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1.
Cell ; 153(4): 910-8, 2013 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-23643243

RESUMO

Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty--8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.


Assuntos
Marcação de Genes/métodos , Camundongos/genética , Animais , Sequência de Bases , Células-Tronco Embrionárias/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Pequeno RNA não Traduzido
2.
Nat Immunol ; 16(6): 653-62, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25867473

RESUMO

The methylcytosine dioxygenase TET1 ('ten-eleven translocation 1') is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.


Assuntos
Linfócitos B/fisiologia , Citosina/análogos & derivados , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/fisiologia , Linfoma de Células B/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , 5-Metilcitosina/análogos & derivados , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Instabilidade Cromossômica , Citosina/metabolismo , Metilação de DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Epigênese Genética , Exoma/genética , Perfilação da Expressão Gênica , Humanos , Camundongos , Mutação/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética
3.
Nucleic Acids Res ; 50(6): 3169-3189, 2022 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-35150568

RESUMO

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.


Assuntos
Proteínas de Ligação a DNA , Dioxigenases , Células-Tronco Embrionárias , Proteínas Proto-Oncogênicas , Animais , Diferenciação Celular/genética , DNA/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Células-Tronco Embrionárias/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo
4.
Cell ; 133(1): 103-15, 2008 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-18394993

RESUMO

RanBP2 is a nucleoporin with SUMO E3 ligase activity that functions in both nucleocytoplasmic transport and mitosis. However, the biological relevance of RanBP2 and the in vivo targets of its E3 ligase activity are unknown. Here we show that animals with low amounts of RanBP2 develop severe aneuploidy in the absence of overt transport defects. The main chromosome segregation defect in cells from these mice is anaphase-bridge formation. Topoisomerase IIalpha (Topo IIalpha), which decatenates sister centromeres prior to anaphase onset to prevent bridges, fails to accumulate at inner centromeres when RanBP2 levels are low. We find that RanBP2 sumoylates Topo IIalpha in mitosis and that this modification is required for its proper localization to inner centromeres. Furthermore, mice with low amounts of RanBP2 are highly sensitive to tumor formation. Together, these data identify RanBP2 as a chromosomal instability gene that regulates Topo IIalpha by sumoylation and suppresses tumorigenesis.


Assuntos
Antígenos de Neoplasias/metabolismo , Centrômero/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Anáfase , Aneuploidia , Animais , Carcinógenos , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Camundongos Knockout , Mitose , Chaperonas Moleculares/genética , Mutação , Neoplasias/induzido quimicamente , Neoplasias/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Estrutura Terciária de Proteína , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
6.
Mol Cell ; 49(6): 1023-33, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23453809

RESUMO

Genomic imprinting directs the allele-specific marking and expression of loci according to their parental origin. Differential DNA methylation at imprinted control regions (ICRs) is established in gametes and, although largely preserved through development, can be experimentally reset by fusing somatic cells with embryonic germ cell (EGC) lines. Here, we show that the Ten-Eleven Translocation proteins Tet1 and Tet2 participate in the efficient erasure of imprints in this model system. The fusion of B cells with EGCs initiates pluripotent reprogramming, in which rapid re-expression of Oct4 is accompanied by an accumulation of 5-hydroxymethylcytosine (5hmC) at several ICRs. Tet2 was required for the efficient reprogramming capacity of EGCs, whereas Tet1 was necessary to induce 5-methylcytosine oxidation specifically at ICRs. These data show that the Tet1 and Tet2 proteins have discrete roles in cell-fusion-mediated pluripotent reprogramming and imprint erasure in somatic cells.


Assuntos
Fusão Celular , Proteínas de Ligação a DNA/fisiologia , Impressão Genômica , Proteínas Proto-Oncogênicas/fisiologia , 5-Metilcitosina/análogos & derivados , Animais , Linfócitos B/citologia , Sequência de Bases , Linhagem Celular , Citosina/análogos & derivados , Citosina/metabolismo , Metilação de DNA , Dioxigenases , Células-Tronco Embrionárias/citologia , Expressão Gênica , Células Germinativas/citologia , Proteínas de Fluorescência Verde/biossíntese , Humanos , Fator de Crescimento Insulin-Like II/genética , Camundongos , Dados de Sequência Molecular , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Proteínas/metabolismo , RNA Longo não Codificante/genética , Análise de Sequência de DNA
7.
Commun Biol ; 7(1): 415, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38580843

RESUMO

The ten-eleven-translocation family of proteins (TET1/2/3) are epigenetic regulators of gene expression. They regulate genes by promoting DNA demethylation (i.e., catalytic activity) and by partnering with regulatory proteins (i.e., non-catalytic functions). Unlike Tet1 and Tet2, Tet3 is not expressed in mouse embryonic stem cells (ESCs) but is induced upon ESC differentiation. However, the significance of its dual roles in lineage specification is less defined. By generating TET3 catalytic-mutant (Tet3m/m) and knockout (Tet3-/-) mouse ESCs and differentiating them to neuroectoderm (NE), we identify distinct catalytic-dependent and independent roles of TET3 in NE specification. We find that the catalytic activity of TET3 is important for activation of neural genes while its non-catalytic functions are involved in suppressing mesodermal programs. Interestingly, the vast majority of differentially methylated regions (DMRs) in Tet3m/m and Tet3-/- NE cells are hypomethylated. The hypo-DMRs are associated to aberrantly upregulated genes while the hyper-DMRs are linked to downregulated neural genes. We find the maintenance methyltransferase Dnmt1 as a direct target of TET3, which is downregulated in TET3-deficient NE cells and may contribute to the increased DNA hypomethylation. Our findings establish that the catalytic-dependent and -independent roles of TET3 have distinct contributions to NE specification with potential implications in development.


Assuntos
Dioxigenases , Animais , Camundongos , Diferenciação Celular/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Placa Neural/metabolismo
8.
iScience ; 26(7): 107170, 2023 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-37456851

RESUMO

Tet2 is a member of the Ten-eleven translocation (Tet1/2/3) family of enzymes and is expressed in embryonic stem cells (ESCs). It demethylates DNA (catalytic functions) and partners with chromatin modifiers (noncatalytic functions) to regulate genes. However, the significance of these functions in ESCs is less defined. Using Tet2 catalytic mutant (Tet2m/m) and knockout (Tet2-/-) ESCs, we identified Tet2 target genes regulated by its catalytic dependent versus independent roles. Tet2 was enriched at their active enhancers and promoters to demethylate them. We also identified the histone deacetylase component Sin3a as a Tet2 partner, co-localizing at promoters and active enhancers. Tet2 deficiency diminished Sin3a at these regions. Tet2 and Sin3a co-occupancy overlapped with Tet1. Combined loss of Tet1/2, but not of their catalytic activities, reduced Sin3a at active enhancers. These findings establish Tet2 catalytic and noncatalytic functions as regulators of DNA demethylation and Sin3a recruitment at active enhancers in ESCs.

9.
Exp Hematol ; 124: 45-55.e2, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37225048

RESUMO

TET2 is a member of the Ten-eleven translocation (Tet) family of DNA dioxygenases that regulate gene expression by promoting DNA demethylation (enzymatic activity) and partnering with chromatin regulatory complexes (nonenzymatic functions). TET2 is highly expressed in the hematopoietic lineage, where its molecular functions are the subject of continuous investigations because of the prevalence of TET2 mutations in hematologic malignancies. Previously, we have implicated Tet2 catalytic and noncatalytic functions in the regulation of myeloid and lymphoid lineages, respectively. However, the impact of these functions of Tet2 on hematopoiesis as the bone marrow ages remains unclear. Here, we conducted comparative transplantations and transcriptomic analyses of 3-, 6-, 9-, and 12-month-old Tet2 catalytic mutant (Mut) and knockout (KO) bone marrow. Tet2 Mut bone marrow of all ages exclusively caused hematopoietic disorders of the myeloid lineage. In contrast, young Tet2 KO bone marrow developed both lymphoid and myeloid diseases, whereas older Tet2 KO bone marrow predominantly elicited myeloid disorders with shorter latency than age-matched Tet2 Mut bone marrow. We identified robust gene dysregulation in Tet2 KO Lin- cells at 6 months that involved lymphoma and myelodysplastic syndrome and/or leukemia-causing genes, many of which were hypermethylated early in life. There was a shift from lymphoid to myeloid gene deregulation in Tet2 KO Lin- cells with age, underpinning the higher incidence of myeloid diseases. These findings expand on the dynamic regulation of bone marrow by Tet2 and show that its catalytic-dependent and -independent roles have distinct impacts on myeloid and lymphoid lineages with age.


Assuntos
Dioxigenases , Doenças Hematológicas , Neoplasias Hematológicas , Síndromes Mielodisplásicas , Humanos , Lactente , Medula Óssea/metabolismo , Neoplasias Hematológicas/genética , Síndromes Mielodisplásicas/metabolismo , Hematopoese/genética , Doenças Hematológicas/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Mutação
10.
Cells ; 11(8)2022 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-35456045

RESUMO

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.


Assuntos
Proteínas de Ligação a DNA , Proteínas Proto-Oncogênicas , Animais , Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Translocação Genética
11.
Sci Adv ; 8(9): eabm3470, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35235365

RESUMO

Ten-eleven translocation (Tet) enzymes promote DNA demethylation by oxidizing 5-methylcytosine. They are expressed during development and are essential for mouse gastrulation. However, their postgastrulation functions are not well established. We find that global or endothelial-specific loss of all three Tet enzymes immediately after gastrulation leads to reduced number of hematopoietic stem and progenitor cells (HSPCs) and lethality in mid-gestation mouse embryos. This is due to defects in specification of HSPCs from endothelial cells (ECs) that compromise primitive and definitive hematopoiesis. Mechanistically, loss of Tet enzymes in ECs led to hypermethylation and down-regulation of NFκB1 and master hematopoietic transcription factors (Gata1/2, Runx1, and Gfi1b). Restoring Tet catalytic activity or overexpression of these factors in Tet-deficient ECs rescued hematopoiesis defects. This establishes Tet enzymes as activators of hematopoiesis programs in ECs for specification of HSPCs during embryogenesis, which is distinct from their roles in adult hematopoiesis, with implications in deriving HSPCs from pluripotent cells.


Assuntos
Dioxigenases , Animais , Diferenciação Celular/genética , Desmetilação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Desenvolvimento Embrionário/genética , Células Endoteliais/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Mamíferos/metabolismo , Camundongos
12.
Stem Cell Res ; 61: 102770, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35390758

RESUMO

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.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Neurais , Animais , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/metabolismo , Camundongos , Células-Tronco Neurais/metabolismo
13.
Front Cell Dev Biol ; 9: 645335, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33681230

RESUMO

Studies of tissue-specific epigenomes have revealed 5-hydroxymethylcytosine (5hmC) to be a highly enriched and dynamic DNA modification in the metazoan nervous system, inspiring interest in the function of this epigenetic mark in neurodevelopment and brain function. 5hmC is generated by oxidation of 5-methylcytosine (5mC), a process catalyzed by the ten-eleven translocation (TET) enzymes. 5hmC serves not only as an intermediate in DNA demethylation but also as a stable epigenetic mark. Here, we review the known functions of 5hmC and TET enzymes in neural progenitor cell biology and embryonic and postnatal neurogenesis. We also discuss how TET enzymes and 5hmC regulate neuronal activity and brain function and highlight their implications in human neurodevelopmental and neurodegenerative disorders. Finally, we present outstanding questions in the field and envision new research directions into the roles of 5hmC and TET enzymes in neurodevelopment.

14.
Trends Cancer ; 7(7): 635-646, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33468438

RESUMO

The mechanisms governing the methylome profile of tumor suppressors and oncogenes have expanded with the discovery of oxidized states of 5-methylcytosine (5mC). Ten-eleven translocation (TET) enzymes are a family of dioxygenases that iteratively catalyze 5mC oxidation and promote cytosine demethylation, thereby creating a dynamic global and local methylation landscape. While the catalytic function of TET enzymes during stem cell differentiation and development have been well studied, less is known about the multifaceted roles of TET enzymes during carcinogenesis. This review outlines several tiers of TET regulation and overviews how TET deregulation promotes a cancer phenotype. Defining the tissue-specific and context-dependent roles of TET enzymes will deepen our understanding of the epigenetic perturbations that promote or inhibit carcinogenesis.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Oxigenases de Função Mista/metabolismo , Neoplasias/genética , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinogênese/genética , Carcinogênese/imunologia , Carcinogênese/patologia , Ensaios Clínicos como Assunto , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/imunologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Dioxigenases/antagonistas & inibidores , Dioxigenases/genética , Sinergismo Farmacológico , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/genética , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Resultado do Tratamento
15.
Dev Cell ; 56(22): 3052-3065.e5, 2021 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-34710357

RESUMO

Loss of imprinting (LOI) results in severe developmental defects, but the mechanisms preventing LOI remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) in pluripotent stem cells. We demonstrate that the IG-DMR consists of two antagonistic elements: a paternally methylated CpG island that prevents recruitment of TET dioxygenases and a maternally unmethylated non-canonical enhancer that ensures expression of the Gtl2 lncRNA by counteracting de novo DNA methyltransferases. Genetic or epigenetic editing of these elements leads to distinct LOI phenotypes with characteristic alternations of allele-specific gene expression, DNA methylation, and 3D chromatin topology. Although repression of the Gtl2 promoter results in dysregulated imprinting, the stability of LOI phenotypes depends on the IG-DMR, suggesting a functional hierarchy. These findings establish the IG-DMR as a bipartite control element that maintains imprinting by allele-specific restriction of the DNA (de)methylation machinery.


Assuntos
Alelos , Proteínas de Ligação ao Cálcio/genética , Metilação de DNA/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Animais , Cromossomos/genética , Impressão Genômica/genética , Iodeto Peroxidase/genética , Camundongos , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante/genética
16.
Cell Rep ; 28(10): 2480-2490.e4, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484061

RESUMO

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.


Assuntos
Biocatálise , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Homeostase , Proteínas Proto-Oncogênicas/metabolismo , Animais , Medula Óssea/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Dioxigenases , Regulação da Expressão Gênica , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Camundongos Knockout , Mutação/genética , Fenótipo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética
17.
Cell Rep ; 28(8): 1993-2003.e5, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31433977

RESUMO

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.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Autorrenovação Celular/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/deficiência , Dioxigenases , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Camundongos , Camundongos SCID , Proteína Homeobox Nanog/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/deficiência , Transcrição Gênica
18.
Neuropsychopharmacology ; 42(8): 1657-1669, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28074830

RESUMO

Depression is a leading cause of disease burden, yet current therapies fully treat <50% of affected individuals. Increasing evidence implicates epigenetic mechanisms in depression and antidepressant action. Here we examined a possible role for the DNA dioxygenase, ten-eleven translocation protein 1 (TET1), in depression-related behavioral abnormalities. We applied chronic social defeat stress, an ethologically validated mouse model of depression-like behaviors, and examined Tet1 expression changes in nucleus accumbens (NAc), a key brain reward region. We show decreased Tet1 expression in NAc in stress-susceptible mice only. Surprisingly, selective knockout of Tet1 in NAc neurons of adult mice produced antidepressant-like effects in several behavioral assays. To identify Tet1 targets that mediate these actions, we performed RNAseq on NAc after conditional deletion of Tet1 and found that immune-related genes are the most highly dysregulated. Moreover, many of these genes are also upregulated in the NAc of resilient mice after chronic social defeat stress. These findings reveal a novel role for TET1, an enzyme important for DNA hydroxymethylation, in the brain's reward circuitry in modulating stress responses in mice. We also identify a subset of genes that are regulated by TET1 in this circuitry. These findings provide new insight into the pathophysiology of depression, which can aid in future antidepressant drug discovery efforts.


Assuntos
Ansiedade/fisiopatologia , Proteínas de Ligação a DNA/fisiologia , Depressão/fisiopatologia , Núcleo Accumbens/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Estresse Psicológico/fisiopatologia , Animais , Ansiedade/genética , Comportamento Animal , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Depressão/genética , Modelos Animais de Doenças , Expressão Gênica/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Regulação para Cima
19.
Mol Cell Biol ; 36(3): 452-61, 2016 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-26598602

RESUMO

DNA methylation is a dynamic epigenetic modification with an important role in cell fate specification and reprogramming. The Ten eleven translocation (Tet) family of enzymes converts 5-methylcytosine to 5-hydroxymethylcytosine, which promotes passive DNA demethylation and functions as an intermediate in an active DNA demethylation process. Tet1/Tet2 double-knockout mice are characterized by developmental defects and epigenetic instability, suggesting a requirement for Tet-mediated DNA demethylation for the proper regulation of gene expression during differentiation. Here, we used whole-genome bisulfite and transcriptome sequencing to characterize the underlying mechanisms. Our results uncover the hypermethylation of DNA methylation canyons as the genomic key feature of Tet1/Tet2 double-knockout mouse embryonic fibroblasts. Canyon hypermethylation coincided with disturbed regulation of associated genes, suggesting a mechanistic explanation for the observed Tet-dependent differentiation defects. Based on these results, we propose an important regulatory role of Tet-dependent DNA demethylation for the maintenance of DNA methylation canyons, which prevents invasive DNA methylation and allows functional regulation of canyon-associated genes.


Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/genética , Fibroblastos/citologia , Proteínas Proto-Oncogênicas/genética , Transcriptoma , Adipogenia , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Epigênese Genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/metabolismo
20.
Cell Rep ; 15(8): 1809-21, 2016 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-27184841

RESUMO

Ten-eleven translocation (Tet) family proteins convert 5-methylcytosine to 5-hydroxymethylcytosine. We show that mouse embryonic stem cells (ESCs) depleted of Tet1 and/or Tet2 by RNAi exhibit short telomeres and chromosomal instability, concomitant with reduced telomere recombination. Tet1 and Tet2 double-knockout ESCs also display short telomeres but to a lesser extent. Notably, Tet1/2/3 triple-knockout ESCs show heterogeneous telomere lengths and increased frequency of telomere loss and chromosomal fusion. Mechanistically, Tets depletion or deficiency increases Dnmt3b and decreases 5hmC levels, resulting in elevated methylation levels at sub-telomeres. Consistently, knockdown of Dnmt3b or addition of 2i (MAPK and GSK3ß inhibitors), which also inhibits Dnmt3b, reduces telomere shortening, partially rescuing Tet1/2 deficiency. Interestingly, Tet1/2 double or Tet1/2/3 triple knockout in ESCs consistently upregulates Zscan4, which may counteract telomere shortening. Together, Tet enzymes play important roles in telomere maintenance and chromosomal stability of ESCs by modulating sub-telomeric methylation levels.


Assuntos
Instabilidade Cromossômica/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Telômero/metabolismo , Animais , Cromossomos de Mamíferos/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Deleção de Genes , Camundongos , Camundongos Knockout , Recombinação Genética/genética , Encurtamento do Telômero , DNA Metiltransferase 3B
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