Characterizing the functions of a putative DNA N6-adenine methylation demethylase ATALKBH4 in plants - the long-term goal of this research is to understand the function and mechanism of DNA N6-adenine…

Saint Louis University received a $385,000 Project Grant award from the National Institute of General Medical Sciences (NIGMS) under the Biomedical Research and Research Training program (CFDA 93.859), effective September 1, 2025, through August 31, 2028. The award supports fundamental research to characterize the functions of ATALKBH4, a putative DNA N6-adenine methylation (6MA) demethylase in plants. The research aims to advance understanding of how 6MA and its demethylases regulate gene expression in eukaryotes, addressing a significant gap in epigenetic research given the relative scarcity of existing knowledge regarding 6MA demethylases in eukaryotic systems.

The deliverables include quantitative measurements of DNA 6MA levels in ATALKBH4 mutant and overexpression transgenic plants using ultra-high-performance liquid chromatography-triple-quadrupole mass spectrometry (UHPLC-QQQ-MS/MS), as well as genomic mapping of 6MA locations using single molecule, real-time (SMRT) sequencing technology. Additionally, the research will analyze enzymatic activities of ATALKBH4 through both in vivo studies utilizing mutated constructs and in vitro biochemical characterization of the purified ATALKBH4 protein. These research activities will establish foundational knowledge regarding 6MA-mediated gene regulation mechanisms with implications for broader epigenetic understanding in plants and mammals.

Characterizing the functions of a putative DNA N6-adenine methylation demethylase ATALKBH4 in plants - the long-term goal of this research is to understand the function and mechanism of DNA N6-adenine methylation (6MA) and its demethylases in gene regulation in eukaryotes. Epigenetics has attracted many research efforts in the last two decades due to its importance in regulating many processes in cells, as well as the growth and development of plants and mammals. DNA methylation on the fifth position of cytosine (5MC) is an abundant and essential epigenetic mark in both plants and mammals. However, DNA 6MA is relatively rare in eukaryotes, and the existence and functions of 6MA in eukaryotes remain controversial. With recent progress in next-generation sequencing technology and mass spectrometry, IT has been shown that 6MA plays a critical role in regulating gene expression in eukaryotes. Furthermore, little is currently known about 6MA demethylases in eukaryotes, which remove the methyl group from 6MA residues. We have found an e. Coli alkylation b homolog 4 (ALKBH4) gene in arabidopsis (ATALKBH4) that can serve as a 6MA demethylase in vivo. Here, i propose to 1) measure DNA 6MA levels in the ATALKBH4 mutant and ATALKBH4 overexpression transgenic plants, as well as map the genomic locations of DNA 6MA in plants. I propose to use an ultra-high-performance liquid chromatography-triple-quadrupole mass spectrometer- mass spectrometer (uhplc-QQQ-MS/MS) to measure 6MA levels in the ATALKBH4 mutant and ATALKBH4 overexpression transgenic plants. We will also use single molecule, real-time (smrt) sequencing technology to determine the genomic locations of 6MA in arabidopsis. 2) analyze the enzymatic activities of the putative 6MA demethylase ATALKBH4 in vivo and in vitro. We will mutate the conserved residues of ATALKBH4 and transform the mutated constructs into the ATALKBH4 mutant plant to examine ATALKBH4 activities in vivo. We will also express and purify ATALKBH4 proteins and incubate them with putative DNA substrates to determine ATALKBH4 demethylase activities in vitro. 3) determine the function of the ATALKBH4 gene in arabidopsis. We will examine vegetative and reproductive phenotypes of the ATALKBH4 mutant and ATALKBH4 overexpression transgenic plants under normal and stress conditions in comparison to the wild-type plants. This study is significant because IT responds to recent calls for the reassessment of the existence of 6MA in eukaryotes, and will determine the function and enzyme activities of a putative 6MA demethylase in plants. This research is innovative because IT employs novel approaches to overcome previous limitations in addressing 6MA existence and functionality in eukaryotes. These approaches include using plants grown on sterile mediums without soil bacterial contamination, uhplc-QQQ-MS/MS, and smrt sequencing technology. To achieve the above specific aims, this research will employ genetic, molecular, biochemical, and genomic approaches, revealing the enzymatic activities and functions of the DNA 6MA demethylase ATALKBH4 in plants.