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1.
Nat Commun ; 14(1): 3848, 2023 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-37385984

RESUMO

The Nucleosome Remodeling and Deacetylation (NuRD) complex is a crucial regulator of cellular differentiation. Two members of the Methyl-CpG-binding domain (MBD) protein family, MBD2 and MBD3, are known to be integral, but mutually exclusive subunits of the NuRD complex. Several MBD2 and MBD3 isoforms are present in mammalian cells, resulting in distinct MBD-NuRD complexes. Whether these different complexes serve distinct functional activities during differentiation is not fully explored. Based on the essential role of MBD3 in lineage commitment, we systematically investigated a diverse set of MBD2 and MBD3 variants for their potential to rescue the differentiation block observed for mouse embryonic stem cells (ESCs) lacking MBD3. While MBD3 is indeed crucial for ESC differentiation to neuronal cells, it functions independently of its MBD domain. We further identify that MBD2 isoforms can replace MBD3 during lineage commitment, however with different potential. Full-length MBD2a only partially rescues the differentiation block, while MBD2b, an isoform lacking an N-terminal GR-rich repeat, fully rescues the Mbd3 KO phenotype. In case of MBD2a, we further show that removing the methylated DNA binding capacity or the GR-rich repeat enables full redundancy to MBD3, highlighting the synergistic requirements for these domains in diversifying NuRD complex function.


Assuntos
Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Nucleossomos , Animais , Camundongos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Isoformas de Proteínas/genética , Diferenciação Celular , Células-Tronco Embrionárias Murinas , Mamíferos
2.
Nat Genet ; 54(11): 1702-1710, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36333500

RESUMO

Genomic imprinting is regulated by parental-specific DNA methylation of imprinting control regions (ICRs). Despite an identical DNA sequence, ICRs can exist in two distinct epigenetic states that are memorized throughout unlimited cell divisions and reset during germline formation. Here, we systematically study the genetic and epigenetic determinants of this epigenetic bistability. By iterative integration of ICRs and related DNA sequences to an ectopic location in the mouse genome, we first identify the DNA sequence features required for maintenance of epigenetic states in embryonic stem cells. The autonomous regulatory properties of ICRs further enabled us to create DNA-methylation-sensitive reporters and to screen for key components involved in regulating their epigenetic memory. Besides DNMT1, UHRF1 and ZFP57, we identify factors that prevent switching from methylated to unmethylated states and show that two of these candidates, ATF7IP and ZMYM2, are important for the stability of DNA and H3K9 methylation at ICRs in embryonic stem cells.


Assuntos
Metilação de DNA , Impressão Genômica , Camundongos , Animais , Sequência de Bases , Metilação de DNA/genética , Epigenômica , Cromatina/genética , Proteínas Repressoras/genética
3.
J Cell Sci ; 135(19)2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-36052643

RESUMO

Cell size varies between cell types but is tightly regulated by cell intrinsic and extrinsic mechanisms. Cell size control is important for cell function, and changes in cell size are frequently observed in cancer. Here, we uncover a role for SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription, RNA processing and DNA repair. At the molecular level, SETD2 is best known for associating with RNA polymerase II through its Set2-Rbp1 interacting (SRI) domain and methylating histone H3 on lysine 36 (H3K36) during transcription. Using multiple independent perturbation strategies, we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that overexpression of the H3K36 demethylase KDM4A or the oncohistone H3.3K36M also increase cell size. In addition, ectopic overexpression of a decoy SRI domain increased cell size, suggesting that the relevant substrate is engaged by SETD2 via its SRI domain. These data add a central role of SETD2 in regulating cellular physiology and warrant further studies on separating the different functions of SETD2 in cancer development.


Assuntos
Histonas , Neoplasias , Tamanho Celular , Histona Metiltransferases/metabolismo , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina , Neoplasias/metabolismo , RNA Polimerase II/metabolismo , Proteínas Supressoras de Tumor/metabolismo
4.
Hum Mol Genet ; 31(3): 386-398, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-34494094

RESUMO

Expanded CAG/CTG repeat disorders affect over 1 in 2500 individuals worldwide. Potential therapeutic avenues include gene silencing and modulation of repeat instability. However, there are major mechanistic gaps in our understanding of these processes, which prevent the rational design of an efficient treatment. To address this, we developed a novel system, ParB/ANCHOR-mediated Inducible Targeting (PInT), in which any protein can be recruited at will to a GFP reporter containing an expanded CAG/CTG repeat. Previous studies have implicated the histone deacetylase HDAC5 and the DNA methyltransferase DNMT1 as modulators of repeat instability via mechanisms that are not fully understood. Using PInT, we found no evidence that HDAC5 or DNMT1 modulate repeat instability upon targeting to the expanded repeat, suggesting that their effect is independent of local chromatin structure. Unexpectedly, we found that expanded CAG/CTG repeats reduce the effectiveness of gene silencing mediated by targeting HDAC5 and DNMT1. The repeat-length effect in gene silencing by HDAC5 was abolished by a small molecule inhibitor of HDAC3. Our results have important implications on the design of epigenome editing approaches for expanded CAG/CTG repeat disorders. PInT is a versatile synthetic system to study the effect of any sequence of interest on epigenome editing.


Assuntos
Epigenoma , Expansão das Repetições de Trinucleotídeos , Inativação Gênica , Humanos , Repetições de Trinucleotídeos
5.
Nucleic Acids Res ; 49(1): 145-157, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33290556

RESUMO

Mammalian de novo DNA methyltransferases (DNMT) are responsible for the establishment of cell-type-specific DNA methylation in healthy and diseased tissues. Through genome-wide analysis of de novo methylation activity in murine stem cells we uncover that DNMT3A prefers to methylate CpGs followed by cytosines or thymines, while DNMT3B predominantly methylates CpGs followed by guanines or adenines. These signatures are further observed at non-CpG sites, resembling methylation context observed in specialised cell types, including neurons and oocytes. We further show that these preferences result from structural differences in the catalytic domains of the two de novo DNMTs and are not a consequence of differential recruitment to the genome. Molecular dynamics simulations suggest that, in case of human DNMT3A, the preference is due to favourable polar interactions between the flexible Arg836 side chain and the guanine that base-pairs with the cytosine following the CpG. By exchanging arginine to a lysine, the corresponding side chain in DNMT3B, the sequence preference is reversed, confirming the requirement for arginine at this position. This context-dependent enzymatic activity provides additional insights into the complex regulation of DNA methylation patterns.


Assuntos
Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Camundongos/genética , Substituição de Aminoácidos , Animais , Arginina/química , Sequência de Bases , Cristalografia por Raios X , Citosina/química , DNA Metiltransferase 3A , Conjuntos de Dados como Assunto , Células-Tronco Embrionárias/metabolismo , Técnicas de Inativação de Genes , Guanina/química , Humanos , Lisina/química , Simulação de Dinâmica Molecular , Especificidade por Substrato , Sulfitos , Sequenciamento Completo do Genoma , DNA Metiltransferase 3B
6.
Curr Opin Cell Biol ; 70: 10-17, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33276273

RESUMO

Nucleosomes, the basic structures used to package genetic information into chromatin, are subject to a diverse array of chemical modifications. A large number of these marks serve as interaction hubs for many nuclear proteins and provide critical structural features for protein recruitment. Dynamic deposition and removal of chromatin modifications by regulatory proteins ensure their correct deposition to the genome, which is essential for DNA replication, transcription, chromatin compaction, or DNA damage repair. The spatiotemporal regulation and maintenance of chromatin marks relies on coordinated activities of writer, eraser, and reader enzymes and often depends on complex multicomponent regulatory circuits. In recent years, the field has made enormous advances in uncovering the mechanisms that regulate chromatin modifications. Here, we discuss well-established and emerging concepts in chromatin biology ranging from cooperativity and multivalent interactions to regulatory feedback loops and increased local concentration of chromatin-modifying enzymes.


Assuntos
Cromatina , Reparo do DNA , Epigênese Genética , Replicação do DNA , Nucleossomos , Processamento de Proteína Pós-Traducional
7.
Swiss Med Wkly ; 150: w20329, 2020 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-32920789

RESUMO

DNA methylation is an epigenetic modification that plays a central regulatory role in various biological processes. Methyl groups are coupled to cytosines by the family of DNA methyltransferases (DNMTs), where DNMT1 is the main maintenance enzyme and the DNMT3 branch of the family is mostly responsible for de novo methylation. The regulation and function of DNA methylation are dependent on the genomic and chromatin context, such as binding sites for transcription factors or the presence of histone marks. Yet how local context, especially chromatin marks, influences the recruitment of the different DNMTs to their genomic target sites remains to be completely revealed. Elucidating the crosstalk between different histone modifications and DNA methylation, and their combined effect on the genome-wide epigenetic landscape, is of particular interest. Aberrant distribution of chromatin marks that guide DNMT activity or DNMT mutations that influence their correct recruitment to the genome have a profound impact on the deposition of DNA methylation, with consequences for genome function and gene activity. In this review, we describe the current state of knowledge on this topic and provide an overview on how chromatin marks can guide DNMT recruitment in healthy and diseased cells.


Assuntos
Cromatina , Metilação de DNA , Cromatina/genética , DNA , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Epigênese Genética
8.
Sci Adv ; 6(27): eaaz4012, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32656337

RESUMO

Expanded CAG/CTG repeats underlie 13 neurological disorders, including myotonic dystrophy type 1 (DM1) and Huntington's disease (HD). Upon expansion, disease loci acquire heterochromatic characteristics, which may provoke changes to chromatin conformation and thereby affect both gene expression and repeat instability. Here, we tested this hypothesis by performing 4C sequencing at the DMPK and HTT loci from DM1 and HD-derived cells. We find that allele sizes ranging from 15 to 1700 repeats displayed similar chromatin interaction profiles. This was true for both loci and for alleles with different DNA methylation levels and CTCF binding. Moreover, the ectopic insertion of an expanded CAG repeat tract did not change the conformation of the surrounding chromatin. We conclude that CAG/CTG repeat expansions are not enough to alter chromatin conformation in cis. Therefore, it is unlikely that changes in chromatin interactions drive repeat instability or changes in gene expression in these disorders.

10.
Cell Stem Cell ; 26(3): 431-440.e8, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142663

RESUMO

Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-ß-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-ß resistance and attenuate TGF-ß target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Organoides , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Testes Genéticos , Humanos , Intestinos , Fator de Crescimento Transformador beta
11.
Nat Biotechnol ; 38(6): 728-736, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32123383

RESUMO

Chromatin modifications regulate genome function by recruiting proteins to the genome. However, the protein composition at distinct chromatin modifications has yet to be fully characterized. In this study, we used natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for DNA methylation and histone tri-methylation at H3K4, H3K9 and H3K27 residues. We first demonstrated their utility as selective chromatin binders in living cells by stably expressing eCRs in mouse embryonic stem cells and measuring their subnuclear localization, genomic distribution and histone-modification-binding preference. By fusing eCRs to the biotin ligase BASU, we established ChromID, a method for identifying the chromatin-dependent protein interactome on the basis of proximity biotinylation, and applied it to distinct chromatin modifications in mouse stem cells. Using a synthetic dual-modification reader, we also uncovered the protein composition at bivalently modified promoters marked by H3K4me3 and H3K27me3. These results highlight the ability of ChromID to obtain a detailed view of protein interaction networks on chromatin.


Assuntos
Cromatina , Histonas , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/genética , Proteômica/métodos , Animais , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA/genética , Células-Tronco Embrionárias , Histonas/química , Histonas/genética , Histonas/metabolismo , Camundongos
12.
J Mol Biol ; 432(6): 1599-1601, 2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-32089270
13.
EMBO J ; 39(23): e105606, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33433018

RESUMO

Chromosomes have an intrinsic tendency to segregate into compartments, forming long-distance contacts between loci of similar chromatin states. How genome compartmentalization is regulated remains elusive. Here, comparison of mouse ground-state embryonic stem cells (ESCs) characterized by open and active chromatin, and advanced serum ESCs with a more closed and repressed genome, reveals distinct regulation of their genome organization due to differential dependency on BAZ2A/TIP5, a component of the chromatin remodeling complex NoRC. On ESC chromatin, BAZ2A interacts with SNF2H, DNA topoisomerase 2A (TOP2A) and cohesin. BAZ2A associates with chromatin sub-domains within the active A compartment, which intersect through long-range contacts. We found that ground-state chromatin selectively requires BAZ2A to limit the invasion of active domains into repressive compartments. BAZ2A depletion increases chromatin accessibility at B compartments. Furthermore, BAZ2A regulates H3K27me3 genome occupancy in a TOP2A-dependent manner. Finally, ground-state ESCs require BAZ2A for growth, differentiation, and correct expression of developmental genes. Our results uncover the propensity of open chromatin domains to invade repressive domains, which is counteracted by chromatin remodeling to establish genome partitioning and preserve cell identity.


Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Genoma , Células-Tronco Pluripotentes/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ciclo Celular , Diferenciação Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA Topoisomerases Tipo II/metabolismo , Epigenômica , Regulação da Expressão Gênica , Histonas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Coesinas
14.
Nat Commun ; 10(1): 4986, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676777

RESUMO

Chemotherapy-resistant cancer recurrence is a major cause of mortality. In acute myeloid leukemia (AML), chemorefractory relapses result from the complex interplay between altered genetic, epigenetic and transcriptional states in leukemic cells. Here, we develop an experimental model system using in vitro lineage tracing coupled with exome, transcriptome and in vivo functional readouts to assess the AML population dynamics and associated molecular determinants underpinning chemoresistance development. We find that combining standard chemotherapeutic regimens with low doses of DNA methyltransferase inhibitors (DNMTi, hypomethylating drugs) prevents chemoresistant relapses. Mechanistically, DNMTi suppresses the outgrowth of a pre-determined set of chemoresistant AML clones with stemness properties, instead favoring the expansion of rarer and unfit chemosensitive clones. Importantly, we confirm the capacity of DNMTi combination to suppress stemness-dependent chemoresistance development in xenotransplantation models and primary AML patient samples. Together, these results support the potential of DNMTi combination treatment to circumvent the development of chemorefractory AML relapses.


Assuntos
Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide/genética , Transcriptoma/genética , Doença Aguda , Antibióticos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Linhagem da Célula/genética , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Decitabina/uso terapêutico , Doxorrubicina/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/patologia
16.
Genome Res ; 29(4): 554-563, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30709850

RESUMO

Most mammalian RNA polymerase II initiation events occur at CpG islands, which are rich in CpGs and devoid of DNA methylation. Despite their relevance for gene regulation, it is unknown to what extent the CpG dinucleotide itself actually contributes to promoter activity. To address this question, we determined the transcriptional activity of a large number of chromosomally integrated promoter constructs and monitored binding of transcription factors assumed to play a role in CpG island activity. This revealed that CpG density significantly improves motif-based prediction of transcription factor binding. Our experiments also show that high CpG density alone is insufficient for transcriptional activity, yet results in increased transcriptional output when combined with particular transcription factor motifs. However, this CpG contribution to promoter activity is independent of DNA methyltransferase activity. Together, this refines our understanding of mammalian promoter regulation as it shows that high CpG density within CpG islands directly contributes to an environment permissive for full transcriptional activity.


Assuntos
Ilhas de CpG , Metilação de DNA , Regiões Promotoras Genéticas , Ativação Transcricional , Animais , Linhagem Celular , Células Cultivadas , Camundongos , Ligação Proteica , Fatores de Transcrição/metabolismo
17.
Mol Cell ; 73(4): 670-683.e12, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30639241

RESUMO

Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability.


Assuntos
Dano ao DNA , Replicação do DNA , Instabilidade Genômica , Neoplasias/genética , Clivagem do RNA , Precursores de RNA/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Transporte Ativo do Núcleo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Poliadenilação , Precursores de RNA/biossíntese , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas de Ligação a RNA
18.
Methods Mol Biol ; 1766: 157-174, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29605852

RESUMO

Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is currently the method of choice to determine binding sites of chromatin-associated factors in a genome-wide manner. Here, we describe a method to investigate the binding preferences of mammalian DNA methyltransferases (DNMT) based on ChIP-seq using biotin-tagging. Stringent ChIP of DNMT proteins based on the strong interaction between biotin and avidin circumvents limitations arising from low antibody specificity and ensures reproducible enrichment. DNMT-bound DNA fragments are ligated to sequencing adaptors, amplified and sequenced on a high-throughput sequencing instrument. Bioinformatic analysis gives valuable information about the binding preferences of DNMTs genome-wide and around promoter regions. This method is unconventional due to the use of genetically engineered cells; however, it allows specific and reliable determination of DNMT binding.


Assuntos
Metilação de DNA , Metilases de Modificação do DNA/genética , DNA/genética , Estudo de Associação Genômica Ampla , Análise Serial de Proteínas , Animais , Avidina/química , Sítios de Ligação , Biotina/química , Cromatina/química , Cromatina/genética , DNA/química , Metilases de Modificação do DNA/química , Humanos , Regiões Promotoras Genéticas , Software
19.
PLoS Genet ; 13(12): e1007102, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29267285

RESUMO

Genomic location can inform on potential function and recruitment signals for chromatin-associated proteins. High mobility group (Hmg) proteins are of similar size as histones with Hmga1 and Hmga2 being particularly abundant in replicating normal tissues and in cancerous cells. While several roles for Hmga proteins have been proposed we lack a comprehensive description of their genomic location as a function of chromatin, DNA sequence and functional domains. Here we report such a characterization in mouse embryonic stem cells in which we introduce biotin-tagged constructs of wild-type and DNA-binding domain mutants. Comparative analysis of the genome-wide distribution of Hmga proteins reveals pervasive binding, a feature that critically depends on a functional DNA-binding domain and which is shared by both Hmga proteins. Assessment of the underlying queues instructive for this binding modality identifies AT richness, defined as high frequency of A or T bases, as the major criterion for local binding. Additionally, we show that other chromatin states such as those linked to cis-regulatory regions have little impact on Hmga binding both in stem and differentiated cells. As a consequence, Hmga proteins are preferentially found at AT-rich regions such as constitutively heterochromatic regions but are absent from enhancers and promoters arguing for a limited role in regulating individual genes. In line with this model, we show that genetic deletion of Hmga proteins in stem cells causes limited transcriptional effects and that binding is conserved in neuronal progenitors. Overall our comparative study describing the in vivo binding modality of Hmga1 and Hmga2 identifies the proteins' preference for AT-rich DNA genome-wide and argues against a suggested function of Hmga at regulatory regions. Instead we discover pervasive binding with enrichment at regions of higher AT content irrespective of local variation in chromatin modifications.


Assuntos
Sequência Rica em At , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Animais , Composição de Bases , Sequência de Bases , Cromatina/genética , Cromatina/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Histonas/genética , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico
20.
EMBO J ; 36(23): 3421-3434, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29074627

RESUMO

DNA methylation is a prevalent epigenetic modification involved in transcriptional regulation and essential for mammalian development. While the genome-wide distribution of this mark has been studied to great detail, the mechanisms responsible for its correct deposition, as well as the cause for its aberrant localization in cancers, have not been fully elucidated. Here, we have compared the activity of individual DNMT3A isoforms in mouse embryonic stem and neuronal progenitor cells and report that these isoforms differ in their genomic binding and DNA methylation activity at regulatory sites. We identify that the longer isoform DNMT3A1 preferentially localizes to the methylated shores of bivalent CpG island promoters in a tissue-specific manner. The isoform-specific targeting of DNMT3A1 coincides with elevated hydroxymethylcytosine (5-hmC) deposition, suggesting an involvement of this isoform in mediating turnover of DNA methylation at these sites. Through genetic deletion and rescue experiments, we demonstrate that this isoform-specific recruitment plays a role in de novo DNA methylation at CpG island shores, with potential implications on H3K27me3-mediated regulation of developmental genes.


Assuntos
Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Animais , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo
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