Transient expression of inactive RB in mesenchymal stem cells impairs their adipogenic potential and is associated with hypermethylation of the PPARγ2 promoter

Baryshev, Mikhail; Петров, Н. С.; Ryabov, Vladimir; Попов, Б. В.

doi:10.1016/j.gendis.2020.11.001

Cited by 4 publications

(3 citation statements)

References 25 publications

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“…S1, S2). All plasmids used in the analysis of DNA methylation of distal and proximal Oct4 enhancers (Baryshev et al 2018 ) and the proximal PPARg2 promoter (Baryshev et al 2022 ) were obtained by the DM miniprep method. We compared the presence of host genomic DNA in the plasmids obtained by DM, SM minipreps and commercial non-miniprep kits recommended for purification of high-quality plasmid DNA.…”

Section: Resultsmentioning

confidence: 99%

A new device-mediated miniprep method

Baryshev

Dmitrijs²,

Ivan³

2022

AMB Expr

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Small-scale plasmid DNA preparation or miniprep is a fundamental technique in estimation cloning experiments and is widely used for DNA methylation analysis in epigenetic research. Current plasmid DNA minipreps use the alkali-SDS-based method in a three-solution format and require spin column-based purification steps. This procedure requires the vortexing or pipetting of pelleted bacteria by centrifugation and manual mixing of the solutions. Here, we describe a centrifuge/mixer-based instrument with the ability to perform centrifugation, vibration, and rotor oscillation in order to perform all steps of plasmid DNA isolation by device only. We found that by applying rotor oscillation-driven mixing of solutions added in the lysis and neutralization steps, homogeneous mixing was achieved within 5 s at a rotor oscillation amplitude of 45° and oscillation frequency of 400 ± 30 rpm, yielding the maximal quantity and quality of plasmid DNA. No increase in host chromosome presence purified by this approach occurs for high-copy-number plasmids compared to manually performed miniprep, and indeed, there is a significant decrease in the presence of the chromosomal fraction in low-copy-number plasmids. The supercoiled form of plasmid DNA purified at a rotor oscillation amplitude of 45° does not turn into an open circular (OC) isoform when the plasmid is stored for 1 year at plus four degrees, in contrast to the plasmid purified with rotor oscillation amplitudes of 270°, 180° and 90°. The programmed time-work-efficient protocol of plasmid miniprep installed in the device gives the extreme simplicity of plasmid minipreps speeding up and facilitating the isolation of plasmid DNAs.

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Section: Resultsmentioning

confidence: 99%

A new device-mediated miniprep method

Baryshev

Dmitrijs²,

Ivan³

2022

AMB Expr

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show abstract

“…S1, S2). All plasmids used in the analysis of DNA methylation of distal and proximal Oct4 enhancers (Baryshev et al, 2018) and the proximal PPARg2 promoter (Baryshev et al, 2020) were obtained by the DM miniprep method. We have compared the presence of host genomic DNA in the plasmids obtained both by DM, SM minipreps and commercial nonminiprep kits recommended for puri cation of high quality plasmid DNA.…”

Section: Plasmid Yield By Dm Miniprep Depends On Oscillation-driving Mixing Parameters and Exceeds That By Sm Methodsmentioning

confidence: 99%

A new devise-mediated miniprep method

BAryshev¹

2022

Preprint

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Small-scale plasmid DNA preparation or miniprep is a fundamental technique in estimation cloning experiments and is widely used for DNA methylation analysis in epigenetic research. Current plasmid DNA minipreps use alkali - SDS based method in a three-solution format and require the spin column-based purification steps. This procedure demands the vortexing or pipetting of pelleted by centrifugation bacteria and mixing of solutions manually. Here we describe a centrifuge/mixer-based instrument having an ability to perform centrifugation, vibration, and rotor oscillation in order to perform all steps of plasmid DNA isolation by device only. We found that applying rotor oscillation-driving mixing of solutions added in the lysis and neutralization steps, the homogeneous mixing is achieved within 5 second at rotor oscillation amplitude 45 degrees and oscillation frequency 400±30 rpm, yielding maximal quantity and quality of plasmid DNA. No increase of host chromosome presence in purified by this approach occurs for high copy number plasmids as compared to manually performed miniprep, and indeed, there is a significant decrease in the presence of chromosomal fraction in low copy number plasmids. The supercoiled form of plasmid DNA purified at a rotor oscillation amplitude of 45 degrees does not turn into open circular (OC) isoform when the plasmid is stored for one year at plus four degrees, in contrast to the plasmid purified with rotor oscillation amplitude 270, 180 and 90 degrees. The programmed time-work-efficient protocol of plasmid miniprep installed in the device gives the extreme simplicity of plasmid minipreps speeding up and facilitating the isolation of plasmid DNAs.

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“…Exploring the altered status of DNA methylation to use it as a disease-speci c biomarker, some researchers focus on CpG islands (CGIs) and do not attach importance to the methylation of several CpGs located in proximal promoters [6]. However, it has been shown that methylation of even a single CpG in proximity of transcription start site (TSS) of Pparg2, a major regulator of adipogenesis, can silence the gene and thus prevent its expression, thereby blocking the adipocyte differentiation pathway [7,8]. Given the general clinical utility of the PSA gene in PCa testing, methylation of the CpG and CCWGG (W = A/T) proximal promoter was studied in PSA-expressing or nonexpressing LNCaP and PC3 cancer cell lines, as well as in non-cancerous BPH1 and HPrEpiC cells.…”

Section: Introductionmentioning

confidence: 99%

Allele-specific methylation of the PSA promoter in prostate cells: a new translational marker for the differential diagnosis of prostate cancer

Baryshev,

Maksimova,

Sasoveca

2023

Preprint

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Background DNA methylation is one of the mechanisms of epigenetic control of gene expression and a change in the intrinsic pattern can lead to various diseases and disorders. At the same time, this makes DNA methylation a disease-specific biomarker. Here, we show that the acquisition of biallelic methylation status or even biallelic lack of methylation by the PSA promoter is a characteristic feature of cancer cells, while the monoallelic distribution of CpG\CCWGG methylation in the PSA promoter is a hallmark of non-cancerous conditions. Methods We performed PCR bisulfite sequencing analysis of the proximal PSA promoter, which has six potential CpG and five CCWGG methylation marks that are targets of DNMTs and therefore have the ability to epigenetically influence gene expression and be a disease-specific biomarker. SNP G-158A, located in the AREI of the PSA promoter, was taken into account when genotyping prostate cell lines for allele-specific methylation analysis. To clarify the differences in PSA promoter methylation we applied RT-qPCR to estimate the level expression of the DNMTs by comparing that with data of gene expression in 54 tissues from GTEx RNA-seq of donors. A PCR cloning sequencing approach and detection of SNPs in exon 3 were used to identify biallelic expression of PSA in the LNCaP cell line. Results According to our study of prostate cancer cell lines, the CG/CCWGG methylation in PSA promoter has a great translational ability to be a disease-specific biomarker. Elevated PSA level along with monoallelic promoter methylation would reflect BPH or other non-malignant conditions, whereas a significantly elevated PSA level due to biallelic PSA expression and status of the unmethylated PSA promoter will be consistent with the PCa. The detection of biallelic PSA promoter methylation at the given PSA level will indicate the aggressive PCa. We propose that the CCWGG motif represents an allele-specific methylation signal, which is a novel functionality for this mark involved in the allele-specific methylation mechanism and/or imprinting, as well as its possible role in regulating monoallelic gene expression. Significance According to our study in prostate cell line models, the determination of allele-specific methylation in the PSA promoter can be translated into a disease-specific state and can refine PSA-based PCa testing.

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Transient expression of inactive RB in mesenchymal stem cells impairs their adipogenic potential and is associated with hypermethylation of the PPARγ2 promoter

Cited by 4 publications

References 25 publications

A new device-mediated miniprep method

A new device-mediated miniprep method

A new devise-mediated miniprep method

Allele-specific methylation of the PSA promoter in prostate cells: a new translational marker for the differential diagnosis of prostate cancer

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