Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry

Zhang, Junjie; Zhang, Lijian; Zhou, Keyuan; Ye, Xiaoxia; Chun-an, Liu; Zhang, Liangtao; Kang, Jing‐Xuan; Cai, Chun

doi:10.1016/j.ab.2011.01.029

Cited by 44 publications

(30 citation statements)

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“…The solution was then evaporated under nitrogen. The residue was dissolved in methanol and centrifuged at 15,000 ϫ g for 5 min, and the supernatant was extracted for analysis by high-pressure liquid chromatography tandem mass spectrometry (HPLC-MS) (18).…”

Section: Methodsmentioning

confidence: 99%

“…HBP1 is a member of the sequence-specific high-mobilitygroup (HMG) family of transcription factors (17)(18)(19), and in most, but not all, cases, HBP1 acts as a transcriptional inhibitor (16,17,20,21). HBP1 was first identified in a screen that complemented a potassium channel defect (22), but we and others rediscovered HBP1 as a binding partner of pRB (21,23), which itself has integral functions for premature senescence (24,25).…”

mentioning

confidence: 99%

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HBP1-Mediated Transcriptional Regulation of DNA Methyltransferase 1 and Its Impact on Cell Senescence

Pan

Chen

Roth

et al. 2013

Molecular and Cellular Biology

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bThe activity of DNA methyltransferase 1 (DNMT1) is associated with diverse biological activities, including cell proliferation, senescence, and cancer development. In this study, we demonstrated that the HMG box-containing protein 1 (HBP1) transcription factor is a new repressor of DNMT1 in a complex mechanism during senescence. The DNMT1 gene contains an HBP1-binding site at bp ؊115 to ؊134 from the transcriptional start site. HBP1 repressed the endogenous DNMT1 gene through sequencespecific binding, resulting in both gene-specific (e.g., p16INK4 ) and global DNA hypomethylation changes. The HBP1-mediated repression by DNMT1 contributed to replicative and premature senescence, the latter of which could be induced by Ras and HBP1 itself. A detailed investigation unexpectedly revealed that HBP1 has dual and complex transcriptional functions, both of which contribute to premature senescence. HBP1 both repressed the DNMT1 gene and activated the p16 gene in premature senescence. The opposite transcriptional functions proceeded through different DNA sequences and differential protein acetylation. While intricate, the reciprocal partnership between HBP1 and DNMT1 has exceptional importance, since its abrogation compromises senescence and promotes tumorigenesis. Together, our results suggest that the HBP1 transcription factor orchestrates a complex regulation of key genes during cellular senescence, with an impact on overall DNA methylation state. Epigenetic alterations have essential roles in determining gene expression patterns and in setting the environment for activators or repressors to function appropriately. DNA methylation has been associated with cancer and senescence (1-4). Cellular senescence is characterized by a permanent cell cycle arrest and the acquisition of distinct morphological, physiological, and epigenetic changes in response to events such as telomere attrition, aberrant oncogene activation, or abrogation of tumor suppressor gene functions. Senescence is a tumor-suppressive process the abrogation of which enables the path to tumorigenesis (5-8). Although seemingly two distinct phenomena, cellular senescence and cancer share similarly altered global epigenetic profiles comprising complex changes in DNA methylation, involving both hypomethylation and hypermethylation of certain genes and sequences. The establishment of DNA methylation during DNA replication and DNA repair is catalyzed by a family of DNA methyltransferases (e.g., DNMT1, DNMT3A, and DNMT3B). In particular, DNMT1 mRNA expression is significantly elevated in different cancers and is regarded as a maintenance methylase (9-11). In senescence, the levels of DNA methylation and DNMT1 protein decline in concert with aging (12-15). Yet, the mechanism of age-dependent DNA methylation changes remains unknown. In this study, we found an unexpected connection to the HBP1 transcription factor, which our previous studies had linked to premature senescence (16).HBP1 is a member of the sequence-specific high-mobilitygroup (HMG) family of transcript...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

HBP1-Mediated Transcriptional Regulation of DNA Methyltransferase 1 and Its Impact on Cell Senescence

Pan

Chen

Roth

et al. 2013

Molecular and Cellular Biology

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“…The extent of global DNA methylation was determined by slight modification of the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (20). The analyses were conducted in a system comprising a Waters Acquity UltraPerformance Liquid Chromatograph (UPLC; Waters Corporation, Milford, MA), coupled with a Waters Quattro Premier XE triple quadrupole mass spectrometer operating in positive ion electrospray mode.…”

Section: Quantitative Reverse Transcription Real-time Pcr (Qrt-pcr)mentioning

confidence: 99%

Modulation of intracellular iron metabolism by iron chelation affects chromatin remodeling proteins and corresponding epigenetic modifications in breast cancer cells and increases their sensitivity to chemotherapeutic agents

Pogribny

Tryndyak

Pogribna

et al. 2013

International Journal of Oncology

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Abstract. Iron plays a vital role in the normal functioning of cells via the regulation of essential cellular metabolic reactions, including several DNA and histone-modifying proteins. The metabolic status of iron and the regulation of epige netic mechanisms are well-balanced and tightly controlled in normal cells; however, in cancer cells these processes are profoundly disturbed. Cancer-related abnormalities in iron metabolism have been corrected through the use of iron-chelating agents, which cause an inhibition of DNA synthesis, G 1 -S phase arrest, an inhibition of epithelial-to-mesenchymal transition, and the activation of apoptosis. In the present study, we show that, in addition to these well-studied molecular mechanisms, the treatment of wild-type TP53 MCF-7 and mutant TP53 MDA-MB-231 human breast cancer cells with desferrioxamine (DFO), a model iron chelator, causes significant epigenetic alterations at the global and gene-specific levels. Specifically, DFO treatment decreased the protein levels of the histone H3 lysine 9 demethylase, Jumonji domain-containing protein 2A (JMJD2A), in the MCF-7 and MDA-MB-231 cells and down-regulated the levels of the histone H3 lysine 4 demethylase, lysine-specific demethylase 1 (LSD1), in the MDA-MB-231 cells. These changes were accompanied by alterations in corresponding metabolically sensitive histone marks. Additionally, we demonstrate that DFO treatment activates apoptotic programs in MCF-7 and MDA-MB-231 cancer cells and enhances their sensitivity to the chemotherapeutic agents, doxorubicin and cisplatin; however, the mechanisms underlying this activation differ. The induction of apoptosis in wild-type TP53 MCF-7 cells was p53-dependent, triggered mainly by the down-regulation of the JMJD2A histone demethylase, while in mutant TP53 MDA-MB-231 cells, the activation of the p53-independent apoptotic program was driven predominantly by the epigenetic up-regulation of p21. IntroductionA growing body of evidence indicates the existence of an intimate link between the metabolic status and epigenetic regulation of cells (1,2). This is exemplified by the fact that a variety of small molecules involved in intercellular metabolism, including adenosine triphosphate, S-adenosylmethionine, nicotinamide adenine dinucleotide, flavin adenine dinucleotide, folate, acetyl coenzyme A, α-ketoglutarate and iron, are essential for the proper maintenance of the cellular epigenome.Iron is an essential trace element for normal cellular function. In addition to its significance in controlling a variety of cellular processes, including proliferation, DNA synthesis and repair, and mitochondrial electron transport, which are essential for the accurate maintenance of normal cellular homeostasis, iron plays a key regulatory role in the functioning of DNA and histone-modifying proteins (3,4). Specifically, the Jumonji domain-containing histone demethylase (JHDM) family, which catalyzes the demethylation of tri-and dimethylated lysine 9 and lysine 36 residues in histone H3 (5,6) and ten-eleven tra...

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“…The range of the calibration curves was based on the published concentrations in clinical samples. 24 …”

Section: Stock and Working Standard Solutionsmentioning

confidence: 99%

Simultaneous Determination of Global DNA Methylation and Hydroxymethylation Levels by Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry

Zhang

Zhou

et al. 2012

SLAS Discovery

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Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry

Cited by 44 publications

References 28 publications

HBP1-Mediated Transcriptional Regulation of DNA Methyltransferase 1 and Its Impact on Cell Senescence

HBP1-Mediated Transcriptional Regulation of DNA Methyltransferase 1 and Its Impact on Cell Senescence

Modulation of intracellular iron metabolism by iron chelation affects chromatin remodeling proteins and corresponding epigenetic modifications in breast cancer cells and increases their sensitivity to chemotherapeutic agents

Simultaneous Determination of Global DNA Methylation and Hydroxymethylation Levels by Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry

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