Functional Analysis of DNMT3A DNA Methyltransferase Mutations Reported in Patients with Acute Myeloid Leukemia

In mammals, DNA methylation is necessary for the maintenance of genomic stability, gene expression regulation, and other processes. During malignant diseases progression, changes in both DNA methylation patterns and DNA methyltransferase (MTase) genes are observed. Human de novo MTase DNMT3A is most frequently mutated in acute myeloid leukemia (AML) with a striking prevalence of R882H mutation, which has been extensively studied. Here, we investigate the functional role of the missense mutations (S714C, R635W, R736H, R771L, P777R, and F752V) found in the catalytic domain of DNMT3A in AML patients. These were accordingly mutated in the murine Dnmt3a catalytic domain (S124C, R45W, R146H, R181L, P187R, and F162V) and in addition, one-site CpG-containing DNA substrates were used as a model system. The 3-15-fold decrease (S124C and P187R) or complete loss (F162V, R45W, and R146H) of Dnmt3a-CD methylation activity was observed. Remarkably, Pro 187 and Arg 146 are not located at or near the Dnmt3a functional motives. Regulatory protein Dnmt3L did not enhance the methylation activity of R45W, R146H, P187R, and F162V mutants. The key steps of the Dnmt3a-mediated methylation mechanism, including DNA binding and transient covalent intermediate formation, were examined. There was a complete loss of DNA-binding affinity for R45W located in the AdoMet binding region and for R146H. Dnmt3a mutants studied in vitro suggest functional impairment of DNMT3A during pathogenesis

Une âme de jeune fille [ : Gabrielle***] / Jean Vaudon,...

<p>A. Distribution of CpG-motifs within rDNA (transcribed region of human ribosomal repeat). The digits indicate the nucleotide order number, the vertical bar shows the motif location. Red color is used to mark region A and region B, that were analyzed for the presence of methylated CCGG sites. B. Determination of methylation index of three genes in DNA from cells treated with 20 μM DBP(1–4), 72 h (description is given in Methods).</p

DNA methylation in nuclei of the control MCF-7 cells.

<p>A. Cells were processed for immunofluorescence staining with anti 5-mC antibody. (1)—Magnification X60; (2)—For analysis of the nuclei the photo was enlarged by an order using computer processing. The arrow points to the methylated DNA block close to the nucleolus. B. (1)—DNA methylation in nuclei of the control MCF-7 cells and the cells treated with 10 μM AzaC or 20μM DBP(1–4), 72 h. Background: Control cells were only treated with secondary antibodies conjugated with FITC. (2) The photo was enlarged considerably for the analysis of nuclei size. The nucleus of the cell cultivated with DBP(4) is shown for an example. C. An example of the different DNA methylation level in cells’ nuclei that were cultivated in the presence of 20μM DBP(4), 72 h. A,B(1) and C—Cell nuclei were additionally colored with DAPI.</p

The influence of DBP(1–4) on the MCF-7mitochondria.

<p>A. (1)—control cells (stained with MitoTracker TMRM) in visible light (VIS), in λex = 520 nm (TMRM) and in λex = 380 nm (background). (2)—control cells stained with MitoTracker TMRM and Hoechst 33258 (10μM, 0.5 h). Specially selected field shows single cells with a weak mitochondria staining. Particularly these cells accumulate Hoechst 33258 in the nuclei. B. MCF-7 cells treated with 20μM DBP(2) for 0.5–48 h after incubation with MitoTracker TMRM (15 min). For 0.5 h time point we specially selected a field that shows cells with a weak mitochondria staining. DBP(2) quickly penetrates into these cells, similar to Hoechst 33258. Magnification X 60. The cell in a yellow square is enlarged several times for demonstration.</p

A (FL-reader).

<p>(1)–Example of reaction rate constant determination for DCF formation when DCFH reacts with ROS. Cells were cultivated for 24 hours in the presence of 40 μM DBP(1). The cultivation environment was replaced by 5 μm H2DCFH-DA in PBS solution and a relative fluorescence intensity increase was detected. I_t,I0 –sample’s signal at time t and immediately after H2DCFH-DA addition respectively. The slope of the line– <a href="http://universal_ru_en.academic.ru/378785/reaction_rate_constant" target="_blank">reaction rate constant</a> for DCF formation (k). ROS index Δk/k₀ = (k_DBP−k₀)×100/k₀ (%). (2)—Relationship between ROS index Δk/k₀ and DBP(1–4) concentration. Time of cultivation is shown on the figure. B (FCA). Proliferation of MCF-7 cells exposed to DBP(1–4) at final concentration 20 μM for 24 hours. (1)—Distribution of fluorescence of fixed cells stained with anti-Ki-67 antibodies (control—dark green color, DBP(2) as example—blue color). Background fluorescence was quantified using FITC-conjugated secondary antibodies (green color). (2)—The median signal intensity of FL1 (Ki-67+) (gate R). Each experiment was repeated at least three times. Results represent the average of the medians of the FL parameter in independent experiments. C (FCA). (1)—Distribution of fluorescence of fixed cells stained with propidium iodide. The fractions of (1–4) cells with different DNA amounts are shown. (2)–Proportion MCF-7 culture cells with DNA amount corresponding to the G1-, S—and G2/M phases of the cell cycle. The data shown is an average value from three independent experiments. (3) The total number of cells in culture. (4) The number hypodiploid cells (fraction SubG0/G1).D. The proportion of cells in culture with signs of apoptotic nuclei (condensed chromatin, the irregular nucleus shape, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189826#pone.0189826.g004" target="_blank">Fig 4</a>). B, C, D–cells were cultivated in the presence of 20 μM DBP(1–4) for 24 hours.</p

Overall 5-methylcytosine level in DNA of cells treated with 20 μM DBP(1–4) for 72 hours.

<p>A (ELISA). (1, 2)–example of calibration dependence between methylated DNA in samples and integral stain density (I). (3) example of DNA samples analysis from cells treated with DBP(1–4). 10 ng of DNA was applied. pBR322 plasmid was used as a negative control. (4) Relative signal change reflecting difference in DNA methylation level. B (<i>UPLC/MS/MS</i>). Average 5mC content in DNA samples.</p

Incorporation of DBP(1–4) in MCF-7 cells.

<p>Excitation of fluorescence at 380 nm. Magnification X 20. MCF-7 were incubated with DBP(1–4) (7, 10 or 20 μM) for 24 h and the fluorescence was analyzed in unfixed cells. Staining of the cells was heterogeneous and practically did not depend on the concentrations of DBP(1–4). The 20 μM concentrations of DBP(1–4) were used for an example. The photos of cells were taken with identical exposure. For example the most common nuclei staining types are shown with arrows and numbers: 1 –nucleus is stained only, 2 –nucleus and the cytoplasm are stained. 3- cells show strong blue fluorescence signal while nuclei do not contrast. Background: the photo of control cells in visible light (VIS) and at λex = 380 nm (FL), the exposure is increased two times.</p

DBP(n) (20μM, 72h) effect on RNA level in MCF-7.

<p>DBP(n) (20μM, 72h) effect on RNA level in MCF-7.</p