Trayectoria sobre rieles

Compared to enhancers, the majority of bivalent promoters do not show dramatic changes in DNA methylation during development (Jones, 2012; Smith and Meissner, 2013).As a result it was unclear whether the methylation state of bivalent promoters is actively regulated and if it has functional relevance for cell differentiation. Previous studies have shown that the TET proteins regulate methylation at enhancers in developing mouse embryos and mESCs (Bogdanovic, 2017; Lu et al., 2014). Here we show that the TET proteins are also critical for maintaining a hypomethylated state at bivalent promoters in hESCs. In the absence of the TET proteins,

aberrant hypermethylation of bivalent promoters leads to impaired gene activation upon exposure to differentiation signals. Importantly, the alteration of DNA methylation at bivalent promoters does not cause immediate

changes in transcription but significantly impacts hESC differentiation. This suggests the need to revise the predominant approach that focuses on DNA methylation and other epigenetic changes with immediate impact on gene expression, as epigenetic changes may predict future cell behavior during embryonic development or adult stem cell differentiation. Broadly, the competitive balance between the TET proteins and de novo

methyltransferases at bivalent promoters could facilitate rapid changes of their methylation state to either activate or silence transcription in a cell- lineage and gene dependent manner. Although we focused on lineage regulators, such as PAX6, bivalent promoters may also regulate signaling pathways during development. Furthermore, we observed hypermethylation at enhancer regions after TET inactivation. TET activity at a variety of

lineage regulators, all of which contribute to proper embryonic development and cellular differentiation.

Previous studies have indicated that genomic regions marked by the H3K4me3 modification are refractory to de novo DNA methylation (Guo et al., 2014; Ooi et al., 2007). TET deletion causes bivalent promoter

hypermethylation without causing apparent changes in H3K4me3

occupancy. Thus TET or additional TET-dependent factors (including 5hmC) that frequently co-occur with H3K4me3 may mediate some of the effects reported to be associated with the H3K4me3 mark. Also intriguingly, this aberrant hypermethylation, once established in TKO hESCs, is not adequately maintained by DNMT1. Upon inactivation of DNMT3B in the TKO background we observed a global reduction of DNA methylation at bivalent promoters. Based on the >99.7% overall fidelity of DNMT1 in preserving methylation (Liao et al., 2015), DNMT1 would be expected to largely preserve the hypermethylation seen in TKO hESCs during the approximately 6 passages it took to generate and expand the QKO cells for analysis. The significant reversal of the hypermethylation phenotype at PAX6 and other bivalent promoters in QKO cells indicates the

hypermethylation phenotype requires continuous DNMT3B activity. It also suggests that additional mechanisms, such as transient transcription (Di Ruscio et al., 2013), may inhibit DNMT1 activity at these loci. Alternatively, though not mutually exclusively, DNMT3B may function as both a de novo and maintenance methyltransferase at particular loci in ESCs, as previously suggested (Chen et al., 2003; Liang et al., 2002). Previous studies have shown that DNMT3A and DNMT3B share largely overlapping targets (Liao et al., 2015), yet deletion of DNMT3B alone in TKO hESCs was sufficient to

partially reverse bivalent promoter hypermethylation along with the associated NE differentiation defect. DNMT3B may have stronger

preference or activity compared to DNMT3A at bivalent promoters such as PAX6, and such loci have been identified previously in hESCs (Liao et al., 2015). Perhaps more likely, the rescue by DNMT3B deletion alone could be due to the relatively low DNMT3A expression compared to DNMT3B

(Supplementary Fig. 9a) and the lack of compensatory increase of DNMT3A expression upon DNMT3B deletion (Supplementary Fig. 11d). Similarly a recent paper of TKO mouse embryos found that inactivating either DNMT3A or DNMT3B was able to rescue the gastrulation phenotype (Dai et al.,

2016). It is possible that in other cell types DNMT3A, along with or instead of DNMT3B, may counteract the TET proteins to regulate methylation (Rinaldi et al., 2016; Zhang et al., 2016).

Our work highlights the utility of locus-specific epigenome editing tools to directly probe the functional consequences of epigenetic changes and to distinguish direct, locus-specific effects from indirect effects. The observation of specific instead of global hypermethylation in TKO hESCs suggests that the antagonistic relationship between TET and DNMTs may be regulated through recruitment of TETs and/or DNMTs in a locus-specific manner. Such competitive interplay and potential cooperative interactions between TETs and DNMTs may also occur at later stages of differentiation as recently shown in the hematopoietic system (Zhang et al., 2016). The expression of the individual TET genes can vary at different stages of development (Rasmussen and Helin, 2016), and each TET protein may have unique targets so that distinct sets of promoters are maintained in a hypomethylated/5hmC rich state depending on the cellular context. Similarly

the recruitment or enzymatic activity of the DNMTs may be locus-specific or influenced by the genomic distribution of transcription factors and histone modifications, which can change dramatically between cell types. It would be of great interest to investigate what factors determine whether the

methylation state of a particular regulatory region is dynamically regulated or is relatively static, how different factors cooperate or counteract in different genomic contexts to establish the unique methylation state of regulatory regions, and ultimately how methylation states not only regulate but also predict (in the context of cell differentiation) cell-type specific transcriptional programs.