Real-time dynamics of methyl-CpG-binding domain protein 3 and its role in DNA demethylation by fluorescence correlation spectroscopy

Cui, Yi; Cho, Il Hoon; Chowdhury, Basudev; Irudayaraj, Joseph

doi:10.4161/epi.25958

Cited by 23 publications

(28 citation statements)

References 83 publications

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“…The intensity of the signal loss was greater with the higher VPA concentration (Fig 2A and 2B), suggesting that VPA elicits a dose-dependent response [4, 7, 34]. Representative images analyzed using the ImageJ 3D plugin [35] reinforced the progressive loss of fluorescence intensity for 5mC as the VPA concentration increased compared with the untreated control (Fig 2B and 2C). …”

Section: Resultsmentioning

confidence: 94%

DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy

et al. 2017

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Valproic acid (VPA), a well-known histone deacetylase inhibitor, has been reported to affect the DNA methylation status in addition to inducing histone hyperacetylation in several cell types. In HeLa cells, VPA promotes histone acetylation and chromatin remodeling. However, DNA demethylation was not checked in this cell model for standing effects longer than those provided by histone acetylation, which is a rapid and transient phenomenon. Demonstration of VPA-induced DNA demethylation in HeLa cells would contribute to understanding the effect of VPA on an aggressive tumor cell line. In the present work, DNA demethylation in VPA-treated HeLa cells was assessed by image analysis of chromatin texture, the abundance of 5-methylcytosine (5mC) immunofluorescence signals and Fourier transform-infrared (FT-IR) microspectroscopy centered on spectral regions related to the vibration of–CH3 groups. Image analysis indicated that increased chromatin unpacking promoted by a 4-h-treatment with 1.0 mM VPA persisted for 24 h in the absence of the drug, suggesting the occurrence of DNA demethylation that was confirmed by decreased 5mC immunofluorescence signals. FT-IR spectra of DNA samples from 1 mM or 20 mM VPA-treated cells subjected to a peak fitting analysis of the spectral window for–CH3 stretching vibrations showed decreased vibrations and energy of these groups as a function of the decreased abundance of 5mC induced by increased VPA concentrations. Only the 20 mM-VPA treatment caused an increase in the ratio of -CH3 bending vibrations evaluated at 1375 cm-1 in relation to in-plane vibrations of overall cytosines evaluated at 1492 cm-1. CH3 stretching vibrations showed to be more sensitive than–CH3 bending vibrations, as detected with FT-IR microspectroscopy, for studies aiming to associate vibrational spectroscopy and changes in DNA 5mC abundance.

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Section: Resultsmentioning

confidence: 94%

DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy

et al. 2017

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“…Ten 10-s fluorescence trace measurements ª 2019 The Authors EMBO reports 21: e48211 | 2020 were taken per cell with an average laser power of~0.2 lW (measured at the back aperture of the objective) to minimize photobleaching effects in each segment of the data, and the data were averaged per cell for FCS analysis [46,47]. FCS analysis is described elsewhere in our previous work [46,48,49] For FCS measurement, the autocorrelation function G(t) is defined as follows: FCS analysis is described elsewhere in our previous work [46,48,49] For FCS measurement, the autocorrelation function G(t) is defined as follows:…”

Section: Fluorescence Correlation Spectroscopy In Living Yeastmentioning

confidence: 99%

Monomeric cohesin state revealed by live‐cell single‐molecule spectroscopy

et al. 2019

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The cohesin complex plays an important role in the maintenance of genome stability. Cohesin is composed of four core subunits and a set of regulatory subunits that interact with the core subunits. Less is known about cohesin dynamics in live cells and on the contribution of individual subunits to the overall complex. Understanding the tethering mechanism of cohesin is still a challenge, especially because the proposed mechanisms are still not conclusive. Models proposed to describe tethering depend on either the monomeric cohesin ring or a cohesin dimer. Here, we investigate the role of cohesin dynamics and stoichiometry in live yeast cells at single-molecule resolution. We explore the effect of regulatory subunit deletion on cohesin mobility and found that depletion of different regulatory subunits has opposing effects. Finally, we show that cohesin exists mostly as a canonical monomer throughout the cell cycle, and its monomeric form is independent of its regulatory factors. Our results demonstrate that single-molecule tools have the potential to provide new insights into the cohesin mechanism of action in live cells.

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“…39–41 By utilizing ultrashort pulsed laser and SPAD system, the fluorescence fluctuation in an extremely small volume (~0.6 femtoliter under 465 nm excitation) can be extracted and interpreted with FCS and PCH as presented in Figure 2. 42 In order for the fluorescence fluctuation to be detected, the concentration of fluorescent molecules should be in the pM to nM range to avoid signal deficiency or saturation.…”

Section: Resultsmentioning

confidence: 99%

Decitabine Nanoconjugate Sensitizes Human Glioblastoma Cells to Temozolomide

Cui¹,

Naz

Thompson³

et al. 2015

Mol. Pharmaceutics

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In this study we developed and characterized a delivery system for the epigenetic demethylating drug, decitabine, to sensitize temozolomide-resistant human glioblastoma multiforme (GBM) cells to alkylating chemotherapy. A poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) based nano-conjugate was fabricated to encapsulate decitabine and achieved a better therapeutic response in GBM cells. After synthesis, the highly efficient uptake process and intracellular dynamics of this nano-conjugate was monitored by single-molecule fluorescence tools. Our experiments demonstrated that, under an acidic pH due to active glycolysis in cancer cells, the PLGA-PEG nano-vector could release the conjugated decitabine at a faster rate, after which the hydrolyzed lactic acid and glycolic acid would further acidify the intracellular microenvironment, thus providing a “positive feedback” to increase the effective drug concentration and realize growth inhibition. In temozolomide-resistant GBM cells, decitabine can potentiate the cytotoxic DNA alkylation by counteracting cytosine methylation and reactivating tumor suppressor genes, such as p53 and p21. Owing to excellent internalization and endo-lysosomal escape enabled by the PLGA-PEG backbone, the encapsulated decitabine exhibited a better anti-GBM potential than free drug molecules. Hence, the synthesized nano-conjugate and temozolomide could act in synergy to deliver a more potent and long-term anti-proliferation effect against malignant GBM cells.

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Real-time dynamics of methyl-CpG-binding domain protein 3 and its role in DNA demethylation by fluorescence correlation spectroscopy

Cited by 23 publications

References 83 publications

DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy

DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy

Monomeric cohesin state revealed by live‐cell single‐molecule spectroscopy

Decitabine Nanoconjugate Sensitizes Human Glioblastoma Cells to Temozolomide

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