Title: The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity
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Vazyme product: ChamQ Universal SYBR qPCR Master Mix (Vazyme #Q711, for SYBR Green qPCR detection)
Research Background Introduction
N6-methyladenosine (m6A) is the most prevalent internal mRNA modification, which is first discovered in the early 1970s1. m6A participates in almost all processes involving mRNA metabolism, including RNA transcription, translation, and degradation2. Therefore, the m6A plays a key role in many biological processes3. However, we still know very little about the function of m6A in early mammalian development.
Figure 1: Structure of N6-Methyladenosine
For the research on this issue, Dr. Chen Jiekai’s research group published an article entitled “The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity” in the international journal Nature.
The research group found that the RNA m6A reader YT521-B homology-domain-containing protein 1 (YTHDC1) silences retrotransposons and defends embryonic stem (ES) cell identity. The m6A readeYTHDC is required for the maintenance of mouse ES cells in an m6A-dependent manner, and its deletion initiates cellular reprogramming to a two-cell-like (2C) state. The research group further demonstrated that YTHDC1 and its target m6A RNA are major inducers of the 2C stage-like program. They are able to repress retrotransposons and Dux. This study reveals an essential role for m6A RNA and YTHDC1 in chromatin modification and retrotransposon repression.
Figure 2: RNA m6A reader YTHDC1 guards heterochromatin formation
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Methods and Results
The researchers performed a conditional knockout of exons 7-9 of YTHDC1, which encodes an important part of the YTH domain, and found that the ability of cells to proliferate decreased rapidly after knockout. According to gene expression profiles, 2C-like genes were up-regulated, and 2C-like signature genes and retrotransposable elements were activated. YTHDC1 relies on its m6A-binding activity to maintain the ES cell state, limiting its transformation into 2C-like cells. This demonstrates that the m6A reader YTHDC1 maintains mouse ES cells in an m6A-dependent manner, and that loss of YTHDC1 initiates cell reprogramming to 2C-like cells.
Figure 3: Gene set enrichment analysis (GSEA)
H3K9me3 is a common repressive histone modification mainly related to the formation of heterochromatin. It is abundantly present in retrotransposons and some gene promoter regions in adult cells and silences these regions. SETDB1 is a methyltransferase of H3K9me3, and H3K9me3 on retrotransposons is catalyzed by SETDB1. In a previous study, researchers found that SETDB1-mediated H3K9 methylation inhibits pluripotency-to-pluripotency transition mainly by inhibiting Dux (Cell Reports 2020). Because of the consistent phenotype, we further investigated whether RNA m6A is involved in the regulation of SETDB1-H3K9me3. The study found:
• YTHDC1 binds to transcripts of retrotransposons such as IAP, ERVK, and LINE1 in mouse ES cells.
• YTHDC1 knockdown resulted in reactivation of these silenced retrotransposons and an overall reduction in SETDB1-mediated histone H3K9me3.
• Knockdown of SETDB1 resulted in reactivation of retrotransposon and transformed ES cells into 2C-like cells with totipotency characteristics.
• Co-immunoprecipitation confirmed that YTHDC1 interacts with SETDB1, indicating that m6A targeting YTHDC1 recruits SETDB1 to methylate H3K9 at designated retrotransposons.
• YTHDC1 binds to transcripts of retrotransposons (e.g. IAP, ERVK and LINE1) in mouse ES cells.
These conclusions support the co-regulation of retrotransposon silencing by YTHDC1 and SETDB1 via H3K9me3.
Figure 4: YTHDC1 mediates SETDB1 H3K9me3-dependent retrotransposon repression
The researcher investigated how YTHDC1 binds to chromatin to mediate SETDB1-dependent H3K9me3. Through ChIP-seq analysis, it was found that the YTHDC1-H3K9me3 co-binding peak was mainly located in the retrotransposon region. The binding of YTHDC1 was also found to affect H3K9me3-enriched chromatin and subsequent repression of retrotransposons.
METTL3 is an m6A methylase, and METTL3 knockout reduced SETDB1-dependent H3K9me3, enhancing the expression of SETDB1-dependent H3K9me3-modified retrotransposons, and overlapped with the major expressed genes up-regulated after YTHDC1 knockdown. These results suggest that YTHDC1-mediated chromatin regulation is functionally regulated by H3K9me3 and retrotransposon repression, requiring m6A modification of retrotransposon RNA.
Further study found that m6A-YTHDC1 inhibits retrotransposon and Dux (Dux is the main inducer of 2C-like transition) through SETDB1-mediated H3K9me3, thereby inhibiting cell reprogramming to 2C-like cells. Important roles of m6A RNA and YTHDC1 in chromatin modification and retrotransposon repression are revealed.
In conclusion, YTHDC1 can directly bind to m6A modifications on RNAs transcribed from transposon elements (TEs) and recruit SETDB1 to the corresponding chromatin locations. Subsequently, H3K9me3 on TEs is catalyzed by SETDB1 to form heterochromatin and silence these transposable elements. Knockdown of YTHDC1 results in a significant decrease in SETDB1-dependent H3K9me3 signaling, demonstrating that YTHDC1 is an important mechanistic link of SETDB1-mediated retrotransposon silencing, and also revealing the function of RNA m6A in regulating chromatin.
Vazyme Product Support
Vazyme’s ChamQ Universal SYBR qPCR Master Mix (Q711) facilitated this study. Researchers used this product for qPCR assays when studying the expression differences of genes such as YTHDC1 and SETDB1 knockouts and 2C genes and retrotransposons. ChamQ Universal SYBR qPCR Master Mix (Q711) is a dedicated master mix for qPCR reactions using SYBR Green I chimeric fluorescence method. It is very suitable for high specificity and high sensitivity qPCR reactions. This product contains a special ROX Passive Reference Dye, which is suitable for all qPCR instruments. There is no need to adjust the concentration of ROX on different instruments. It is only necessary to add primers and templates when preparing the reaction system for amplification.
ChamQ Universal SYBR qPCR Master Mix (Q711)
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1. Dubin, D. T., and Taylor, R. H. (1975). The methylation state of poly A-containing messenger RNA from cultured hamster cells. Nucleic Acids Res. 2, 1653–1668. doi: 10.1093/nar/2.10.1653
2. Roundtree, I. A., Luo, G.-Z., Zhang, Z., Wang, X., Zhou, T., Cui, Y., et al. (2017b). YTHDC1 mediates nuclear export of N(6)-methyladenosine methylated mRNAs. Elife 6:e31311. doi: 10.7554/eLife.31311
3. Fu Y, Dominissini D, Rechavi G, He C. (2014) Gene expression regulation mediated through reversible m⁶A RNA methylation. Nat Rev Genet. 15(5):293-306. doi:10.1038/nrg3724