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Title: Functional evaluation of transposable elements as enhancers in mouse embryonic and trophoblast stem cells}
Article:
Abstract:
Transposable elements( TEs) are thought to have helped establish gene regulatory networks. Both the embryonic and extraembryonic lineages of the early mouse embryo have seemingly co-opted TEs as enhancers, but there is little evidence that they play significant roles in gene regulation. Here we tested a set of long terminal repeat TE families for roles as enhancers in mouse embryonic and trophoblast stem cells. Epigenomic and transcriptomic data suggested that a large number of TEs helped to establish tissue-specific gene expression programmes. Genetic editing of individual TEs confirmed a subset of these regulatory relationships. However, a wider survey via CRISPR interference of RLTR13D6 elements in embryonic stem cells revealed that only a minority play significant roles in gene regulation. Our results suggest that a subset of TEs are important for gene regulation in early mouse development, and highlight the importance of functional experiments when evaluating gene regulatory roles of TEs.
Introduction:
Our knowledge of the tissue-specific regulatory landscape of genomes has vastly increased over the last 10 years, thanks in part to large efforts from consortia such as ENCODE and NIH Roadmap( ENCODE Project Consortium, 2012; Kundaje et al., 2015). But whilst such mapping efforts have been instrumental in categorising the non-coding genome into different types of biochemical activity, our understanding of the associated functional roles remains limited. One of the grand challenges of the post-ENCODE era has been to ascribe regulatory function to the biochemically active non-coding portion of genomes. This question is particularly pertinent to transposable elements( TEs)( Elliott et al., 2014; Doolittle and Brunet, 2017), which often display marks of regulatory activity in a species-specific manner( Jacques et al., 2013; Carninci, 2014). On one hand, successful TEs are expected to display such active profiles, which serve the selfish interests of TEs but may act neutrally with respect to host fitness. On the other, TEs can be co-opted( or exapted) by the host to serve gene regulatory roles, such as alternative promoters or enhancers( Chuong et al., 2017). The distinction between these two scenarios relies on approaches that query the causal links between TEs, gene expression and phenotype. Primarily based on epigenomic data, evidence to date suggests that particular TE families have contributed to the evolution of tissue-specific gene regulatory networks in contexts such as early development( Kunarso et al., 2010), placentation( Chuong et al., 2013), pregnancy( Lynch et al., 2011) and innate immunity( Chuong et al., 2016), amongst others. Transcription factor( TF) binding motifs hosted within the regulatory portion of TEs enable their expression in specific tissues( Sundaram et al., 2014; Sundaram et al., 2017), presumably in a manner that enables vertical inheritance of new TE insertions via the germline. In this respect, mammalian preimplantation development is a seemingly well exploited context for TE expansion, driving genetic conflicts with the host, as well as creating opportunities for TE exaptation( Rodriguez-Terrones and Torres-Padilla, 2018). In the mouse, TE-derived regulatory activity has been implicated at multiple stages of preimplantation development. Namely, MERVL elements become highly activated upon zygotic genome activation and are thought to play a role in the establishment of the 2-cell stage expression programme( Macfarlan et al., 2012). Transition from the 2-cell stage and development progression to the blastocyst stage appear to depend on LINE-1 expression( Jachowicz et al., 2017; Percharde et al., 2018). Finally, work from embryonic and trophoblast stem cells( ESCs and TSCs, respectively), suggests a divergence in TE regulatory activity that is concomitant with the separation of the embryonic and extraembryonic lineages at the blastocyst stage( Kunarso et al., 2010; Chuong et al., 2013). In ESCs, TE families such as RLTR13D6 bind key ESC TFs( e. g., OCT4, NANOG), whereas a distinct subset( e. g., RLTR13D5) bind factors essential for the maintenance of the TSC state( CDX2, ELF5, EOMES). These elements are enriched for histone marks that are characteristic of distal enhancers and lie near genes that are expressed in the lineages where they are active( Kunarso et al., 2010; Chuong et al., 2013). TE enhancer activity depends on the cooperative action of multiple TFs, whose binding motifs appear to have been already present in the corresponding ancestral TE insertions( Sundaram et al., 2017). However, it remains unclear to what extent such lineage-specific TEs are important for maintaining gene expression programmes during preimplantation development. Here we have tested the gene regulatory function of specific TE families in ESCs and TSCs using genetic and epigenetic editing approaches, and compared them with predictions from extensive analyses of epigenomic and transcriptomic data. We identify a number of TEs that are important to drive expression of lineage-specific genes. However, our data suggest that these constitute a minority of all the putative TE-derived enhancers identified through bioinformatic analyses, highlighting the importance of functional tests when assessing the contribution of TEs to gene regulatory networks.
Discussion:
TEs are increasingly being presented as major contributors to gene regulatory networks in a variety of contexts( Chuong et al., 2013; Chuong et al., 2016; Kunarso et al., 2010; Jacques et al., 2013; Sundaram et al., 2014; Lynch et al., 2011; Fuentes et al., 2018; Imbeault et al., 2017). Yet most of these studies have relied largely on the idea that biochemical activity at the chromatin level is indicative of function, a concept famously associated with the findings of the ENCODE project( ENCODE Project Consortium, 2012) that triggered a still ongoing debate( Graur et al., 2013; Ponting, 2017; Doolittle and Brunet, 2017). The use of genetic and epigenetic editing tools as presented here, and also used by other labs( Chuong et al., 2016; Jachowicz et al., 2017; Fuentes et al., 2018), initiate a much needed move to evaluating causal roles for TEs in gene regulation. Our work has revealed that a set of TEs with regulatory potential in ESCs act mostly neutrally with respect to their effects on gene expression, which contrasts with earlier suggestions from analyses of chromatin profiling experiments( Kunarso et al., 2010). In the absence of a substantial contribution of ESC TE+ enhancers to gene regulation, the striking correlations emerging from profiling efforts most likely reflect the fact that TE insertions are best tolerated in regions where their tissue-specific enhancer action matches the expression profiles of nearby genes. Whilst the enhancer activity of these TEs could be inconsequential for gene expression, this genomic ‘safe niche’ would be permissive for fixation by genetic drift. Additionally, we cannot exclude the possibility that some TE+ enhancers act redundantly with TE- enhancers, despite our attempt to isolate the effects of each of these enhancer groups. Such TEs could still be important to ensure regulatory robustness and, indeed, enhancer redundancy is a seemingly common feature of enhancer-gene networks( Osterwalder et al., 2018). Extensive genetic work will be necessary to evaluate what proportion of TE+ enhancers act in a redundant fashion with other regulatory elements. In a contrasting example to our findings, epigenetic editing work by the Wysocka lab has revealed that a large proportion of LTR5Hs elements play significant roles in the regulation of nearly 300 genes in a human embryonal carcinoma cell line( Fuentes et al., 2018). Notably, in the latter study virtually all H3K27ac-marked LTR5Hs elements were inactivated by CRISPRi( 257 copies, according to our own analysis), probably due to the use of twelve sgRNAs simultaneously( Fuentes et al., 2018). It is therefore possible that more efficient targeting of all H3K27ac-marked RLTR13D6 elements( 76 copies) in mouse ESCs would uncover additional regulatory elements. However, the effects seen here from silencing 34( 44%) of these copies suggest that regulatory RLTR13D6 elements would likely remain a minority. Another explanation to the differences between LTR5Hs and RLTR13D6 action is that the regulatory effects of TEs are variable between families and cellular contexts. Indeed, other TEs considered here and whose effects we did not test by CRISPRi( RLTR9, RLTR13D5, RLTR13B), may play important roles in ESC and TSC gene regulation. These considerations further emphasise the need to perform functional experiments on a case-by-case basis. Despite the neutral action of most TEs analysed here, we have uncovered a number of elements that act as key enhancers of gene expression in ESCs( Tdrd12, Smarcad1, Spp1 and Hook3) and TSCs( Map3k8 and Scarf2). But do these TE insertions impact on cellular and organismal phenotypes, ultimately affecting host fitness? TDRD12 is a protein essential for secondary piRNA production in mice and is essential for male fertility( Pandey et al., 2013; Pandey et al., 2018). It is therefore possible that the RLTR13D6 element we identified is also active during male germ line development, which would ironically implicate it in genome defence against the mobility of younger, piRNA-targeted TEs. Smarcad1 knockout mice are subviable, displaying growth defects and low fertility( Schoor et al., 1999). It remains to be seen whether the activity of the RLTR9E element we identified plays any role in these phenotypes, possibly by affecting early embryonic differentiation( Hong et al., 2009). Other genes that we found to be regulated by TEs are not essential for development: both Map3k8( Dumitru et al., 2000) or Spp1( Rittling et al., 1998) knockout mice are viable and appear to develop normally. In these cases it is likely that the action of the associated TEs is inconsequential to the organism, although there could be more subtle embryonic phenotypes or these TEs could play additional roles outside of the early developmental context. Notably, most exemplars of phenotypically relevant TE-derived regulatory elements have been uncovered by analysing naturally occurring phenotypes( Lisch, 2013; Chuong et al., 2017). The reverse approach of searching for phenotypes linked with putative cis-acting TEs has the potential to reveal a wide array of adaptive TE insertions, although this is nonetheless challenging and examples to date are limited( Chuong et al., 2017). At a time when epigenomic data are providing abundant indications that TEs play functional regulatory roles, our findings place these observations into perspective and provide a reminder that a large proportion of mammalian genomic sequences are neutrally evolving. Yet evolutionary tinkering( Jacob, 1977) may still benefit from a large amount of ‘junk DNA’ that occasionally can be put to good use. As was fittingly put by Goodier and Kazazian, ‘evolution has been adept of turning some ‘junk’ into treasure’( Goodier and Kazazian, 2008).
Much of what is known about genetics has come from studying only a tiny fraction of the genome’s sequence , the part that primarily codes for proteins . But the genome has many other features outside these regions , some of which play an important biological role . Transposable elements – repetitive sequences that are present in many species – make up around half of the mouse genome . They are ‘selfish’ elements , in that the spread of them within the genome does not necessarily benefit the host organism . But sometimes transposable elements can be ‘domesticated’ , and used to the host’s advantage . For example , transposable elements can generate new genes . In other cases , their non-coding sequences can regulate the activity of other nearby genes or even those elsewhere in the genome . It remains unclear to what extent transposable elements have shaped genome regulation throughout evolution . One idea is that the spread of transposable elements can help to establish large regulatory networks – whereby many genes are collectively regulated to produce a specific output . But it has not been fully explored how effective transposable elements are at regulating gene expression . Now , Todd et al . investigate whether particular transposable elements , that are suspected to boost the activity of other genes , are essential for normal gene expression in early mouse development . Todd et al . genetically edited stem cells from the inner and outer layer of the early mouse embryo to find transposable elements that promote gene expression . Whilst some transposable elements were found to be important for gene regulation , not all of the candidates tested were needed to maintain expression levels . To widen the search , several transposable elements were turned off simultaneously by compacting specific stretches of DNA so that they could no longer be activated . When 34 transposable elements were inactivated at once , it emerged that only three transposable elements had a significant impact on gene expression . These findings suggest that whether or not a given transposable element regulates gene expression cannot be predicted solely from profiling the structure and sequence of the genome . This highlights why it is important to interrogate the effect transposable elements have ona gene's role within a cell . Transposable elements are largely disregarded in genomics due to technical difficulties in analysing these repetitive stretches of DNA . But characteristic variations within a population may in part be driven by differences in these parts of the genome , which may also be implicated in diseases such as cancer . Identifying which transposable elements are important for driving gene expression , and linking their actions to specific traits could aid the discovery of important genetic variants .