Lysines Acetylome and Methylome Profiling of H3 and H4 Histones in Trichostatin A—Treated Stem Cells

Cozzolino, Flora; Iacobucci, Ilaria; Monaco, Vittoria; Angrisano, Tiziana; Monti, Maria

doi:10.3390/ijms22042063

Cited by 7 publications

(6 citation statements)

References 42 publications

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“…S3 ). Most of these findings are in line with the recently described effects of TSA on mouse ESCs 29 . Moreover, we recently described for the first time that the histone code changes in a very similar way between mouse and human ESC during differentiation, making mouse a potential model for developmental toxicoepigenetics 38 .…”

Section: Resultssupporting

confidence: 89%

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

Verhelst

Puyvelde

Willems

et al. 2022

Sci Rep

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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).

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Section: Resultssupporting

confidence: 89%

“…the pronounced decrease in H3K9 methylation levels, which was already established for other HDACis (e.g. TSA) 29 . However, not all other hPTMs decline in response to a rise in acetylation.…”

Section: Resultssupporting

confidence: 58%

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

Verhelst

Puyvelde

Willems

et al. 2022

Sci Rep

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“…For TSA, a pan-HDAC inhibitor originally known as an antifungal antibiotic, the results are very similar to those already discussed for VPA, with an overall distinct increase in acetylation levels, along with a rise in H31K27me2, H33K27me2, and H31K27me3 and a decrease in methylation of H31K9 and H31K36me2. Most of these ndings are in line with the recently described effects of TSA on mouse ESCs (Cozzolino et al 2021). Moreover, we recently described for the rst time that the histone code changes in a very similar way between mouse and human ESC during differentiation, making mouse a potential model for developmental toxicoepigenetics (De Clerck et al 2019a).…”

Section: Vpa Treatment Results In a Hyperacetylation Of Histone H3 And H4supporting

confidence: 80%

“…the pronounced decrease in H3K9 methylation levels, which was already established for other HDACis (e.g. TSA) (Cozzolino et al 2021). However, not all other hPTMs decline in response to a rise in acetylation.…”

Section: Vpa Treatment Results In a Hyperacetylation Of Histone H3 And H4supporting

confidence: 57%

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

Verhelst

Puyvelde

Willems

et al. 2021

Preprint

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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.

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“…The impairment of either process, the modulation of histones modifications, or the recruitment time- and space-specific of protein complexes on their nucleotide-binding site, is often involved in the onset of particularly serious pathologies. − Already in 1940, the biologist Conrad Hal Waddington, introducing the term epigenetics, emphasized the importance of the interactions of genes with the surrounding environment for the understanding of a specific phenotype. Hence the need to elucidate the mechanisms underlying these complex phenomena and the development of techniques to be applied for their investigation. , Therefore, in these past years, we have assisted in the implementation of a large number of analytical methods for studying both the profiling of epigenetics modification in different conditions (i.e., physiological vs pathological) − and the isolation and identifications of specific DNA/RNA binding protein complexes. − …”

Section: Introductionmentioning

confidence: 99%

Protein–DNA/RNA Interactions: An Overview of Investigation Methods in the -Omics Era

et al. 2021

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The fields of application of functional proteomics are not limited to the study of protein–protein interactions; they also extend to those involving protein complexes that bind DNA or RNA. These interactions affect fundamental processes such as replication, transcription, and repair in the case of DNA, as well as transport, translation, splicing, and silencing in the case of RNA. Analytical or preparative experimental approaches, both in vivo and in vitro, have been developed to isolate and identify DNA/RNA binding proteins by exploiting the advantage of the affinity shown by these proteins toward a specific oligonucleotide sequence. The present review proposes an overview of the approaches most commonly employed in proteomics applications for the identification of nucleic acid-binding proteins, such as affinity purification (AP) protocols, EMSA, chromatin purification methods, and CRISPR-based chromatin affinity purification, which are generally associated with mass spectrometry methodologies for the unbiased protein identification.

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Lysines Acetylome and Methylome Profiling of H3 and H4 Histones in Trichostatin A—Treated Stem Cells

Cited by 7 publications

References 42 publications

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

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

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

Protein–DNA/RNA Interactions: An Overview of Investigation Methods in the -Omics Era

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