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).

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

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...

Watch this Scientific Journal Video about Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue at JoVE.com

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

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

3.8K Views.  Albert Einstein College of Medicine. 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.

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

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

Chemistry

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Genetics

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

Developmental Biology

Histone Proteomic Analysis and Post-Translational Modification Characterization

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

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF

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 proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

2D Gel Separation for Histone Isoform Enrichment and PTM Characterization

Unveiling Histone Proteoforms using 2D-TAU Gel Electrophoresis

Histones undergo various post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, acylation, and ubiquitination, which control nucleosome dynamics and determine cell fate. The nucleosome, which is the functional unit of chromatin, comprises DNA, four pairs of histones (H3, H4, H2A, and H2B) making up the globular core, and the linker histone H1, which stabilizes the chromatin structure. The amino (N)-terminal tails of the histones protrude from the globular core domains and undergo distinct PTMs that influence the chromatin landscape. Some evidence suggests that histone PTM homeostasis is crucial for preserving all physiological activities. The deregulation of histone PTMs is the primary cause of abnormal cellular proliferation, invasion, and metastasis. Therefore, developing methods for characterizing histone PTMs is crucial. Here, we describe an effective technique for isolating and analyzing histone isoforms. The method, based on the combination of two orthogonal separations, allows the enrichment of histone isoforms and the following mass spectrometry identification. The technique, originally described by Shechter et al., combines acid-urea polyacrylamide gels (TAU-GEL), which can separate basic histone proteins based on size and charge, and Sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), which can separate proteins by molecular weight. The result is a two-dimensional map of histone isoforms, suitable for in-gel digestion followed by mass spectrometry identification and western blot analysis. The result is a two-dimensional map of histone isoforms, suitable for both in-gel digestion followed by mass spectrometry identification and western blot analysis. This proteomic approach is a robust method that allows the enrichment of a single histone isoform and the characterization of new histone PTMs.

Author Spotlight: Epigenetic Modifications and Metabolic Rewiring as Targets for Cancer Therapy

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

Histones undergo various post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, acylation, and ubiquitination, which ...

Biochemistry

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

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array of diverse posttranslational modifications (PTMs) that mostly occur in the amino termini of histone proteins (i.e., histone tail) and regulate the accessibility and functional state of the underlying DNA. Histone tails extend from the core of the nucleosome and are subject to the addition of acetyl groups by histone acetyltransferases (HATs) and the removal of acetyl groups by histone deacetylases (HDACs) during cellular growth and differentiation. Specific acetylation patterns on lysine (K) residues on histone tails determine a dynamic homeostasis between transcriptionally active or transcriptionally repressed chromatin by (1) influencing the core histone assembly and (2) recruiting synergistic or antagonistic chromatin-associated proteins to the transcription site. The fundamental regulatory mechanism of the complex nature of histone tail PTMs influences the majority of chromatin-templated processes and results in changes in cell maturation and differentiation in both normal and pathological development. The goal of the current report is to provide novices with an efficient method to purify core histone proteins from cells and brain tissue and to reliably quantify acetylation marks on histones H3 and H4.

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.

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array ...

Neuroscience

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

Medicine

Mouse Brain Histone Modification Quantification Using Flow Cytometry

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

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

Author Spotlight: Enhancements in Gene Expression Regulation Research

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.

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...