Lineage-specific functions of TET1 in the postimplantation mouse embryo

Khoueiry, Rita; Sohni, Abhishek; Thienpont, Bernard; Luo, Xinlong; Velde, Joris Vande; Bartoccetti, Michela; Boeckx, Bram; Zwijsen, An; Rao, Anjana; Lambrechts, Diether; Koh, Kian Peng

doi:10.1038/ng.3868

Cited by 99 publications

(142 citation statements)

References 66 publications

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“…Further, TET2 gene mutation has been reported to negatively impact patient survival in CMML (Kosmider, Gelsi-Boyer, Ciudad, et al, 2009) as well as in the most common peripheral T-cell lymphomas (Lemonnier et al, 2012). Furthermore, the nonepigenetic effects of TET2 variants may also be of some relevance in hematologic neoplasms, as it has been recently shown for other TET family members (Khoueiry et al, 2017).…”

Section: Ibmentioning

confidence: 94%

TET2 missense variants in human neoplasia. A proposal of structural and functional classification

Bussaglia¹,

Antón

Nomdedéu³

et al. 2019

Molec Gen & Gen Med

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Background The human TET2 gene plays a pivotal role in the epigenetic regulation of normal and malignant hematopoiesis. Somatic TET2 mutations have been repeatedly identified in age‐related clonal hematopoiesis and in myeloid neoplasms ranging from acute myeloid leukemia (AML) to myeloproliferative neoplasms. However, there have been no attempts to systematically explore the structural and functional consequences of the hundreds of TET2 missense variants reported to date. Methods We have sequenced the TET2 gene in 189 Spanish AML patients using Sanger sequencing and NGS protocols. Next, we performed a thorough bioinformatics analysis of TET2 protein and of the expected impact of all reported TET2 missense variants on protein structure and function, exploiting available structure‐and‐function information as well as 3D structure prediction tools. Results We have identified 38 TET2 allelic variants in the studied patients, including two frequent SNPs: p.G355D (10 cases) and p.I1762V (28 cases). Four of the detected mutations are reported here for the first time: c.122C>T (p.P41L), c.4535C>G (p.A1512G), c.4760A>G (p.D1587G), and c.5087A>T (p.Y1696F). We predict a complex multidomain architecture for the noncatalytic regions of TET2, and in particular the presence of well‐conserved α+β globular domains immediately preceding and following the actual catalytic unit. Further, we provide a rigorous interpretation of over 430 missense SNVs that affect the TET2 catalytic domain, and we hypothesize explanations for ~700 additional variants found within the regulatory regions of the protein. Finally, we propose a systematic classification of all missense mutants and SNPs reported to date into three major categories (severe, moderate, and mild), based on their predicted structural and functional impact. Conclusions The proposed classification of missense TET2 variants would help to assess their clinical impact on human neoplasia and may guide future structure‐and‐function investigations of TET family members.

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Section: Ibmentioning

confidence: 94%

TET2 missense variants in human neoplasia. A proposal of structural and functional classification

Bussaglia¹,

Antón

Nomdedéu³

et al. 2019

Molec Gen & Gen Med

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“…We generated two independent murine transgenic strains harboring mCherry reporters to distinguish developmental stage-specific expression patterns of Tet1 25,27 (Figure 1A). The first strain, named Tg( Tet1- mCherry)B, contains a randomly integrated mCherry-2A-puromycin resistance cassette driven by a minimal 6-kb distal promoter region of Tet1 (previously denoted as genomic region B6 in ref 25).…”

Section: Resultsmentioning

confidence: 99%

“…To mark Tet1 gene activity from both proximal and distal promoter regions, we generated a second independent Tet1- mCherry strain, the B6- Tet1 tm1.1Koh line, in which the mCherry reporter was targeted after the ATG start codon of the coding sequence in exon 2 (Figure 1A). This strain – named “total” Tet1- mCherry - was generated by reporter exchange in the original Tet1- loxP-lacZ-loxP-mCherry knock-in cassette of the Tet1 tm1Koh allele 27 following breeding with Cre-deleter mice to obtain a Tet1- loxP-mCherry allele. In agreement with Tet1 expression in the post-implantation epiblast 25,27 , transgenic embryos showed detectable signals in the E6.5 epiblast (Figure 1K).…”

Section: Resultsmentioning

confidence: 99%

“…This strain – named “total” Tet1- mCherry - was generated by reporter exchange in the original Tet1- loxP-lacZ-loxP-mCherry knock-in cassette of the Tet1 tm1Koh allele 27 following breeding with Cre-deleter mice to obtain a Tet1- loxP-mCherry allele. In agreement with Tet1 expression in the post-implantation epiblast 25,27 , transgenic embryos showed detectable signals in the E6.5 epiblast (Figure 1K).…”

Section: Resultsmentioning

confidence: 99%

“…In these previous studies, the Tet1 gene was inactivated respectively by deleting exon 5 or exons 11-13 to disrupt expression of the catalytic domain. Because these earlier genetic knockouts (KOs) of Tet1 may not have disrupted non-catalytic functions provided by the N-terminal domain of TET1, we assessed reprogramming using Tet1 KO MEFs generated from our B6- Tet1 tm1Koh strain, which disrupts Tet1 expression using a knock-in (KI) cassette after the ATG start codon 27 . We crossed the Tet1 tm1Koh allele to the TgOG2 strain to assess whether naive-state activation of Oct4 DE as reflected by the GFP reporter could be affected by complete loss of TET1.…”

Section: Resultsmentioning

confidence: 99%

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Temporal resolution of global gene expression and DNA methylation changes in the final phases of reprogramming towards induced pluripotency

Bartoccetti

Luo

Veer

et al. 2019

Preprint

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The generation of induced pluripotent stem cells (iPSCs) involves activation of the endogenous 1 pluripotency circuitry and global DNA demethylation late in reprogramming, but temporal 2 resolution of these events using existing markers is insufficient. Here, we generated murine 3 transgenic lines harboring dual fluorescent reporters reflecting cell-state specific expression of 4 the master pluripotency factor Oct4 and the 5-methylcytosine dioxygenase Tet1. By assessing 5 reprogramming intermediates based on dual reporter patterns, we identified a sequential order of 6 Tet1 and Oct4 gene activation at proximal and distal regulatory elements following pluripotency 7 entry. Full induction of Tet1 marks a pivotal late intermediate stage occurring after a phase of 8 global gene repression, and preceding full activation of Oct4 along with late naive pluripotency 9 2 and germline-specific genes. Sequential activation of Tet1 further distinguishes two waves of 10 global DNA demethylation, targeting distinct genomic features and largely uncoupled from 11 transcriptional changes, with dynamics unique to iPSC reprogramming. Moreover, we 12 demonstrate that loss of Tet1 is compatible with reprogramming towards full Oct4 gene 13 activation, but generates iPSCs with aberrant DNA methylation, chromosomal instability during 14 lineage priming and defective differentiation potential. Therefore, the transcriptional logic of Tet1 15 expression signals a deterministic epigenetic roadmap towards generation of high quality iPSCs. 16 65 by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation 66 products at CpG dinucleotides 21-23 . Of the three TET genes, Tet1 expression is barely detectable 67 in mouse embryonic fibroblasts (MEFs), but robustly up-regulated in iPSCs concomitantly with 68 5 endogenous Oct4 late in reprogramming 8,24 . Because Tet1 is a target gene regulated by OCT4 69 24,25 , a positive feedback logic involving Tet1 and Oct4 may function to jump-start the 70 pluripotency circuitry during reprogramming 26 . However, the sequential patterns of endogenous 71 Oct4 and Tet1 gene activation during reprogramming remain unknown. 72 During early embryogenesis, Tet1 expression is driven by two state-specific promoter-73 enhancer regions, using a similar cis-regulatory logic as for Oct4, where a distal promoter region 74 spanning 6 kb drives an alternative transcription start site (TSS) at exon 1b which is activated 75 exclusively in pre-implantation embryos and naive ESCs 25 . A proximal promoter-enhancer 76 coupling at exon 1a sustains Tet1 expression through primed pluripotency. Here, we generated 77 MEFs harboring distinct dual-fluorescent transgenic reporters for state-specific expression of 78 Oct4 and Tet1. By using live cell imaging and flow cytometry coupled with genome-scale 79 analyses, our studies revealed a distinct trajectory of epigenetic events marking cell state 80 transitions from early acquisition of pluripotency to terminal establishment of clonal iPSCs. We 81 further clar...

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TET1 Directs Chondrogenic Differentiation by Regulating SOX9 Dependent Activation of Col2a1 and Acan In Vitro

et al. 2020

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Skeletal development is a tightly orchestrated process in which cartilage and bone differentiation are intricately intertwined. Recent studies have highlighted the contribution of epigenetic modifications and their writers to skeletal development. Methylated cytosine (5mC) can be oxidized to 5‐hydroxymethylcytosine (5hmC) by the Ten‐eleven‐translocation (TET) enzymes leading to demethylation. We have previously demonstrated that 5hmC is stably accumulated on lineage‐specific genes that are activated during in vitro chondrogenesis in the ATDC5 chondroprogenitors. Knockdown (KD) of Tet1 via short‐hairpin RNAs blocked ATDC5 chondrogenic differentiation. Here, we aimed to provide the mechanistic basis for TET1 function during ATDC5 differentiation. Transcriptomic analysis of Tet1 KD cells demonstrated that 54% of downregulated genes were SOX9 targets, suggesting a role for TET1 in mediating activation of a subset of the SOX9 target genes. Using genome‐wide mapping of 5hmC during ATDC5 differentiation, we found that 5hmC is preferentially accumulated at chondrocyte‐specific class II binding sites for SOX9, as compared with the tissue‐agnostic class I sites. Specifically, we find that SOX9 is unable to bind to Col2a1 and Acan after Tet1 KD, despite no changes in SOX9 levels. Finally, we compared this KD scenario with the genetic loss of TET1 in the growth plate using Tet1−/− embryos, which are approximately 10% smaller than their WT counterparts. In E17.5 Tet1−/− embryos, loss of SOX9 target gene expression is more modest than upon Tet1 KD in vitro. Overall, our data suggest a role for TET1‐mediated 5hmC deposition in partly shaping an epigenome conducive for SOX9 function. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

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Lineage-specific functions of TET1 in the postimplantation mouse embryo

Cited by 99 publications

References 66 publications

TET2 missense variants in human neoplasia. A proposal of structural and functional classification

TET2 missense variants in human neoplasia. A proposal of structural and functional classification

Temporal resolution of global gene expression and DNA methylation changes in the final phases of reprogramming towards induced pluripotency

TET1 Directs Chondrogenic Differentiation by Regulating SOX9 Dependent Activation of Col2a1 and Acan In Vitro

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