Name: Identification of Modified Histones as Binding Substrates of Human Spindlin Family Member 4 (SPIN4) by Peptide Arrays and Native Nucleosome Pulldown (Video) | JoVE Journal
Uploaded: 2026-03-27T14:40:00.000+00:00
Duration: 9 min 34 s
Description: Learn to identify histone binding substrates of SPIN4 using peptide arrays and native nucleosome pulldown assays. This protocol enables rapid characterization of epigenetic reader protein interactions.

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The authors thank all past and current members of the Yu and Jee Lab for their technical support. J.-R.Y. was supported by NIH NIGMS Maximizing Investigators&#8217; Research Award (MIRA) R35GM160046.&#160;YH.J was supported by the Children&#8217;s National Hospital and the Children&#8217;s Research Institute.

<ol><li>Lui, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss-of-function+variant+in+SPIN4+causes+an+X-linked+overgrowth+syndrome%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Loss-of-function variant in SPIN4 causes an X-linked overgrowth syndrome.</a> JCI Insight. 8 (9), e167074(2023).</li>
<li>Chawla, N., Rajput, M., Shriyan, R., Bachani, S., Gupta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+observation+of+a+rare+Binder+phenotype+with+fetal+overgrowth+due+to+a+SPIN4+mutation%5BTitle%5D)+AND+%22Matern+Fetal+Med%22%5BJournal%5D)">Clinical observation of a rare Binder phenotype with fetal overgrowth due to a SPIN4 mutation.</a> Matern Fetal Med. 7 (1), e0030(2025).</li>
<li>Lu, R., Wang, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tudor%3A+a+versatile+family+of+histone+methylation+readers%5BTitle%5D)+AND+%22Trends+Biochem+Sci%22%5BJournal%5D)">Tudor: a versatile family of histone methylation readers.</a> Trends Biochem Sci. 38 (11), 546-555 (2013).</li>
<li>Ruthenburg, A. J., Li, H., Patel, D. J., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multivalent+engagement+of+chromatin+modifications+by+linked+binding+modules%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Multivalent engagement of chromatin modifications by linked binding modules.</a> Nat Rev Mol Cell Biol. 8 (12), 983-994 (2007).</li>
<li>Mauser, R., Jeltsch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+modified+histone+peptide+arrays+in+chromatin+research%5BTitle%5D)+AND+%22Arch.+Biochem+Biophys%22%5BJournal%5D)">Application of modified histone peptide arrays in chromatin research.</a> Arch. Biochem Biophys. 661, 31-38 (2019).</li>
<li>Hsu, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paraspeckle+protein+NONO+regulates+active+chromatin+by+allosterically+stimulating+NSD1%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Paraspeckle protein NONO regulates active chromatin by allosterically stimulating NSD1.</a> Cell Rep. 44 (1), 116247(2025).</li>
<li>Hsu, C. I., Yeh, E., Chen, C. C. L., Sahn, M., Yu, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+for+reconstituting+enzymatic+activities+for+ultra-large+histone+methyltransferases+NSD1+and+SETD2+using+a+baculovirus+expression+system%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Protocol for reconstituting enzymatic activities for ultra-large histone methyltransferases NSD1 and SETD2 using a baculovirus expression system.</a> STAR Protoc. 6 (1), 103963(2025).</li>
<li>Plazas-Mayorca, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One-pot+shotgun+quantitative+mass+spectrometry+characterization+of+histones%5BTitle%5D)+AND+%22J+Proteome+Res%22%5BJournal%5D)">One-pot shotgun quantitative mass spectrometry characterization of histones.</a> J Proteome Res. 8 (11), 5367-5374 (2009).</li>
<li>Fuhrmann, J., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+arginine+methylation+and+citrullination+in+epigenetic+regulation%5BTitle%5D)+AND+%22ACS+Chem+Biol%22%5BJournal%5D)">Protein arginine methylation and citrullination in epigenetic regulation.</a> ACS Chem Biol. 11, 654(2015).</li>
<li>Zhao, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+basis+for+histone+H3+%E2%80%9CK4me3-K9me3%2F2%E2%80%9D+methylation+pattern+readout+by+Spindlin1%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Molecular basis for histone H3 “K4me3-K9me3/2” methylation pattern readout by Spindlin1.</a> J Biol Chem. 295 (49), 16877(2021).</li>
<li>Padeken, J., Methot, S. P., Gasser, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+H3K9-methylated+heterochromatin+and+its+functions+in+tissue+differentiation+and+maintenance%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Establishment of H3K9-methylated heterochromatin and its functions in tissue differentiation and maintenance.</a> Nat Rev Mol Cell Biol. 23 (9), 623-640 (2022).</li>
<li>Greschik, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+histone+code+reader+Spin1+controls+skeletal+muscle+development%5BTitle%5D)+AND+%22Cell+Death+Dis%22%5BJournal%5D)">The histone code reader Spin1 controls skeletal muscle development.</a> Cell Death Dis. 8 (11), e3173(2017).</li>
</ol>

The authors declare no conflicts of interest.

A time- and cost-effective method for biochemically pinpointing specific histone PTMs as targets of a poorly characterized protein, and for assessing how its disease-associated mutation affects the target&#39;s binding affinity, was presented. The advantage of this method is that it requires no expensive or time-consuming reconstitution of synthetic nucleosomes. This approach leveraged the convenience of commercialized histone PTM peptide arrays while ensuring that SPIN4 is a bona fide H3K4me reader by using native monon...

Spindlin Family Member 4 (SPIN4) is an epigenetic reader and has been recently implicated in overgrowth syndrome, presenting with a constellation of pre- and postnatal overgrowth and tall stature1,2. Spin4 knockout (KO) mice recapitulated the exact human phenotype, supporting the causality. To date, SPIN4 is the only identified exclusive epigenetic reader that negatively regulates bone growth. However, the biochemical properties of SPIN4, a critical epigenetic reader for growth, have not been characterized, leaving a critical knowledge gap in understanding the fundamental, disease-causing mechanisms in this novel developmental disorder. SPIN4 contains three tandem repeats of Tudor domains, which are generally considered methylated arginine or lysine histone reader protein domains3. A SPIN4D82H missense variant has recently been identified in a patient with prenatal and postnatal bone overgrowth syndrome. This mutation affects the first Tudor domain of SPIN4; however, the relevant binding substrates of these Tudor domains remain unclear, limiting functional dissection of SPIN4-dependent regulation of chromatin and gene expression during bone development.
Identification of new histone-binding substrates of reader protein domains does not rely on a single, standardized approach. Post-translational modifications (PTMs) of histone mostly occur at histone tails that protrude outwards from the core nucleosome particles. The recognition of PTMs on histone tails by reader protein domains is highly variable and often requires multivalent, electrostatic interactions involving nucleosomal DNA, linker DNA, and/or other histone domains to collectively stabilize the reader-target interaction4. Currently, PTMs of histone tail peptides have been constructed in the form of microarrays and commercialized by several vendors5. While histone tail peptide-based PTM screening for binding targets of reader protein domains is high-throughput, it often yields false-positive hits in the absence of nucleosomes. Further, the experimental conditions for such screens are highly empirical and vary from one protein to another, making them technically difficult to address. Synthetic nucleosome-based PTM screening or pulldown provides a much more robust readout, but it is much more costly and low-throughput, since reconstituting nucleosomes carrying variable combinations of PTMs is inherently challenging.
This protocol describes a rapid approach for identifying histone PTMs in nucleosomes as binding targets of SPIN4. Recombinant SPIN4 was reconstituted and screened against a commercial histone PTM peptide array to identify potential binding targets. Native mono-nucleosomes containing diverse endogenous histone PTMs were subsequently generated from live cells. SPIN4 pulldown of mono-nucleosomes enabled antibody-based targeted validation of histone PTMs following the initial screening. This integrated biochemical strategy demonstrates a proof-of-principle approach for time- and cost-efficient target discovery and can be readily applied to identify binding substrates of other poorly characterized epigenetic reader proteins.

<table><tbody><tr><td>1.5 mL Microcentrifuge Tubes</td><td>Sarstedt</td><td>72.690.001</td><td>General experiments</td></tr><tr><td>15 cm Tissue Culture Dish</td><td>Sarstedt</td><td>83.3903.300</td><td>General experiments</td></tr><tr><td>15 mL Conical Centrifuge Tubes</td><td>Sarstedt</td><td>62.554.101</td><td>General experiments</td></tr><tr><td>50 mL Conical Centrifuge Tubes</td><td>Sarstedt</td><td>62.548.004</td><td>General experiments</td></tr><tr><td>500 mL Erlenmeyer Flask</td><td>Stellar Scientific</td><td>CT229809</td><td>Sf9 cell culture</td></tr><tr><td>Amersham ImageQuant 800 CCD Imager</td><td>Cytiva</td><td>IMQ800</td><td>CCD imaging system</td></tr><tr><td>Anti-FLAG M2 Affinity Gel</td><td>Sigma-Aldrich</td><td>A2220</td><td>Biochemical pulldown</td></tr><tr><td>Anti-FLAG M2 antibody</td><td>Sigma-Aldrich</td><td>F1804</td><td>Western blot</td></tr><tr><td>Aprotinin</td><td>MedChemExpress</td><td>HY-P0017</td><td>Protease inhibitor</td></tr><tr><td>Array Analyze Sofrware</td><td>Active Motif</td><td>https://www.activemotif.com/documents/Array_Analyze_Software_v16.1.zip</td><td>MODified peptide array data analysis</td></tr><tr><td>ASI Agarose LE</td><td>Alkali Scientific</td><td>A7700</td><td>DNA gel</td></tr><tr><td>Bac-to-Bac Baculovirus Expression System</td><td>Thermo Scientific</td><td>10359016</td><td>Baculoviral expression</td></tr><tr><td>Benzamidine hydrochloride</td><td>MedChemExpress</td><td>HY-W018781</td><td>Protease inhibitor</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A9647</td><td>Protein quantification standard</td></tr><tr><td>Bromophenol Blue</td><td>Thermo Scientific</td><td>A18469.08</td><td>Lammeli buffer</td></tr><tr><td>Coomassie Brilliant Blue R-250</td><td>Thermo Scientific</td><td>20278</td><td>Protien gel staining</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D5545</td><td>Lysis buffer</td></tr><tr><td>EDTA</td><td>RPI research products</td><td>E58100</td><td>Lysis buffer</td></tr><tr><td>Enhanced Chemiluminescence (ECL) substrate</td><td>Cytiva</td><td>RPN2232</td><td>Western blot</td></tr><tr><td>ESF 921 Insect Cell Culture Medium</td><td>Expression Systems</td><td>96-001</td><td>Sf9 cell culture</td></tr><tr><td>Ethanol (200 proof)</td><td>Spectrum Chemical</td><td>ET107</td><td>DNA precipitation</td></tr><tr><td>EVOS M5000 Imaging System</td><td>Invitrogen</td><td>M5000</td><td>General imaging</td></tr><tr><td>FLAG peptide</td><td>MedChemExpress</td><td>HY-P7006</td><td>Pulldown elution</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G77893</td><td>Storage buffer</td></tr><tr><td>H3K4me1 antibody&amp;nbsp;</td><td>Active Motif</td><td>39300</td><td>Western blot</td></tr><tr><td>H3K4me2 antibody&amp;nbsp;</td><td>Cell Signaling</td><td>9725T</td><td>Western blot</td></tr><tr><td>H3K4me3 antibody&amp;nbsp;</td><td>Cell Signaling</td><td>9751T</td><td>Western blot</td></tr><tr><td>HEK293FT cells</td><td>Thermo Scientific</td><td>R70007</td><td>Human embryonic kidney fibroblastcell line</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich&amp;nbsp;</td><td>H3375</td><td>Lysis buffer</td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td>https://imagej.net/ij/download.html</td><td>&amp;nbsp;Java-based image processing program</td></tr><tr><td>Leupeptin hemisulfate</td><td>MedChemExpress</td><td>HY-18234A</td><td>Protease inhibitor</td></tr><tr><td>Micrococcal Nuclease (MNase)</td><td>NEB&amp;nbsp;</td><td>M0247S</td><td>Endonuclease for chromatin digestion</td></tr><tr><td>Model 120 Sonic Dismembrator</td><td>Fisher Scientific</td><td>FB120110</td><td>Sonication</td></tr><tr><td>MODified Histone Peptide Array</td><td>Active Motif</td><td>13005</td><td>Protein binding assay</td></tr><tr><td>Pepstatin A</td><td>MedChemExpress</td><td>HY-P0018</td><td>Protease inhibitor</td></tr><tr><td>Peroxidase IgG Fraction Monoclonal Goat Anti-Mouse IgG, light chain specific</td><td>Jackson ImmunoResearch</td><td>115-035-174</td><td>Western blot</td></tr><tr><td>pFastBac1-Flag-SPIN4</td><td>Thermo Scientific</td><td>10360014</td><td>Baculoviral expression vector</td></tr><tr><td>pFastBac1-Flag-SPIN4-D82H</td><td>Thermo Scientific</td><td>10360014</td><td>Baculoviral expression vector</td></tr><tr><td>Phenol:Chloroform:Isoamyl Alcohol (25:24:1)</td><td>Sigma-Aldrich</td><td>P2069</td><td>DNA purification</td></tr><tr><td>Pierce Rapid Gold BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>PIA53226</td><td>Protein quantificaiton</td></tr><tr><td>Pierce Silver Stain Kit</td><td>Thermo Scientific</td><td>24612</td><td>Protein detection</td></tr><tr><td>PMSF Protease Inhibitor</td><td>Thermo Scientific</td><td>36978</td><td>Protease inhibitor</td></tr><tr><td>Polyacrylamide Gel</td><td>Made in-house</td><td>N/A</td><td>Protein gel</td></tr><tr><td>Sf9 insect cells</td><td>ATCC</td><td>CRL-1711</td><td>Insect cell line for baculovirus expression</td></tr><tr><td>Slide-A-Lyzer MINI Dialysis Device, 10K MWCO</td><td>Thermo Scientific</td><td>69572</td><td>Protein dialysis</td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S8750</td><td>DNA precipitation</td></tr><tr><td>Sodium Chloride&amp;nbsp;</td><td>Fischer&amp;nbsp;</td><td>BP358-1</td><td>General buffers</td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L3771</td><td>General buffers</td></tr><tr><td>Trypan Blue Solution (0.4%)</td><td>Thermo Scientific</td><td>15250061</td><td>Nuclei staining</td></tr></tbody></table>

identification of modified histones as binding substrates of human spindlin family member 4 (spin4) by peptide arrays and native nucleosome pulldown

Identification of Modified Histones as Binding Substrates of Human Spindlin Family Member 4 (SPIN4) by Peptide Arrays and Native Nucleosome Pulldown

Human Spindlin Family Member 4 (SPIN4) has just emerged as a novel regulator of bone growth. Loss-of-function variants in SPIN4 were identified in patients exhibiting a pre- and post-natal overgrowth syndrome, which is inherited in an X-linked dominant fashion. SPIN4 contains three tandem repeats of Tudor domains, each forming an individual aromatic cage. These Tudor domains are considered epigenetic reader domains for methylated arginine and lysine residues of histones based on sequence homology. However, the exact binding substrates of SPIN4 remain unclear. Human SPIN4, in both its wild-type and disease-causing mutant forms, was successfully generated via an insect cell expression system. By a histone modification peptide array, the differential binding affinities between wild-type and mutant SPIN4 were determined. Functional validations further demonstrated that SPIN4 is a bona fide reader of histone H3 Lysine 4 methylation (H3K4me), as evidenced by biochemical pulldown of native mononucleosomes. This integrated approach facilitates a rapid binding target identification of epigenetic reader proteins.

To investigate the binding substrates of human SPIN4, wild-type SPIN4 and a patient-derived mutant associated with overgrowth syndrome were reconstituted using a Sf9 insect cell expression system. Specifically, this is an aspartic acid (D) to histidine (H) missense mutation at residue 82 (D82H), which resides within the first Tudor domain of SPIN4, implying its role in regulating the substrate binding of SPIN4. A baculoviral vector encoding a single FLAG-tagged SPIN4 was used for protein expression in Sf9 cells, followed...

Genetic variants in human Spindlin Family Member 4 (SPIN4) have recently been identified in patients with bone overgrowth, a novel developmental disorder. A protocol and new results describing the biochemical identification of modified histones in the context of nucleosomes as SPIN4 binding substrates are presented.

Human Spindlin Family Member 4 (SPIN4) has just emerged as a novel regulator of bone growth. Loss-of-function variants in SPIN4 were identified in ...

SPIN4 is a bone development regulating histone reader. SPIN4 mutations cause overgrowth, but its pathogenic role remains unclear. This protocol can be adapted and applied to study binding substrates of histone-binding proteins across all spaces.To begin, prepare the blocking buffer by weighing five grams of bovine serum albumin, or BSA, and dissolving it into 100 milliliters of BC-150 protein storage buffer. Next, use a hydrophobic pen to outline the reaction area on the peptide array to conserve the reagents. Then place filter papers in a 15 centimeter Petri dish and moisten them with water.Position the arrays on moistened filter papers and add 300 microliters of blocking buffer to each array area carefully. Cover and incubate the Petri dish at room temperature for one to three hours. Prepare one micromolar of recombinant FLAG tagged Spindlin-4, or FLAG SPIN4 protein, and 1X FLAG peptide control, each in blocking buffer.Transfer the reagents and array dish to a cold room to reduce background and improve the signal to noise ratio. Remove the excess blocking buffer from the arrays. Add the FLAG SPIN4 protein, or FLAG peptide control, onto each block.Cover the Petri dish for overnight incubation in the cold room. Add 50 milliliters of peptide array wash buffer to a wash box. Discard the protein solution from the arrays and transfer them into the wash buffer with the reaction side up.Wash the arrays five times. Perform the first two washes for one minute each, followed by three subsequent washes of five minutes each. Then rinse with blocking buffer to remove residual detergents.Place the arrays back into the Petri dish. Add 300 microliters of anti-FLAG antibody to each block and incubate for one hour at room temperature. Discard the antibody solution, transfer the arrays to the wash buffer, and repeat the washes as demonstrated previously.Add 300 microliters of HRP-conjugated goat anti-mouse IgG secondary antibody solution to each block and incubate for one hour at room temperature. Discard the antibody solution and wash the array five times as demonstrated previously. Place the arrays inside a transparent sheet protector and add one milliliter of enhanced chemiluminescence, or ECL substrate, ensuring even coverage.Remove trapped air bubbles. Capture chemiluminescent in bright-field images using a digital imager. After uploading the raw data file to the MODified Histone Peptide Array Analysis software, in the Spot Alignment tab, confirm each grid is centered on its peptide spot.In Spot Statistics, check duplicate spot correlation and ensure that the error is less than 5%Use the Reactivity tab to assess binding across various histone modifications. Then use the Specificity tab to determine the target peptide's relative binding preference among different histone modifications. To prepare 2X bead blocking buffer, add one gram of BSA into 10 milliliters of BC-150 lysis buffer and vortex to dissolve.Transfer FLAG SPIN4 wild-type and the FLAG SPIN4 D82H mutant beads containing equimolar concentrations into separate 1.5 milliliter tubes. To the wild-type sample, add an equal volume of 100 millimolar 1X FLAG peptide negative control beads. Adjust all samples to uniform volumes using an appropriate amount of BC-150 lysis buffer.Add an equal volume of 2X bead blocking buffer to each tube to make up the final volume to one milliliter. Incubate overnight on a tube rotator set at four degrees Celsius and 20 RPM. To initiate nucleosome binding, thaw 1X mononucleosomes extracted from HEK293FT cells on ice.For initial optimization, calculate one in one molar ratio of 1X nucleosome to SPIN4 protein. Mix 1X nucleosome with an equal volume of 2X bead blocking buffer. Then combine the mixture with pre-blocked beads in a 15 milliliter tube and incubate for four hours on a tube rotator at four degrees Celsius and 20 RPM for complexation.Centrifuge the mixture at 3000G for three minutes at four degrees Celsius. After discarding the supernatant, resuspend the beads in one milliliter of BC-150 lysis buffer and transfer to 1.5 milliliter tubes. To wash the beads three times, centrifuge at 3000G for one minute.After discarding the supernatant, resuspend the pellet in one milliliter of BC-150 lysis buffer. Agitate the tube occasionally for uniform distribution of beads between repeat washes. Then perform two additional washes, incorporating a 10-minute incubation step on a rotator at 20 RPM and four degrees Celsius between the resuspension and centrifugation steps.After the final wash, resuspend the beads in nine column volumes of BC-150 lysis buffer. Mix 160 microliters of beads from each sample with 40 microliters of 5X Laemmli buffer. Heat at 95 degrees Celsius for five minutes for denaturation.Optionally, to obtain cleaner results with reduced background, perform a FLAG peptide illusion of the protein nucleosome complex. Centrifuge at 3000G for five minutes at room temperature. Then perform silver staining and Western blotting to validate and detect the modified histone SPIN4 binding.SPIN4 significantly binds to histone H3 Lysine 4, or H3K4, monomethyl, dimethyl, and trimethyl peptides. Conversely, the SPIN4 D82H loss of function mutant showed reduced binding to all targets, confirming that Tudor domain 1 is essential for substrate binding. The binding affinity of SPIN4 is negatively impacted by other post-translational modifications insist confirmations, such as H3R2me2s and H3R2me2a.Consistent peptide array observations, SPIN4 D82H exhibited an overall reduction of nucleosome binding relative to wild-type SPIN4. Western blot analysis comparing the pulldown samples of SPIN4 and SPIN4 D82H further showed no quantifiable binding of SPIN4 D82H to monomethyl, dimethyl, or trimethyl H3K4 peptides, validating these dual approaches for rapidly identifying relative histone post-translational modification substrates. Fast and targeted validation of the binding substrates of histone binding proteins.The conditions required for expression and purification of each candidate's histone-binding proteins may vary. Histone mass spectrometry following native mononucleosome pulldown will give the full scope of histone PTMs.

identification-modified-histones-as-binding-substrates-human-spindlin

Research

JoVE Journal

Biochemistry

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Quantitative Analysis of Chromatin Proteomes in Disease

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.

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

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Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

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

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

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Analysis of Histone Antibody Specificity with Peptide Microarrays

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

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

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

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Enrichment and sequencing of protein-associated nascent DNA (eSPAN) was developed to detect the relative abundance of a chromatin-associated protein on two replicating DNA strands, thereby revealing molecular insight into chromatin replication and its coupled processes. This protocol describes eSPAN procedures in yeast and mouse embryonic stem (ES) cells.

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Learn to identify histone binding substrates of SPIN4 using peptide arrays and native nucleosome pulldown assays. This protocol enables rapid characterization of epigenetic reader protein interactions.

Identification of Modified Histones as Binding Substrates of Human Spindlin Family Member 4 (SPIN4) by Peptide Arrays and Native Nucleosome Pulldown (Video) | JoVE Journal

Identification of Modified Histones as Binding Substrates of Human Spindlin Family Member 4 (SPIN4) by Peptide Arrays and Native Nucleosome Pulldown

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