The authors express their gratitude to the Florida Department of Health Ed and Ethel Moore Alzheimer&#39;s Research Program (grants 6AZ08 and 7AZ26), the NIH-NIAAA (grant 5R01AA023781-03), and the American Heart Association (grant 17PRE33660831).

All mice were housed in a humidity- and temperature-controlled, AAALAC-accredited animal facility at the University of Miami Miller School of Medicine. All experiments were approved by the University of Miami Miller School of Medicine Institutional Animal Care and Use Committee (IACUC) and conducted according to specifications of the NIH. 
1. Preparation of the Sample Extract
<ol>
	<li>Adherent cells
	<ol>
		<li>Plate cells in 10 cm dishes in the appropriate culture media (1 x 106 to 1 x 109 cells per dish for cell lines, such as BV2, HEK-293, and SH-SY5Y, but ~1 x 1015 cells per dish for primary cells, such as primary cortical neurons). Assure that the cells are equally distributed on the entire surface of the plate and allow the cells to grow for 48 h to reach ~100% confluence (37 &#176;C, 5% CO2).</li>
		<li>Once the cells have reached the desired confluency, gently aspirate the culture media and wash the cells 2x with prewarmed serum-free media under a tissue culture hood.</li>
		<li>Aspirate the serum-free media from the dish and add 1 mL of ice-cold extraction buffer (0.4 M sulfuric acid, 1 mM KCl, 1 mM MgCl2, 50 mM Tris-HCl [pH 8.0] and 1x protease inhibitor cocktail) to each dish.</li>
		<li>Use a plastic cell scraper to collect all cells in the extraction buffer (by scraping) and transfer them to a 1.5 mL labeled tube with a 1,000 &#181;L pipette. Pipet the cells up and down 3x to facilitate homogenization.</li>
		<li>Close all tubes and immediately put them on ice.</li>
	</ol>
	</li>
	<li>Brain tissue 
	<ol>
		<li>If frozen tissue is being used, place the tissue in a prechilled 1.5 mL tube and briefly thaw it on ice. If fresh tissue is being used, proceed immediately to step 1.2.2. 
		NOTE: The current protocol describes procedures using frozen mouse brain and mouse prefrontal cortex samples.</li>
		<li>Homogenize the tissue using a handheld Dounce homogenizer using the appropriate amount of extraction buffer and the recommended number of strokes (Table 1). To avoid excessive chromatin shredding, do not exceed the number of recommended strokes.</li>
		<li>Using a single channel 1,000 &#181;L pipette, transfer the homogenate to a prechilled 1.5 mL tube. Close all tubes and immediately put them on ice.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Crude Histone Extract
<ol>
	<li>Place the 1.5 mL tubes containing the cells or tissue suspended in extraction buffer on a rotating platform and rotate at 15 rpm at 4 &#176;C to allow the extraction of crude histones. 
	NOTE: The extraction time may vary for different cell and tissue types and must be optimized for each procedure. The current protocol presents results obtained following 15 min, 2 h, and 24 h of extraction (Figure 2, Figure 3, Figure 4, and Figure 5).</li>
	<li>Prechill the microcentrifuge to 4 &#176;C. After the desired extraction time has passed, centrifuge the tubes at maximum speed for 10 min at 4 &#176;C.</li>
	<li>Transfer the supernatant including the crude histones to a new, prechilled 1.5 mL tube. Discard the pellet.</li>
	<li>Store the supernatant at -80 &#176;C (the extraction can be stopped at this step, see &#8220;Stop step&#8221; in Figure 1) or immediately proceed to the next step.</li>
	<li>Neutralize the crude histones with a 1/4 volume of 5x neutralization buffer (e.g., add 250 &#181;L of 5x neutralization buffer to 1 mL of crude histones). Mix well by pipetting up and down 6x.</li>
	<li>Check the pH of the mixture with pH strips. Adjust accordingly by adding more neutralization buffer to reach a pH of 7.</li>
	<li>Evaluate the presence of histone and nonhistone protein in the crude histone extract as follows (Figure 2).
	<ol>
		<li>Add 37.5 &#181;L of the sample to 12.5 &#181;L of 4x (Laemmli) sample buffer and denature for 10 min at 99 &#176;C.</li>
		<li>Load the sample onto an SDS-PAGE gel and run the gel for 1 h at 100 V.</li>
		<li>Stain the gel overnight with Coomassie Brilliant Blue R-250 staining solution and destain during three consecutive washes (1 h/wash) with Coomassie Brilliant Blue R-250 destaining solution. 
		NOTE: Crude histones (Figure 2) can be compared with eluted and purified histones (i.e., column input [Figure 5A]).</li>
	</ol>
	</li>
</ol>
3. Purification of Core Histones
<ol>
	<li>Spin column equilibration 
	<ol>
		<li>Add 500 &#181;L of equilibration buffer to each spin column being used. Do not touch the column membrane.</li>
		<li>Centrifuge at 4 &#176;C for 3 min at 800 x g. Discard the flow-through. Repeat 1x.</li>
	</ol>
	</li>
	<li>Histone Purification 
	<ol>
		<li>Add 500 &#181;L of the sample of interest from step 2.6 to the column. Centrifuge at 4 &#176;C for 3 min at 800 x g. Collect the flow-through.</li>
		<li>Repeat the previous step as many times as necessary to load the entire sample onto the column. Do not overfill the spin column.</li>
		<li>Combine the flow-through from each centrifugation step to analyze the column-binding efficiency (Figure 3).</li>
		<li>Follow step 2.7 to analyze the column flow-through.</li>
	</ol>
	</li>
	<li>Column wash 
	<ol>
		<li>Add 500 &#181;L of the wash buffer to each column. Centrifuge at 4 &#176;C for 3 min at 800 x g. Collect the flow-through wash (wash #1).</li>
		<li>Repeat step 3.3.1 for a total of three washes. Collect the flow-through washes #2 and #3. Do not pool the consecutive column flow-through washes.</li>
		<li>To further evaluate the column&#8217;s histone-binding efficiency, analyze the three column washes by following step 2.7 (Figure 4).</li>
	</ol>
	</li>
	<li>Histone elution 
	<ol>
		<li>Transfer the column into a new labelled 1.5 mL tube.</li>
		<li>Add 50 &#181;L of the histone elution buffer. Centrifuge at 4 &#176;C for 3 min at 800 x g. Save the flow-through containing histone proteins.</li>
		<li>For an additional elution, repeat step 3.4.2. Do not combine the first and second flow-through eluates as they differ in histone quantity and purity.</li>
	</ol>
	</li>
</ol>
4. Precipitation of Core Histones
<ol>
	<li>Add perchloric acid (PCA) to the purified histones to a final concentration of 4% PCA (e.g., add 3 &#181;L of 70% PCA to 50 &#181;L of purified histones from step 3.4.2.</li>
	<li>Centrifuge for 3 s to collect all residual liquid from the tube wall. Mix by pipetting up and down 6x.</li>
	<li>Place the tubes in a rack and incubate for 24 h at 4 &#176;C.</li>
	<li>The next day, prechill a microcentrifuge to 4 &#176;C and centrifuge the samples for 75 min at maximum speed at 4 &#176;C.</li>
	<li>After the centrifugation is complete, a small white pellet containing precipitated histones will be visible on the bottom of the tube. Do not vortex the sample.</li>
	<li>Carefully aspirate the supernatant and, without disturbing the pellet, add 500 &#181;L of ice-cold 4% PCA to the sample.</li>
	<li>Centrifuge at 4 &#176;C for 10 min at maximum speed. Carefully aspirate the supernatant.</li>
	<li>Repeat step 4.7 2x.</li>
	<li>Without disturbing the pellet, add 500 &#181;L of ice-cold acetone. Centrifuge at 4 &#176;C for 10 min at maximum speed. Carefully aspirate the supernatant.</li>
	<li>Repeat step 4.9 2x.</li>
	<li>Carefully aspirate the supernatant, leave the tubes uncapped, and allow the sample to dry on ice for 30 min. Check if all residual acetone has evaporated.</li>
	<li>Leave the tubes uncapped and allow the sample to dry at room temperature (RT) for 5 min.</li>
	<li>Resuspend the pellet in 30 &#181;L of sterile water. Do not pipet up and down. Flick the tube gently with a finger.</li>
	<li>Cap all tubes and allow the histones to reconstitute on ice for 30 - 50 min, depending on the pellet size. Check if the pellet is resuspended.</li>
	<li>Cap all tubes and allow the pellet to further resuspend at RT for 5 min. 
	NOTE: This solution (first and second elution from step 3.4.3) is constituted of purified and desalted histones and may be used for further quantification and histone acetylation analysis.</li>
</ol>
5. Quantification of Eluted Histone Proteins
<ol>
	<li>Use a spectrophotometer according to the manufacturer&#8217;s protocol to quantify the total histone proteins obtained after the final elution in step 4.15. Measure the absorbance at 230 nm. Record the A260/A280 ratio indicative of the sample contamination with nucleic acid.</li>
	<li>Use the following formula to calculate the histone concentration (x): 
	<img alt="Equation" src="/files/ftp_upload/58648/58648eq1.jpg" /> 
	Here, OD is the optical density measured at A230 nm.</li>
	<li>A histone concentration of ~1.5 mg/mL is considered an average yield for cell lines, while a histone concentration of ~5.0 mg/mL is considered an average yield for 30 mg of tissue.</li>
</ol>
6. Western Blot Analysis
<ol>
	<li>Adjust the purified and eluted histone from step 4.15 to ~10 &#181;g of histone protein/sample.</li>
	<li>Add the appropriate volume of water and 4x Laemmli sample buffer to adjust the loading volumes.</li>
	<li>Denature the samples for 10 min at 99 &#176;C. Cool them on ice. Centrifuge for 3 s to collect all residual liquid and condensation from the tube wall.</li>
	<li>Load the samples onto an SDS-PAGE gel and run the gel for 1 h at 100 V.</li>
	<li>To visualize total histone protein, stain the gel overnight with Coomassie Brilliant Blue R-250 staining solution and destain during three consecutive washes (1 h/wash) with Coomassie Brilliant Blue R-250 destaining solution. 
	NOTE: The first elution of histone proteins contains high-quality histones (Figure 5A) while the second elution contains low to no levels of histones (Figure 5B).</li>
	<li>To quantify histone PTMs, use a transfer system (see Table of Materials) to transfer the histone protein from the SDS-PAGE gel (step 6.4) onto a PVDF membrane.
	<ol>
		<li>To assemble the transfer sandwich, open the transfer system cassette and place the PVDF membrane stack (labelled as Bottom+) on the bottom of the cassette with the membrane facing up. Roll the membrane gently with a blot roller to remove any air from between the stack and the membrane.</li>
		<li>Lay the gel on top of the membrane, roll the gel gently with a blot roller to remove any air from between the membrane and the gel, and place the top stack on the gel. Roll gently again and place the cassette cover on top of the sandwich, press down firmly, and turn the nob clockwise to lock.</li>
		<li>Insert the cassette to the transfer system slot. On the apparatus screen, select Turbo Protocol. Use a 3 min protocol for a single mini gel or a 7 min protocol for more than two mini gels.</li>
		<li>Stain the membrane with Ponceau S stain for 5 min and visualize the total histone proteins.</li>
		<li>Wash them in 1x Tris-buffered saline (TBS) with 0.1% Tween 20 for 2 h and block them in 5% milk for 1 h. Incubate with primary and secondary antibody (overnight at 4 &#176;C or 1 h at RT, respectively) or according to a previously optimized protocol. 
		NOTE: In the current protocol, antibodies against acetylated histones H4K12 (Figure 6 and Figure 7) and H3K27 (Figure 8) were used.</li>
	</ol>
	</li>
</ol>

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<li>Sartor, G. C., Powell, S. K., Brothers, S. P., Wahlestedt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetic+Readers+of+Lysine+Acetylation+Regulate+Cocaine-Induced+Plasticity%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Epigenetic Readers of Lysine Acetylation Regulate Cocaine-Induced Plasticity.</a> The Journal of Neuroscience. 35, 15062-15072 (2015).</li>
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<li>Roh, T. -Y., Cuddapah, S., Zhao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Active+chromatin+domains+are+defined+by+acetylation+islands+revealed+by+genome-wide+mapping%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping.</a> Genes & Development. 19, 542-552 (2005).</li>
<li>Mutskov, V., Felsenfeld, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Silencing+of+transgene+transcription+precedes+methylation+of+promoter+DNA+and+histone+H3+lysine+9%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9.</a> The EMBO Journal. 23, 138-149 (2004).</li>
<li>Howe, L., Brown, C. E., Lechner, T., Workman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+acetyltransferase+complexes+and+their+link+to+transcription%5BTitle%5D)+AND+%22Critical+Reviews+in+Eukaryotic+Gene+Expression%22%5BJournal%5D)">Histone acetyltransferase complexes and their link to transcription.</a> Critical Reviews in Eukaryotic Gene Expression. 9, 231-243 (1999).</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, 1074-1080 (2001).</li>
<li>Bowman, G. D., Poirier, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Post-Translational+Modifications+of+Histones+That+Influence+Nucleosome+Dynamics%5BTitle%5D)+AND+%22Chemical+Reviews%22%5BJournal%5D)">Post-Translational Modifications of Histones That Influence Nucleosome Dynamics.</a> Chemical Reviews. 115, 2274-2295 (2015).</li>
<li>Volmar, C. -H., Wahlestedt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+deacetylases+%28HDACs%29+and+brain+function%5BTitle%5D)+AND+%22Neuroepigenetics%22%5BJournal%5D)">Histone deacetylases (HDACs) and brain function.</a> Neuroepigenetics. 1, 20-27 (2015).</li>
<li>Plagg, B., Ehrlich, D., Kniewallner, K. M., Marksteiner, J., Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+Acetylation+of+Histone+H4+at+Lysine+12+%28H4K12%29+in+Monocytes+of+Transgenic+Alzheimer%27s+Mice+and+in+Human+Patients%5BTitle%5D)+AND+%22Current+Alzheimer+Research%22%5BJournal%5D)">Increased Acetylation of Histone H4 at Lysine 12 (H4K12) in Monocytes of Transgenic Alzheimer's Mice and in Human Patients.</a> Current Alzheimer Research. 12, 752-760 (2015).</li>
<li>Bhaskara, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hdac3+is+essential+for+the+maintenance+of+chromatin+structure+and+genome+stability%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">Hdac3 is essential for the maintenance of chromatin structure and genome stability.</a> Cancer Cell. 18, 436-447 (2010).</li>
<li>Mottamal, M., Zheng, S., Huang, T. L., Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+Deacetylase+Inhibitors+in+Clinical+Studies+as+Templates+for+New+Anticancer+Agents%5BTitle%5D)+AND+%22Molecules%22%5BJournal%5D)">Histone Deacetylase Inhibitors in Clinical Studies as Templates for New Anticancer Agents.</a> Molecules. 20, 3898-3941 (2015).</li>
<li>Ramakrishnan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HDAC+1+and+6+modulate+cell+invasion+and+migration+in+clear+cell+renal+cell+carcinoma%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">HDAC 1 and 6 modulate cell invasion and migration in clear cell renal cell carcinoma.</a> BMC Cancer. 16, 617(2016).</li>
<li>Wapenaar, H., Dekker, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+acetyltransferases%3A+challenges+in+targeting+bi-substrate+enzymes%5BTitle%5D)+AND+%22Clinical+Epigenetics%22%5BJournal%5D)">Histone acetyltransferases: challenges in targeting bi-substrate enzymes.</a> Clinical Epigenetics. 8, 59(2016).</li>
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The authors have nothing to disclose.

In the current work, we demonstrated an optimized method to purify core histone proteins and quantify histone H3 and H4 PTMs (e.g., acetylation). The presented protocol is a comprehensive workflow that incorporates optimized procedures regarding cells and brain tissue preparation, crude histone purification, and detailed histone precipitation, elution, and quantification, which are followed by histone electrophoresis and robust histone PTM quantification. The large amount of details provided here allows for a replicable generation of high-quality data, despite the need for lengthy manipulations of the histone samples.
Many currently published protocols require the use of HPLC to isolate pure fractions of histones H3 and H419. Although HPLC is a powerful technique, its complexity and low-throughput deter most molecular biologists and nonexperts from its frequent use. Indeed, HPLC is not available to many labs, and highly-skilled personnel is required to operate the instrument. HPLC is oftentimes time-consuming, expensive, and potentially hazardous. Presented here is an inexpensive, medium-throughput strategy to achieve results of similar quality which bypasses HPLC. The reported strategy is also more practical and suitable for use in almost any lab as it uses a simple spin column approach that does not require specialized instrument operation skills. Additionally, histone H3/H4 tetramer is extracted as a single, pure, and abundant fraction, enabling a reliable quantification of preserved PTMs on each of the proteins.
PTMs are extremely sensitive to changes in oxidative stress and changes in pH20,21. Thus, in contrast to previously published methods18, we report an efficient strategy of rinsing the cells in serum-free media to ensure a minimum metabolic disturbance of the cells and to avoid the interference of the native PTMs with serum components. The current protocol not only bypasses the traditional nuclei isolation but also provides optimal times for cell lysis and the exact tissue homogenization procedure that allows for the preservation of the nuclear envelope while avoiding nuclear aggregation. Although the extraction time can be manipulated based on the number of cells, the cell type used, the tissue size, etc., extended lysis is not desirable as it might lead to the lysis of the nuclei and DNA release, making the sample difficult to handle. Importantly, multiple checkpoints within the protocol exist for the validation of successful histone purification (e.g.,&#160;steps 2.7 and 3.2.3). This strategy also facilitates troubleshooting throughout the lengthy procedure.
Another important and unique feature of the presented protocol is its full compatibility with downstream western blot analysis tools and others if so desired. Histone proteins are detected at ~15 kDa13,22,23 and, similarly to other small molecular weight proteins, have been proven challenging to detect by standard immunoblotting techniques. The use of a high-performance and high-throughput transfer system in combination with optimal resolution protein gels allows for the maintenance of protein native confirmation (in the absence of SDS) and activity in the absence of SDS and high transfer efficiency of the low molecular weight histone proteins, thus assuring a reliable histone PTM quantification.

The term epigenetics refers to heritable changes in gene activity that occur independently from changes in the DNA sequence1,2. Gene transcription and repression are determined by (1) the accessibility of the chromosomal DNA wrapped around an octamer of core histone proteins (two copies each of H2A, H2B, H3, and H4) and by (2) the availability of transcription factors and scaffold proteins recruited to specific promoter sites3,4. Gene transcription is regulated by enzyme-mediated modifications of specific DNA promoter sites and the PTMs of histone tails5,6,7. The N-termini of histone H3 and H4 are among the most highly conserved sequences known in eukaryotic organisms3, and their posttranslational modifications have been extensively documented to play a central role in determining chromatin structure and function8,9. PTMs at the histone tails (i.e., acetylation, methylation, phosphorylation, and ubiquitination) change the interaction potential of the tails, influence the structural state and folding of chromatin fiber, and thereby, regulate DNA accessibility and processing4,10,11,12. Acetyl groups are added to and removed from K residues on histone tails by a set of specific histone-interacting epigenetic enzymes, namely HATs and HDACs, respectively13. For example, the acetylation of histone H4 at lysine 12 (H4K12ac) has been previously shown to activate the transcription of genes related to memory acquisition and consolidation14. Additionally, several lines of evidence suggest that the enzyme-mediated epigenetic control of gene transcription is a crucial aspect of healthy cellular growth and differentiation6,15. Alternation in the epigenetic regulation of gene expression, either by epigenetic modifications of DNA or by a mutation of the epigenetic enzymes themselves, has been shown to be dysregulated in human diseases where change in a particular gene activity is a hallmark of the pathology (e.g.,&#160;cancer)6,16,17. Thus, the evaluation of changes in core histone PTMs is emerging as a high-value target for potential therapeutic interventions. However, determining the abundance, interacting partners, and specific roles of histones PTMs has been proven challenging18.
In the current report, an optimized, medium-throughput strategy to purify core histones from cells and brain tissues in a single fraction, and a complete protocol for the quantification of histones H3 and H4 PTMs is described. Of note, although currently published acid-based purification techniques and antibody-based histone detection strategies have been widely adopted for histone characterization, they lack descriptive details regarding critical steps of the procedure, thus hindering quick and replicable histone extraction and quantification. For example, the processing of cell extract and tissue biopsies requires different tools and technologies for successful extraction. Moreover, the optimized protocol presented in the current manuscript demonstrates a practical, medium-throughput approach. Core histones are extracted as a single, pure fraction, which enables reliable downstream antibody-mediated PTM detection without any interference from impurities. Furthermore, in the current manuscript, the challenges regarding histone detection due to their small molecular weight have been circumvented. Typically, the lack of compatibility between purification, quantification, and gel electrophoresis protocols hinder scientists from obtaining replicable and conclusive results. Here, an optimized workflow to purify core histones from cells and tissue and prepare them for downstream PTM analyses via western blot is presented.
The current protocol enables the purification of core histone proteins while preserving their native posttranslational modifications (i.e., acetylation, methylation, and phosphorylation). Figure 1 depicts the timeline of the histone purification protocol.

<table><tbody><tr><td>1.5 mL microcentrifuge tubes</td><td>ThermoFiser Scientific</td><td>05-408-129</td><td>or equivalent from other sources</td></tr><tr><td>Sterile water</td><td>Gibco</td><td>15-230-204</td><td>or equivalent from other sources</td></tr><tr><td>70% perchloric acid</td><td>Sigma Aldrich</td><td>311421</td><td>or equivalent from other sources</td></tr><tr><td>100% acetone</td><td>Sigma Aldrich</td><td>270725</td><td>or equivalent from other sources</td></tr><tr><td>pH-indicator strips, non-bleeding</td><td>Milliipore Sigma</td><td>1095310001</td><td></td></tr><tr><td>4x SDS sample buffer</td><td>BIO-RAD</td><td>161-0747</td><td></td></tr><tr><td>Benchtop rotor</td><td>Cole-Parmer</td><td>UX-04397-34</td><td>or equivalent from other sources</td></tr><tr><td>1.5 mL tube rack</td><td>ThermoFiser Scientific</td><td>05-541</td><td>or equivalent from other sources</td></tr><tr><td>Histone purification mini kit</td><td>Active Motif</td><td>40026</td><td>spin columns included in the kit</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>ThermoFiser Scientific</td><td>78430</td><td>or equivalent from other sources</td></tr><tr><td>Nanodrop instrument</td><td>ThermoFiser Scientific</td><td>ND-2000</td><td></td></tr><tr><td>Tissue culture dishes</td><td>VWR</td><td>10062-880</td><td>required for histone extraction from cultured cells</td></tr><tr><td>Tissue culute media</td><td>varies based on cell line used</td><td>varies based on cell line used</td><td>required for histone extraction from cultured cells</td></tr><tr><td>Low-serum media</td><td>ThermoFiser Scientific</td><td>51985091</td><td>required for histone extraction from cultured cells</td></tr><tr><td>Plastic cell scraper</td><td>Falcon</td><td>353086</td><td>required for histone extraction from cultured cells</td></tr><tr><td>SDS-PAGE gradient gel</td><td>BIO-RAD</td><td>456-9035</td><td>required for histone extraction from cultured cells</td></tr><tr><td>Coomassie Brilliant Blue R-250 Staining Solution</td><td>BIO-RAD</td><td>1610436</td><td>required for histone extraction from cultured cells</td></tr><tr><td>Coomassie Brilliant Blue R-250 Destaining Solution</td><td>BIO-RAD</td><td>1610438</td><td>required for histone extraction from cultured cells</td></tr><tr><td>Trans-Blot Turbo Mini PVDF Transfer Packs</td><td>BIO-RAD</td><td>1704156</td><td>required for histone extraction from cultured cells</td></tr><tr><td>Trans-Blot Turbo Transfer System</td><td>RIO-RAD</td><td>1704150</td><td>required for histone extraction from cultured cells</td></tr><tr><td>Ponceau S stain</td><td>CellSignalling</td><td>59803S</td><td>required for histone extraction from cultured cells</td></tr><tr><td>Dounce homogenizer (size/cap sc 7mL) with a small size clearance</td><td>Kimble Chase</td><td>885302-0007</td><td>required for histone extraction from tissues</td></tr><tr><td>100% bleach</td><td>Clorox</td><td>68973</td><td>required for histone extraction from tissues</td></tr><tr><td>H4K12ac antibody</td><td>Active Motif</td><td>39166</td><td>required for PTMs quantification via WB</td></tr><tr><td>H3K27ac antibody</td><td>Active Motif</td><td>39134</td><td>required for PTMs quantification via WB</td></tr></tbody></table>

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.

To illustrate the progression of the histone purification protocol and composition of all analyzed fractions, we evaluated different histone extracts from human microglial BV2 cells. To demonstrate the quantification of histones H3 and H4 PTMs (i.e.,&#160;acetylation), we used brain tissue lysates.
BV2 cells were plated at 5 x 106 cells per dish in 10 cm tissue culture-treated dishes and allowed to grow to confluency for 48 h. Cells were then collected, and histones were released from chromatin by an incubation in extraction buffer containing 0.4 M sulfuric acid, 1 mM KCl, 1 mM MgCl2, 50 mM Tris-HCl (pH 8.0), and 1x protease inhibitor. The extraction time, between 15 min and 24 h, did not affect the overall composition of crude histone extracts as determined by Coomassie Brilliant Blue staining (Figure 2). Next, crude histones were passed through the equilibrated histone columns and the flow-through was analyzed. High column-binding efficiency is determined by the absence of histone protein in the flow-through when analyzed by Coomassie Brilliant Blue staining. We determined the column-binding efficiency to be 100% as there were no detectable histone proteins present in the analyzed flow-through (Figure 3). All membranes with bound histones were then washed three times with wash buffer to remove any remaining impurities, leaving only histone proteins bound to the silica gel. We determined that, for all histone extraction times (i.e.,15 min, 2 h, and 24 h), the first membrane wash was the most important to remove nonhistone contaminations from the columns, while the second and third washes did not influence the sample purity. Thus, depending on the sample type, the last two washes might be omitted. Following the first elution of histone protein from the column (using elution buffer containing 1mM NaCl and EDTA), histones were precipitated overnight with 4% perchloric acid and then pelleted, washed, and analyzed for the enrichment of purified histones H3 and H4. We observed that 24 h of extraction time increases the amount of H3 and H4 histones in the purified fraction compared to 15 min and 2 h of extraction time (Figure 5A). The second elution from the column did not result in high-quality or high-quantity histones (Figure 5B).
Next, we used brain tissue homogenates to quantify histone H3 and H4 PTMs, namely acetylation. Wild-type (C57BL6/J) male mice were administered a broadly acting HDAC inhibitor (tributyrin) at a dose of 5 g/kg orally for 3 d. Whole-brain tissue was collected on day 4 and crude histones were extracted according to the described protocol. Using an unpaired t-test, we determined that tributyrin increases the acetylation of histones in the crude extract (t(6) = 6.184, P = 0.0004); however, impurities are detected in the extract (the histone bands are not clearly defined). Thus, H4K12ac antibody does not have a high specificity (Figure 6). To further evaluate the applicability of the presented protocol to smaller tissue sections, we collected the prefrontal cortex from triple transgenic Alzheimer&#39;s disease (3 x Tg-AD) mice treated daily with 10 mg/kg M344, a class I and IIb HDAC inhibitor, for four months. Histone purification and precipitation was performed according to the herein described protocol. Using the purified histone H3 and H4 fraction, we determined that M344 increases H4K12 acetylation 2.4-fold (t(6) = 13.03, P &#60; 0.0001), with a high specificity of H4K12ac antibody (Figure 7). Similarly, we observed an increase in histone H3 acetylation in BV2 cells in response to another HDAC inhibitor, namely the selective HDAC3 inhibitor, RGFP-966. 10 &#181;M of RGFP-966 causes an approximately twofold increase of acetylation at histone H3K27 after 24 h of treatment. Student&#39;s unpaired t-test was used to compare control versus treated cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58648/58648fig1.jpg" /> 
Figure 1: Timeline of the histone purification protocol. All steps for histone analyses are shown below along with the estimated time needed for each step. Figures depicting the outcome of particular steps and presented within the manuscript are referred to in parentheses. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58648/58648fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58648/58648fig2.jpg" /> 
Figure 2: Representative Coomassie Brilliant Blue-stained gel demonstrating crude histones extracted from BV2 cells. BV2 cells were cultured for 48 h before the histone extraction protocol began. Crude histones were extracted for 15 min, 2 h, and 24 h, with three replicates for each time point (this is also the case in Figure 3, Figure 4, and Figure 5). Both nonhistone and histone proteins are present in the crude histone extract. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58648/58648fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58648/58648fig3.jpg" /> 
Figure 3: Representative Coomassie Brilliant Blue-stained gel demonstrating column flow-through following histone purification steps from BV2 cells. Following crude histone passing through the histone binding column, only nonhistone proteins are present in the flow-through. Histone proteins are absent in this fraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58648/58648fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58648/58648fig4.jpg" /> 
Figure 4: Representative Coomassie Brilliant Blue-stained gel demonstrating a column wash following a histone purification step from BV2 cells. Regardless of the histone extraction times, which were (A) 15 min, (B) 2 h, or (C) 24 h, low quantities of nonhistone proteins were only present in the first-wash histonebinding column. Histone proteins were absent in all washes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58648/58648fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58648/58648fig5.jpg" /> 
Figure 5: Representative Coomassie Brilliant Blue-stained gel demonstrating elutions following a histone purification step from BV2 cells. (A) High-quality purified and desalted histone H3 and H4 were detected after the first elution from the histone purification column. (B) The second elution from the histone purification column did not yield a high quality or quantity of histones H3 or H4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58648/58648fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58648/58648fig6.jpg" /> 
Figure 6: The broadly acting HDAC inhibitor, tributyrin, increases H4K12 acetylation in the whole brain of wild-type mice. (A) This panel shows a representative western blot depicting an increase of H4K12 acetylation in the crude histone extract collected from the whole brain of wild-type mice in response to the broadly acting HDAC inhibitor, tributyrin. (B) This panel shows the quantification of the increase in H4K12 acetylation in vivo. Unpaired t-test was used to compare groups (t(6) = 6.076, P = 0.0005). The bars represent the mean &#177; the standard error of the mean (SEM). N = 8. ***P &#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58648/58648fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58648/58648fig7.jpg" /> 
Figure 7: The class I and IIb HDAC inhibitor, M344, increases H4K12 acetylation in the prefrontal cortex of triple transgenic Alzheimer&#39;s disease (3 x Tg-AD) mice. (A) This panel shows a representative western blot depicting an increase of H4K12 acetylation in the purified and desalted histone extract collected from the prefrontal cortex of 3 x Tg-AD mice in response to the inhibition of HDACs by M344. (B) This panel shows the quantification of the increase of H4K12 acetylation in response to M344 administered at a 10 mg/kg daily dose for four months. Unpaired t-test was used to compare groups (t(6) = 13.30, P &#60; 0.0001). The bars represent the mean &#177; the SEM. N = 8. ****P &#60; 0.00001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58648/58648fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58648/58648fig8.jpg" /> 
Figure 8: The selective HDAC3 inhibitor, RGFP-966, increases H3K27 acetylation in BV2 microglial cells. (A) This panel shows a representative western blot depicting an increase of H3K27 acetylation in the purified and desalted histone extract collected from BV2 cells in response to the HDAC3 inhibition by RGFP-966. (B) RGFP-966 causes an approximately twofold increase of acetylation at histone H3 and lysine (K) 27 after 24 h of treatment. Unpaired t-test was used to compare control versus treated cells (t(4) = 5.981, P = 0.002). The bars represent the mean &#177; the SEM. N = 6. **P &#60; 0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58648/58648fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Representative tissue (one hemisphere) *</td>
			<td>Average tissue weight (mg)</td>
			<td>Extraction Buffer (mL)</td>
			<td>Number of strokes</td>
		</tr>
		<tr>
			<td>Mouse cerebellum</td>
			<td>40</td>
			<td>1</td>
			<td>40</td>
		</tr>
		<tr>
			<td>Mouse frontal cortex</td>
			<td>30</td>
			<td>0.3</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Mouse hippocampus</td>
			<td>27</td>
			<td>0.3</td>
			<td>18</td>
		</tr>
		<tr>
			<td>Mouse entorhinal cortex</td>
			<td>19</td>
			<td>0.3</td>
			<td>17</td>
		</tr>
		<tr>
			<td colspan="3">* All experiments were performed on adult male mice.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Average age: 16 months. Average weight: 30 grams.</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Optimized condition for brain tissue homogenization.

Watch this Scientific Journal Video about Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue at JoVE.com

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

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 ...

purification-h3-h4-histone-proteins-quantification-acetylated-histone

Nanyang Technological University

Research

JoVE Journal

Neuroscience

21.3K Views.  University of Miami Miller School of Medicine. Deze methode maakt nauwkeurige kwantificering van de kern histonen H3 en H4 en hun post-translationele wijzigingen uit cel-en weefselextracten die bijgevolg leiden tot het genereren van hoge kwaliteit, reproduceerbare resultaten. De gepresenteerde techniek heeft drie belangrijke voordelen. Ten eerste behoudt het inheemse post-translationele wijzigingen van histonen.Ten tweede omzeilt het dure methoden voor lage doorvoer. Ten slotte is het volledig compatibel met downstream medium-throu...

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

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Biology

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

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.

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Medicine

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

Cancer Research

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 ...

Chemistry

Development of a Click-Based Assay for Screening HAT1 Enzyme Activity

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

Biochemistry

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 ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

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 ...

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 ...

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

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

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.

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

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

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