important findings from this experiment was the identification of YTHDC2, an m6A RNA reader, as a key protein associated with LTR7 regions5. YTHDC2 is preferentially found near LTR7 copies that were marked by accessible chromatin, high levels of H3K27ac (an active chromatin mark), and low levels of H3K9me3 (a repressive chromatin mark). These observations suggested that YTHDC2 is implied in the regulation of HERV-H transcription.
To further validate this interaction, the authors utilized several techniques, including RIP-seq (RNA Immunoprecipitation Sequencing), eCLIP-seq (enhanced crosslinking and immunoprecipitation sequencing), and motif analysis, to explore how YTHDC2 is recruited to HERV-H RNA. Motif analysis identified that the YTHDC2-binding sites in HERV-H RNAs have a significant enrichment of the GGACU sequence, which is the RRACH motif. This motif is the primary site for adenine methylation. These data confirm that YTHDC2 binds to m6A-modified HERV-H transcripts in a motif-dependent manner.
Biochemical assays, including RNA pull-down assays and electrophoretic mobility shift assays (EMSA), further confirmed that the recruitment of YTHDC2 to the LTR7 regions of HERV-H depends on the presence of m6A modifications on the RNA, with m6A inhibition or m6A erasure disrupting this interaction.
The ChIP-seq data showed the co-localization of YTHDC2 and TET1 at LTR7 loci. For this reason, the study next explored how YTHDC2 modulates chromatin dynamics at LTR7 loci by recruiting the DNA demethylase enzyme TET1.
TET1 reduces 5-methylcytosine (5mC) levels while increasing 5-hydroxymethylcytosine (5hmC), leading to an active chromatin state.
In YTHDC2 KO cells, there is not the TET1 recruitment to LTR7, leading to an accumulation of CpG methylation at these loci. This indicates that TET1 is essential for maintaining HERV-H transcription by preventing DNA methylation and allowing these regions to remain transcriptionally active.This suggests that YTHDC2 acts as a bridge, facilitating the recruitment of TET1 to LTR7 regions.
This establishes a clear RNA-to-DNA regulatory pathway, where m6A-modified HERV-H RNA recruits YTHDC2, which in turn recruits TET1 to maintain LTR7 regions in an active, demethylated state, sustaining HERV-H transcription.
LTR7/HERV-H Governs Cell Fate Decisions and Pluripotency in hESC
The functional consequences of these regulatory interactions were then explored, particularly in the context of pluripotency and differentiation. The study found that YTHDC2 and LTR7 silencing did not directly impact the maintenance of pluripotency, as evidenced by the continued expression of pluripotency markers6. However, both YTHDC2 KO and LTR7 silencing promoted early neural differentiation, indicating that the regulation of HERV-H activity is important for maintaining pluripotency and controlling cellular fate7. This suggests that HERV-H RNA, through the YTHDC2/LTR7 axis, inhibits neural differentiation.
Among the downstream targets regulated by this axis, the HERV-H-derived lncRNA LINC-ROR emerged as a critical effector. The expression of LINC-ROR depends on the YTHDC2/LTR7 axis, and its activity contributes to the prevention of neurogenic differentiation. The loss of LINCROR expression in YTHDC2 KO and LTR7 silenced cells led to an increased expression of neural markers, supporting the role of TE-derived RNAs in regulating stem cell fate and differentiation.
Limitations
One of the main limitations is that most experiments were performed in a single human embryonic stem cell line (H1). For this reason, it is unclear whether the proposed mechanism also applies to other pluripotent stem cell models, such as H9 cells or iPSC. In addition, the analysis of LTR7 elements was conducted at a bulk level. Since the human genome contains more than 1,800 LTR7/HERV-H copies with differences in sequence, chromatin context, and transcriptional activity, these approaches cannot determine whether YTHDC2 regulates all LTR7 loci or only a specific subset. Another limitation is that functional m6A editing was performed only on the LINC-ROR transcript. This restricts the conclusions that can be drawn for the wider HERV-H family. Moreover, although the study suggests a link between YTHDC2 and TET1, the nature of this interaction is not fully defined, and it remains unclear whether it is direct or mediated by other proteins.
Future studies should test this mechanism in multiple pluripotent stem cell lines and use methods with singlelocus resolution to identify the specific LTR7 elements that are functionally important. Extending m6A editing to additional HERV-H transcripts would help to determine how much this regulatory mechanism can be generalized. Finally, examining different differentiation pathways, beyond neuronal differentiation, and exploring whether similar RNA– DNA regulatory mechanisms apply to other transposable elements will clarify the wider role of this pathway in pluripotency and cell fate regulation.
Discussion
This study reveals the complex regulatory network involving m6A modification on RNA, YTHDC2, and TET1 in maintaining the transcriptional activity of HERV-H retrotransposons. The findings highlight the cooperative interaction between RNA and DNA epigenetic modifications as a fundamental mechanism for maintaining genome stability and pluripotency in hESCs. The results show the importance of YTHDC2 in regulating HERV-H activity, as its presence prevents the epigenetic silencing of LTR7 regions, crucial for pluripotency. The m6A modification of RNA serves as a key factor in regulating DNA modification and chromatin structure, thus influencing cell fate.
Understanding the crosstalk between RNA modifications and DNA in regulating pluripotency could provide valuable insights for regenerative medicine, potentially allowing for better control over stem cell differentiation and reprogramming.
References
- Sun, T., Xu, Y., Xiang, Y., Ou, J., Soderblom, E. J., & Diao,Y. (2023). Crosstalk between RNA m6A and DNA methylation regulates transposable element chromatin activation and cell fate in human pluripotent stem cells. Nature genetics, 55(8), 1324–1335. https://doi.org/10.1038/s41588-023-01452-5
- Bourque, G., Burns, K. H., Gehring, M., Gorbunova, V.,Seluanov, A., Hammell, M., Imbeault, M., Izsvák, Z., Levin, H. L., Macfarlan, T. S., Mager, D. L., & Feschotte, C. (2018). Ten things you should know about transposable elements. Genome biology, 19(1), 199. https://doi.org/10.1186/s13059-018-1577-z
- Padeken, J., Methot, S. P., & Gasser, S. M. (2022). Establishment of H3K9-methylated heterochromatin and its functions in tissue differentiation and maintenance. Nature reviews. Molecular cell biology, 23(9), 623–640. https://doi.org/10.1038/s41580-022-00483-w
- Liu, J., Dou, X., Chen, C., Chen, C., Liu, C., Xu, M. M., Zhao, S., Shen, B., Gao, Y., Han, D., & He, C. (2020). N6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription. Science (New York, N.Y.), 367(6477), 580–586. https://doi.org/10.1126/science.aay6018
- Shi, H., Wei, J., & He, C. (2019). Where, When, and How: Context-Dependent Functions of RNA Methylation Writers, Readers, and Erasers. Molecular cell, 74(4), 640–650. https://doi.org/10.1016/j.molcel.2019.04.025
- Zheng, Y., Yan, R. Z., Sun, S., Kobayashi, M., Xiang, L., Yang, R., Goedel, A., Kang, Y., Xue, X., Esfahani, S. N., Liu, Y., Resto Irizarry, A. M., Wu, W., Li, Y., Ji, W., Niu, Y., Chien, K. R., Li, T., Shioda, T., & Fu, J. (2022). Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development. Cell stem cell, 29(9), 1402–1419.e8. https://doi.org/10.1016/j.stem.2022.08.009
- Batista, P. J., Molinie, B., Wang, J., Qu, K., Zhang, J., Li, L., Bouley, D. M., Lujan, E., Haddad, B., Daneshvar, K., Carter, A. C., Flynn, R. A., Zhou, C., Lim, K. S., Dedon, P., Wernig, M., Mullen, A. C., Xing, Y., Giallourakis, C. C., & Chang, H. Y. (2014). m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell stem cell, 15(6), 707–719. https://doi.org/10.1016/j.stem.2014.09.019