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

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.