Epigenetic regulation by TET1 in gene-environmental interactions influencing susceptibility to congenital malformations.

The etiology of neural tube defects (NTDs) involves complex gene-environmental interactions. Folic acid (FA) prevents NTDs, but the mechanisms remain poorly understood and at least 30% of human NTDs resist the beneficial effects of FA supplementation. Here, we identify the DNA demethylase TET1 as a nexus of folate-dependent one-carbon metabolism and genetic risk factors post-neural tube closure. We determine that cranial NTDs in Tet1 -/- embryos occur at two to three times higher penetrance in genetically heterogeneous than in homogeneous genetic backgrounds, suggesting a strong impact of genetic modifiers on phenotypic expression. Quantitative trait locus mapping identified a strong NTD risk locus in the 129S6 strain, which harbors missense and modifier variants at genes implicated in intracellular endocytic trafficking and developmental signaling. NTDs across Tet1 -/- strains are resistant to FA supplementation. However, both excess and depleted maternal FA diets modify the impact of Tet1 loss on offspring DNA methylation primarily at neurodevelopmental loci. FA deficiency reveals susceptibility to NTD and other structural brain defects due to haploinsufficiency of Tet1. In contrast, excess FA in Tet1 -/- embryos drives promoter DNA hypermethylation and reduced expression of multiple membrane solute transporters, including a FA transporter, accompanied by loss of phospholipid metabolites. Overall, our study unravels interactions between modified maternal FA status, Tet1 gene dosage and genetic backgrounds that impact neurotransmitter functions, cellular methylation and individual susceptibilities to congenital malformations, further implicating that epigenetic dysregulation may underlie NTDs resistant to FA supplementation.

Osmolar Modulation Drives Reversible Cell Cycle Exit and Human Pluripotent Cell Differentiation via NF-κВ and WNT Signaling.

Terminally differentiated cells are commonly regarded as the most stable cell state in adult organisms, characterized by growth arrest while fulfilling their specialized functions. A better understanding of the mechanisms involved in promoting cell cycle exit will improve the ability to differentiate pluripotent cells into mature tissues for both pharmacological and therapeutic use. Here, it demonstrates that a hyperosmolar environment enforces a protective p53-independent quiescent state in immature hepatoma cells and in pluripotent stem cell-derived models of human hepatocytes and endothelial cells. Prolonged culture in hyperosmolar conditions stimulates changes in gene expression promoting functional cell maturation. Interestingly, hyperosmolar conditions do not only trigger growth arrest and cellular maturation but are also necessary to maintain this maturated state, as switching back to plasma osmolarity reverses the changes in expression of maturation and proliferative markers. Transcriptome analysis revealed sequential stages of osmolarity-regulated growth arrest followed by cell maturation, mediated by activation of NF-κВ, and repression of WNT signaling, respectively. This study reveals that a modulated increase in osmolarity serves as a biochemical signal to promote long-term growth arrest and cellular maturation into different lineages, providing a practical method to generate differentiated hiPSCs that resemble their mature counterpart more closely.

VEGF overexpressed mesoangioblasts enhance urethral and vaginal recovery following simulated vaginal birth in rats.

Vaginal birth causes pelvic floor injury which may lead to urinary incontinence. Cell therapy has been proposed to assist in functional recovery. We aim to assess if intra-arterial injection of rat mesoangioblasts (MABs) and stable Vascular Endothelial Growth Factor (VEGF)-expressing MABs, improve recovery of urethral and vaginal function following simulated vaginal delivery (SVD). Female rats (n = 86) were assigned to either injection of saline (control), allogeneic-MABs (MABsallo), autologous-MABs (MABsauto) or allogeneic-MABs transduced to stably expressed VEGF (MABsallo-VEGF). One hour after SVD, 0.5 × 106 MABs or saline were injected into the aorta. Primary outcome was urethral (7d and 14d) and vaginal (14d) function; others were bioluminescent imaging for cell tracking (1, 3 and 7d), morphometry (7, 14 and 60d) and mRNAseq (3 and 7d). All MABs injected rats had external urethral sphincter and vaginal function recovery within 14d, as compared to only half of saline controls. Functional recovery was paralleled by improved muscle regeneration and microvascularization. Recovery rate was not different between MABsallo and MABsauto. MABsallo-VEGF accelerated functional recovery and increased GAP-43 expression at 7d. At 3d we detected major transcriptional changes in the urethra of both MABsallo and MABsallo-VEGF-injected animals, with upregulation of Rho/GTPase activity, epigenetic factors and dendrite development. MABSallo also upregulated transcripts that encode proteins involved in myogenesis and downregulated pro-inflammatory processes. MABsallo-VEGF also upregulated transcripts that encode proteins involved in neuron development and downregulated genes involved in hypoxia and oxidative stress. At 7d, urethras of MABsallo-VEGF-injected rats showed downregulation of oxidative and inflammatory response compared to MABSallo. Intra-arterial injection of MABsallo-VEGF enhances neuromuscular regeneration induced by untransduced MABs and accelerates the functional urethral and vaginal recovery after SVD.

Dual functions of TET1 in germ layer lineage bifurcation distinguished by genomic context and dependence on 5-methylcytosine oxidation.

Gastrulation begins when the epiblast forms the primitive streak or becomes definitive ectoderm. During this lineage bifurcation, the DNA dioxygenase TET1 has bipartite functions in transcriptional activation and repression, but the mechanisms remain unclear. By converting mouse embryonic stem cells (ESCs) into neuroprogenitors, we defined how Tet1-/- cells switch from neuroectoderm fate to form mesoderm and endoderm. We identified the Wnt repressor Tcf7l1 as a TET1 target that suppresses Wnt/ß-catenin and Nodal signalling. ESCs expressing catalytic dead TET1 retain neural potential but activate Nodal and subsequently Wnt/ß-catenin pathways to generate also mesoderm and endoderm. At CpG-poor distal enhancers, TET1 maintains accessible chromatin at neuroectodermal loci independently of DNA demethylation. At CpG-rich promoters, DNA demethylation by TET1 affects the expression of bivalent genes. In ESCs, a non-catalytic TET1 cooperation with Polycomb represses primitive streak genes; post-lineage priming, the interaction becomes antagonistic at neuronal genes, when TET1's catalytic activity is further involved by repressing Wnt signalling. The convergence of repressive DNA and histone methylation does not inhibit neural induction in Tet1-deficient cells, but some DNA hypermethylated loci persist at genes with brain-specific functions. Our results reveal versatile switching of non-catalytic and catalytic TET1 activities based on genomic context, lineage and developmental stage.

The Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency.

Early during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, TCF7L1 is required for PE specification as deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage specification in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.

Wnt/ß-Catenin Inhibition Disrupts Carboplatin Resistance in Isogenic Models of Triple-Negative Breast Cancer.

Triple-Negative Breast Cancer (TNBC) is the most aggressive breast cancer subtype, characterized by limited treatment options and higher relapse rates than hormone-receptor-positive breast cancers. Chemotherapy remains the mainstay treatment for TNBC, and platinum salts have been explored as a therapeutic alternative in neo-adjuvant and metastatic settings. However, primary and acquired resistance to chemotherapy in general and platinum-based regimens specifically strongly hampers TNBC management. In this study, we used carboplatin-resistant in vivo patient-derived xenograft and isogenic TNBC cell-line models and detected enhanced Wnt/ß-catenin activity correlating with an induced expression of stem cell markers in both resistant models. In accordance, the activation of canonical Wnt signaling in parental TNBC cell lines increases stem cell markers' expression, formation of tumorspheres and promotes carboplatin resistance. Finally, we prove that Wnt signaling inhibition resensitizes resistant models to carboplatin both in vitro and in vivo, suggesting the synergistic use of Wnt inhibitors and carboplatin as a therapeutic option in TNBC. Here we provide evidence for a prominent role of Wnt signaling in mediating resistance to carboplatin, and we establish that combinatorial targeting of Wnt signaling overcomes carboplatin resistance enhancing chemotherapeutic drug efficacy.

Coordination of germ layer lineage choice by TET1 during primed pluripotency.

Gastrulation in the early postimplantation stage mammalian embryo begins when epiblast cells ingress to form the primitive streak or develop as the embryonic ectoderm. The DNA dioxygenase Tet1 is highly expressed in the epiblast and yet continues to regulate lineage specification during gastrulation when its expression is diminished. Here, we show how Tet1 plays a pivotal role upstream of germ layer lineage bifurcation. During the transition from naive pluripotency to lineage priming, a global reconfiguration redistributes Tet1 from Oct4-cobound promoters to distal regulatory elements at lineage differentiation genes, which are distinct from high-affinity sites engaged by Oct4. An altered chromatin landscape in Tet1-deficient primed epiblast-like cells is associated with enhanced Oct4 expression and binding to Nodal and Wnt target genes, resulting in collaborative signals that enhance mesendodermal and inhibit neuroectodermal gene expression during lineage segregation. A permissive role for Tet1 in neural fate induction involves Zic2-dependent engagement at neural target genes at lineage priming, is dependent on the signaling environment during gastrulation, and impacts neural tube closure after gastrulation. Our findings provide mechanistic information for epigenetic integration of pluripotency and signal-induced differentiation cues.

Regulatory Dynamics of Tet1 and Oct4 Resolve Stages of Global DNA Demethylation and Transcriptomic Changes in Reprogramming.

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) involves the reactivation of endogenous pluripotency genes and global DNA demethylation, but temporal resolution of these events using existing markers is limited. Here, we generate murine transgenic lines harboring reporters for the 5-methylcytosine dioxygenase Tet1 and for Oct4. By monitoring dual reporter fluorescence during pluripotency entry, we identify a sequential order of Tet1 and Oct4 activation by proximal and distal regulatory elements. Full Tet1 activation marks an intermediate stage that accompanies predominantly repression of somatic genes, preceding full Oct4 activation, and distinguishes two waves of global DNA demethylation that target distinct genomic features but are uncoupled from transcriptional changes. Tet1 knockout shows that TET1 contributes to both waves of demethylation and activates germline regulatory genes in reprogramming intermediates but is dispensable for Oct4 reactivation. Our dual reporter system for time-resolving pluripotency entry thus refines the molecular roadmap of iPSC maturation.

Patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug screening.

Endometrial disorders represent a major gynaecological burden. Current research models fail to recapitulate the nature and heterogeneity of these diseases, thereby hampering scientific and clinical progress. Here we developed long-term expandable organoids from a broad spectrum of endometrial pathologies. Organoids from endometriosis show disease-associated traits and cancer-linked mutations. Endometrial cancer-derived organoids accurately capture cancer subtypes, replicate the mutational landscape of the tumours and display patient-specific drug responses. Organoids were also established from precancerous pathologies encompassing endometrial hyperplasia and Lynch syndrome, and inherited gene mutations were maintained. Endometrial disease organoids reproduced the original lesion when transplanted in vivo. In summary, we developed multiple organoid models that capture endometrial disease diversity and will provide powerful research models and drug screening and discovery tools.

Lineage-specific functions of TET1 in the postimplantation mouse embryo.

The mammalian TET enzymes catalyze DNA demethylation. While they have been intensely studied as major epigenetic regulators, little is known about their physiological roles and the extent of functional redundancy following embryo implantation. Here we define non-redundant roles for TET1 at an early postimplantation stage of the mouse embryo, when its paralogs Tet2 and Tet3 are not detectably expressed. TET1 regulates numerous genes defining differentiation programs in the epiblast and extraembryonic ectoderm. In epiblast cells, TET1 demethylates gene promoters via hydroxymethylation and maintains telomere stability. Surprisingly, TET1 represses a majority of epiblast target genes independently of methylation changes, in part through regulation of the gene encoding the transcriptional repressor JMJD8. Dysregulated gene expression in the absence of TET1 causes embryonic defects, which are partially penetrant in an inbred strain but fully lethal in non-inbred mice. Collectively, our study highlights an interplay between the catalytic and non-catalytic activities of TET1 that is essential for normal development.

PDGFRα+ Cells in Embryonic Stem Cell Cultures Represent the In Vitro Equivalent of the Pre-implantation Primitive Endoderm Precursors.

In early mouse pre-implantation development, primitive endoderm (PrE) precursors are platelet-derived growth factor receptor alpha (PDGFRα) positive. Here, we demonstrated that cultured mouse embryonic stem cells (mESCs) express PDGFRα heterogeneously, fluctuating between a PDGFRα+ (PrE-primed) and a platelet endothelial cell adhesion molecule 1 (PECAM1)-positive state (epiblast-primed). The two surface markers can be co-detected on a third subpopulation, expressing epiblast and PrE determinants (double-positive). In vitro, these subpopulations differ in their self-renewal and differentiation capability, transcriptional and epigenetic states. In vivo, double-positive cells contributed to epiblast and PrE, while PrE-primed cells exclusively contributed to PrE derivatives. The transcriptome of PDGFRα+ subpopulations differs from previously described subpopulations and shows similarities with early/mid blastocyst cells. The heterogeneity did not depend on PDGFRα but on leukemia inhibitory factor and fibroblast growth factor signaling and DNA methylation. Thus, PDGFRα+ cells represent the in vitro counterpart of in vivo PrE precursors, and their selection from cultured mESCs yields pure PrE precursors.

Dynamic switching of active promoter and enhancer domains regulates Tet1 and Tet2 expression during cell state transitions between pluripotency and differentiation.

The Tet 5-methylcytosine dioxygenases catalyze DNA demethylation by producing 5-hydroxymethylcytosine and further oxidized products. Tet1 and Tet2 are highly expressed in mouse pluripotent cells and downregulated to different extents in somatic cells, but the transcriptional mechanisms are unclear. Here we defined the promoter and enhancer domains in Tet1 and Tet2. Within a 15-kb "superenhancer" of Tet1, there are two transcription start sites (TSSs) with different activation patterns during development. A 6-kb promoter region upstream of the distal TSS is highly active in naive pluripotent cells, autonomously reports Tet1 expression in a transgenic system, and rapidly undergoes DNA methylation and silencing upon differentiation in cultured cells and native epiblast. A second TSS downstream, associated with a constitutively weak CpG-rich promoter, is activated by a neighboring enhancer in naive embryonic stem cells (ESCs) and primed epiblast-like cells (EpiLCs). Tet2 has a CpG island promoter with pluripotency-independent activity and an ESC-specific distal intragenic enhancer; the latter is rapidly downregulated in EpiLCs. Our study reveals distinct modes of transcriptional regulation at Tet1 and Tet2 during cell state transitions of early development. New transgenic reporters using Tet1 and Tet2 cis-regulatory domains may serve to distinguish nuanced changes in pluripotent states and the underlying epigenetic variations.

Modulation of TET2 expression and 5-methylcytosine oxidation by the CXXC domain protein IDAX.

TET (ten-eleven-translocation) proteins are Fe(ii)- and α-ketoglutarate-dependent dioxygenases that modify the methylation status of DNA by successively oxidizing 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, potential intermediates in the active erasure of DNA-methylation marks. Here we show that IDAX (also known as CXXC4), a reported inhibitor of Wnt signalling that has been implicated in malignant renal cell carcinoma and colonic villous adenoma, regulates TET2 protein expression. IDAX was originally encoded within an ancestral TET2 gene that underwent a chromosomal gene inversion during evolution, thus separating the TET2 CXXC domain from the catalytic domain. The IDAX CXXC domain binds DNA sequences containing unmethylated CpG dinucleotides, localizes to promoters and CpG islands in genomic DNA and interacts directly with the catalytic domain of TET2. Unexpectedly, IDAX expression results in caspase activation and TET2 protein downregulation, in a manner that depends on DNA binding through the IDAX CXXC domain, suggesting that IDAX recruits TET2 to DNA before degradation. IDAX depletion prevents TET2 downregulation in differentiating mouse embryonic stem cells, and short hairpin RNA against IDAX increases TET2 protein expression in the human monocytic cell line U937. Notably, we find that the expression and activity of TET3 is also regulated through its CXXC domain. Taken together, these results establish the separate and linked CXXC domains of TET2 and TET3, respectively, as previously unknown regulators of caspase activation and TET enzymatic activity.

DNA methylation and methylcytosine oxidation in cell fate decisions.

Changes in cellular phenotypes and identities are fundamentally regulated by epigenetic mechanisms including DNA methylation, post-translational histone modifications and chromatin remodeling. Recent genome-wide profiles of the mammalian DNA 'methylome' suggest that hotspots of dynamic DNA methylation changes during cell fate transitions occur at distal regulatory regions with low or intermediate CpG densities. These changes are most prevalent early during the course of cellular differentiation and can be locally influenced by binding of cell-type specific transcription factors. With the advent of next-generation quantitative base-resolution maps of 5-methylcytosine and its oxidized derivatives and better coverage of the genome, we expect to learn more about the true significance of these DNA modifications in the regulation of cell fate choices.

Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells.

TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.

Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.

TET2 is a close relative of TET1, an enzyme that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. The gene encoding TET2 resides at chromosome 4q24, in a region showing recurrent microdeletions and copy-neutral loss of heterozygosity (CN-LOH) in patients with diverse myeloid malignancies. Somatic TET2 mutations are frequently observed in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukaemia (CMML), acute myeloid leukaemias (AML) and secondary AML (sAML). We show here that TET2 mutations associated with myeloid malignancies compromise catalytic activity. Bone marrow samples from patients with TET2 mutations displayed uniformly low levels of 5hmC in genomic DNA compared to bone marrow samples from healthy controls. Moreover, small hairpin RNA (shRNA)-mediated depletion of Tet2 in mouse haematopoietic precursors skewed their differentiation towards monocyte/macrophage lineages in culture. There was no significant difference in DNA methylation between bone marrow samples from patients with high 5hmC versus healthy controls, but samples from patients with low 5hmC showed hypomethylation relative to controls at the majority of differentially methylated CpG sites. Our results demonstrate that Tet2 is important for normal myelopoiesis, and suggest that disruption of TET2 enzymatic activity favours myeloid tumorigenesis. Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool, to tailor therapies and assess responses to anticancer drugs.

Domain requirements and sequence specificity of DNA binding for the forkhead transcription factor FOXP3.

The forkhead, winged-helix transcription factor FOXP3 is preferentially expressed in T regulatory (Treg) cells and is critical for their immunosuppressive function. Mutations that abolish FOXP3 function lead to systemic autoimmunity in mice and humans. However, the manner by which FOXP3 recognizes cognate DNA elements is unclear. Here we identify an in vitro optimized DNA sequence to assess FOXP3 DNA binding by electrophoretic mobility shift assay (EMSA). The optimized sequence contains two tandem copies of a core DNA element resembling, but not identical to, the canonical forkhead (FKH) binding element. The tandem nature of this optimized FOXP3-binding oligonucleotide suggests a requirement for multimerization, and EMSA experiments confirm that both the DNA-binding FKH domain and an intact leucine-zipper domain, which mediates homo-multimerization of FOXP3, are required for DNA binding. These results establish a practical framework for understanding the molecular basis by which FOXP3 regulates gene transcription and programs Treg suppressive function.

Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.

DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference-mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.

Human allograft arterial injury is ameliorated by sirolimus and cyclosporine and correlates with suppression of interferon-gamma.

BACKGROUND: Chronic allograft dysfunction may result from arterial injury, manifest as transplant arteriosclerosis (TA). This represents an important factor limiting long-term outcomes after heart and kidney transplantation; a relationship between acute allograft arterial injury and TA has been suggested. We have used SCID/bg mice bearing transplanted human artery, inoculated with allogeneic human PBMC to study arteriopathy in human vessels. Earlier work demonstrated arteriopathy similar to that observed clinically, and identified interferon-gamma as a mediator of the process. This study evaluated whether sirolimus (SRL), with cyclosporine A (CsA) or alone, affects TA, and examined possible mechanisms of action. METHODS: CB17/SCID/bg mice were transplanted with human arteries replacing the abdominal aorta; reconstituted with allogeneic human PBMC. Controls received vehicle alone for comparison with mice given CsA (5 mg/kg/d), SRL (0.1 or 0.5 mg/kg/d), or CsA (5 mg/kg/d) plus SRL (0.1 mg/kg/d). Transplant arteries were examined 28 days later by histology and immunohistochemistry; circulating human interferon-gamma was evaluated by ELISA, and intragraft interferon-gamma mRNA by qRT-PCR. RESULTS: The characteristic TA was modestly reduced by CsA or low-dose SRL, but eliminated by combination CsA plus SRL or higher dose SRL alone. Circulating interferon-gamma was reduced by CsA, but inhibition was dramatic with SRL alone or combined with CsA. Intragraft interferon-gamma and HLA-DR expression were moderately reduced by CsA or SRL, and eliminated with combined CsA plus SRL. CONCLUSIONS: SRL plus CsA prevented allograft arteriopathy, correlating with suppression of intragraft interferon-gamma, suggesting that SRL effects may result from anti-inflammatory consequences from inhibiting interferon-gamma.