Decoding the role of TET family dioxygenases in lineage specification

Maintenance of hypomethylated promoters of developmental genes

Accumulated evidence indicates that DNA methylation status at promoter regions influences gene transcription [50]. Promoter hypermethylation is believed to contribute to the establishment of a transcriptionally poised/inactive state [50]. For example, pluripotent genes, such as OCT4, are actively expressed in human ESCs and suppressed by promoter hypermethylation upon differentiation [44]. It has been widely documented that genetic ablation of TET induces promoter hypermethylation and aberrant gene expression in multiple lineage differentiation systems [6, 25, 27, 38, 51–53]. For example, TET2 is required for the maintenance of NANOG expression in the spontaneous differentiation of mesodermal lineage cells from human ESCs. TET2 ChIP-seq revealed that TET2 associated with the NANOG promoter prevents DNA methylation [27]. In helper T cell differentiation, Tet2 is recruited to the promoters of cytokine genes in a lineage-specific transcriptional factor-dependent manner, which stimulates active DNA demethylation and expression of these cytokine genes [38]. TET1 and TET3 also regulate DNA methylation status in the promoter regions. For instance, Tet3 directly binds to the promoters of genes critical for neural development in Xenopus, such as Pax6, Rx, and Ngn2, and sustains high levels of 5hmC at promoters [6]. Furthermore, simultaneous deletion of Tet2 and Tet3 downregulates P2rX7 expression along with reduction of 5hmC at the P2rX7 promoter during bone marrow mesenchymal stem cell differentiation [53], indicating that TET-mediated rapid and specific oxidation of 5mC at promoter loci is biologically relevant.

More interestingly, TET-mediated DNA demethylation has been suggested in association with the establishment and/or maintenance of bivalent promoters of developmental genes, in which H3K4me3 and H3K27me3 histone modifications take place simultaneously [14, 54]. In general, bivalent promoters of developmental genes are hypomethylated in ESCs [55]. They are preferentially repressed by trimethylation of histone H3 at lysine 27, which is easier to be reversed than DNA methylation. These low-methylated genetic loci can extend beyond promoter regions, forming H3K27me3-marked DNA methylation valleys (DMVs) that provide binding sites for a large set of transcription factors to mediate complex regulation during development [56]. It has been recently shown that TET proteins and Polycomb Repressive Complex 2 (PRC2), which is responsible for H3K27 methylation, can recruit each other to maintain a hypomethylated status at bivalent promoters and DMVs [57–59]. In addition, TETs can interact with OGT (O-linked β-N-acetylglucosamine transferase), which enhances methylation of histone H3 at lysine 4 by promoting the binding of a component of the H3K4 methyltransferase SET1/COMPASS complex to active promoters [60]. It has been further demonstrated that overexpression of TET2, but not the catalytically inactive TET2, results in an increase in 5hmC at a particular set of key developmental gene promoters, which is sufficient to promote DNA demethylation and de novo bivalent modifications [54]. This discovery was subsequently supported by a recent report which illustrated that TETs safeguard bivalent promoters of many lineage determinants, such as FOXA2, GATA2, PAX6, and SOX17, by preventing their aberrant hypermethylation to ensure developmental competency [14]. Together, these studies suggest that TET-mediated DNA demethylation retains developmental plasticity at the promoters and/or DMVs of developmentally important genes, and therefore ensure robust induction of lineage-specific transcription upon differentiation (Fig. 1).

Regulation of chromatin accessibility and enhancer architecture

In addition to facilitate promoter hypomethylation, TET proteins also play critical roles in regulating chromatin accessibility and enhancer architecture (Fig. 1). 5hmC co-localizes with enhancers and open chromosome regions during different biological processes, such as B cell differentiation and pancreatic endocrine differentiation [7, 28, 61]. By examining genome-wide DNA methylation and hydroxymethylation in the context of Tet2 deletion in mESCs, Ren and colleagues have found that depletion of Tet2 leads to enhancer hypermethylation, accompanied by the loss of active enhancer mark H3K27ac and delayed the induction of Slit3, Lmo4, and Irx3 upon differentiation to a neural progenitor fate [62]. A similar observation has been made in cardiomyocytes where deletion of Tet2 causes loss of 5hmC at enhancers and is accompanied by extensive elevation of DNA methylation, reduction of H3K27ac, and impaired gene expression during heart development [10]. Re-expression of Tet2 catalytic domain in Tet2/3 double knockout pro-B cells restores chromatin accessibility at a genome-wide level as well as at the Igκ enhancer [7]. In agreement with the above studies, application of TET inhibitor dimethyloxalylglycine (DMOG) reduces chromatin accessibility at specific enhancers of P19 embryonic carcinoma cells when differentiated to NPCs [63]. These data implicate the functional relevance of TETs and TET-mediated 5hmC to chromatin accessibility at distal regulatory elements, particularly enhancers. Although the precise molecular mechanisms remain unclear, epigenetic readers of 5hmC, such as MeCP2 (methyl-CpG-binding protein 2), might constitute a mechanism of TET-regulated chromatin opening [64].

In addition to chromatin accessibility, TETs may also contribute to enhancer priming. Prior to activation, the enhancer region is methylated and not accessible to general transcription factors. However, pioneer transcription factors, such as FOXA1, MEIS1, and PBX1, preferentially bind to oligonucleotide probes which contain methylated cytosines [63, 65]. It has also been suggested that pioneer transcription factors can physically interact with TETs which catalyze oxidation of 5mC at gene regulatory regions [7, 66, 67]. For example, pioneer transcription factors, PU.1, recruit Tet2 proteins to the Igκ enhancer to facilitate DNA demethylation during early B cell maturation [7]. Furthermore, removal of 5mC or deposition of 5hmC coincides with increased accessibility of enhancers and monomethylation of H3K4 [12, 28, 63]. Accordingly, H3K4 methyltransferases are repelled by 5mC [68, 69], indicating that TET-mediated DNA demethylation is necessary for the recruitment of H3K4 methyltransferases to prime enhancers. Additionally, it has been demonstrated that TETs can recruit SET1/COMPASS H3K4 methyltransferase as well as histone acetyltransferase p300 to gene regulatory regions [38, 60]. Therefore, H3K4 monomethyltransferases MLL3/4, which are COMPASS family members, might also interact with TETs and become recruited to enhancer regions [70].

Despite their plausible contribution to chromatin architecture, TETs may regulate gene transcription by interfering with RNA polymerization and RNA splicing. In CD4⁺ T cells, TET1 and TET2 alter CTCF-dependent alternative pre-mRNA splicing through oxidation of 5mC to 5hmC and 5caC at corresponding intragenic CTCF-binding sites in the PTPRC (protein tyrosine phosphatase CD45) locus [71, 72]. Additionally, it has been suggested that oxidation derivatives of 5hmC are concentrated on the gene bodies of transcribed genes and support transcriptional consistency [35, 47].

Neuroectoderm lineage specification

The brain is one of the places in mammals with the most abundant 5hmC, suggesting that TET proteins and TET-mediated 5hmC might have significant impacts in neurogenesis. Tet1 knockout mice exhibit defects in neuron function including learning, memory consolidation, storage, and extinction [52, 75, 76]. Ablation of Tet1 leads to downregulation of neuronal activity-regulated genes such as Npas4 [75]. Although Tet1 is highly expressed in multiple regions and cells of the brain like hippocampus, isocortex, and cerebellar granule cells [77, 78], Tet1 knockout does not alter brain morphology. It has been revealed that compensatory upregulations of Tet2 and Tet3 were observed in the Tet1 knockout mouse brain [76]. In addition, overexpression of Tet3 can facilitate the reprogramming of MEFs (mouse embryo fibroblasts) into neuronal cells, which is accompanied with an active demethylation process at the promoters of genes encoding neuron-specific transcription factors such Ascl1, Brn2, and Ngn2 [79]. Therefore, TET family members are functionally redundant in neurogenesis. In the absence of Tet1, other Tet family members can compensate for the activity of Tet1 to regulate neurogenesis.

A recent study from Huangfu’s group has nicely demonstrated that TET1/2/3 triple knockout human ESCs lose their ability to differentiate into PAX6⁺/SOX1⁺ neuroectoderm cells, which is mainly caused by aberrant hypermethylation at the PAX6 promoter [14]. In human ESCs, TET1 binding at PAX6 bivalent promoter leads to the progressive oxidation of 5-methylcytosine. In the absence of 5hmC, de novo DNA methyltransferase DNMT3B anchors at PAX6 bivalent promoter P0 and induces promoter hypermethylation, which suppresses PAX6 induction and subsequent neuroectoderm differentiation. Targeted demethylation of the PAX6 P0 promoter by a catalytically inactive Cas9 (dCas9) fused with a TET1 catalytic domain partially restored PAX6 expression and rescued neuron differentiation defects [14]. These results clearly illustrate that TET-mediated hydroxymethylation prevents repressive DNA methylation and ensures key lineage-specific transcription factor expression in neuron differentiation.

Mesoderm lineage specification

TET2 is ubiquitously expressed in multiple hematopoietic cells, and its mutation is frequently found in hematological malignancies [31, 80, 81]. It has been well documented that TET2 is critical for hematopoiesis. In patients who develop chronic myelomonocytic leukemia and carry TET2 mutations in CD34⁺ hematopoietic progenitor cells, the TET2-mutated CD34⁺ progenitors preferentially develop into myeloid instead of erythroid cells upon differentiation [82]. Furthermore, it has been shown that single deletion of Tet2 or double deletion of Tet2/3 or Tet1/2 alters T cell, B cell, NKT cell, red blood cell, or mast cell differentiation and maturation [38, 61, 74, 83–85]. For instance, genetic depletion of Tet2 perturbs the induction of signature cytokine genes upon differentiation of CD4⁺ T cells toward helper T (Th) cells, which is associated with differential enrichment of 5hmC and p300 at the promoters of Ifng and Il17 [38]. In contrast, Tet1/Tet2 or Tet2/Tet3 double knockout mice contain less regulatory T (Treg) cells in the spleen. It has been further shown that Tet proteins can demethylate the conserved noncoding sequences (CNS) in Foxp3 loci to maintain the expression of Foxp3 [83, 86]. One of the Foxp3 CNS functions as a super enhancer and docking site for chromatin organizer Satb1 binding [87].

Moreover, TET dioxygenases have also been implicated to play crucial roles during cardiomyocytes and skeletal myoblast differentiation [10, 51, 88]. Knockdown of Tet2 in undifferentiated C2C12 myoblasts significantly reduces the induction of myoblast differentiation-associated genes Myog and myoM with elevated methylation at their promoters [88]. Additionally, TET2 is shown to control the expression of contractile genes, such as SRF, MYOCD, and MYH11, in human coronary artery smooth muscle cells [51]. Similarly, loss of gene expression in smooth muscle cells is correlated with a significant decrease in TET2 binding and 5hmC levels at the promoters of corresponding genes. In cardiomyocyte differentiation, Tet2 knockdown deregulates a large number of genes associated with heart development and contraction. In particular, induction of the key cardiac gene Myh7 is suppressed upon cardiomyocyte differentiation in Tet2 knockdown cells, presumably due to the aberrant methylation at its enhancer [10].

Endoderm lineage specification

To date, functional analyses of TETs and 5hmC in the context of endodermal lineage specification are still limited. In the intestine, Tet1 is highly expressed in intestinal stem cells and positively regulates the expression of Wnt target genes such as Axin2 and Lgr5 [25]. In a model of hepatocyte differentiation from the human hepatic progenitor HepaRG cells, Hernandez-Vargas and colleagues demonstrated that TET1 dioxygenase is necessary for HNF4A promoter P1 demethylation and activation upon hepatocyte differentiation [89]. They found that TET1 binds to the P1 locus via the pioneer transcription factor FOXA2, which is required for the establishment of the hepatocyte program [89]. Additionally, to understand the precise role of TET-mediated hydroxymethylation during pancreatic lineage progression, we recently characterized each lineage intermediate, including definitive endoderm, primitive gut tube, posterior foregut, and pancreatic progenitors, in great detail during their stepwise differentiation from human ESCs [28]. We developed genome-wide maps for each stage, encompassing 5mC/5hmC, gene expression, and chromatin architecture/accessibility. We identified that 5hmC is positively correlated with enhancer activities and chromatin accessibility during pancreatic differentiation [28], and further discovered that TET dioxygenases promoted pancreatic endocrine differentiation but had less effect on the early endoderm formation (unpublished results).