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

Abstract

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.

Impact of G-Quadruplex Structures on Methylation of Model Substrates by DNA Methyltransferase Dnmt3a

“…We selected the C-terminal catalytic domain, Dnmt3a-CD, which is known to interact with DNA [ 19 ], as well as the PWWP domain, which is a part of the N-terminal regulatory region of the enzyme [ 23 ] ( Figure 1 ). PWWP was recently reported to also interact with DNA, albeit with K d an order of magnitude weaker (3.5 µM [ 26 ]) as compared to Dnmt3a-CD ( K d 0.2 µM [ 35 , 36 ]). As part of our study, model recombinant proteins were obtained: full-length murine AIR-Dnmt3a2 with substitutions in the ADD domain to remove autoinhibition, as well as catalytic (Dnmt3a-CD) and regulatory (PWWP) domains.…”

Light‐Activation of DNA‐Methyltransferases

“…[17] Conflicting results have been reported with DNMT3a CD for different substrates in vitro,ranging from 4-fold increased to completely abolished activity. [17,18] Our light activation measurements revealed ar eduction in activity to ~60 %( Figure 2c). Another frequently mutated residue of the same interface is R771, being engaged in asalt bridge to DNMT3L (Figure 2b).…”

“…This mutant exhibited ∼50% 5mC signal compared to wt pcDNMT3a3L after light activation, being in a similar range as observed for the DNMT3a CD in vitro but different to observations in murine ES cells, where this mutant is inactive (Figure 2 c). [11, 17, 18] …”

Variants in the ASXL1 and DMNT3A genes are potential markers of the effectiveness of tyrosine kinase inhibitors therapy in chronic myeloid leukemia

“…Варианты в генах ASXL1 и DNMT3A часто выявляются при миелопролиферативных заболеваниях и ассоциированы с плохим прогнозом. Существует ряд работ, показывающих функциональную значимость вариантов, найденных в ASXL1 и DNMT3A [3,4].…”