Immunoprecipitation of NPAS4–NuA4 complexes

To isolate protein complexes associated with NPAS4, 8–12 hippocampi were isolated from 6-week-old Npas4–Flag-HA and Arnt2–Flag-HA mice injected with 15 mg kg^–1 KA (Sigma-Aldrich, K0250) to induce high levels of NPAS4 expression. Males and females were used in equal proportions in pooled replicate samples. Wild-type mice of the same age and sex distributions were processed in parallel to serve as controls. Hippocampal regions were dissected 3 h after KA injection to allow sufficient time for NPAS4 expression and assembly into potential complexes. Hippocampi were collected and dounced 20× in 10 ml of NE1 buffer (20 mM HEPES pH 7.9, 10 mM KCl, 3 mM MgCl₂, 0.1% Triton and 0.1 mM EDTA) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, P5726 and P0044). Nuclei were released through 10 min of incubation in NE1 buffer and then pelleted by gentle centrifugation (1,000–1,500g). Nuclear pellets were resuspended in 1 packed nuclear volume of NE1 buffer. To facilitate release of chromatin-associated proteins, nuclei were incubated for 30 min at 4 °C with benzonase endonuclease (Sigma-Aldrich, E1014, >25 KU (1 µl per 10 million nuclei)) followed by the addition of NaCl to a final concentration of 300 mM. Following high-speed centrifugation to remove insoluble material, lysates were diluted to achieve a final salt concentration of 200 mM. To preclear nonspecific interactions, lysates were incubated for 30 min at 4 °C with 50 µl of ms-IgG-coated agarose beads (Sigma-Aldrich, A0919). Following preclear, lysates were incubated for 1.5 h with 60 µl of anti-M2-Flag resin (Sigma-Aldrich, A2220) per 1 ml of diluted lysate. Samples were washed 4× in NE1 buffer containing 250 mM NaCl for 5 min with rotation at 4 °C. NPAS4-interacting or ARNT2-interacting proteins were competitively eluted off M2 resin by incubation in 50–100 µl of 500 µg ml^–1 3× Flag peptide (Sigma-Aldrich, F4799) diluted in NE1 buffer for 30 min at room temperature. For mass spectrometry analysis, eluted proteins were precipitated with trichloroacetic acid. Replicates shown in Fig. ​Fig.1c1c consisted of 3 independent pools of 8–12 mice collected from wild-type or Npas4–FH lines and processed on separate days. Validation immunoprecipitation assays using either Npas4–Flag-HA mice or Tip60-H3F mice followed by immunoblotting analysis were conducted under the same conditions as described above. For validation experiments in mouse visual cortex following light stimulation, 20 hippocampi were isolated from the V1 cortices of Npas4–Flag-HA or wild-type controls. Mice were housed in the dark for 1 week followed by 2 h of light stimulation. Data are shown in Extended Data Fig. ​Fig.1f1f.

To isolate high molecular weight complexes containing intact NPAS4–NuA4 for mass spectrometry, 8–10 6-week-old Npas4–Flag-HA, Tip60-H3F and wild-type control mice were injected with KA and dissected 2 h after injection. Males and females were used in equal proportions in pooled replicate samples. Replicates consisted of independent pools of 8–10 mice run on independent gradients and days. Two replicates were performed. Whole forebrain, minus hippocampus and striatum, were minced and transferred NE1 buffer, and nuclear lysates were prepared as described above. Approximately 6 ml gradients were prepared using a BIOCOMP Gradient Master 108 (Science Services) using a 10–40% long glycerol gradient for the SW41 Beckman rotor. Sample concentrations were normalized to around 2 mg ml^–1, and 1 ml was loaded on top of the 10–40% glycerol gradient. Samples were centrifuged for 16 h at 37,000 r.p.m. at 4 °C. One millilitre fractions were collected at 4 °C. Fractions were run on SDS–PAGE gels to assess which fractions contained components of the NuA4 complex. Immunoprecipitation assays were then performed from fractions 1–3, which contained the majority of TRRAP and EP400 in the lysates. To preclear nonspecific interactions from fractions, lysates were incubated for 30 min at 4 °C with 100 µl of ms-IgG-coated agarose beads (Sigma-Aldrich, A0919). Following preclear, lysates were incubated for 1.5 h with 100 µl of anti-M2-Flag resin (Sigma-Aldrich, A2220) per 1 ml fraction. Samples were washed 4× in NE1 containing 250 mM NaCl for 5 min with rotation at 4 °C. Interacting proteins were eluted off M2 resin by incubation in 500 µg ml^–1 3× Flag peptide (Sigma-Aldrich, F4799). Eluates from immunoprecipitation assays performed on fractions 1–3 were pooled to have sufficient material for mass spectrometry, and eluted proteins were precipitated with trichloroacetic acid.