RNA m⁶A Reader YTHDC1 Governs Heterochromatin Formation

N⁶-methyladenosine (m⁶A) is the most abundant modification on RNA molecules present in eukaryotes, and recognized byYTH-domain containing readers.  

Among five YTH family m⁶A readers, nuclear reader YTHDC1 is the sole protein leading to early embryonic lethality upon deletion in mice, resembling the phenotype of Mettle3, an m⁶A transferase, knockout (KO). By contrast, mice deficient in other YTH readers including Ythdf1-3 or known YTHDC1-interacting genes such as Zcchc8 survive at the early embryonic stage, suggesting m⁶A has unidentified role besides post-transcriptional regulation.  

The formation of H3K9me3-associated heterochromatin is a prominent mechanism in cell fate determination. H3K9me3 predominantly labels and represses the retrotransposon regions across the genome in mouse embryonic stem (ES) cells, in addition to the constitutive heterochromatin including centromere and telomere. SETDB1 catalyzes retrotransposon H3K9me3, as its ablation reactivates these elements, triggering the transition of ES cells into a 2C-like totipotency state. However, the mechanism towards the selective recruitment of SETDB1 to retrotransposon warrants further investigations. 

The researchers first constructed the Ythdc1 conditional KO mouse ES cell line by excising Ythdc1 exons 7–9 which spanning the m⁶A-binding site of the YTH domain. Ythdc1-KO cells showed severe defects in proliferation. Intriguingly, Ythdc1-KO cells programed into a 2C-like state, as validated by transcriptome analysis, MERVL-reporter induction, pluripotency NANOG suppression, and chimera assay. Further, only the wild type but not YTH m⁶A-binding-site mutants of YTHDC1 rescues the proliferation deficiency and 2C-like transition, demonstrating that Ythdc1 relies on its m⁶A-binding activity to maintain ES cell identity. 

Concomitantly, the researchers discovered that deletion of Ythdc1 upregulated the expression of retrotransposons such as IAP and LINE1, and identified these m⁶A marked transposable element (TE) transcripts as YTHDC1-binding targets. They then investigated how YTHDC1 associates with chromatin and mediates SETDB1-dependent H3K9me3, and identified Ythdc1-KO strikingly dampened SETDB1-dependent H3K9me3 deposition on TEs. In addition, co-immunoprecipitation revealed physical interactions between YTHDC1 and SETDB1. These observations suggest that SETDB1 mediated retrotransposon silencing requires YTHDC1 and RNA m⁶A modulates chromatin function. 

To determine whether YTHDC1 is recruited onto chromatin by m⁶A-marked TE-derived RNAs, the researchers interrogated the chromatin occupancy of m⁶A-modified TE RNAs using ChIRP-seq and GRID-seq. The results showed that IAP and LINE1 RNAs predominantly target the chromatin regions of their respective subfamilies.  

Subsequently, they constructed Mettl3-KO mouse ES cells by knocking out exons of 5-7 of Mettl3, which disrupted the core MT-A70 domain starts from exon 6, and found that Mettl3-KO abolishes RNA m6A modification on mRNAs and m⁶A-marked TE transcripts. Consistently, Mettl3-KO also induces the reactivation of TEs and the reduction of H3K9me3, demonstrating YTHDC1 targets chromatin via m⁶A TE RNA. 

To explore the relationship between TE reactivation and 2C-like transition, ChIRP and GRID-seq revealed that LINE1 RNA occupied the Dux locus. Dux is a known 2C-like regulator. The researchers found despite the blockage of the 2C-like program,Dux-KO cells retained the ability to reactivate many TEs upon Ythdc1 depletion. These results emphasize that YTHDC1-mediated repression of TE is independent of Dux and Dux-regulated cell-fate decisions, but is mediated by YTHDC1-SETDB1 chromatin regulation. 

RNA-induced transcriptional silencing is known to mediate heterochromatin formation in fission yeast, plants,Caenorhabditis elegans, Drosophila melanogaster and ciliates, but it remains unclear whether this occurs in mammalian cells. Notably, H3K9 methylation at fission yeast meiotic genes involves not only the RNAi-dependent mechanism that is central to constitutive heterochromatin, but also the YTH-domain-containing protein Mmi1. Nonetheless, Mmi1 targets meiotic RNAs by recognizing their determinant of selective removal motif through its YTH domain, but in an m⁶A-independent manner. The evidence suggests both evolutionarily conserved and divergent mechanisms of chromatin regulation. 

This study provided insight into m⁶A function and mechanisms of heterochromatin regulation in mammals. 

Fig. 1 RNA m⁶A reader YTHDC1 guards heterochromatin formation (Image by GIBH)  

Contact:

CHEN Jiekai

E-mail: chen_jiekai@gibh.ac.cn