Metalúrgicos, entre tornos y vagones

ESCs are used as a platform to study early development in a cell culture system. ESC lines have been generated from cells of the inner cell mass in mouse and human embryos. Despite having originated from similar stages in development, mESCs and hESCs show differences in growth, genetic requirements and epigenetic features. mESCs are characterized as displaying a “naïve” pluripotency that is similar to the inner cell mass of the preimplantation blastocyst. In contrast hESCs reflect a “primed” pluripotency stage that is similar to the post-implantation blastocyst. Regardless of these differences both mESCs and hESCs display the properties of indefinite self- renewal and the ability to differentiate into all somatic cell types (Ginis et al.,

2004; Schnerch et al., 2010). Thus they are an ideal platform to study the role of the TET proteins in the cell fate conversion that occurs during the formation of somatic tissues.

The DNMT genes have been inactivated in mESCs and hESCs. Inactivation of DNMT3A and DNMT3B individually or in combination has no effect on self-renewal or pluripotency marker expression in mESCs and hESCs. Dnmt3a-/-Dnmt3b-/- mESCs progressively lose DNA methylation. Interestingly early passage Dnmt3a-/-Dnmt3b-/- mESCs, which still contain significant levels of DNA methylation are able to differentiate into the germ layer and form teratomas. This ability is lost in later passages, upon greater loss of DNA methylation, indicating that although DNA methylation is not required for ESC proliferation, a certain level is required for proper

differentiation of mESCs (Okano et al., 1999). Inactivation of DNMT1 has different phenotypes in mESCs versus hESCs. Dnmt1-/- mESCs are significantly hypomethylated but are still viable (Lei et al., 1996). In fact Dnmt1-/-Dnmt3a-/-Dnmt3b-/- mESCs have been generated that display normal stem cell proliferation and euploidy (Tsumura et al., 2006). In contrast knockout of DNMT1 in hESCs is lethal and produces global DNA demethylation, DNA damage and cell cycle arrest (Liao et al., 2015). It is possible that because hESCs show properties of a later developmental stage they are more sensitive to loss of DNMT1. As all somatic cells require DNMT1 for survival hESCs may represent the first developmental period at which DNA methylation maintenance becomes essential.

The three Tet genes have been inactivated individually and in

different combinations in mESCs. Thus far none of the TET genes has been inactivated in hESCs. Notably inactivation of the individual Tet genes, or all

3 Tet genes together, has no impact on the self-renewal capacity of mESCs. However inactivation of the Tet genes does produce differentiation defects that become more severe as a greater number of Tet genes are mutated (Dawlaty et al., 2014; Dawlaty et al., 2013; Dawlaty et al., 2011; Lu et al., 2014). Depletion of Tet1 singly or together with Tet2 showed mild

differentiation defects. For example teratomas from these cells contain tissues from all three germ layers but are also enriched for extraembryonic trophoblastic cells. This suggests that in wildtype cells TET1 may function to suppress extra embryonic differentiation. Tet1 depletion leads to increased expression of key extra embryonic markers such as caudal-type homeobox 2 (CDX2), eomesodermin (EOMES) and E74 Like ETS Transcription Factor 5 (ELF5). At the same time Tet1-/- and Tet1-/-Tet2-/- mESCs contribute efficiently to chimeras when injected into blastocysts showing that the increased extraembryonic potential can be suppressed in the normal developmental environment. Furthermore upon in-vitro differentiation, Tet1- /- and Tet1-/-Tet2-/- mESCs can form all three germ layers (Dawlaty et al., 2013; Dawlaty et al., 2011). It is possible that in these DKO mESCs, Tet3 can compensate for the loss of Tet1 and Tet2. In fact TKO mESCs, in which all three Tet genes have been inactivated, show strong impairment in their ability to differentiate by spontaneous embryoid body differentiation and teratoma formation assays. Furthermore TKO mESCs show poor chimeric contribution to embryos and cannot support embryonic development by tetraploid complementation assays (Dawlaty et al., 2014). The overall

conclusion from these studies is that the TET proteins may not be necessary to support ESC proliferation and self-renewal but are required for the proper activation of differentiation programs.

The mechanisms underlying the observed differentiation defect of TKO mESCs are currently unclear. Measurement of total 5mC show a modest increase in total methylation upon Tet inactivation in TKO mESCs (Dai et al., 2016; Dawlaty et al., 2014; Lu et al., 2014). Although the overall increase in methylation is small it is possible that concentration of this hypermethylation in regulatory regions could explain the dramatic

differentiation defects of TKO mESCs. Genome-wide methylation analysis of TKO mESCs has thus been performed to uncover specific loci that gain methylation upon Tet inactivation. Interestingly a number of regulatory regions gained hypermethylation in TKO mESCs including enhancers and promoters. Hypermethylation of enhancers leads to a decrease in

expression of associated genes (Lu et al., 2014). A more recent study found that TET1 and 5hmC localize to similar developmental enhancers in

xenopus, zebrafish and mouse embryos during early embryonic

development. This 5hmC signal is associated with increased chromatin accessibility and activation of enhancers as determined by H3K27ac and p300 enrichment. Knockdown of tet1 in zebrafish was associated with increased methylation at these particular enhancers and reduced chromatin accessibility (Bogdanovic, 2017).