Gene-Targeted DNA Methylation: Towards Long-Lasting Reprogramming of Gene Expression?

Cortés-Mancera, Fabián; Sarno, Federica; Goubert, Désirée; Rots, Marianne G.

doi:10.1007/978-3-031-11454-0_18

Cited by 9 publications

(6 citation statements)

References 102 publications

(185 reference statements)

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“…Consistently, acquisition of histone methylation alone is not enough for stably silencing the transcriptional expression of UCHL1, as also reported for, e.g., HER2, for which persistent silencing required both a DNA methyltransferase and EZH2 [ 46 , 51 ]. Although not in H1299 cells, cotargeting of dCas-MSssI (Q147L) and dCas-SKD did result in prolonged downregulation of UCHL1 in HEK293T cells, confirming other reports [ 17 , 27 , 29 ]. Straightforward long-term reprogramming of gene expression by epigenetic editing would provide a powerful and flexible approach to study the role of any given gene in various contexts.…”

Section: Discussionsupporting

confidence: 88%

“…We explored the possible role of UCHL1 in smoking-related lung diseases via chemical inhibitor treatment and conventional plasmid-based upregulation in cell lines. Furthermore, CRISPR/dCas9-mediated epigenetic editing of UCHL1 (using a catalytically inactivated Cas9) [ 17 ] – was employed to up- and downregulate the expression of UCHL1 to further validate these findings.…”

Section: Introductionmentioning

confidence: 99%

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Targeted epigenetic silencing of UCHL1 expression suppresses collagen-1 production in human lung epithelial cells

Lau

et al. 2023

Epigenetics

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Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is highly expressed in smokers, but little is known about the molecular mechanism of UCHL1 in airway epithelium and its possible role in affecting extracellular matrix (ECM) remodelling in the underlying submucosa. Since cigarette smoking is a major cause of lung diseases, we studied its effect on UCHL1 expression and DNA methylation patterns in human bronchial epithelial cells, obtained after laser capture micro-dissection (LCM) or isolated from residual tracheal/main stem bronchial tissue. Targeted regulation of UCHL1 expression via CRISPR/dCas9 based-epigenetic editing was used to explore the function of UCHL1 in lung epithelium. Our results show that cigarette smoke extract (CSE) stimulated the expression of UCHL1 in vitro . The methylation status of the UCHL1 gene was negatively associated with UCHL1 transcription in LCM-obtained airway epithelium at specific sites. Treatment with a UCHL1 inhibitor showed that the TGF-β1-induced upregulation of the ECM gene COL1A1 can be prevented by the inhibition of UCHL1 activity in cell lines. Furthermore, upon downregulation of UCHL1 by epigenetic editing using CRISPR/dCas-EZH2, mRNA expression of COL1A1 and fibronectin was reduced. In conclusion, we confirmed higher UCHL1 expression in current smokers compared to non- and ex-smokers, and induced downregulation of UCHL1 by epigenetic editing. The subsequent repression of genes encoding ECM proteins suggest a role for UCHL1 as a therapeutic target in fibrosis-related disease.

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Section: Discussionsupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Targeted epigenetic silencing of UCHL1 expression suppresses collagen-1 production in human lung epithelial cells

Lau

et al. 2023

Epigenetics

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“…Indeed, DNA-targeted approaches are currently explored to remove mutated mitochondrial DNA from diseased cells ( 68 ). To modulate DNA methylation, DNMT and TET enzymes can be targeted to loci of interest already reaching mainstream applications for nuclear DNA ( 69 ). Since DNA (de)methylating enzymes also localize to mitochondria ( 70 ), these approaches might turn out effective in treating mitochondrial dysfunction for a range of diseases.…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial DNA methylation in metabolic associated fatty liver disease

et al. 2023

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IntroductionHepatic lipid accumulation and mitochondrial dysfunction are hallmarks of metabolic associated fatty liver disease (MAFLD), yet molecular parameters underlying MAFLD progression are not well understood. Differential methylation within the mitochondrial DNA (mtDNA) has been suggested to be associated with dysfunctional mitochondria, also during progression to Metabolic Steatohepatitis (MeSH). This study further investigates whether mtDNA methylation is associated with hepatic lipid accumulation and MAFLD.MethodsHepG2 cells were constructed to stably express mitochondria-targeted viral and prokaryotic cytosine DNA methyltransferases (mtM.CviPI or mtM.SssI for GpC or CpG methylation, respectively). A catalytically inactive variant (mtM.CviPI-Mut) was constructed as a control. Mouse and human patients’ samples were also investigated. mtDNA methylation was assessed by pyro- or nanopore sequencing.Results and discussionDifferentially induced mtDNA hypermethylation impaired mitochondrial gene expression and metabolic activity in HepG2-mtM.CviPI and HepG2-mtM.SssI cells and was associated with increased lipid accumulation, when compared to the controls. To test whether lipid accumulation causes mtDNA methylation, HepG2 cells were subjected to 1 or 2 weeks of fatty acid treatment, but no clear differences in mtDNA methylation were detected. In contrast, hepatic Nd6 mitochondrial gene body cytosine methylation and Nd6 gene expression were increased in mice fed a high-fat high cholesterol diet (HFC for 6 or 20 weeks), when compared to controls, while mtDNA content was unchanged. For patients with simple steatosis, a higher ND6 methylation was confirmed using Methylation Specific PCR, but no additional distinctive cytosines could be identified using pyrosequencing. This study warrants further investigation into a role for mtDNA methylation in promoting mitochondrial dysfunction and impaired lipid metabolism in MAFLD.

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“…DNA methylation is found to be strongly associated with gene expression regulation and therefore in the development of different pathologies ( 52 ). In the current study, the mechanism of elevated PLEKHA4 expression in LGG was investigated, and results showed that PLEKHA4 overexpression was negatively correlated with the methylation of cg06339706, cg01093065, cg06705122 and cg23002721, which were located in the promotor or near the TSS of PLEKHA4 gene.…”

Section: Discussionmentioning

confidence: 99%

PLEKHA4 is a novel prognostic biomarker that reshapes the tumor microenvironment in lower-grade glioma

Zhi,

Wang,

Jiang

et al. 2023

Front. Immunol.

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BackgroundLower-grade glioma (LGG) is a primary intracranial tumor that carry a high risk of malignant transformation and limited therapeutic options. Emerging evidence indicates that the tumor microenvironment (TME) is a superior predictor for tumor progression and therapy response. PLEKHA4 has been demonstrated to be a biomarker for LGG that correlate with immune infiltration. However, the fundamental mechanism by which PLEKHA4 contributes to LGG is still poorly understood.MethodsMultiple bioinformatic tools, including Tumor Immune Estimation Resource (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA2), Shiny Methylation Analysis Resource Tool (SMART), etc., were incorporated to analyze the PLEKHA4. ESTIMATE, ssGSEA, CIBERSORT, TIDE and CellMiner algorithms were employed to determine the association of PLEKHA4 with TME, immunotherapy response and drug sensitivities. Immunohistochemistry (IHC)-based tissue microarrays and M2 macrophage infiltration assay were conducted to verify their associations.ResultsPLEKHA4 expression was found to be dramatically upregulated and strongly associated with unfavorable overall survival (OS) and disease-specific survival (DSS) in LGG patients, as well as their poor clinicopathological characteristics. Cox regression analysis identified that PLEKHA4 was an independent prognostic factor. Methylation analysis revealed that DNA methylation correlates with PLEKHA4 expression and indicates a better outcome in LGG. Moreover, PLEKHA4 was remarkably correlated with immune responses and TME remodeling, as evidenced by its positive correlation with particular immune marker subsets and the putative infiltration of immune cells. Surprisingly, the proportion of M2 macrophages in TME was strikingly higher than others, inferring that PLEKHA4 may regulate the infiltration and polarization of M2 macrophages. Evidence provided by IHC-based tissue microarrays and M2 macrophage infiltration assay further validated our findings. Moreover, PLEKHA4 expression was found to be significantly correlated with chemokines, interleukins, and their receptors, further supporting the critical role of PLEKHA4 in reshaping the TME. Additionally, we found that PLEKHA4 expression was closely associated with drug sensitivities and immunotherapy responses, indicating that PLEKHA4 expression also had potential clinical significance in guiding immunotherapy and chemotherapy in LGG.ConclusionPLEKHA4 plays a pivotal role in reshaping the TME of LGG patients, and may serve as a potential predictor for LGG prognosis and therapy.

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Gene-Targeted DNA Methylation: Towards Long-Lasting Reprogramming of Gene Expression?

Cited by 9 publications

References 102 publications

Targeted epigenetic silencing of UCHL1 expression suppresses collagen-1 production in human lung epithelial cells

Targeted epigenetic silencing of UCHL1 expression suppresses collagen-1 production in human lung epithelial cells

Mitochondrial DNA methylation in metabolic associated fatty liver disease

PLEKHA4 is a novel prognostic biomarker that reshapes the tumor microenvironment in lower-grade glioma

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