Study of ALKBH4 negatively regulating the expression level of DNMT1 by conducting a quantitative proteomic experiment to reveal the differential expression of proteins in HEK293T cells upon genetic ablation of ALKBH4

ABSTRACT: ALKBH4 is a versatile demethylase that catalyzes the removal of methyl group from monomethylated lysine-84 on actin and N6-methyladenine in DNA. We conducted a quantitative proteomic experiment to reveal the differential expression of proteins in HEK293T cells upon genetic ablation of ALKBH4. Our results revealed markedly diminished levels of GSTP1 and HSPB1 proteins in ALKBH4-depleted cells, which emanate from an augmented expression level of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and the ensuing elevated cytosine methylation in the promoter regions of GSTP1 and HSPB1 genes in ALKBH4-deficient cells. Together, our results revealed a role of ALKBH4 in modulating DNA cytosine methylation through regulating the expression level of DNMT1 protein. Yeast two-hybrid screening identified several proteins that are associated with chromatin and/or involved with transcription, as interaction partners of human ALKBH4, though global gene expression was only marginally changed upon overexpression of ALKBH4. We reason that a comprehensive assessment of how genetic depletion of ALKBH4 modulates protein expression at the global proteome scale may provide new insights into the biological function of ALKBH4 protein. In the present study, we conducted such an analysis and we found that CRISPR-mediated ablation of ALKBH4 led to substantial changes in expression of a large number of proteins, including the markedly diminished expression of GSTP1 and HSPB1 proteins. Mechanistically, these changes arise from elevated expression of DNMT1 and the ensuing epigenetic silencing of these two genes.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Kailin Yu  

LAB HEAD: Yinsheng Wang

PROVIDER: PXD028148 | Pride | 2021-12-30

REPOSITORIES: Pride