A practical guide for analysis of histone post-translational modifications by mass spectrometry: Best practices and pitfalls

Thomas, Sydney P.; Haws, Spencer A.; Borth, Laura; Denu, John M.

doi:10.1016/j.ymeth.2019.12.001

Cited by 17 publications

(16 citation statements)

References 72 publications

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“…To determine the broad effects of extracellular SCFAs on histone PTMs, we treated HCT116 cells with acetate, propionate, and butyrate individually and then performed histone proteomics ( Karch et al, 2013 ; Krautkramer et al, 2015 ). This mass spectrometry-based method allows for simultaneous analysis of >70 histone PTMs, which significantly improves on traditional Western blotting ( Thomas et al, 2020 ). While the impact of SCFAs on a few of these PTMs has been studied previously, to our knowledge this is the first study to show how SCFAs regulate a range of both canonical and non-canonical PTMs ( Hinnebusch et al, 2002 ; Kiefer et al, 2006 ; Silva et al, 2018 ; Wang et al, 2018 ).…”

Section: Resultsmentioning

confidence: 99%

“…After performing a Bradford assay to determine protein concentrations, 5 µg of each sample was dried down and resuspended in 100 mM triethylammonium bicarbonate buffer. Unmodified lysines were labeled with d 6 -acetic anhydride, a labeling method that allows for quantification of histone acylations ( Thomas et al, 2020 ). Labeled histones were digested with trypsin and labeled with N-termini phenyl isocyanate before stage tip desalting and resuspension sample diluent (5% ACN, 0.1% acetic acid in H 2 O).…”

Section: Methodsmentioning

confidence: 99%

“…Data analysis -Thermo RAW files were loaded into EpiProfile 2.0 and run using the AcD3 module (Yuan et al, 2018). Normalization and statistics were then performed using our previously published Histone Analysis Workflow (Thomas et al, 2019). RAW files, peak tables, and processing scripts have been uploaded to the MassIVE database and can be accessed with username: MSV000087800_reviewer and password: SCFAp300.…”

Section: Scfa Cell Line Treatmentsmentioning

confidence: 99%

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Short-chain fatty acids activate acetyltransferase p300

Thomas

Denu

2021

eLife

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Short-chain fatty acids (SCFAs) acetate, propionate, and butyrate are produced in large quantities by the gut microbiome and contribute to a wide array of physiological processes. While the underlying mechanisms are largely unknown, many effects of SCFAs have been traced to changes in the cell’s epigenetic state. Here, we systematically investigate how SCFAs alter the epigenome. Using quantitative proteomics of histone modification states, we identified rapid and sustained increases in histone acetylation after addition of butyrate or propionate, but not acetate. While decades of prior observations would have suggested that hyperacetylation induced by SCFAs are attributed to inhibition of histone deacetylases (HDACs), we found that propionate and butyrate instead activate the acetyltransferase p300. Propionate and butyrate are rapidly converted to the corresponding acyl-CoAs which are then used by p300 to catalyze auto-acylation of the autoinhibitory loop, activating the enzyme for histone/protein acetylation. This data challenges the long-held belief that SCFAs mainly regulate chromatin by inhibiting HDACs, and instead reveals a previously unknown mechanism of HAT activation that can explain how an influx of low levels of SCFAs alters global chromatin states.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Scfa Cell Line Treatmentsmentioning

confidence: 99%

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Short-chain fatty acids activate acetyltransferase p300

Thomas

Denu

2021

eLife

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“…EpiProfile 2.0 was used for quantification of histone PTMs [100]. The R script provided by Denu lab (https://github.…”

Section: Histone Ptm Quantificationmentioning

confidence: 99%

Gut microbiome variation modulates the effects of dietary fiber on host metabolism

et al. 2021

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Background There is general consensus that consumption of dietary fermentable fiber improves cardiometabolic health, in part by promoting mutualistic microbes and by increasing production of beneficial metabolites in the distal gut. However, human studies have reported variations in the observed benefits among individuals consuming the same fiber. Several factors likely contribute to this variation, including host genetic and gut microbial differences. We hypothesized that gut microbial metabolism of dietary fiber represents an important and differential factor that modulates how dietary fiber impacts the host. Results We examined genetically identical gnotobiotic mice harboring two distinct complex gut microbial communities and exposed to four isocaloric diets, each containing different fibers: (i) cellulose, (ii) inulin, (iii) pectin, (iv) a mix of 5 fermentable fibers (assorted fiber). Gut microbiome analysis showed that each transplanted community preserved a core of common taxa across diets that differentiated it from the other community, but there were variations in richness and bacterial taxa abundance within each community among the different diet treatments. Host epigenetic, transcriptional, and metabolomic analyses revealed diet-directed differences between animals colonized with the two communities, including variation in amino acids and lipid pathways that were associated with divergent health outcomes. Conclusion This study demonstrates that interindividual variation in the gut microbiome is causally linked to differential effects of dietary fiber on host metabolic phenotypes and suggests that a one-fits-all fiber supplementation approach to promote health is unlikely to elicit consistent effects across individuals. Overall, the presented results underscore the importance of microbe-diet interactions on host metabolism and suggest that gut microbes modulate dietary fiber efficacy.

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“…At present, there are, however, only limited experimental schemes that can identify and characterize metabolites with chromatin function. The most developed approach is the mass spectrometry characterization of covalent histone modifications (186)(187)(188). Not surprisingly, the majority of uncommon metabolites that we discussed here form non-enzymatic or enzymatic histone PTMs.…”

Section: Perspectivementioning

confidence: 99%

Unconventional metabolites in chromatin regulation

2022

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Chromatin, the complex of DNA and histone proteins, serves as a main integrator of cellular signals. Increasing evidence links cellular functional to chromatin state. Indeed, different metabolites are emerging as modulators of chromatin function and structure. Alterations in chromatin state are decisive for regulating all aspects of genome function and ultimately have the potential to produce phenotypic changes. Several metabolites such as acetyl-CoA, S-adenosyl methionine (SAM) or adenosine triphosphate (ATP) have now been well characterized as main substrates or cofactors of chromatin modifying enzymes. However, there are other metabolites that can directly interact with chromatin influencing its state or that modulate the properties of chromatin regulatory factors. Also, there is a growing list of atypical enzymatic and non-enzymatic chromatin modifications that originate from different cellular pathways that have not been in the limelight of chromatin research. Here, we summarize different properties and functions of uncommon regulatory molecules originating from intermediate metabolism of lipids, carbohydrates and amino acids. Based on the various modes of action on chromatin and the plethora of putative, so far not described chromatin regulating metabolites, we propose that there are more links between cellular functional state and chromatin regulation to be discovered. We hypothesize that these connections could provide interesting starting points for interfering with cellular epigenetic states at a molecular level.

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A practical guide for analysis of histone post-translational modifications by mass spectrometry: Best practices and pitfalls

Cited by 17 publications

References 72 publications

Short-chain fatty acids activate acetyltransferase p300

Short-chain fatty acids activate acetyltransferase p300

Gut microbiome variation modulates the effects of dietary fiber on host metabolism

Unconventional metabolites in chromatin regulation

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