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1.
Mol Cell ; 72(4): 739-752.e9, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30392929

RESUMO

The RING E3 ubiquitin ligase UHRF1 controls DNA methylation through its ability to target the maintenance DNA methyltransferase DNMT1 to newly replicated chromatin. DNMT1 recruitment relies on ubiquitylation of histone H3 by UHRF1; however, how UHRF1 deposits ubiquitin onto the histone is unknown. Here, we demonstrate that the ubiquitin-like domain (UBL) of UHRF1 is essential for RING-mediated H3 ubiquitylation. Using chemical crosslinking and mass spectrometry, biochemical assays, and recombinant chromatin substrates, we show that the UBL participates in structural rearrangements of UHRF1 upon binding to chromatin and the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1. Similar to ubiquitin, the UBL exerts its effects through a hydrophobic patch that contacts a regulatory surface on the "backside" of the E2 to stabilize the E2-E3-chromatin complex. Our analysis of the enzymatic mechanism of UHRF1 uncovers an unexpected function of the UBL domain and defines a new role for this domain in DNMT1-dependent inheritance of DNA methylation.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Células HEK293 , Histonas/metabolismo , Humanos , Masculino , Camundongos , Células-Tronco Embrionárias Murinas , Proteínas Nucleares/metabolismo , Ligação Proteica , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
2.
Nucleic Acids Res ; 50(15): 8491-8511, 2022 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-35904814

RESUMO

DNA methylation (5-methylcytosine (5mC)) is critical for genome stability and transcriptional regulation in mammals. The discovery that ten-eleven translocation (TET) proteins catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized our perspective on the complexity and regulation of DNA modifications. However, to what extent the regulatory functions of TET1 can be attributed to its catalytic activity remains unclear. Here, we use genome engineering and quantitative multi-omics approaches to dissect the precise catalytic vs. non-catalytic functions of TET1 in murine embryonic stem cells (mESCs). Our study identifies TET1 as an essential interaction hub for multiple chromatin modifying complexes and a global regulator of histone modifications. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. In particular, we show that TET1 is critical for the establishment of H3K9me3 and H4K20me3 at endogenous retroviral elements (ERVs) and their silencing that is independent of its canonical role in DNA demethylation. Furthermore, we provide evidence that this repression of ERVs depends on the interaction between TET1 and SIN3A. In summary, we demonstrate that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Retrovirus Endógenos , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/metabolismo , Animais , Citosina/metabolismo , Desmetilação do DNA , Metilação de DNA , Proteínas de Ligação a DNA/genética , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Expressão Gênica , Mamíferos/genética , Camundongos , Proteínas Proto-Oncogênicas/genética
3.
Nucleic Acids Res ; 50(21): 12527-12542, 2022 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-36420895

RESUMO

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Dedos de Zinco PHD , Camundongos , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Cromatina , Metilação de DNA , Proteínas Cromossômicas não Histona/metabolismo
4.
Nucleic Acids Res ; 49(13): 7406-7423, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34214177

RESUMO

Heterochromatin binding protein HP1ß plays an important role in chromatin organization and cell differentiation, however the underlying mechanisms remain unclear. Here, we generated HP1ß-/- embryonic stem cells and observed reduced heterochromatin clustering and impaired differentiation. We found that during stem cell differentiation, HP1ß is phosphorylated at serine 89 by CK2, which creates a binding site for the pluripotency regulator KAP1. This phosphorylation dependent sequestration of KAP1 in heterochromatin compartments causes a downregulation of pluripotency factors and triggers pluripotency exit. Accordingly, HP1ß-/- and phospho-mutant cells exhibited impaired differentiation, while ubiquitination-deficient KAP1-/- cells had the opposite phenotype with enhanced differentiation. These results suggest that KAP1 regulates pluripotency via its ubiquitination activity. We propose that the formation of subnuclear membraneless heterochromatin compartments may serve as a dynamic reservoir to trap or release cellular factors. The sequestration of essential regulators defines a novel and active role of heterochromatin in gene regulation and represents a dynamic mode of remote control to regulate cellular processes like cell fate decisions.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Embrionárias/metabolismo , Heterocromatina/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Caseína Quinase II/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/fisiologia , Cricetinae , Células-Tronco Embrionárias/citologia , Técnicas de Inativação de Genes , Humanos , Camundongos , Fosforilação , Serina/metabolismo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/fisiologia
5.
Nucleic Acids Res ; 43(17): e112, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26007658

RESUMO

Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3 and Uhrf1, we generated MIN-tagged embryonic stem cell lines and created a toolbox of prefabricated modules that can be integrated via Bxb1-mediated recombination. We used these functional modules to study protein interactions and their spatio-temporal dynamics as well as gene expression and specific mutations during cellular differentiation and in response to external stimuli. Our genome engineering strategy provides a versatile open platform for efficient generation of multiple isogenic cell lines to study gene function under physiological conditions.


Assuntos
Engenharia Celular/métodos , Animais , Anticorpos Monoclonais , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Loci Gênicos , Genômica/métodos , Integrases/genética , Integrases/imunologia , Integrases/metabolismo , Mutação , Ratos , Recombinação Genética
6.
Sci Rep ; 10(1): 12066, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694513

RESUMO

Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation.


Assuntos
Diferenciação Celular , Citosina/metabolismo , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Oxirredução , Proteínas Proto-Oncogênicas/genética , Animais , Sistemas CRISPR-Cas , Cromatografia Líquida de Alta Pressão , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Epigênese Genética , Camundongos , Camundongos Knockout , Proteoma , Proteômica , Proteínas Proto-Oncogênicas/metabolismo , Espectrometria de Massas em Tandem
7.
Nat Commun ; 11(1): 1222, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144273

RESUMO

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/genética , Ubiquitinação , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/metabolismo , Humanos , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Espermatozoides/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Xenopus laevis
8.
Nat Commun ; 11(1): 5972, 2020 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-33235224

RESUMO

Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.


Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona , Desmetilação do DNA , Mamíferos/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Evolução Biológica , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA , DNA Polimerase Dirigida por DNA/metabolismo , Epigenômica , Evolução Molecular , Regulação da Expressão Gênica , Genes Reguladores , Células Germinativas/metabolismo , Camundongos , Ubiquitina-Proteína Ligases/metabolismo
9.
Epigenetics Chromatin ; 11(1): 41, 2018 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-30045751

RESUMO

BACKGROUND: Epigenome-wide association studies (EWAS) based on human brain samples allow a deep and direct understanding of epigenetic dysregulation in Alzheimer's disease (AD). However, strong variation of cell-type proportions across brain tissue samples represents a significant source of data noise. Here, we report the first EWAS based on sorted neuronal and non-neuronal (mostly glia) nuclei from postmortem human brain tissues. RESULTS: We show that cell sorting strongly enhances the robust detection of disease-related DNA methylation changes even in a relatively small cohort. We identify numerous genes with cell-type-specific methylation signatures and document differential methylation dynamics associated with aging specifically in neurons such as CLU, SYNJ2 and NCOR2 or in glia RAI1,CXXC5 and INPP5A. Further, we found neuron or glia-specific associations with AD Braak stage progression at genes such as MCF2L, ANK1, MAP2, LRRC8B, STK32C and S100B. A comparison of our study with previous tissue-based EWAS validates multiple AD-associated DNA methylation signals and additionally specifies their origin to neuron, e.g., HOXA3 or glia (ANK1). In a meta-analysis, we reveal two novel previously unrecognized methylation changes at the key AD risk genes APP and ADAM17. CONCLUSIONS: Our data highlight the complex interplay between disease, age and cell-type-specific methylation changes in AD risk genes thus offering new perspectives for the validation and interpretation of large EWAS results.


Assuntos
Envelhecimento/genética , Doença de Alzheimer/genética , Metilação de DNA , Estudo de Associação Genômica Ampla/métodos , Neuroglia/citologia , Neurônios/citologia , Proteína ADAM17/genética , Precursor de Proteína beta-Amiloide/genética , Autopsia , Separação Celular , Epigênese Genética , Epigenômica , Predisposição Genética para Doença , Humanos , Neuroglia/química , Neurônios/química , Especificidade de Órgãos , Transcriptoma
10.
Blood Adv ; 2(23): 3447-3461, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30518537

RESUMO

Mesenchymal stromal cells (MSCs) are crucial components of the bone marrow (BM) microenvironment essential for regulating self-renewal, survival, and differentiation of hematopoietic stem/progenitor cells (HSPCs) in the stem cell niche. MSCs are functionally altered in myelodysplastic syndromes (MDS) and exhibit an altered methylome compared with MSCs from healthy controls, thus contributing to disease progression. To determine whether MSCs are amenable to epigenetic therapy and if this affects their function, we examined growth, differentiation, and HSPC-supporting capacity of ex vivo-expanded MSCs from MDS patients in comparison with age-matched healthy controls after direct treatment in vitro with the hypomethylating agent azacitidine (AZA). Strikingly, we find that AZA exerts a direct effect on healthy as well as MDS-derived MSCs such that they favor support of healthy over malignant clonal HSPC expansion in coculture experiments. RNA-sequencing analyses of MSCs identified stromal networks regulated by AZA. Notably, these comprise distinct molecular pathways crucial for HSPC support, foremost extracellular matrix molecules (including collagens) and interferon pathway components. Our study demonstrates that the hypomethylating agent AZA exerts its antileukemic activity in part through a direct effect on the HSPC-supporting BM niche and provides proof of concept for the therapeutic potential of epigenetic treatment of diseased MSCs. In addition, our comprehensive data set of AZA-sensitive gene networks represents a valuable framework to guide future development of targeted epigenetic niche therapy in myeloid malignancies such as MDS and acute myeloid leukemia.


Assuntos
Azacitidina/farmacologia , Hematopoese/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/citologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Imunofenotipagem , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Síndromes Mielodisplásicas , Osteogênese/efeitos dos fármacos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Adulto Jovem
11.
J Mol Biol ; 429(24): 3814-3824, 2017 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-29055779

RESUMO

Ubiquitination is a multifunctional posttranslational modification controlling the activity, subcellular localization and stability of proteins. The E3 ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) is an essential epigenetic factor that recognizes repressive histone marks as well as hemi-methylated DNA and recruits DNA methyltransferase 1. To explore enzymatic functions of UHRF1 beyond epigenetic regulation, we conducted a comprehensive screen in mouse embryonic stem cells to identify novel ubiquitination targets of UHRF1 and its paralogue UHRF2. We found differentially ubiquitinated peptides associated with a variety of biological processes such as transcriptional regulation and DNA damage response. Most prominently, we identified PCNA-associated factor 15 (PAF15; also known as Pclaf, Ns5atp9, KIAA0101 and OEATC-1) as a specific ubiquitination target of UHRF1. Although the function of PAF15 ubiquitination in translesion DNA synthesis is well characterized, the respective E3 ligase had been unknown. We could show that UHRF1 ubiquitinates PAF15 at Lys 15 and Lys 24 and promotes its binding to PCNA during late S-phase. In summary, we identified novel ubiquitination targets that link UHRF1 to transcriptional regulation and DNA damage response.


Assuntos
Proteínas de Transporte/fisiologia , Dano ao DNA , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas Nucleares/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitina/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Células Cultivadas , Reparo do DNA , Replicação do DNA , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Epigênese Genética , Camundongos , Camundongos Knockout , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Fase S/fisiologia , Ubiquitinação
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