Functional role of Tet-mediated RNA hydroxymethylcytosine in mouse ES cells and during differentiation

Abstract

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.

TET-Mediated Epigenetic Regulation in Immune Cell Development and Disease

“…The PSPC1-TET2 mediated 5hmC deposition on MERVL transcripts resulted in their destabilization and subsequent degradation (Guallar et al, 2018 ). In addition, both TET1 and TET2 deposit 5hmC in mRNAs in genes that are fundamental for pluripotency, such as Jarid2 and Eed , and can result in reduced mRNA stability (Lan et al, 2020 ). As a consequence of the transcript destabilization, pluripotency genes that would be expressed too highly acquire appropriate expression levels and adequately repress the expression of lineage-specifying factors (Lan et al, 2020 ).…”

“…In addition, both TET1 and TET2 deposit 5hmC in mRNAs in genes that are fundamental for pluripotency, such as Jarid2 and Eed , and can result in reduced mRNA stability (Lan et al, 2020 ). As a consequence of the transcript destabilization, pluripotency genes that would be expressed too highly acquire appropriate expression levels and adequately repress the expression of lineage-specifying factors (Lan et al, 2020 ). As the ES cells receive differentiation cues, the pluripotency factors are rapidly turned off and the lineage-specifying factors are upregulated to drive the differentiation process with efficiency (Lan et al, 2020 ).…”

The Complexity of TET2 Functions in Pluripotency and Development

“…Besides its binding to DNA, more recently TET2 has also been shown to interact with RNA in vitro and in vivo (Fu et al, 2014 ; Guallar et al, 2018 ; Shen et al, 2018 ; He et al, 2020 ; Lan et al, 2020 ). Indeed, and in line with the observation that TET2 is less strongly associated to chromatin than TET1 (Vella et al, 2013 ), its recruitment to chromatin was recently shown to be facilitated by RNA and decreased upon global transcriptional inhibition (Guallar et al, 2018 ).…”

“…Although TET2 DNA-binding domain (DBD) and RBR domain have been mapped to different regions of the protein ( Figure 1A ) both were found to fold in proximity in human TET2 (Hu et al, 2013 ). Thus, it will be important to address how TET2 affinity and preference for RNA or DNA is regulated in the context of chromatin, where both nascent RNAs and genomic DNA can be oxidized (Chen et al, 2013 ; Hon et al, 2014 ; Guallar et al, 2018 ; Lan et al, 2020 ). Importantly, overexpression of full-length TET2 in HEK293T cells showed no catalytic activity in m 5 C in RNA, in contrast to TET2 catalytic domain, suggesting negative regulatory functions of the N-terminal domain on TET2 function on RNA oxidation (Fu et al, 2014 ).…”

“…TET2 plays thus key roles in shaping the methylome of a cell and establishing novel 5hmC-enriched genomic regions, including enhancers, for chromatin and transcriptional regulation. Moreover, recently we and others have shown that TET2 can also oxidize m 5 C (to distinguish it from 5mC in DNA) in RNAs of ESCs and during myelopoiesis for the regulation of RNA stability (Guallar et al, 2018 ; Shen et al, 2018 ; He et al, 2020 ; Lan et al, 2020 ) Here we review the current understanding of TET2 functions, providing an overview of its roles in development and pluripotency contexts. Although multiple studies have approached 5hmC function through double and triple Tet1/2/3 depletion (Dawlaty et al, 2013 , 2014 ; Lu et al, 2014 ; Kang et al, 2015 ; Li et al, 2016 ; Verma et al, 2018 ; Charlton et al, 2020 ) we focus on Tet2 single mutants to try to better dissect TET2 specific functions on both DNA and RNA by integrating structure and function, providing a comprehensive overview of this versatile epigenetic and epitranscriptomic regulator.…”