Journal: The Journal of Biological Chemistry
Article Title: HMGN1 and HMGN2 are recruited to acetylated and histone variant H2A.Z-containing nucleosomes to regulate chromatin state and transcription
doi: 10.1016/j.jbc.2025.110997
Figure Lengend Snippet: HMGN1 and HMGN2 preferentially bind to nucleosomes containing H2A.Z and acetylated histone tails . A , titration of GST-HMGN1 protein with each nucleosome-bead conjugate, expressed as relative fluorescence units before normalization. B , titration of GST-HMGN2 protein with each nucleosome-bead conjugate, expressed as relative fluorescence units before normalization. One outlier data point was excluded from the H2A.Z variant at the 5 nM protein concentration. C , GST-HMGN1 binding relative to the canonical nucleosome with background subtracted. Background signal captured by negative control bead conjugates (average signal of 50 mM BSA-bead, 100 mM BSA-bead, and 200 mM BSA-bead conjugates) and wells containing 0 mM GST-HMGN1 protein were subtracted from raw values for each nucleosome-bead conjugate at 0.625 nM GST-HMGN1 concentration. A t test was used to assess statistical significance, with one asterisk (∗) denoting a p value less than 0.05, ∗∗ indicating a p value less than 0.01, and ∗∗∗ representing a p value less than 0.001. Error bars represent the standard deviation calculated from three technical replicates. D , GST-HMGN2 binding relative to the canonical nucleosome with background subtracted. Background signal captured by negative control bead conjugates (average signal of 50 mM BSA-bead, 100 mM BSA-bead, and 200 mM BSA-bead conjugates) and wells containing 0 mM GST-HMGN2 protein were subtracted from raw values for each nucleosome-bead conjugate at 0.625 nM GST-HMGN2 concentration. A t test was used to assess statistical significance, with one asterisk (∗) denoting a p value less than 0.05, ∗∗ indicating a p value less than 0.01, and ∗∗∗ representing a p value less than 0.001. Error bars represent the standard deviation calculated from three technical replicates. E , GST-HMGN1ΔC binding relative to the canonical nucleosome with background subtracted. Background signal captured by negative control bead conjugates (average signal of 50 mM BSA-bead, 100 mM BSA-bead, and 200 mM BSA-bead conjugates) and wells containing 0 mM GST-HMGN1ΔC protein were subtracted from raw values for each nucleosome-bead conjugate at 0.625 nM GST-HMGN1ΔC concentration. A t test was used to assess statistical significance, with one asterisk (∗) denoting a p value less than 0.05, ∗∗ indicating a p value less than 0.01, and ∗∗∗ representing a p value less than 0.001. Error bars represent the standard deviation calculated from three technical replicates. F , GST-HMGN2ΔC binding relative to the canonical nucleosome with background subtracted. Background signal captured by negative control bead conjugates (average signal of 50 mM BSA-bead, 100 mM BSA-bead, and 200 mM BSA-bead conjugates) and wells containing 0 mM GST-HMGN2ΔC protein were subtracted from raw values for each nucleosome-bead conjugate at 0.625 nM GST-HMGN2ΔC concentration. A t test was used to assess statistical significance, with one asterisk (∗) denoting a p value less than 0.05, ∗∗ indicating a p value less than 0.01, and ∗∗∗ representing a p value less than 0.001. Error bars represent the standard deviation calculated from three technical replicates. G , bar graph of normalized GST-HMGN1ΔC nucleosome binding data over GST-HMGN1 nucleosome binding data relative to unmodified H3.1 mononucleosome-bead conjugate. H , bar graph of normalized GST-HMGN2ΔC nucleosome binding data over GST-HMGN2 nucleosome binding data relative to unmodified H3.1 mononucleosome-bead conjugate. BSA, bovine serum albumin; GST, glutathione- S -transferase; HMGN, High Mobility Nucleosome-binding protein.
Article Snippet: Optimal p300 enzyme concentration for endpoint analysis was identified through an enzyme titration (0–500 nM) against 300 nM recombinant human H3.1 mononucleosome (Epicypher, 16-0006) at 37 °C for 30 min. Nucleosome substrate, purified HMGN protein, and HAT buffer were first incubated for 30 min on ice prior to the addition of purified p300 to allow for HMGN–nucleosome complexes to form.
Techniques: Titration, Fluorescence, Variant Assay, Protein Concentration, Binding Assay, Negative Control, Concentration Assay, Standard Deviation
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