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Binding of 14-3-3 reader proteins to phosphorylated DNMT1 facilitates aberrant DNA methylation and gene expression

ABSTRACT: Mammalian DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for maintenance methylation. Phosphorylation of Ser143 (pSer143) stabilizes DNMT1 during DNA replication. Here, we show 14-3-3 is a reader protein of DNMT1pSer143. In mammalian cells 14-3-3 colocalizes and binds DNMT1pSer143 post-DNA replication. The level of DNMT1pSer143 increased with overexpression of 14-3-3 and decreased by its depletion. Binding of 14-3-3 proteins with DNMT1pSer143 resulted in inhibition of DNA methylation activity in vitro. In addition, overexpression of 14-3-3 in NIH3T3 cells led to decrease in DNMT1 specific activity and was rescued by transfection of DNMT1. Decrease in DNMT1 specific activity led to hypomethylation of the genome. Genes representing cell migration, mobility, proliferation and focal adhesion pathway were hypomethylated and overexpressed. Furthermore, 14-3-3 overexpression also resulted in enhanced cell invasion. Therefore, we suggest that 14-3-3 is a crucial reader of DNMT1pSer143 that regulates DNMT1 level, DNA methylation and altered gene expression that contributes to cell invasion.

ORGANISM(S): Mus musculus

PROVIDER: GSE72860 | GEO | 2015/09/10

SECONDARY ACCESSION(S): PRJNA295204

REPOSITORIES: GEO

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Binding of 14-3-3 reader proteins to phosphorylated DNMT1 facilitates aberrant DNA methylation and gene expression

Project description:Mammalian DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for maintenance methylation. Phosphorylation of Ser143 (pSer143) stabilizes DNMT1 during DNA replication. Here, we show 14-3-3 is a reader protein of DNMT1pSer143. In mammalian cells 14-3-3 colocalizes and binds DNMT1pSer143 post-DNA replication. The level of DNMT1pSer143 increased with overexpression of 14-3-3 and decreased by its depletion. Binding of 14-3-3 proteins with DNMT1pSer143 resulted in inhibition of DNA methylation activity in vitro. In addition, overexpression of 14-3-3 in NIH3T3 cells led to decrease in DNMT1 specific activity and was rescued by transfection of DNMT1. Decrease in DNMT1 specific activity led to hypomethylation of the genome. Genes representing cell migration, mobility, proliferation and focal adhesion pathway were hypomethylated and overexpressed. Furthermore, 14-3-3 overexpression also resulted in enhanced cell invasion. Therefore, we suggest that 14-3-3 is a crucial reader of DNMT1pSer143 that regulates DNMT1 level, DNA methylation and altered gene expression that contributes to cell invasion. Examine of the DNA methylation and RNA expression profiles of stable NIH3T3 clones overexressing the 14-3-3ε homologue

2015-09-10 | E-GEOD-72860 | biostudies-arrayexpress

S-adenosylhomocysteine hydrolase participates in DNA methylation inheritance

Project description:DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for mammalian development and maintenance of DNA methylation following DNA replication in cells. The DNA methylation process generates S-adenosyl-L-homocysteine, a strong inhibitor of DNMT1. Here we report that S-adenosylhomocysteine hydrolase (SAHH/AHCY), the only mammalian enzyme capable of hydrolyzing S-adenosyl-L-homocysteine binds to DNMT1 during DNA replication. SAHH activates DNMT1 in vitro and its overexpression in mammalian cells leads to hypermethylation of the genome, whereas its inhibition by adenosine periodate resulted in hypomethylation of the genome. Hypermethylation was consistent in both gene bodies and repetitive DNA elements leading to both down- and up-regulation of genes. Similarly, hypomethylation led to both up- and down-regulation of genes suggesting methylated regions influence gene expression either positively or negatively. Cells overexpressing SAHH specifically up-regulated metabolic pathway genes and down-regulated PPAR and MAPK signaling pathways genes. Therefore, we suggest that alteration of SAHH level in the cell leads to aberrant DNA methylation, altered metabolite levels and gene expression.

2018-06-06 | PXD006476 | Pride

Small RNA mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation

Project description:Mammalian cells contain copious amounts of RNA including both coding and non-coding RNA (ncRNA). Generally the ncRNAs function to regulate gene expression at the transcriptional and post-transcriptional level. Among ncRNA, the long ncRNA and small ncRNA can affect histone modification, DNA methylation targeting and gene silencing. Here we show that endogenous DNA methyltransferase 1 (DNMT1) co-purifies with inhibitory ncRNAs. MicroRNAs (miRNAs) binds directly to DNMT1 with high affinity. The binding of miRNAs, such as miR-155, leads to inhibition of DNMT1 enzyme activity. Exogenous miR-155 in cells induces aberrant DNA methylation of the genome, resulting in hypomethylation of low to moderately methylated regions. And small shift of hypermethylation of previously hypomethylated region was also observed. Furthermore, hypomethylation led to activation of genes. Based on these observations, we propose that overexpression of specific miRNAs in human cancer may lead to aberrant DNA methylation and altered gene-expression. Examine of the DNA methylation and mRNA profile of HCT 116 cells transfected by random 23-mer or miR-155 RNA

2015-04-21 | E-GEOD-68027 | biostudies-arrayexpress

Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

Project description:The DNA methyltransferase activity of DNMT1 is vital for genomic maintenance of DNA methylation. We report here that DNMT1 function is regulated by O-GlcNAcylation, a protein modification that is sensitive to glucose levels, and that elevated O-GlcNAcylation of DNMT1 from high glucose environment leads to alterations to the epigenome. Using mass spectrometry and complementary alanine mutation experiments, we identified S878 as the major residue that is O-GlcNAcylated on DNMT1. Functional studies further revealed that O-GlcNAcylation of DNMT1-S878 results in an inhibition of methyltransferase activity, resulting in a general loss of DNA methylation that is preferentially at partially methylated domains (PMDs). This loss of methylation corresponds with an increase in DNA damage and apoptosis. These results establish O-GlcNAcylation of DNMT1 as a mechanism through which the epigenome is regulated by glucose metabolism and implicates a role for glycosylation of DNMT1 in metabolic diseases characterized by hyperglycemia.

2023-07-26 | PXD043031 | Pride

Dnmt1 has de novo activity targeted to transposable elements

Project description:DNA methylation plays a critical role in development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation depleted embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that Dnmt1's de novo methylation activity depends on Uhrf1 and its genomic recruitment overlaps with targets that enrich for Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can de novo add and maintain DNA methylation, especially at retrotransposons and that this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.

2021-06-29 | PXD025736 | Pride

Small RNA mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation

2015-04-21 | GSE68027 | GEO

Neuronal activation modulates enhancer activity of genes for excitatory synaptogenesis through de novo DNA methylation

Project description:Post-mitotic neurons exhibit DNA methylation changes, contrary to the longstanding belief that the epigenetic pattern in terminally differentiated cells is essentially unchanging. While the mechanism and physiological significance of DNA demethylation in neurons have been extensively elucidated, occurrence of de novo DNA methylation and its impacts have been much less investigated. Here we show that neuronal activation induces global de novo DNA methylation at enhancer regions that can repress target genes in primary cultured hippocampal neurons. The functional significance of this de novo DNA methylation was underpinned by the demonstration that inhibition of DNA methyltransferase (DNMT) activity decreased neuronal activity-induced excitatory synaptogenesis. Overexpression of WW and C2 Domain containing 1 (Wwc1), a representative target gene of de novo DNA methylation, could phenocopy this DNMT inhibition-induced decrease in the synaptogenesis. We found that both DNMT1 and DNMT3a were required for the neuronal activity-induced de novo DNA methylation of Wwc1 enhancer. Taking these findings altogether, we concluded that activity-induced de novo DNA methylation affecting gene expression has impacts on neuronal physiology comparable to those of DNA demethylation.

2020-04-26 | GSE149342 | GEO

Identificaiton of novel acetylation sites in UHRF1 mediated by acetyltransferase MOF

Project description:The multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 for DNA methylation maintenance during DNA replication. Here, we show that MOF (Males absent On the First) is an acetyltransferase of UHRF1 to acetylate UHRF1 at Lys670 in the pre-RING linker region whereas HDAC1 is a deacetylase of UHRF1 at the same site. The MOF/HDAC1-mediated acetylation in UHRF1 is cell-cycle regulated and peaks at G1/S phase, in line with the function of UHRF1 in recruiting DNMT1 to maintain DNA methylation. In addition, UHRF1 acetylation significantly enhances its E3 ligase activity and elimination of UHRF1 acetylation at these sites attenuates UHRF1-mediated H3 ubiquitination, which in turn impairs the DNMT1 recruitment and DNA methylation. Taken together, these findings not only identify MOF as a new acetyltransferase for UHRF1 but also reveal a novel mechanism underlying the regulation of DNA methylation maintenance through MOF-mediated UHRF1 acetylation.

2024-03-06 | PXD040426 | Pride

DNMT1 IP-MS alalysis to identify ubiquitin-dependent interacting partners

Project description:Stable inheritance of DNA methylation is critical for maintaining the differentiated phenotypes in multicellular organisms. However, the molecular basis ensuring high fidelity of maintenance DNA methylation is largely unknown. Here, we demonstrate that two distinct modes of DNMT1 recruitment, one is DNA replication-coupled and the other is uncoupled mechanism, regulate the stable inheritance of DNA methylation. PCNA-associated factor 15 (PAF15) represents a primary target of UHRF1 and undergoes dual mono-ubiquitylation (PAF15Ub2) on chromatin. PAF15Ub2 specifically interacts with DNMT1 and controls the recruitment of DNMT1 in a DNA replication-coupled manner. Thus, loss of PAF15Ub2 results in impaired DNA methylation at sites replicating during early S phase. In contrast, outside of S phase or when PAF15 ubiquitylation is perturbed, UHRF1 ubiquitylates histone H3 to promote DNMT1 recruitment. Together, we identify replication-coupled and uncoupled mechanisms of maintenance DNA methylation, both of which collaboratively ensure the stable DNA methylation.

2020-02-25 | PXD015282 | Pride

Study of ALKBH4 negatively regulating the expression level of DNMT1 by conducting a quantitative proteomic experiment to reveal the differential expression of proteins in HEK293T cells upon genetic ablation of ALKBH4

Project description:ALKBH4 is a versatile demethylase that catalyzes the removal of methyl group from monomethylated lysine-84 on actin and N6-methyladenine in DNA. We conducted a quantitative proteomic experiment to reveal the differential expression of proteins in HEK293T cells upon genetic ablation of ALKBH4. Our results revealed markedly diminished levels of GSTP1 and HSPB1 proteins in ALKBH4-depleted cells, which emanate from an augmented expression level of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and the ensuing elevated cytosine methylation in the promoter regions of GSTP1 and HSPB1 genes in ALKBH4-deficient cells. Together, our results revealed a role of ALKBH4 in modulating DNA cytosine methylation through regulating the expression level of DNMT1 protein. Yeast two-hybrid screening identified several proteins that are associated with chromatin and/or involved with transcription, as interaction partners of human ALKBH4, though global gene expression was only marginally changed upon overexpression of ALKBH4. We reason that a comprehensive assessment of how genetic depletion of ALKBH4 modulates protein expression at the global proteome scale may provide new insights into the biological function of ALKBH4 protein. In the present study, we conducted such an analysis and we found that CRISPR-mediated ablation of ALKBH4 led to substantial changes in expression of a large number of proteins, including the markedly diminished expression of GSTP1 and HSPB1 proteins. Mechanistically, these changes arise from elevated expression of DNMT1 and the ensuing epigenetic silencing of these two genes.

2021-12-30 | PXD028148 | Pride

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