C ARACTERIZAR LAS ACTIVIDADES REALIZADAS DENTRO DEL PROCESO

The role of DNA methylation in transcriptional regulation has been investigated in a number of systems, with the broad conclusion being that this mark is associated with transcriptional repression (Bird & Wolffe 1999; Bird 2002; Klose & Bird 2006; Moore et al. 2013). DNA methylation occurs primarily at cytosines in the context of CpG dinucleotides. Non-CpG modification is also possible, although this is a rarer event (Lister et al. 2009). The frequency of CpGs in the genome is overall lower than would be expected by chance, partly due to the tendency of 5mC to mutate into thymine. However, the majority of CpG dinucleotides (70-80%) throughout the mammalian genome are methylated (Bird 2002). DNA methylation is associated with various processes, including X chromosome inactivation and genomic imprinting, and a large proportion of DNA methylation is thought to be involved in repression of endogenous retrotransposons and viral elements (Beard et al. 1995; Jaenisch et al. 1985; Mohandas et al. 1981; Schulz et al. 2006). Of key relevance to the role in 5mC in transcription, most (60-70%) promoters contain CpG islands (CGIs). These are stretches of DNA that contain a higher frequency of CpG dinucleotides than the surrounding genome, but are largely protected from DNA methylation (Illingworth et al. 2010). Whilst most promoters are therefore depleted of 5mC irrespective of the transcriptional activity of the gene they control, where methylation of CGI-containing promoters does occur, this is negatively correlated with gene expression (Bird & Wolffe, 1999).

Two main mechanisms have been proposed for 5mC-directed transcriptional repression. Firstly, the presence of 5mC is thought to have a destabilising influence on the DNA binding of most transcription factors and DNA-binding proteins, hindering transcriptional activation (Yin et al. 2017). Secondly, of the proteins shown to interact directly with 5mC, the earliest of these identified were transcriptional repressors, which recruit histone deacetylase complexes and catalyse chromatin condensation (Bird & Wolffe 1999; Jones et al. 1998; Nan et al. 1998). Examples of these methyl-CpG-binding domain (MBD)- containing proteins include MeCP1, MeCP2, and MBD1, 2, and 4 (Klose & Bird 2006). In addition, the transcriptional repressor Kaiso (ZBTB33) has been shown to bind methylated CpG dinucleotides through its zinc-finger domains (Prokhortchouk et al. 2001; Yoon et al.

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2003), and it is possible that other members of this large protein family may possess similar functions (Filion et al. 2006).

Nevertheless, despite this mechanistic evidence for the repressive activity of 5mC, recent studies have identified a range of additional proteins capable of interacting with 5mC, not all of which are associated with transcriptional repression, and which may have more diverse regulatory functions (Iurlaro et al. 2013; Spruijt et al. 2013; Spruijt & Vermeulen 2014). In addition, the fact that a strong transcriptional activator can trigger transcription from a previously silenced, methylated promoter (Thompson et al. 1986), and that genes possessing methylated CGIs are expressed in some developmental contexts (Fouse et al. 2008), implies that a balance between 5mC and the profile of transcriptional regulators that interact with this mark may “fine-tune” gene expression at different loci. It therefore appears that further work is required to precisely define the potentially complex relationship between 5mC and gene expression.

In terms of levels of 5mC at other regulatory elements, overall, enhancers also appear to be depleted of this mark, although less so than promoters (Luo et al. 2018; Stadler et al. 2011), and at these sites 5mC loss is similarly correlated with positive transcriptional activity (Hon et al. 2013; Lister et al. 2009; Stadler et al. 2011; Ziller et al. 2013). Importantly, Ziller et al. (2013) examined genome-wide DNA methylation across 30 human cell and tissue types through investigation of 42 separate whole genome bisulfite sequencing (WGBS) datasets. This analysis showed that dynamic regulation of methylation occurred at only 21.8% of CpG sites within a normal developmental context. The majority of these dynamic CpGs co-localised with gene regulatory elements, particularly enhancers and transcription factor binding sites linked to cell type-specific genes. This indicates that regulation of 5mC at enhancers may be a key process in the definition of cell phenotypes, with interplay between DNMT-mediated methylation and TET-mediated demethylation potentially central to this. As the studies cited in the previous section highlight, TET depletion in several systems corroborates this notion, with the finding that regulatory elements appear to be the main targets of TET-mediated regulation of DNA modifications (Hon et al. 2014; Lu et al. 2014; Stadler et al. 2011).

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In contrast to 5mC, which is notable in its distribution by the regions from which it is excluded, 5hmC is specifically enriched at various genomic regions, including promoters, gene bodies, and enhancers (Hahn et al. 2013; Johnson et al. 2016; Pastor et al. 2011; Stroud et al. 2011; Wu et al. 2011). Whilst studies show 5hmC at enhancers is typically associated with transcriptional activation, its role at promoters remains unclear, with several studies proposing 5hmC as an activating mark at these sites (Hahn et al. 2013; Madzo et al. 2014; Taylor et al. 2016), and others suggesting it may have a repressive role (Neri et al. 2013; Wu et al. 2011). However, as 5mC is required as a substrate for conversion to 5hmC, these conflicting findings may be partly due to different basal levels of 5mC at these promoters that confound measurement of the effects of 5hmC (Pastor et al. 2013). Interestingly, screens conducted by Iurlaro et al. (2013) and Spruijt et al. (2013) to identify readers of 5mC and its oxidised derivatives demonstrated that 5hmC interacts both with proteins thought to be involved in active DNA demethylation pathways, and those involved in transcriptional regulation. This reinforces the idea of 5hmC as a stable transcriptional regulatory mark, in addition to being a potential DNA demethylation intermediate. Furthermore, in these studies, 5mC and 5hmC were found to attract the binding of distinct proteins, with very few proteins capable of interacting with both marks. This further fuels the notion of distinct transcriptional roles for these two modifications.

Compared to 5mC and 5hmC, the specific functions of the final products of TET metabolism, 5fC and 5caC, have been much less investigated. Nevertheless, the in vitro screen performed by Iurlaro et al. (2013) demonstrated that far more specific readers bound uniquely to 5fC than to 5mC or 5hmC, and the functions of these proteins strongly suggest transcriptional regulatory capabilities. Moreover, in a recent study by the same group, genome-wide profiling of 5fC was performed in mouse embryos, through biotin labelling of 5fC followed by pull-down and sequencing (Iurlaro et al. 2016). This showed that 5fC is enriched at active enhancers in mouse embryos in a tissue-specific manner, suggesting a role for this mark in embryonic development. Work in additional systems, and the development of further methods to profile both 5fC and 5caC, may reveal further insights into the potential transcriptional roles of these products of TET activity.

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