Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity
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ABSTRACT:
PROVIDER: PRJNA1206753 | ENA |
REPOSITORIES: ENA
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Project description:Here we show a novel role for arsenic in regulating the abundance and function of the demethylase activity of ALKBH1 in arsenic-induced skin cancer. We demonstrate that ALKBH1 is upregulated in response to acute and chronic arsenic exposure. In chronic arsenic-exposed keratinocytes, ALKBH1 promotes tumorigenicity, as knockdown of ALKBH1 leads to decreased tumorigenicity. ALKBH1 as a demethylase has several substrates and knockdown of ALKBH1 in arsenic-induced keratinocytes lead to increased m6A in mRNA. We identify that ALKBH1 demethylates m6A in vitro in an arsenic-dependent manner. Arsenic binds to the ALKBH1 protein via two cysteine residues to regulate the m6A RNA demethylase activity of ALKBH1. m6A sequencing identifies that ALKBH1 regulates m6A demethylation on the NR2C2 transcript to modulate NR2C2 protein translation in a YTHDF1-dependent manner. Knockdown of ALKBH1 leads to increased NR2C2 expression, which, in turn, leads to decreased mTOR signaling and global translation. Our findings demonstrate a novel role for arsenic in regulating ALKBH1 abundance and activity and add new mechanistic insights into arsenic-induced skin tumorigenicity.
2025-08-15 | GSE285900 | GEO
Project description:AlkB homolog 1 (ALKBH1) has been reported to act as a DNA 6mA demethylase to remove 6mA modificatione in ukaryotic genomes. Our previous data showed downregalted ALKBH1 in mouse and human fatty liver and increased DNA 6mA levels in mouse fatty liver. We hypothesize that ALKBH1 may be involved in hepatic lipid metabolism. To test this hypothesis, we generated liver-specific ALKBH1 knockout (Alkbh1 f/f, Albumine-cre; AKO) mice and used high-throughput RNA sequence and other assays to directly study the role of ALKBH1 during the development of hepatic steatosis.
2022-10-26 | GSE193497 | GEO
Project description:Cytochrome P450s (CYPs) are a family of enzymes responsible for metabolizing nearly 80% of small molecule drugs. Variants in CYPs can substantially alter drug metabolism, which may result in improper dosing and severe adverse drug reactions. CYPs have low sequence conservation, making it difficult to anticipate whether variant effects measured in one CYP may extend to others based on sequence alone. Even closely related CYPs, like CYP2C9 and its closest homolog CYP2C19, have distinct phenotypic properties despite sharing 92% of their sequences. Thus, we used Variant Abundance by Massively Parallel sequencing (VAMP-seq) to measure the protein abundance of 7,660 missense variants in CYP2C19 expressed in cultured human cells. Our results confirmed positions and structural features critical for CYP function and revealed how variants at positions conserved across all eukaryotic CYPs influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core, and that substitutions in some regions reduced abundance in CYP2C19 but not CYP2C9. We also measured the abundance of all single and some multiple WT amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 (SRS4) reduced abundance in CYP2C19. When nearby amino acids were exchanged in double and triple mutants, we found distinct interactions between the sites in CYP2C19 and CYP2C9, revealing a region that is partially responsible for the difference in thermodynamic stability between the two homologs. Since these positions are also important for determining substrate specificity, there may be an evolutionary tradeoff between stability and altered enzymatic function. Finally, we used our data to analyze 368 previously unannotated human variants, finding that 43% had decreased abundance. Thus, by comparing variant effects between two closely related, important human genes, we have uncovered regions underlying their functional differences and paved the way for a more complete understanding of one of the most versatile families of enzymes.
2024-10-02 | GSE244489 | GEO
Project description:DNA N6-methyladenosine (6mA) in eukaryotic genomes has recently been observed in diverse species. 6mA has been reported to associate with multiple physiological processes including embryonic development and tumorigenesis, and ALKBH1 is a primary 6mA demethylase in mouse and human cells. To research the roles of 6mA and its putative regulators such as putative ALKBH1 in the homeostatic maintenance of human stem cells and their differentiated derivatives, We generated ALKBH1-deficient human embryonic stem cells (hESCs) via CRISPR/Cas9-mediated non-homologous end joining (NHEJ). We next differentiated ALKBH1+/+ and ALKBH1-/- hESCs into human mesenchymal stem cells (hMSCs) and vascular smooth muscle cells (hVSMCs). Early-onset growth arrest of ALKBH1-/- hMSCs was observed, and increased apoptosis and enhanced migration ability were observed in ALKBH1-/- hVSMCs. However, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis revealed no change in 6mA levels between ALKBH1+/+ and ALKBH1-/- hESCs, between ALKBH1+/+ and ALKBH1-/- hMSCs, and between ALKBH1+/+ and ALKBH1-/- hVSMCs, respectively. These data demonstrate that depletion of ALKBH1 exerts minimal impact on 6mA levels in hESCs and their differentiated derivatives and that ALKBH1 regulates the homeostasis of hMSCs and hVSMCs possibly in a DNA 6mA-independent manner.
2020-05-22 | GSE150936 | GEO
Project description:AlkB homolog 1 (ALKBH1) has been reported to act as a DNA 6mA demethylase to remove 6mA modification in eukaryotic genomes. Our previous data showed downregulated ALKBH1 in mouse and human fatty liver and increased DNA 6mA levels in mouse fatty liver. We developed liver-specific ALKBH1 knockout (AKO) mice to directly study the role of ALKBH1 mediated DNA 6mA modification during the development of hepatic steatosis. Deletion of liver ALKBH1 promoted hepatic steatosis and insulin resistance. Overexpression of ALKBH1 resulted in opposite phenotypes. To investigate the mechanism of ALKBH1 in regulating target gene expression. We performed ChIP-seq for identifying ALKBH1 binding sites on mouse hepatocytes genomes and validated its demethylase activity for DNA 6mA modification on the target genes via ChIP-qPCR assays.
2022-10-26 | GSE197185 | GEO
Project description:DNA 6-adenine methylation (6mA), as a novel adenine modification existing in eukaryotes, shows essential functions in embryogenesis and mitochondrial transcriptions. ALKBH1 is a demethylase of 6mA and plays critical roles in osteogenesis, tumorigenesis, and adaptation to stress. However, the integrated biological functions of ALKBH1 still require further exploration. Here, we demonstrate that knockdown of ALKBH1 inhibits adipogenic differentiation in both human mesenchymal stem cells (hMSCs) and 3T3-L1 preadipocytes, while overexpression of ALKBH1 leads to increased adipogenesis. Using a combination of RNA-seq and N6-mA-DNA-IP-seq analyses, we identify hypoxia-inducible factor-1 (HIF-1) signaling as a crucial downstream target of ALKBH1 activity. Depletion of ALKBH1 leads to hypermethylation of both HIF-1α and its downstream target GYS1. Simultaneous overexpression of HIF-1α and GYS1 restores the adipogenic commitment of ALKBH1-deficient cells. Taken together, our data indicate that ALKBH1 is indispensable for adipogenic differentiation, revealing a novel epigenetic mechanism that regulates adipogenesis.
2021-12-12 | GSE177059 | GEO
Project description:The aim of the study was to elucidate the role of Alkbh1 by targeted deletion in C57/BL6 mice. Alkb deficiency results in sex-ratio distortion of offspring and apoptosis in adult testes, most likely caused by defects in the pachytene stage during spermatogenesis. Due to the pivotal role of Alkbh1 in mouse survival and potentially in germ cells, we searched for Alkbh1-regulated genes in adult testes. Testes from 12 week old mice were selected for RNA extraction and hybridization on Affymetrix microarrays. We used testes from 3 wild type mice and 3 KO mice
2010-06-08 | E-GEOD-22073 | biostudies-arrayexpress
Project description:The aim of the study was to elucidate the role of Alkbh1 by targeted deletion in C57/BL6 mice. Alkb deficiency results in sex-ratio distortion of offspring and apoptosis in adult testes, most likely caused by defects in the pachytene stage during spermatogenesis. Due to the pivotal role of Alkbh1 in mouse survival and potentially in germ cells, we searched for Alkbh1-regulated genes in adult testes.
2010-06-02 | GSE22073 | GEO
Project description:AlkB homolog 1 (ALKBH1) is one of nine members of the AlkB homologs in mammals. Most Alkbh1-deficient mice die during embryonic development, and survivors have severe defects in tissues originating from the ectodermal lineage. We hypothesized the phenotype to rely upon aberrant epigenetic regulation and provided evidence for ALKBH1 to be a histone H2A demethylase. We used a whole genome expression microarray to detail differentially expressed genes in embryonic stem cells lacking the Alkbh1 gene and identified distinct classes of up- and down-regulated genes during this process.
2011-11-01 | GSE30561 | GEO
Project description:Deep mutational scanning of CYP2C19 reveals a substrate specificity-abundance tradeoff
| PRJNA1023263 | ENA