Investigation on DNMT1 Orchestrated-G2/M Transition and Chromatin Segregation by Interaction with MKI67 and CDK1 in Lung Adenocarcinoma Cells

Abstract

Background: According to the prevailing views, the chromatin organization pattern influences the microenvironment of a gene within a chromosome; transcription factors and epigenetic modifications of chromatin dictate the possibility of its expression and progression through the cell cycle. Gene expression mechanisms and their relationship with cell cycle dynamics remain unexplored till today. The theoretical background and conceptual logics suggest that epigenetic modifications, specifically DNA methyltransferase (DNMTs)-mediated DNA methylation, might orchestrate cell cycle dynamics by modulating chromatin accessibility and gene expression at key checkpoints to ensure precise coordination between proliferation and genomic fidelity. Among several DNMTs, DNMT1 is the principal custodian of maintaining the methylation pattern during DNA replication, ensuring epigenetic inheritance. Overexpression of DNMT1, in association with genome-wide hypermethylation, is frequently observed in several cancers, suggesting its involvement in critical cellular processes beyond its canonical function. Methods: Lung adenocarcinoma (LUAD) cells (A549) and normal lung cells (L132) were cultured in DMEM media, supplemented with 10% FBS along with 100 units/ml penicillin and 0.1 mg/ml streptomycin and used as a model experimental system throughout this study. The Viability of cells was checked to determine the sublethal concentrations (Inhibitory Concentration 30 or IC30) of 5-AZA-2′-deoxycytidine using the MTT assay on A549 and L132 cells. At first, flow cytometric analysis was performed to check the cell cycle distribution patterns of the control and treated cells after staining with Propidium Iodide (PI). Further to check the mRNA level expression of the target genes, initially the total RNA was extracted from both control and treated cells by using the TRIzol method, followed by cDNA synthesis using cDNA synthesis kit to perform quantitative RT-PCR. Subsequently, protein level expressions of the target genes were quantified by western blot analysis using the extracted cytosolic and nuclear proteins. Later, Co-Immunoprecipitation (Co-IP), followed by Western blotting, was performed to check the physical interactions between the proteins of interest. After that, colocalization of proteins was visualized by using confocal microscopy. Wound closure percentage was quantified to check cell proliferation before and after treatment by performing wound healing assay. Apart from in vitro investigations, Biogrid and STRING databases were used to examine the interaction of DNMT1 with other cell cycle regulatory proteins. Enrichment analysis of DNMT1 gene was performed using the Enrichr tool. Furthermore, to investigate the correlation between DNMT1 and G2/M phase specific genes, correlation analysis was performed by using cBioPortal. Molecular docking was also performed to check the molecular interactions between DNMT1 MKI67 and DNMT1-CDK1. Results: This study reveals the precise mechanism by which DNMT1 contributes to the G2/M phase transition in LUAD cells. Flow cytometric analysis reveals that the inhibition of DNMT1 by 5-AZA-2′-deoxycytidine induces the G2/M phase arrest in A549 cells, which is further supported by the protein-level expression of CCNB1 and CDK1. Decreased wound closure percentage in the cell migration assay depicts lower proliferation rates of treated cells. Protein-protein interaction network draws a coherent linkage among DNMT1 and cell cycle regulatory genes. Correlation analyses reveal a strong positive correlation between DNMT1 and UHRF1, as well as CCNB1, CDK1, MKI67, and CENPA. Among these genes, Co immunoprecipitation further confirms the physical association of DNMT1 with MKI67 and CDK1, which is also confirmed by confocal microscopy. Furthermore, confocal microscopy of synchronized cells by serum starvation reveals the spatiotemporal colocalization of DNMT1 with MKI67 and CDK1 throughout different phases of the cell cycle; whereas inhibition of DNMT1 results in mitotic arrest and disrupts chromatin segregation. Implications: Collectively, our findings demonstrate, for the first time, a critical non canonical role of DNMT1 in regulating the G2/M phase transition by interacting with MKI67 and CDK1 while ensuring mitotic progression in LUAD.