Name: Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue (Video) | JoVE Journal
Uploaded: 2022-05-05T14:00:00.000+00:00
Duration: 8 min 12 s
Description: Learn global quantification of histone post-translational modifications in 3D hepatic spheroid cultures using C3A hepatocytes and liquid chromatography mass spectrometry. This method enables comprehensive analysis of chromatin dynamics.

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5 minutes

The Sidoli lab gratefully acknowledges the Leukemia Research Foundation (Hollis Brownstein New Investigator Research Grant), AFAR (Sagol Network GerOmics award), Deerfield (Xseed award), Relay Therapeutics, Merck, and the NIH Office of the Director (1S10OD030286-01).

<ol><li>Kapalczynska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((2D+and+3D+cell+cultures+-+a+comparison+of+different+types+of+cancer+cell+cultures%5BTitle%5D)+AND+%22Archives+of+Medical+Science%22%5BJournal%5D)">2D and 3D cell cultures - a comparison of different types of cancer cell cultures.</a> Archives of Medical Science. 14 (4), 910-919 (2018).</li>
<li>Breslin, S., O'Driscoll, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+cell+culture%3A+the+missing+link+in+drug+discovery%5BTitle%5D)+AND+%22Drug+Discovery+Today%22%5BJournal%5D)">Three-dimensional cell culture: the missing link in drug discovery.</a> Drug Discovery Today. 18 (5-6), 240-249 (2013).</li>
<li>Kim, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+tissue+culture+models+in+cancer+biology%5BTitle%5D)+AND+%22Seminars+in+Cancer+Biology%22%5BJournal%5D)">Three-dimensional tissue culture models in cancer biology.</a> Seminars in Cancer Biology. 15 (5), 365-377 (2005).</li>
<li>Duval, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+physiological+events+in+2D+vs.+3D+cell+culture%5BTitle%5D)+AND+%22Physiology+%28Bethesda%29%22%5BJournal%5D)">Modeling physiological events in 2D vs. 3D cell culture.</a> Physiology (Bethesda). 32 (4), 266-277 (2017).</li>
<li>Wrzesinski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cultural+divide%3A+exponential+growth+in+classical+2D+and+metabolic+equilibrium+in+3D+environments%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The cultural divide: exponential growth in classical 2D and metabolic equilibrium in 3D environments.</a> PLoS One. 9 (9), 106973(2014).</li>
<li>Wrzesinski, K., Fey, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+reprogramming+and+the+recovery+of+physiological+functionality+in+3D+cultures+in+micro-bioreactors%5BTitle%5D)+AND+%22Bioengineering+%28Basel%29%22%5BJournal%5D)">Metabolic reprogramming and the recovery of physiological functionality in 3D cultures in micro-bioreactors.</a> Bioengineering (Basel). 5 (1), 22(2018).</li>
<li>Gonda, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+transgenic+cell+model+to+quantify+genotoxic+effects+of+space+environment%5BTitle%5D)+AND+%22Advances+in+Space+Research%22%5BJournal%5D)">Three-dimensional transgenic cell model to quantify genotoxic effects of space environment.</a> Advances in Space Research. 27 (2), 421-430 (2001).</li>
<li>Yamada, K. M., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+tissue+morphogenesis+and+cancer+in+3D%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Modeling tissue morphogenesis and cancer in 3D.</a> Cell. 130 (4), 601-610 (2007).</li>
<li>Tvardovskiy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Top-down+and+middle-down+protein+analysis+reveals+that+intact+and+clipped+human+histones+differ+in+post-translational+modification+patterns%5BTitle%5D)+AND+%22Molecular+and+Cellular+Proteomics%22%5BJournal%5D)">Top-down and middle-down protein analysis reveals that intact and clipped human histones differ in post-translational modification patterns.</a> Molecular and Cellular Proteomics. 14 (12), 3142-3153 (2015).</li>
<li>Azad, G. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modifying+chromatin+by+histone+tail+clipping%5BTitle%5D)+AND+%22Journal+of+Molecular+Biology%22%5BJournal%5D)">Modifying chromatin by histone tail clipping.</a> Journal of Molecular Biology. 430 (18), 3051-3067 (2018).</li>
<li>Kragesteen, B. K., Amit, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heads+or+tails%3A+histone+tail+clipping+regulates+macrophage+activity%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">Heads or tails: histone tail clipping regulates macrophage activity.</a> Nature Immunology. 22 (6), 678-680 (2021).</li>
<li>Dhaenens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+clipping%3A+the+punctuation+in+the+histone+code%5BTitle%5D)+AND+%22EMBO+Reports%22%5BJournal%5D)">Histone clipping: the punctuation in the histone code.</a> EMBO Reports. 22 (8), 53440(2021).</li>
<li>Anderson, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analyses+of+histone+proteoforms+using+front-end+electron+transfer+dissociation-enabled+orbitrap+instruments%5BTitle%5D)+AND+%22Molecular+and+Cellular+Proteomics%22%5BJournal%5D)">Analyses of histone proteoforms using front-end electron transfer dissociation-enabled orbitrap instruments.</a> Molecular and Cellular Proteomics. 15 (3), 975-988 (2016).</li>
<li>Morgan, M. A. J., Shilatifard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reevaluating+the+roles+of+histone-modifying+enzymes+and+their+associated+chromatin+modifications+in+transcriptional+regulation%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">Reevaluating the roles of histone-modifying enzymes and their associated chromatin modifications in transcriptional regulation.</a> Nature Genetics. 52 (12), 1271-1281 (2020).</li>
<li>Chan, J. C., Maze, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nothing+is+yet+set+in+%28hi%29stone%3A+novel+post-translational+modifications+regulating+chromatin+function%5BTitle%5D)+AND+%22Trends+in+Biochemical+Sciences%22%5BJournal%5D)">Nothing is yet set in (hi)stone: novel post-translational modifications regulating chromatin function.</a> Trends in Biochemical Sciences. 45 (10), 829-844 (2020).</li>
<li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translating+the+histone+code%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Translating the histone code.</a> Science. 293 (5532), 1074-1080 (2001).</li>
<li>Fischle, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+basis+for+the+discrimination+of+repressive+methyl-lysine+marks+in+histone+H3+by+Polycomb+and+HP1+chromodomains%5BTitle%5D)+AND+%22Genes+and+Development%22%5BJournal%5D)">Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains.</a> Genes and Development. 17 (15), 1870-1881 (2003).</li>
<li>Onder, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progress+in+epigenetic+histone+modification+analysis+by+mass+spectrometry+for+clinical+investigations%5BTitle%5D)+AND+%22Expert+Review+of+Proteomics%22%5BJournal%5D)">Progress in epigenetic histone modification analysis by mass spectrometry for clinical investigations.</a> Expert Review of Proteomics. 12 (5), 499-517 (2015).</li>
<li>Sidoli, S., Cheng, L., Jensen, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomics+in+chromatin+biology+and+epigenetics%3A+Elucidation+of+post-translational+modifications+of+histone+proteins+by+mass+spectrometry%5BTitle%5D)+AND+%22Journal+of+Proteomics%22%5BJournal%5D)">Proteomics in chromatin biology and epigenetics: Elucidation of post-translational modifications of histone proteins by mass spectrometry.</a> Journal of Proteomics. 75 (12), 3419-3433 (2012).</li>
<li>Egelhofer, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+assessment+of+histone-modification+antibody+quality%5BTitle%5D)+AND+%22Nature+Structural+and+Molecular+Biology%22%5BJournal%5D)">An assessment of histone-modification antibody quality.</a> Nature Structural and Molecular Biology. 18 (1), 91-93 (2011).</li>
<li>Moradian, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+top-down%2C+middle-down%2C+and+bottom-up+mass+spectrometry+approaches+for+characterization+of+histone+variants+and+their+post-translational+modifications%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">The top-down, middle-down, and bottom-up mass spectrometry approaches for characterization of histone variants and their post-translational modifications.</a> Proteomics. 14 (4-5), 489-497 (2014).</li>
<li>Zheng, Y., Huang, X., Kelleher, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epiproteomics%3A+quantitative+analysis+of+histone+marks+and+codes+by+mass+spectrometry%5BTitle%5D)+AND+%22Current+Opinion+in+Chemical+Biology%22%5BJournal%5D)">Epiproteomics: quantitative analysis of histone marks and codes by mass spectrometry.</a> Current Opinion in Chemical Biology. 33, 142-150 (2016).</li>
<li>Sidoli, S., Garcia, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Middle-down+proteomics%3A+a+still+unexploited+resource+for+chromatin+biology%5BTitle%5D)+AND+%22Expert+Review+of+Proteomics%22%5BJournal%5D)">Middle-down proteomics: a still unexploited resource for chromatin biology.</a> Expert Review of Proteomics. 14 (7), 617-626 (2017).</li>
<li>Stampar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatocellular+carcinoma+%28HepG2%2FC3A%29+cell-based+3D+model+for+genotoxicity+testing+of+chemicals%5BTitle%5D)+AND+%22Science+of+the+Total+Environment%22%5BJournal%5D)">Hepatocellular carcinoma (HepG2/C3A) cell-based 3D model for genotoxicity testing of chemicals.</a> Science of the Total Environment. 755, 143255(2021).</li>
<li>Calitz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toxicity+and+anti-prolific+properties+of+Xysmalobium+undulatum+water+extract+during+short-term+exposure+to+two-dimensional+and+three-dimensional+spheroid+cell+cultures%5BTitle%5D)+AND+%22Toxicology+Mechanisms+and+Methods%22%5BJournal%5D)">Toxicity and anti-prolific properties of Xysmalobium undulatum water extract during short-term exposure to two-dimensional and three-dimensional spheroid cell cultures.</a> Toxicology Mechanisms and Methods. 28 (9), 641-652 (2018).</li>
<li>Garcia, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+derivatization+of+histones+for+facilitated+analysis+by+mass+spectrometry%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Chemical derivatization of histones for facilitated analysis by mass spectrometry.</a> Nature Protocols. 2 (4), 933-938 (2007).</li>
<li>Sidoli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequential+window+acquisition+of+all+theoretical+mass+spectra+%28SWATH%29+analysis+for+characterization+and+quantification+of+histone+post-translational+modifications%5BTitle%5D)+AND+%22Molecular+and+Cellular+Proteomics%22%5BJournal%5D)">Sequential window acquisition of all theoretical mass spectra (SWATH) analysis for characterization and quantification of histone post-translational modifications.</a> Molecular and Cellular Proteomics. 14 (9), 2420-2428 (2015).</li>
<li>Sidoli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low+resolution+data-independent+acquisition+in+an+LTQ-orbitrap+allows+for+simplified+and+fully+untargeted+analysis+of+histone+modifications%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Low resolution data-independent acquisition in an LTQ-orbitrap allows for simplified and fully untargeted analysis of histone modifications.</a> Analytical Chemistry. 87 (22), 11448-11454 (2015).</li>
<li>Karch, K. R., Sidoli, S., Garcia, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+quantification+of+histone+PTMs+using+high-resolution+mass+spectrometry%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Identification and quantification of histone PTMs using high-resolution mass spectrometry.</a> Methods in Enzymology. 574, 3-29 (2016).</li>
<li>Yuan, Z. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EpiProfile+quantifies+histone+peptides+with+modifications+by+extracting+retention+time+and+intensity+in+high-resolution+mass+spectra%5BTitle%5D)+AND+%22Molecular+and+Cellular+Proteomics%22%5BJournal%5D)">EpiProfile quantifies histone peptides with modifications by extracting retention time and intensity in high-resolution mass spectra.</a> Molecular and Cellular Proteomics. 14 (6), 1696-1707 (2015).</li>
<li>Yuan, Z. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EpiProfile+2.0%3A+a+computational+platform+for+processing+epi-proteomics+mass+spectrometry+data%5BTitle%5D)+AND+%22Journal+of+Proteome+Research%22%5BJournal%5D)">EpiProfile 2.0: a computational platform for processing epi-proteomics mass spectrometry data.</a> Journal of Proteome Research. 17 (7), 2533-2541 (2018).</li>
<li>MacLean, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skyline%3A+an+open+source+document+editor+for+creating+and+analyzing+targeted+proteomics+experiments%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.</a> Bioinformatics. 26 (7), 966-968 (2010).</li>
<li>Pino, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Skyline+ecosystem%3A+Informatics+for+quantitative+mass+spectrometry+proteomics%5BTitle%5D)+AND+%22Mass+Spectrometry+Reviews%22%5BJournal%5D)">The Skyline ecosystem: Informatics for quantitative mass spectrometry proteomics.</a> Mass Spectrometry Reviews. 39 (3), 229-244 (2020).</li>
<li>Aguilan, J. T., Kulej, K., Sidoli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guide+for+protein+fold+change+and+p-value+calculation+for+non-experts+in+proteomics%5BTitle%5D)+AND+%22Molecular+Omics%22%5BJournal%5D)">Guide for protein fold change and p-value calculation for non-experts in proteomics.</a> Molecular Omics. 16 (6), 573-582 (2020).</li>
<li>Lex, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((UpSet%3A+Visualization+of+intersecting+sets%5BTitle%5D)+AND+%22IEEE+Transactions+on+Visualization+and+Computer+Graphics%22%5BJournal%5D)">UpSet: Visualization of intersecting sets.</a> IEEE Transactions on Visualization and Computer Graphics. 20 (12), 1983-1992 (2014).</li>
<li>Shah, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HISTome2%3A+a+database+of+histone+proteins%2C+modifiers+for+multiple+organisms+and+epidrugs%5BTitle%5D)+AND+%22Epigenetics+and+Chromatin%22%5BJournal%5D)">HISTome2: a database of histone proteins, modifiers for multiple organisms and epidrugs.</a> Epigenetics and Chromatin. 13 (1), 31(2020).</li>
<li>Xie, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lysine+succinylation+and+lysine+malonylation+in+histones%5BTitle%5D)+AND+%22Molecular+and+Cellular+Proteomics%22%5BJournal%5D)">Lysine succinylation and lysine malonylation in histones.</a> Molecular and Cellular Proteomics. 11 (5), 100-107 (2012).</li>
<li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+succinylation+and+its+function+on+the+nucleosome%5BTitle%5D)+AND+%22Journal+of+Cellular+and+Molecular+Medicine%22%5BJournal%5D)">Histone succinylation and its function on the nucleosome.</a> Journal of Cellular and Molecular Medicine. 25 (15), 7101-7109 (2021).</li>
<li>Howe, F. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Is+H3K4me3+instructive+for+transcription+activation%5BTitle%5D)+AND+%22Bioessays%22%5BJournal%5D)">Is H3K4me3 instructive for transcription activation.</a> Bioessays. 39 (1), 1-12 (2017).</li>
<li>Nicetto, D., Zaret, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+H3K9me3+heterochromatin+in+cell+identity+establishment+and+maintenance%5BTitle%5D)+AND+%22Current+Opinion+in+Genetics+and+Development%22%5BJournal%5D)">Role of H3K9me3 heterochromatin in cell identity establishment and maintenance.</a> Current Opinion in Genetics and Development. 55, 1-10 (2019).</li>
<li>Wojcik, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+H3K27+dimethyl+loss+is+highly+specific+for+malignant+peripheral+nerve+sheath+tumor+and+distinguishes+true+PRC2+loss+from+isolated+H3K27+trimethyl+loss%5BTitle%5D)+AND+%22Modern+Pathology%22%5BJournal%5D)">Histone H3K27 dimethyl loss is highly specific for malignant peripheral nerve sheath tumor and distinguishes true PRC2 loss from isolated H3K27 trimethyl loss.</a> Modern Pathology. 32 (10), 1434-1446 (2019).</li>
<li>Sellers, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Next-generation+characterization+of+the+cancer+cell+line+encyclopedia%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Next-generation characterization of the cancer cell line encyclopedia.</a> Nature. 569 (7757), 503-508 (2019).</li>
<li>Zhang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+regulation+of+gene+expression+by+histone+lactylation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Metabolic regulation of gene expression by histone lactylation.</a> Nature. 574 (7779), 575-580 (2019).</li>
<li>Farrelly, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+serotonylation+is+a+permissive+modification+that+enhances+TFIID+binding+to+H3K4me3%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Histone serotonylation is a permissive modification that enhances TFIID binding to H3K4me3.</a> Nature. 567 (7749), 535-539 (2019).</li>
<li>Chen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ADP-ribosylation+of+histone+variant+H2AX+promotes+base+excision+repair%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">ADP-ribosylation of histone variant H2AX promotes base excision repair.</a> The EMBO Journal. 40 (2), 104542(2021).</li>
<li>Zheng, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reversible+histone+glycation+is+associated+with+disease-related+changes+in+chromatin+architecture%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Reversible histone glycation is associated with disease-related changes in chromatin architecture.</a> Nature Communications. 10 (1), 1289(2019).</li>
</ol>

The authors have no competing financial interests.

The analysis of histone PTMs is fundamentally different from the typical proteomics analysis pipeline. Most histone PTMs still have enigmatic biological functions; as a result, annotations such as Gene Ontology or pathway databases are not available. Several resources exist that associate histone modifications with the enzyme responsible for their catalysis or proteins containing domains that bind these PTMs (e.g., HISTome36). As well, it is possible to speculate on the overall state of the chroma...

Since the late 19th century, cell culture systems have been used as a model to study the growth and development of cells outside of the human body1,2. Their use has also been extended to study how tissues and organs function in both healthy and diseased contexts1,3. Suspension cells (e.g., blood cells) grow in Petri dishes or flasks seamlessly and interchangeably as they do not assemble in three-dimensional (3D) structures in vivo. Cells derived from solid organs can grow in either two-dimensional (2D) or 3D culture systems. In 2D culture, cells are grown in a monolayer that adheres to a flat surface2,4. 2D cell culture systems are characterized by exponential growth and a fast doubling time, typically 24 h to a few days5. Cells in 3D systems grow to form intricate cell-cell interactions modeling tissue-like conglomerates more closely, and they are characterized by their ability to reach a dynamic equilibrium where their doubling time can reach 1 month or longer5.
Presented in this paper is an innovative methodology to grow 3D spheroids in rotating cell culture systems that simulate reduced gravity6. This is a simplified derivative of a cell culture system introduced by NASA in the 1990s7. This approach minimizes shearing forces, which occur in existing methods like spinning flasks, and increases spheroid reproducibility6. In addition, the rotating bioreactor increases active nutrient diffusion, minimizing necrotic formation that occurs in systems like hanging drop cell culture where media exchange is impractical6. This way, cells grow mostly undisturbed, allowing for the formation of structural and physiological characteristics associated with cells growing in tissue. C3A hepatocytes (HepG2/C3A) cultured in this manner not only had ultrastructural organelles, but also produced amounts of ATP, adenylate kinase, urea, and cholesterol comparable to levels observed in vivo1,2. In addition, cells grown in 2D vs. 3D cell culture systems exhibit different gene expression patterns8. Gene expression analysis of C3A hepatocytes grown as 3D spheroids showed that these cells expressed a wide range of liver-specific proteins, as well as genes involved in key pathways that regulate liver function8. Prior publications demonstrated the differences between proteomes of exponentially growing cells in 2D culture vs. cells at dynamic equilibrium in 3D spheroid cultures5. These differences include cellular metabolism, which in turn affects the structure, function, and physiology of the cell5. The proteome of cells grown in 2D culture was more enriched in proteins involved in cell replication, while the proteome of 3D spheroids was more enriched in liver functionality5.
The slower replication rate of cells grown as 3D spheroids more accurately models specific phenomena associated with chromatin state and modifications&#160;(e.g. histone clipping9). Histone clipping is an irreversible histone post-translational modification (PTM) that causes proteolytic cleavage of part of the histone N-terminal tail. While its biological function is still under discussion10,11,12,13, it is clear that its presence in primary cells and liver tissue is modeled by HepG2/C3A cells grown as spheroids, but not as flat cells9. This is critical, as chromatin state and modifications regulate DNA readout mostly by modulating accessibility to genes and thus their expression14. Histone PTMs either influence chromatin state directly by affecting the net charge of the nucleosomes where histones are assembled, or indirectly by recruiting chromatin writers, readers, and erasers14. Hundreds of histone PTMs have been identified to date15, reinforcing the hypothesis that chromatin hosts a &#34;histone code&#34; used by the cell to interpret DNA16. However, the identification of a myriad of PTM combinations15, and the discovery that combinations of histone PTMs frequently have different biological functions from PTMs present in isolation (e.g. Fischle, et al.17), highlights that more work is required to decrypt the &#34;histone code&#34;.
Currently, histone PTM analysis is either based on techniques utilizing antibodies (e.g., western blots, immunofluorescence, or chromatin immunoprecipitation followed by sequencing [ChIP-seq]) or mass spectrometry (MS). Antibody-based techniques have high sensitivity and can provide detailed information about the genome-wide localization of histone marks but are frequently limited in studying rare PTMs or PTMs present in combinations18,19,20. MS is more suitable for high-throughput and unbiased identification and quantification of single and co-existing protein modifications, in particular histone proteins18,19,20. For these reasons, this and several other laboratories have optimized the MS pipeline for the analysis of histone peptides (bottom-up MS), intact histone tails (middle-down MS), and full-length histone proteins (top-down MS)21,22,23.
Detailed below is a workflow for growing HepG2/C3A spheroids and preparing them for histone peptide analysis (bottom-up MS) via nano-liquid chromatography, coupled online with tandem mass spectrometry (nLC-MS/MS). A 2D cell culture was grown and the cells were harvested and transferred to a bioreactor where they would start to form spheroids (Figure 1). After 18 days in culture, spheroids were treated with sodium butyrate or sodium succinate to increase the relative abundances of histone acetylation and succinylation. Notably, 3D cultures can be treated with genotoxic compounds just as well as their flat culture equivalents; in fact, recent publications highlight that the toxicology response of cells in 3D culture is more similar to primary tissues than those in 2D flat culture24,25. Cells were then collected at specified time points and nuclear isolation was performed. Then, histones were extracted and derivatized with propionic anhydride before and after trypsin digestion according to a protocol first developed by Garcia et al.26. This procedure generates peptides of an appropriate size for online separation with reversed-phase chromatography (C18) and detection with MS. Finally, histone peptides were identified and quantified, and the generated data was represented in multiple ways for a more complete biological interpretation.

<table><tbody><tr><td>0.05% trypsin-EDTA solution</td><td>Gibco</td><td>25300054</td><td></td></tr><tr><td>0.5-20 &amp;micro;L pipet tips</td><td>BRAND</td><td>13-889-172 (Fisher Scientific)</td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>Bio-Rad</td><td>2239480</td><td></td></tr><tr><td>10 &amp;micro;L multi-channel pipette</td><td>BRAND</td><td>BR7059000 (Millipore Sigma)</td><td></td></tr><tr><td>10 mL syringe</td><td>Henke Sass Wolf</td><td>14-817-31 (Fisher Scientific)</td><td>Luer lock tip, graduated to 12 mL</td></tr><tr><td>10, 20, 200, and 1000 &amp;micro;L single-channel pipettes</td><td>Eppendorf</td><td>14-285-904 (Fisher Scientific)</td><td></td></tr><tr><td>1000 &amp;micro;L pipet tips</td><td>Rainin</td><td>30389164</td><td></td></tr><tr><td>18 G syringe needle</td><td>Air-Tite</td><td>14-817-100 (Fisher Scientific)</td><td>3&quot; length, 0.05&quot; diameter</td></tr><tr><td>200 &amp;micro;L multi-channel pipette</td><td>Corning</td><td>4082</td><td></td></tr><tr><td>2-200 &amp;micro;L pipet tips</td><td>BRAND</td><td>Z740118 (Millipore Sigma)</td><td></td></tr><tr><td>24-well ultra-low attachment microplate</td><td>Corning</td><td>07-200-602</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt;&amp;nbsp; U-shaped cell culture flask</td><td>Corning</td><td>461464U</td><td>Untreated, with vent cap</td></tr><tr><td>96-well skirted plate</td><td>Axygen</td><td>PCR-96-FS-C (Corning)</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A949-1</td><td>Acetone should be used cold</td></tr><tr><td>Ammonium bicarbonate (NH&lt;sub&gt;4&lt;/sub&gt;HCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>A6141-25G</td><td></td></tr><tr><td>Ammonium hydroxide solution</td><td>Fisher Scientific</td><td>AC423300250</td><td></td></tr><tr><td>Cell culture grade water</td><td>Corning</td><td>25-055-CV</td><td></td></tr><tr><td>ClinoReactor</td><td>CelVivo</td><td>10004-12</td><td>Bioreactor for 3D cell culture</td></tr><tr><td>ClinoStar</td><td>CelVivo</td><td>N/A</td><td>Clinostat CO&lt;sub&gt;2&lt;/sub&gt; incubator for 3D cell culture</td></tr><tr><td>Control unit</td><td>CelVivo</td><td>N/A</td><td>Tablet for ClinoStar settings</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Corning</td><td>17-205-CV</td><td>1X solution with 4.5 g/L glucose and sodium pyruvate, without L-glutamine and phenol red</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Corning</td><td>35-010-CV</td><td></td></tr><tr><td>Formic acid</td><td>Thermo Scientific</td><td>28905</td><td></td></tr><tr><td>Fume hood</td><td>Mott</td><td>N/A</td><td>Model 7121000</td></tr><tr><td>Glass Pasteur pipette</td><td>Fisher Scientific</td><td>13-678-8B</td><td>9&quot;, cotton-plugged, borosilicate glass, non-sterile</td></tr><tr><td>Glutagro supplement</td><td>Corning</td><td>25-015-CI</td><td>200 mM L-ananyl-L-glutamine</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS)</td><td>Corning</td><td>21-022-CV</td><td>1X solution without calcium, magnesium, and phenol red</td></tr><tr><td>HPLC grade acetonitrile</td><td>Fisher Scientific</td><td>A955-4</td><td></td></tr><tr><td>HPLC grade water</td><td>Fisher Scientific</td><td>W6-1</td><td></td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Fisher Scientific</td><td>A481-212</td><td></td></tr><tr><td>Ice</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>MEM non-essential amino acids</td><td>Corning</td><td>25-025-CI</td><td>100X solution</td></tr><tr><td>Oasis HLB resin</td><td>Waters</td><td>186007549</td><td>Hydrophilic-Lipophilic-Balanced (HLB) Resin with 30&amp;micro;m particle size</td></tr><tr><td>Orbitrap Fusion Lumos Tribrid mass spectrometer</td><td>Thermo Fisher Scientific</td><td>IQLAAEGAAPFADBMBHQ</td><td>High resolution mass spectrometer</td></tr><tr><td>Oro-Flex I polypropylene filter plate</td><td>Orochem</td><td>OF1100</td><td>96-well polypropylene filter plate w/ 10 &amp;micro;M PE frit</td></tr><tr><td>Penicillin-Streptomycin</td><td>Corning</td><td>30-002-CI</td><td>100X solution</td></tr><tr><td>pH paper</td><td>Hydrion</td><td>Z111848 (Sigma-Aldrich)</td><td>0-13 pH test paper</td></tr><tr><td>Pipette gun</td><td>Eppendorf</td><td>Z666467 (Millipore Sigma)</td><td></td></tr><tr><td>Polymicro capillary</td><td>Molex</td><td>50-110-7740 (Fisher Scientific)</td><td>Flexible fused silica capillary tubing with polymide coating, 75 &amp;micro;M ID x 363 &amp;micro;M OD</td></tr><tr><td>Polystyrene 10 mL serological pipets, sterile</td><td>Fisher Scientific</td><td>1367549</td><td></td></tr><tr><td>Propionic anhydride</td><td>Sigma-Aldrich</td><td>240311-50G</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td>Thermo Scientific</td><td>75-217-420</td><td></td></tr><tr><td>Reprosil-Pur resin</td><td>MSWIL</td><td>R13.AQ.0003</td><td>120 &amp;Aring; pore size, C18-AQ phase, 3 &amp;micro;M bead size</td></tr><tr><td>Rotator</td><td>Clay Adams</td><td>25477 (American Laboratory Trading)</td><td>Nutator Mixer 1105</td></tr><tr><td>Sequencing grade modified trypsin</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>Sodium butyrate</td><td>Thermo Scientific</td><td>A11079</td><td></td></tr><tr><td>Sodium succinate dibasic</td><td>Sigma-Aldrich</td><td>14160-100G</td><td></td></tr><tr><td>SpeedVac vacuum concentrator (1.5 mL microcentrifuge tubes)</td><td>Savant</td><td>20249 (American Laboratory Trading)</td><td></td></tr><tr><td>SpeedVac vacuum concentrator (96-well)</td><td>Thermo Scientific</td><td>15308325</td><td>Savant SPD1010</td></tr><tr><td>Sterile hood</td><td>Thermo Scientific</td><td>1375</td><td>Class II, Type A2</td></tr><tr><td>Sulfuric acid (H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>02-004-375</td><td>Baker Analyzed ACS reagent</td></tr><tr><td>Tissue-culture treated 100 mm x 20 mm dish</td><td>Fisher Scientific</td><td>08-772-23</td><td></td></tr><tr><td>Trichloroacetic acid (TCA)</td><td>Thermo Scientific</td><td>AC421451000</td><td>Resuspend 100% w/v in HPLC grade water</td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>Fisher Scientific</td><td>PI28904</td><td>Sequencing grade</td></tr><tr><td>Vacuum manifold 96-well</td><td>Millipore</td><td>MAVM0960R</td><td></td></tr><tr><td>Vortex</td><td>Sigma-Aldrich</td><td>Z258415</td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific</td><td>FSGPD10</td><td></td></tr><tr><td>Wide bore pipet tips 1000 &amp;micro;L</td><td>Axygen</td><td>14-222-703 (Fisher Scientific)</td><td></td></tr><tr><td>Wide bore pipet tips 200 &amp;micro;L</td><td>Axygen</td><td>14-222-730 (Fisher Scientific)</td><td></td></tr></tbody></table>

global level quantification of histone post-translational modifications in a 3d cell culture model of hepatic tissue

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

In this protocol, HepG2/C3A spheroids were treated with 20 mM NaBut and 10 mM NaSuc, both of which affected global levels of histone PTMs (Figure 3A). Histone PTMs were then identified and quantified at the single residue level via MS/MS acquisition (Figure 3B).
When samples are run in replicates, statistical analysis can be performed to assess the fold change enrichment of a PTM between samples, as well as the reproducibilit...

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid ...

global-level-quantification-histone-post-translational-modifications

Research

JoVE Journal

Biology

We describe a set of assays to analyze expression levels of H1 linker histones.  mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Expression Analysis of Mammalian Linker-histone Subtypes

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

Quantitative Analysis of Chromatin Proteomes in Disease

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

The purpose of this article is to provide a comprehensive, systematic guide to the efficient purification of histones H3 and H4 and the quantification of acetylated histone residues.

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue

This protocol focuses on chromatin preparation from snap frozen tissues and it is suitable for Crosslinking Chromatin Immunoprecipitation (X-ChIP) followed by either quantitative PCR analysis (X-ChIP-qPCR) or next generation sequencing approaches (X-ChIP-seq).

Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

This method describes a simple and fast strategy for histone proteoform characterization and investigation.

Unveiling Histone Proteoforms using 2D-TAU Gel Electrophoresis

This protocol enables efficient histone extraction from various clinical samples for subsequent post-translational modification analysis by LC-MS/MS. By facilitating robust detection of key histone modifications, it serves as a valuable tool for comprehensive epigenetic profiling in large clinical cohorts.

Extraction of Histones from Clinical Specimens for Epigenetic Profiling by Mass Spectrometry

Here, we present a protocol to automate single-cell histone post-translational modification analysis using an isotopic two-plex labeling strategy and ArgC digestion. This workflow enables quantitative, reproducible, and high-sensitivity profiling of chromatin heterogeneity and epigenetic responses at single-cell resolution.

Automated Sample Preparation for the Multiplexed Analysis of Single-Cell Histone Post-Translational Modifications (sc-hPTM2)

This protocol presents a comprehensive imaging pipeline to visualize and model hepatic stellate cells morphology in intact liver tissue. This approach combines fluorescent labeling of fibroblasts using mouse genetics, in situ liver perfusion-based tissue preparation, and a modified iDISCO tissue clearing method to achieve optical transparency.

Mapping Hepatic Stellate Cell Morphology in Mouse Models of Liver Fibrosis

Learn global quantification of histone post-translational modifications in 3D hepatic spheroid cultures using C3A hepatocytes and liquid chromatography mass spectrometry. This method enables comprehensive analysis of chromatin dynamics.

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue

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