Comprehensive histone epigenetics: A mass spectrometry based screening assay to measure epigenetic toxicity

Willems

et al. 2021

Preprint

Self Cite

Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms, such as histone post-translational modifications (hPTMs). Due to substantial progress in the large-scale study of hPTMs, integration into the field of toxicology is promising and offers the opportunity to gain novel insights into toxicological phenomena. Moreover, there is a growing demand for high-throughput human-based in vitro assays for toxicity testing, especially for developmental toxicity. Consequently, we developed a mass spectrometry-based proof-of-concept to assess a histone code screening assay capable of simultaneously detecting multiple hPTM-changes in human embryonic stem cells. To prove the applicability and performance, we first validated the untargeted workflow with valproic acid (VPA), a histone deacetylase inhibitor. These results demonstrate that our workflow is capable of mapping the hPTM-dynamics, with a general increase in acetylations as an internal control. To illustrate the scalability, a dose-response study was performed on a proof-of-concept library of ten compounds i) with a known effect on the hPTMs (BIX-01294, 3-Deazaneplanocin A, Trichostatin A, and VPA), ii) classified as highly embryotoxic by the European Centre for the Validation of Alternative Methods (ECVAM) (Methotrexate, and All-trans retinoic acid), iii) classified as non-embryotoxic by ECVAM (Penicillin G), and iv) compounds of abuse with a presumed developmental toxicity (ethanol, caffeine, and nicotine). In conclusion, we show that toxicoepigenetic screening on histones is feasible and yields very rich data that holds potential, not only for applications in the pharmaceutical industry, but also for environmental toxicity and food safety.

Section: Discussionmentioning

confidence: 99%

Large Scale Toxicoepigenetics on Histones: A Mass Spectrometry-based Screening Assay Applied to Developmental Toxicity

Verhelst

Willems

et al. 2021

Preprint

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“…Mass spectrometric data analysis was performed as previously described 79 yet some modifications were implemented. For every compound, raw data from all runs were imported in a single experiment and all runs were aligned against a bovine histone standard in Progenesis QIP 4.2.7 (Nonlinear Dynamics, Waters https://www.waters.com/waters/en_US/Progenesis-QI-for-Proteomics/nav.htm?cid=134790665&locale=en_US ).…”

Section: Methodsmentioning

confidence: 99%

A large scale mass spectrometry-based histone screening for assessing epigenetic developmental toxicity

Verhelst

Willems

et al. 2022

Sci Rep

Self Cite

Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms, such as histone post-translational modifications (hPTMs). Due to substantial progress in the large-scale study of hPTMs, integration into the field of toxicology is promising and offers the opportunity to gain novel insights into toxicological phenomena. Moreover, there is a growing demand for high-throughput human-based in vitro assays for toxicity testing, especially for developmental toxicity. Consequently, we developed a mass spectrometry-based proof-of-concept to assess a histone code screening assay capable of simultaneously detecting multiple hPTM-changes in human embryonic stem cells. We first validated the untargeted workflow with valproic acid (VPA), a histone deacetylase inhibitor. These results demonstrate the capability of mapping the hPTM-dynamics, with a general increase in acetylations as an internal control. To illustrate the scalability, a dose–response study was performed on a proof-of-concept library of ten compounds (1) with a known effect on the hPTMs (BIX-01294, 3-Deazaneplanocin A, Trichostatin A, and VPA), (2) classified as highly embryotoxic by the European Centre for the Validation of Alternative Methods (ECVAM) (Methotrexate, and All-trans retinoic acid), (3) classified as non-embryotoxic by ECVAM (Penicillin G), and (4) compounds of abuse with a presumed developmental toxicity (ethanol, caffeine, and nicotine).

“…The most significant genes are indicated with * and included in the protein-protein interaction network. Because RNAseq data revealed the involvement of chromatin remodelers, including FOXA1, NR4A2, and KDM6B, in ferroptotic cell death, we next utilized a LC-MS/MS approach to investigate whether RSL3-induced oxidative stress signals propagate to the nucleus and alter histone post-translational modifications (Supplementary Figure S4a) [45]. First, LC-MS-compatible cell lysis and histone extraction protocols were optimized to obtain pure histone extracts (Supplementary Figure S4b) [46].…”

Section: Glucocorticoid-sensitive and -Resistant Mm1 Cells Exhibit A Similar Transcriptome Stress Response Upon Ferroptotic Cell Death Inmentioning

confidence: 99%

“…After optimization, histone epigenetic profiling was performed using an untargeted, data-dependent acquisition (DDA) MS-based screening method [45]. Briefly, MM1 cells were treated with 5 µM RSL3 for increasing time periods (1h, 2h, 4h, 8h), after which histones were extracted, propionylated, and trypsinized.…”

Section: Glucocorticoid-sensitive and -Resistant Mm1 Cells Exhibit A Similar Transcriptome Stress Response Upon Ferroptotic Cell Death Inmentioning

confidence: 99%

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Ferroptosis Induction in Multiple Myeloma Cells Triggers DNA Methylation and Histone Modification Changes Associated with Cellular Senescence

Logie

Cuypers

et al. 2021

IJMS

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Disease relapse and therapy resistance remain key challenges in treating multiple myeloma. Underlying (epi-)mutational events can promote myelomagenesis and contribute to multi-drug and apoptosis resistance. Therefore, compounds inducing ferroptosis, a form of iron and lipid peroxidation-regulated cell death, are appealing alternative treatment strategies for multiple myeloma and other malignancies. Both ferroptosis and the epigenetic machinery are heavily influenced by oxidative stress and iron metabolism changes. Yet, only a limited number of epigenetic enzymes and modifications have been identified as ferroptosis regulators. In this study, we found that MM1 multiple myeloma cells are sensitive to ferroptosis induction and epigenetic reprogramming by RSL3, irrespective of their glucocorticoid-sensitivity status. LC-MS/MS analysis revealed the formation of non-heme iron-histone complexes and altered expression of histone modifications associated with DNA repair and cellular senescence. In line with this observation, EPIC BeadChip measurements of significant DNA methylation changes in ferroptotic myeloma cells demonstrated an enrichment of CpG probes located in genes associated with cell cycle progression and senescence, such as Nuclear Receptor Subfamily 4 Group A member 2 (NR4A2). Overall, our data show that ferroptotic cell death is associated with an epigenomic stress response that might advance the therapeutic applicability of ferroptotic compounds.