The DNA dioxygenase Tet1 regulates H3K27 modification and embryonic stem cell biology independent of its catalytic activity.

Tet enzymes (Tet1/2/3) oxidize 5-methylcytosine to promote DNA demethylation and partner with chromatin modifiers to regulate gene expression. Tet1 is highly expressed in embryonic stem cells (ESCs), but its enzymatic and non-enzymatic roles in gene regulation are not dissected. We have generated Tet1 catalytically inactive (Tet1m/m) and knockout (Tet1-/-) ESCs and mice to study these functions. Loss of Tet1, but not loss of its catalytic activity, caused aberrant upregulation of bivalent (H3K4me3+; H3K27me3+) developmental genes, leading to defects in differentiation. Wild-type and catalytic-mutant Tet1 occupied similar genomic loci which overlapped with H3K27 tri-methyltransferase PRC2 and the deacetylase complex Sin3a at promoters of bivalent genes and with the helicase Chd4 at active genes. Loss of Tet1, but not loss of its catalytic activity, impaired enrichment of PRC2 and Sin3a at bivalent promoters leading to reduced H3K27 trimethylation and deacetylation, respectively, in absence of any changes in DNA methylation. Tet1-/-, but not Tet1m/m, embryos expressed higher levels of Gata6 and were developmentally delayed. Thus, the critical functions of Tet1 in ESCs and early development are mediated through its non-catalytic roles in regulating H3K27 modifications to silence developmental genes, and are more important than its catalytic functions in DNA demethylation.

ADP-ribosyltransferases Parp1 and Parp7 safeguard pluripotency of ES cells.

Embryonic stem (ES) cells are in a dynamic equilibrium of distinct functional states, characterized by the heterogeneous expression of critical pluripotency factors and regulated by a spectrum of reversible histone modifications. Maintenance of this equilibrium is a hallmark of pluripotency. Here we find that the ADP-ribosyltransferases Parp1 and Parp7 play a critical role in safeguarding this state by occupying key pluripotency genes, notably Nanog, Pou5f1, Sox2, Stella, Tet1 and Zfp42, thereby protecting them from progressive epigenetic repression. In the absence of either Parp1 or Parp7, or upon inhibition of the ADP-ribosylating activity, ES cells exhibit a decrease in ground state pluripotency as they cannot maintain the typical heterogeneity characteristic of the metastable state. As a consequence, they display a higher propensity to differentiate. These findings place Parp1 and Parp7 at the genetic-epigenetic interface of pluripotency networks, fine-tuning the transcriptional heterogeneity and thereby determining the developmental plasticity of ES cells.

Tet1 Suppresses p21 to Ensure Proper Cell Cycle Progression in Embryonic Stem Cells.

Ten eleven translocation 1 (Tet1) is a DNA dioxygenase that promotes DNA demethylation by oxidizing 5-methylcytosine. It can also partner with chromatin-activating and repressive complexes to regulate gene expressions independent of its enzymatic activity. Tet1 is highly expressed in embryonic stem cells (ESCs) and regulates pluripotency and differentiation. However, its roles in ESC cell cycle progression and proliferation have not been investigated. Using a series of Tet1 catalytic mutant (Tet1m/m), knockout (Tet1-/-) and wild type (Tet1+/+) mouse ESCs (mESCs), we identified a non-catalytic role of Tet1 in the proper cell cycle progression and proliferation of mESCs. Tet1-/-, but not Tet1m/m, mESCs exhibited a significant reduction in proliferation and delayed progression through G1. We found that the cyclin-dependent kinase inhibitor p21/Cdkn1a was uniquely upregulated in Tet1-/- mESCs and its knockdown corrected the slow proliferation and delayed G1 progression. Mechanistically, we found that p21 was a direct target of Tet1. Tet1 occupancy at the p21 promoter overlapped with the repressive histone mark H3K27me3 as well as with the H3K27 trimethyl transferase PRC2 component Ezh2. A loss of Tet1, but not loss of its catalytic activity, significantly reduced the enrichment of Ezh2 and H3K27 trimethylation at the p21 promoter without affecting the DNA methylation levels. We also found that the proliferation defects of Tet1-/- mESCs were independent of their differentiation defects. Together, these findings established a non-catalytic role for Tet1 in suppressing p21 in mESCs to ensure a rapid G1-to-S progression, which is a key hallmark of ESC proliferation. It also established Tet1 as an epigenetic regulator of ESC proliferation in addition to its previously defined roles in ESC pluripotency and differentiation.

Idax and Rinf facilitate expression of Tet enzymes to promote neural and suppress trophectodermal programs during differentiation of embryonic stem cells.

The Inhibitor of disheveled and axin (Idax) and its ortholog the Retinoid inducible nuclear factor (Rinf) are DNA binding proteins with nuclear and cytoplasmic functions. Rinf is expressed in embryonic stem cells (ESCs) where it regulates transcription of the Ten-eleven translocation (Tet) enzymes, promoting neural and suppressing mesendoderm/trophectoderm differentiation. Here, we find that Idax, which is not expressed in ESCs, is induced upon differentiation. Like Rinf, Idax facilitates neural and silences trophectodermal programs. Individual or combined loss of Idax and Rinf led to downregulation of neural and upregulation of trophectoderm markers during differentiation of ESCs to embryoid bodies as well as during directed differentiation of ESCs to neural progenitor cells (NPCs) and trophoblast-like cells. These defects resemble those of Tet-deficient ESCs. Consistently, Tet genes are direct targets of Idax and Rinf, and loss of Idax and Rinf led to downregulation of Tet enzymes during ESC differentiation to NPCs and trophoblast-like cells. While Idax and Rinf single and double knockout (DKO) mice were viable and overtly normal, DKO embryos had reduced expression of several NPC markers in embryonic forebrains and deregulated expression of selected trophoblast markers in placentas. NPCs derived from DKO forebrains had reduced self-renewal while DKO placentas had increased junctional zone and reduced labyrinth layers. Together, our findings establish Idax and Rinf as regulators of Tet enzymes for proper differentiation of ESCs.

TET1 and 5-Hydroxymethylation Preserve the Stem Cell State of Mouse Trophoblast.

The ten-eleven translocation factor TET1 and its conferred epigenetic modification 5-hydroxymethylcytosine (5hmC) have important roles in maintaining the pluripotent state of embryonic stem cells (ESCs). We previously showed that TET1 is also essential to maintain the stem cell state of trophoblast stem cells (TSCs). Here, we establish an integrated panel of absolute 5hmC levels, genome-wide DNA methylation and hydroxymethylation patterns, transcriptomes, and TET1 chromatin occupancy in TSCs and differentiated trophoblast cells. We show that the combined presence of 5-methylcytosine (5mC) and 5hmC correlates with transcriptional activity of associated genes. Hypoxia can slow down the global loss of 5hmC that occurs upon differentiation of TSCs. Notably, unlike in ESCs and epiblast cells, most TET1-bound regions overlap with active chromatin marks and TFAP2C binding sites and demarcate putative trophoblast enhancer regions. These chromatin modification and occupancy patterns are highly informative to identify novel candidate regulators of the TSC state.

Non-catalytic Roles of Tet2 Are Essential to Regulate Hematopoietic Stem and Progenitor Cell Homeostasis.

The Ten-eleven translocation (TET) enzymes regulate gene expression by promoting DNA demethylation and partnering with chromatin modifiers. TET2, a member of this family, is frequently mutated in hematological disorders. The contributions of TET2 in hematopoiesis have been attributed to its DNA demethylase activity, and the significance of its nonenzymatic functions has remained undefined. To dissect the catalytic and non-catalytic requirements of Tet2, we engineered catalytically inactive Tet2 mutant mice and conducted comparative analyses of Tet2 mutant and Tet2 knockout animals. Tet2 knockout mice exhibited expansion of hematopoietic stem and progenitor cells (HSPCs) and developed myeloid and lymphoid disorders, while Tet2 mutant mice predominantly developed myeloid malignancies reminiscent of human myelodysplastic syndromes. HSPCs from Tet2 knockout mice exhibited distinct gene expression profiles, including downregulation of Gata2. Overexpression of Gata2 in Tet2 knockout bone marrow cells ameliorated disease phenotypes. Our results reveal the non-catalytic roles of TET2 in HSPC homeostasis.

Rinf Regulates Pluripotency Network Genes and Tet Enzymes in Embryonic Stem Cells.

The Retinoid inducible nuclear factor (Rinf), also known as CXXC5, is a nuclear protein, but its functions in the context of the chromatin are poorly defined. We find that in mouse embryonic stem cells (mESCs), Rinf binds to the chromatin and is enriched at promoters and enhancers of Tet1, Tet2, and pluripotency genes. The Rinf-bound regions show significant overlapping occupancy of pluripotency factors Nanog, Oct4, and Sox2, as well as Tet1 and Tet2. We found that Rinf forms a complex with Nanog, Oct4, Tet1, and Tet2 and facilitates their proper recruitment to regulatory regions of pluripotency and Tet genes in ESCs to positively regulate their transcription. Rinf deficiency in ESCs reduces expression of Rinf target genes, including several pluripotency factors and Tet enzymes, and causes aberrant differentiation. Together, our findings establish Rinf as a regulator of the pluripotency network genes and Tet enzymes in ESCs.

A Critical Role of TET1/2 Proteins in Cell-Cycle Progression of Trophoblast Stem Cells.

The ten-eleven translocation (TET) proteins are well known for their role in maintaining naive pluripotency of embryonic stem cells. Here, we demonstrate that, jointly, TET1 and TET2 also safeguard the self-renewal potential of trophoblast stem cells (TSCs) and have partially redundant roles in maintaining the epithelial integrity of TSCs. For the more abundantly expressed TET1, we show that this is achieved by binding to critical epithelial genes, notably E-cadherin, which becomes hyper-methylated and downregulated in the absence of TET1. The epithelial-to-mesenchymal transition phenotype of mutant TSCs is accompanied by centrosome duplication and separation defects. Moreover, we identify a role of TET1 in maintaining cyclin B1 stability, thereby acting as facilitator of mitotic cell-cycle progression. As a result, Tet1/2 mutant TSCs are prone to undergo endoreduplicative cell cycles leading to the formation of polyploid trophoblast giant cells. Taken together, our data reveal essential functions of TET proteins in the trophoblast lineage.

Divergent wiring of repressive and active chromatin interactions between mouse embryonic and trophoblast lineages.

The establishment of the embryonic and trophoblast lineages is a developmental decision underpinned by dramatic differences in the epigenetic landscape of the two compartments. However, it remains unknown how epigenetic information and transcription factor networks map to the 3D arrangement of the genome, which in turn may mediate transcriptional divergence between the two cell lineages. Here, we perform promoter capture Hi-C experiments in mouse trophoblast (TSC) and embryonic (ESC) stem cells to understand how chromatin conformation relates to cell-specific transcriptional programmes. We find that key TSC genes that are kept repressed in ESCs exhibit interactions between H3K27me3-marked regions in ESCs that depend on Polycomb repressive complex 1. Interactions that are prominent in TSCs are enriched for enhancer-gene contacts involving key TSC transcription factors, as well as TET1, which helps to maintain the expression of TSC-relevant genes. Our work shows that the first developmental cell fate decision results in distinct chromatin conformation patterns establishing lineage-specific contexts involving both repressive and active interactions.

Genetic polymorphisms in warfarin and tacrolimus-related genes VKORC1, CYP2C9 and CYP3A5 in the Greek-Cypriot population.

BACKGROUND: Two variants in the gene encoding the cytochrome P450 2C9 enzyme (CYP2C9) are considered the most significant genetic risk factors associated with bleeding after warfarin prescription. A variant in the vitamin K epoxide reductase (VKORC1) has been also associated by several studies with warfarin response. Another variant in the P450 3A5 enzyme (CYP3A5) gene is known to affect the metabolism of many drugs, including tacrolimus. FINDINGS: We conducted a population genetic study in 148 unrelated healthy Greek-Cypriot volunteers (through PCR-RFLP assays), in order to determine the frequencies of the above pharmacogenetics variants and to compare allele frequencies with those in other major ethnic groups. The allele frequencies of CYP2C9*2, CYP2C9*3 and CYP3A5*3 were found to be 0.162, 0.112 and 0.943 respectively, whereas VKORC1 - 1639A was 0.534. The latter frequency differs significantly when compared with Caucasians, Asians and Africans (p < 0.001) and is still significant when compared with the geographically and culturally closely related to Greek-Cypriots, Hellenes of Greece (p = 0.01). Interestingly ~18% of our population are carriers of four or three risk alleles regarding warfarin sensitivity, therefore they have a high predisposition for bleeding after taking high or even normal warfarin doses. CONCLUSIONS: Our data show no significant difference in the frequency of CYP2C9 and CYP3A5 allelic variants when compared to the Caucasian population, but differ significantly when compared with Africans and Asians (p < 0.001). Also, the frequency of variant VKORC1 - 1639A differs between Greek-Cypriots and every other population we compared. Finally, about 1/5 Greek-Cypriots carry three or four risk alleles and ~50% of them carry at least two independent risk alleles regarding warfarin sensitivity, a potentially high risk for over-anticoagulation.