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 resulting in hypomethylation of the genome that was rescued by transfection of DNMT1. Genes representing cell migration, mobility, proliferation and focal adhesion pathway were hypomethylated and overexpressed. Furthermore, overexpression of 14-3-3 also resulted in enhanced cell invasion. Analysis of TCGA breast cancer patient data showed significant correlation for DNA hypomethylation and reduced patient survival with increased 14-3-3 expressions. Therefore, we suggest that 14-3-3 is a crucial reader of DNMT1pSer143 that regulates DNA methylation and altered gene expression that contributes to cell invasion.

Figure 1.

14-3-3 binds and colocalizes with DNMT1. (A) Binding of DNMT1pSer143 peptide to a phospho-Serine/Threonine protein domain microarray to identify potential readers. The array contains the 14-3-3, 14-3-3-like, WW/SRI, BIR, POLO Box, MH2, WD40, FHA and BRCT domains fused to GST. Binding of biotin-tagged DNMT1pSer143 peptide to the array revealed 14-3-3 homologues as binding partners. The biotinylated peptide binding was detected using streptavidin –Cy3. An image of the full microarray is shown in Supplementary Figure S1A. (B) Recombinant full length DNMT1 binds different GST-14-3-3 homologues. Ponceau staining of transferred proteins from the GST pull-down are shown along with western blot using anti-DNMT1. Densitometry measurements of different 14-3-3 homologues binding to DNMT1 are shown at the bottom of the western blot (arbitrary units). C) Co-immunoprecipitations (Co-IP) of DNMT1 and 14-3-3γ and ϵ. Molecular weight marker, anti-DNMT1, anti-14-3-3γ, and anti-IgG antibodies, are indicated at the top of the western blots (left panel). Anti-FLAG antibody was used for 14-3-3ϵ immunoprecipitation in EP12 stable cell line overexpressing FLAG-14-3-3ϵ (right panel). Antibodies used for protein detection, anti-14-3-3γ, ϵ and anti-DNMT1, are indicated at the right side of the western blots. D) Colocalization studies between DNMT1 and 14-3-3ϵ. Plasmids encoding DsRed-DNMT1 (red) and FLAG-14-3-3ϵ (green) were co-transfected in COS7 cells. Different cell cycle phases are indicated on right side. DNA staining was performed using DAPI (blue). Yellow spots (merge) represent the colocalization.

Figure 2.

14-3-3 protein bind and modulate phosphorylated DNMT1 levels. (A) Protein phosphatase 1 (PP1) impairs DNMT1 binding to GST-14-3-3γ. Presence (+) or absence (−) of PP1 with increasing amount of recombinant full-length DNMT1 (200, 400 or 600 ng) indicated on top of the western blots. Ponceau staining of transferred proteins from the GST pull-down are shown along with western blot using anti-DNMT1 or anti-DNMT1pSer143 antibody. (B) AKT1 kinase mediates DNMT1 binding to 14-3-3. GST-DNMT1 (1–446) phosphorylated (+, with ATP) or control (−, without ATP) with recombinant AKT1 kinase and incubated with different amounts of MBP-14-3-3γ recombinant protein is shown on top of the western blots. Antibodies against DNMT1, MBP and DNMT1pSer143 are indicated on the right side of the western blots. (C) 14-3-3 protein protect against DNMT1 dephosphorylation by PP1. Increasing amounts of recombinant full-length DNMT1 incubated with GST-14-3-3γ or control (GST) in presence of PP1 is shown at the top of the western blots. Ponceau staining of transferred proteins from the GST pull-down are shown along with western blot using anti-DNMT1 and anti-DNMT1pSer143. (D) Exogenous 14-3-3ϵ increases phosphorylation of DNMT1Ser143 by AKT1. Myc-MirAKT was transfected in COS7 cells in the presence (+) or absence (−) of 14-3-3ϵ as indicated on top. Antibodies used are indicated on the right side of the western blot. Densitometry analysis of DNMT1pSer143 was performed and shown as bar graphs below. (E) Depletion of 14-3-3ϵ by siRNA resulted in decrease DNMT1pSer143 protein. siRNA against GFP was used as the control. Antibodies used are indicated at the right side.

Figure 3.

14-3-3 proteins bind and inhibit phosphorylated DNMT1. (A) 14-3-3γ blocks activity of full-length DNMT1 and not DNMT1Δ580 in vitro. DNMT1 activity in the presence of GST (blue) or GST-14-3-3γ (red) is shown. (B) Western blot analysis of DNMT1–14-3-3 complexes after immunoprecipitation with anti-FLAG antibody during cell cycle. Synchronization timing after release is indicated on top. (C) Specific activity of DNMT1 and binding affinity of 14-3-3 and DNMT1 during cell cycle in 14-3-3 overexpression clone. Shaded region indicates the inverse correlation between increased 14-3-3-DNMT1 binding and DNMT1 methyltransferase activity.

Figure 4.

14-3-3 overexpression hypomethylates genome. (A) DNA methylase assay showing reduced DNMT1 specific activity in NIH3T3 stable clonal cell lines overexpressing 14-3-3ϵ. Activity was measured from two control clones (C2 and C3) and two overexpressing clones (EP10 and EP12) using hemimethylated DNA substrate. Asterisk indicates P value less than 0.01 by t-test. Lower panel are western blots showing expression level of DNMT1 in the stable clones. (B) Global methylation level of stable clones measured by LC/MS (left). Data shown are from seven control clones and six 14-3-3ϵ stable clones. Global methylation level of stable clones transfected with full-length DNMT1 (right). P value is obtained by t-test. (C) Volcano plot showing the overall differential methylation profile of 5-kb tiles in the genome determined by RRBS. Differentially methylated region with a q-value < 0.01 are considered significantly different. (D) Differential methylation profile of genes (−5 kb transcription start site (TSS) to +5 kb transcription termination (Ter)). (E) Differential methylation profile of general genomic repetitive elements. Data shown are median value of two clones at each relative position (D and E).

Figure 5.

14-3-3 overexpression affects gene expression. (A) MA plot of RNA-seq data showing upregulated (blue) and downregulated (red) genes. (B) Gene ontology analysis on upregulated genes.

Figure 6.

Correlation of promoter and gene body DNA hypomethylation with elevated gene expression. (A) Genome-wide correlation of DNA methylation and gene expression profile. Correlation analysis was carried out using Spearman's rank correlation test. A linear regression line was also drawn when plotting, and the shaded area represents the 95% confidential area of linear regression. Left, correlation of gene expression with promoter CpG methylation. Right, correlation of gene expression with gene body methylation. (B) CpG methylation (upper half) and gene expression (lower half) track of four representative genes displaying hypomethylation and elevated expression.

Figure 7.

14-3-3 overexpression promotes cell invasion. (A) Images of spheroids formed by control (3T3, C1, C2, C3 and C4) and 14-3-3ϵ overexpressing clones (EP1, EP3, EP10, EP11 and EP12) at 0 h of invasion (48 h post-seeding into spheroid formation matrix) are shown on the left and at 72 h of invasion on the right. (B) Quantitative presentation of increased surface areas of the spheroids at 0 and 72 hrs.

Figure 8.

14-3-3 overexpression is correlated with reduced patient survival time and repetitive elements hypomethylation in breast cancer. (A) Bar plot showing the frequency of 14-3-3 homologues alteration in major cancer types. (B) Kaplan–Meier curve showing the effect of 14-3-3 expression on the survival rates in breast cancer patients. (C) OncoPrint plot showing percentage of breast cancer patients with altered mRNA expression of 14-3-3 homologues. (D and E) Negative correlation of LINE and LTR methylation with 14-3-3ζ expression levels in breast cancer patients.