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TERRA regulate the transcriptional landscape of pluripotent cells through TRF1-dependent recruitment of PRC2

chromosomes and gene expression stem cells and regenerative medicine trf1 telomere pluripotency terra plurycomb lncrna

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#1 Engadin

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Posted 21 August 2019 - 07:27 PM


S O U R C E :    eLife

 

 

 

 

 

Abstract

 

The mechanisms that regulate pluripotency are still largely unknown. Here, we show that Telomere Repeat Binding Factor 1 (TRF1), a component of the shelterin complex, regulates the genome-wide binding of polycomb and polycomb H3K27me3 repressive marks to pluripotency genes, thereby exerting vast epigenetic changes that contribute to the maintenance of mouse ES cells in a naïve state. We further show that TRF1 mediates these effects by regulating TERRA, the lncRNAs transcribed from telomeres. We find that TERRAs are enriched at polycomb and stem cell genes in pluripotent cells and that TRF1 abrogation results in increased TERRA levels and in higher TERRA binding to those genes, coincidental with the induction of cell-fate programs and the loss of the naïve state. These results are consistent with a model in which TRF1-dependent changes in TERRA levels modulate polycomb recruitment to pluripotency and differentiation genes. These unprecedented findings explain why TRF1 is essential for the induction and maintenance of pluripotency.

 

 

Introduction

 

Multiple cellular processes, including pluripotency and the determination of cell fate, are regulated by epigenetic modifications, including genome-wide changes in DNA and histone methylation (Blasco, 2007Theunissen and Jaenisch, 2014). One key histone modification, tri-methylation of lysine 27 in histone 3 (H3K27me3), is a transcription repression mark that it is controlled by the polycomb transcriptional repressor (PRC) proteins (Sparmann and van Lohuizen, 2006). There are two polycomb complexes, PRC1 and PRC2, which cooperate to achieve gene silencing (Lund and van Lohuizen, 2004). PRC2 encompasses EED (Embryonic ectoderm development), EZH2 (Enhancer of zeste), SUZ12 (Suppressor of zeste 12), and ESC (Extra sex combs) (Ringrose and Paro, 2004), and is involved in the initiation of gene repression. In particular, the PRC2 component EZH2 is a SET-domain-containing protein that catalyzes the repressive H3K27me3 mark and to lesser extent also H3K9me3 (Simon and Kingston, 2009). PRC2-mediated H3K27me3 recruits PRC1, which contributes to gene silencing, possibly by blocking transcriptional elongation by the RNA Polymerase II (Stock et al., 2007).

 

Interestingly, polycomb PRC1 and PRC2 complexes have been proposed to have a key role in the maintenance of embryonic stem (ES) cell pluripotency (Pereira et al., 2010). In particular, polycomb proteins are important for restraining the activity of lineage-specifying factors in ES cells, and also have been shown to be essential in establishing the conversion of differentiated cells towards pluripotency (Azuara et al., 2006Boyer et al., 2006Endoh et al., 2008). PRC2 proteins and the H3K27me3 mark are proposed to maintain genes in a state in which they are poised for transcription and contribute to pluripotency (Azuara et al., 2006Boyer et al., 2006Endoh et al., 2008). In agreement with this notion, EZH2 is upregulated during reprogramming and EZH2 knock-down impairs reprogramming. Also, PRC2 is essential for pluripotency but is not essential in human ES cells in the ‘naïve’ state (Shan et al., 2017).

 

In ES cells, a significant proportion of polycomb target genes are repressed genes that encode transcription factors that are required for lineage specification later during development y (Boyer et al., 2006Lee et al., 2006). These genes are also co-occupied by the key pluripotency factors OCT4, NANOG, and SOX2, suggesting that the function of these pluripotency genes in repressing gene expression may be mediated by polycomb (Boyer et al., 2005Boyer et al., 2006). Indeed, downregulation of the PRC1 and PRC2 activity in ES cells leads to global de-repression of these genes and to unscheduled differentiation (Boyer et al., 2006Endoh et al., 2008). Deletion of one of the PRC2 components in mice (EED, SUZ12 or EZH2) results in severe defects in development, suggesting the mis-expression of lineage-specific genes (Faust et al., 1995O'Carroll et al., 2001Pasini et al., 2004). However, the deletion of PRC2 components in ES cells is not lethal (Leeb et al., 2010).

 

Interestingly, polycomb has also been reported to bind to bivalent genes, which are occupied by both the heterochromatic mark H3K27me3 and the active mark H3K4me3. In this scenario, PRC2 is important in maintaining lineage genes in a poised state ready to respond to differentiation cues (Voigt et al., 2013).

 

Telomeres are special heterochromatin structures at chromosome ends, which are formed by tandem repeats of the TTAGGG sequence bound by the so-called shelterin complex (Blackburn, 2005de Lange, 2005Martínez and Blasco, 2011). Telomeric chromatin is enriched in histone-repressive marks including H3K9me3 and H4K20me3 (García-Cao et al., 2002García-Cao et al., 2004Gonzalo et al., 2005Gonzalo et al., 2006Benetti et al., 2007Blasco, 2007). Recently, we also reported that telomeres are enriched for the PRC2-repressive mark H3K27me3 (Montero et al., 2018). The function of telomeres is to protect chromosome ends from degradation and from triggering chromosomal aberrations, such as chromosome end-to-end fusions. Two independent studies showed that the shelterin component TRF1 is also greatly upregulated during reprogramming (Boué et al., 2010Schneider et al., 2013). Indeed, we showed that TRF1 upregulation is an early event during cellular reprogramming, which precedes and is independent of telomere elongation by telomerase (Schneider et al., 2013). The Terf1 gene is a direct target of OCT4, and is also essential for the induction and maintenance of pluripotency. In support of this, deletion of TRF1 causes embryonic lethality at the blastocyst stage (Karlseder et al., 2003). More recently, we showed that TRF1 is also upregulated during in vivo reprogramming, showing a similar pattern of expression to that of OCT4 in reprogrammed tissues (Marión et al., 2017). In spite of this solid evidence that TRF1 has an important role in pluripotency, the mechanisms that allow TRF1 to perform this mediating role have remained unknown until now.

 

PRC2 can interact both in vivo and in vitro with the long non-coding RNAs transcribed from telomeres, or TERRA, and this interaction is essential for the establishment of the H3K27me3 mark at telomeres (Chu et al., 2017Wang et al., 2017Montero et al., 2018). TERRA has also been shown to be associated with polycomb marks in the vicinity of genes and to modulate gene expression (Chu et al., 2017). Thus, there seems to be an interplay between telomere transcriptional status and long-range epigenetic regulation. In fact, PRC2 interacts with several long non-coding RNAs (lncRNAs), and this interaction is thought to regulate gene expression by recruiting PRC2 to specific loci. Some examples of lncRNAs that can physically interact with PRC2 and recruit it to specific loci include Xist (Zhao et al., 2008), Hotair (Rinn et al., 2007) and the antisense non-coding RNA in the Cdkn2alocus (Yap et al., 2010). These lncRNAs play important roles in X chromosome activation and tumorigenesis. However, how a lncRNA is able to provide specificity for PRC2 recruitment is not clear.

 

In addition, TERRA has been previously described to interact with the shelterin component TRF2, which can interact with TRF1, thus opening the possibility that polycomb may also be interacting with shelterin components. In this regard, a recent report showed that the Arabidopsis thaliana telomere-repeat binding factors (TRBs) recruit PRC proteins to different promoters through a telobox motif. In the absence of the three TRB proteins, the PRC2-mediated H3K27me3 mark was altered in a similar manner to that of PRC2 mutants. Indeed, an interaction between TRB1–3 and PRC2 proteins was found (Zhou et al., 2016bZhou et al., 2018).

 

Here, we set to address the mechanisms through which OCT4-mediated TRF1 upregulation functions as an essential process for the induction and maintenance of pluripotency in mouse cells. To this end, we have used an unbiased genome-wide approach, looking for global changes in gene expression in the absence of TRF1. We make the unprecedented finding that TRF1 abrogation has a ‘butterfly effect’ on the transcription of naïve pluripotent cells, altering the epigenetic landscape of these cells through a novel mechanism, which involves TERRA-mediated polycomb recruitment to pluripotency genes and cell-fate genes.

 

 

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Also tagged with one or more of these keywords: chromosomes and gene expression, stem cells and regenerative medicine, trf1, telomere, pluripotency, terra, plurycomb, lncrna

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