ALKBH1-regulated 6mA DNA methylation affects human lineage segregation via TGF-[beta]-signalling pathway

Abstract

DNA methylation is a critical epigenetic modification that regulates various biological processes, including cancer, neurodegeneration, and embryogenesis. While 5-methylcytosine (5mC) has been extensively studied in eukaryotes, the prevalence of 6-methyladenine (6mA) methylation in prokaryotes has attracted attention. Recent advancements in detection methods and high-throughput sequencing have enabled the identification of 6mA methylation signals in eukaryotes, leading to increased research into its role in mammalian cells.

In 2016, the presence of 6mA modification in mammalian stem cells was discovered, even in low abundance within mouse embryonic stem cells (mESCs). This finding highlighted the enrichment of 6mA signals in young LINE1 elements and emphasized the impact of the ALKBH1 demethylase on stemness maintenance and differentiation potential in mESCs. Subsequent studies aimed to unravel additional regulators of 6mA DNA methylation and elucidate its role in mammals.

During early development, 6mA exhibited dynamic patterns, with initial upregulation preceding the pre-implantation phase and subsequent downregulation after the blastocyst stage. This temporal pattern was conserved in zebrafish and pigs, suggesting the evolutionary importance of 6mA. Furthermore, genomic regions displaying 6mA signals often overlapped with histone H3, indicating intricate interactions with other epigenetic markers.

Depletion of ALKBH1 and subsequent accumulation of 6mA signals hindered the differentiation of mouse ESCs into trophoblast-like cells, providing an epigenetic explanation for impaired placental development. To investigate the regulatory mechanisms of 6mA in human stem cells, researchers utilized human expanded potential stem cells (hEPSCs) as an in vitro model for trophoblast differentiation. The study aimed to uncover the mechanisms and shed light on the role of 6mA in early human embryonic development.

The study disrupted ALKBH1's demethylase activity in hEPSCs using the CRISPR-Cas9 system, resulting in increased 6mA signals compared to untargeted cells. ALKBH1 knockout led to downregulation of SMAD2 and SMAD4 protein levels, reduced phosphorylation of the SMAD2/3 protein, and downregulation of STOX2 at the transcriptional level. These findings highlighted ALKBH1's role in modulating the TGF-β-signaling pathway through 6mA levels.

Changes in ALKBH1 expression significantly affected trophoblast and hematopoietic differentiation during cell fate determination. The absence of ALKBH1 hindered these processes, underscoring its importance in regulating developmental pathways. Treatment of ALKBH1 knockout cells with Activin A, an activator of the TGF-β-signaling pathway, partially rescued the observed phenotype, reinforcing the connection between ALKBH1, 6mA regulation, and the TGF-β-signaling pathway in shaping cell fate during early embryonic development. These discoveries provide insights into the molecular mechanisms of 6mA-mediated epigenetic regulation and its significance in coordinating cellular processes during early embryogenesis.