A NPAS4–NuA4 complex couples synaptic activity to DNA repair

Neuronal activity is crucial for adaptive circuit remodelling but poses an inherent risk to the stability of the genome across the long lifespan of postmitotic neurons1–5. Whether neurons have acquired specialized genome protection mechanisms that enable them to withstand decades of potentially damaging stimuli during periods of heightened activity is unknown. Here we identify an activity-dependent DNA repair mechanism in which a new form of the NuA4–TIP60 chromatin modifier assembles in activated neurons around the inducible, neuronal-specific transcription factor NPAS4. We purify this complex from the brain and demonstrate its functions in eliciting activity-dependent changes to neuronal transcriptomes and circuitry. By characterizing the landscape of activity-induced DNA double-strand breaks in the brain, we show that NPAS4–NuA4 binds to recurrently damaged regulatory elements and recruits additional DNA repair machinery to stimulate their repair. Gene regulatory elements bound by NPAS4–NuA4 are partially protected against age-dependent accumulation of somatic mutations. Impaired NPAS4–NuA4 signalling leads to a cascade of cellular defects, including dysregulated activity-dependent transcriptional responses, loss of control over neuronal inhibition and genome instability, which all culminate to reduce organismal lifespan. In addition, mutations in several components of the NuA4 complex are reported to lead to neurodevelopmental and autism spectrum disorders. Together, these findings identify a neuronal-specific complex that couples neuronal activity directly to genome preservation, the disruption of which may contribute to developmental disorders, neurodegeneration and ageing.

For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection VS ASW-FL (Image acquisition software for VS120 Slide Scanner Microscope). FlowJo (10.0.8r1). Sony SH800Z FACS acquisition software.

March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy All sequencing data for RNA-seq, ATAC-seq, ChIP-seq, CUT&RUN, sBLISS-seq, END-seq, snRNA-seq, and amplicon sequencing has been deposited in the Gene Expression Omnibus with accession number GSE175965. Mass spectrometry data has been deposited in PRIDE repository with accession number PXD038718. Raw gel images are provided in Supplementary Information Fig. 1. Additional data is provided as source data throughout the manuscript.

Human research participants
Policy information about studies involving human research participants and Sex and Gender in Research.
Reporting on sex and gender N/A Population characteristics Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
No statistical methods were used to predetermine sample size. Sample sizes were determined according to standards of practice in the field for each assay and generally adhere to guidelines of the ENCODE consortium (https://www.encodeproject.org/about/experimentguidelines/). Sample size details are included in the manuscript with experimental description.
Data exclusions For mutational analysis experiments, to include a given amplicon in our analysis, we required that the amplicon be found in greater than 1/3 of all samples and that the average number of consensus 10 families across all bases in the amplicons was >100. For information on primer pooling and the amplicons included in the final analysis, see Supplementary Table 5. For mutational analysis, extreme outlier points were removed across all conditions using a ROUT's test at 0.1% confidence performed in Prismv8.4.2.

Replication
Replicate details for each assay are provided in the manuscript. All attempts at replication were successful. For specific replicate numbers per assay, please see Supplementary Table 2. In general, in vitro RNA-seq experiments were performed in triplicate. RNA-seq from hippocampal tissue paired with sBLISS-seq data was performed with 8-10 replicates per timepoint. Single-nucleus RNA-seq experiments were performed on 2 independent Npas4fl/fl (Cre vs ΔCre) animals and 3 independent Tip60fl/fl (Cre vs ΔCre) animals. γH2AX ChIP-seq was performed in triplicate. ATAC-seq was performed in triplicate. All CUT&RUN experiments were conducted at least twice and for most antibodies in triplicate, with the exception of one EP400 dataset generated using an antibody from Bethyl Laboratories. This additional dataset corroborates data using a second EP400 antibody from Abcam, which has been conducted in duplicate in wild-type tissue and in triplicate in ΔCre (Control) infected animals. NPAS4 CUT&RUN was performed 5 times in wild-type mice and in duplicate in NPAS4 KO mice. IP-MS experiments on hippocampal tissue were performed in triplicate for the initial isolation of the complex. IP-MS experiments conducted from fractionated lysates were performed in duplicate. BLISS-seq experiments were conducted using 8-10 replicates in wild-type mice and three to five times in our Npas4 and Tip60 cKO (Cre vs ΔCre).
Randomization All our data are derived from mice or cultured HEK293T cells. In general samples were grouped according to mouse age and genotype. Care was taken to include mice of both sexes equally in experimental groups.

Blinding
For genomic assays (ATAC-seq, RNA-seq, ChIP-seq, CUT&RUN, amplicon sequencing for mutational analysis, BLISS-seq, END-seq) and biochemistry, blinding of animal/tissue genotype was not feasible due to constraints in sample processing. All genomic assays are treated to the same bioinformatic pipelines. For electrophysiology experiments, the experimenter was blinded to animal genotype and treatment.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. . For NPAS4 and MRE11, CUT&RUN signal was analyzed relative to knockout conditions. When KO conditions were difficult to obtain, multiple independent antibodies were used for a target protein (e.g. EP400). For immunohistochemistry, the following antibodies were used: rat anti-HA (Sigma-Aldrich, ROAHAHA, 1:250); rabbit anti-NPAS4 (in house, 1:1,000); rabbit anti-ARNT2 (in house, 1:1,000); rabbit anti-KAT5(TIP60) (Proteintech, 10827-1-AP, 1:250); rabbit anti-Cleaved Caspase-3 (Cell Signaling Technology, 9664S, 1:1,000). Antibody staining was validated in conditional knockout tissue or other negative controls (e.g. wild-type mice lacking a transgenic epitope). For FACS with staining, the following antibodies were used: mouse anti-NeuN-Alexa488 (Millipore, MAB377X, clone A60); Mouse IgG-Alexa488 (Life Technologies, MA518167). A non-targeting isotype control antibody was used as a negative control. NeuN staining and sorting was validated by qPCR for neuronal vs non-neuronal markers from sorted populations. For additional validation, see manufacturer's details for specificity using RRID from antibody registry listed above.

Eukaryotic cell lines Policy information about cell lines and Sex and Gender in Research
Cell line source(s) Note that full information on the approval of the study protocol must also be provided in the manuscript.

ChIP-seq Data deposition
Confirm that both raw and final processed data have been deposited in a public database such as GEO.
Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

Data access links
May remain private before publication.
Sequencing data have been deposited in the Gene Expression Omnibus with accession number GSE175965. Additional data are provided in source data files.

Files in database submission
Sequencing data have been deposited in the Gene Expression Omnibus with accession number GSE175965.
Genome browser session (e.g. UCSC) Graph files (bigWig) are included in Gene Expression Omnibus GSE175965.

Methodology
Replicates Replicate information provided in Supplementary Table 2, Methods, and Figure Legends. Briefly, CUT&RUN experiments were conducted at least twice and for most antibodies in triplicate, with the exception of one EP400 dataset generated using an antibody from Bethyl Laboratories A300541A. This additional dataset corroborates data using a second EP400 antibody from Abcam, which has been conducted in duplicate in wild-type tissue and in triplicate in ΔCre (Control) infected animals. NPAS4 CUT&RUN was performed 5 times in wild-type mice and in duplicate in NPAS4 KO mice. γH2AX ChIP-seq was performed in triplicate.

Sequencing depth
Sequencing depth information provided in Supplementary Table 2. Briefly, all CUT&RUN experiments were sequenced on average with 22 million adapter trimmed pairs, with a minimum of 8 million adapter trimmed pairs. anti-γH2AX ChIP-seq samples were sequenced to a minimum depth of 20 million reads.

Software
Peak calling on ChIP samples was performed using MACS2 (macs2/2.1.1). ChIP-seq samples were aligned to the mm10 genome using the Bowtie alignment software(vbowtie2/2.2.9) with the -very-sensitive setting. CUT&RUN peak calling was performed with SEACR_1.1.sh.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology Sample preparation
For GFP and mCherry FACS: Dissected hippocampal tissue was examined under a florescent scope to detect GFP or mCherry. Tissue that was uninfected, or in rare cases showed infection of both fluorophores in a single hemisphere, was discarded. Hippocampi were placed in 0.5 mL of buffer HB (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, pH 7.8, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine) and dounced 5X with a loose pestle and 10X with a tight pestle. 5% IGEPAL CA-630 (32 μL) was added prior to douncing with a tight pestle 5-8 more times and filtering through a 40-μm strainer. DRAQ5 nuclear dye (Abcam; ab108410) was added (1:500), and nuclei expressing either mCherry or GFP were sorted on a SONY SH800. Negative gates were determined using uninfected tissue. Nuclei were collected in 1 mL of CUT&RUN Wash Buffer containing 2 mM EDTA. For NeuN-FACS: To sort neuronal nuclei for DNA isolation, dissected hippocampal tissue was placed in 1 mL of buffer HB (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, pH 7.8, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine) and dounced 5X with a loose pestle and 10X with a tight pestle. 5% IGEPAL CA-630 (32 μL) was added prior to douncing again with a tight pestle 5-8 times. The nuclei suspension in HB was filtered through a 40-μm strainer. Nuclei were then pelleted by centrifugation at 500 g for 5 min and resuspended in 400 μL of FACS Block/Stain Buffer (1% BSA, 0.05% Igepal-630, 3 mM MgCl2 in 1X PBS). Nuclei were incubated for 15 min with gentle rotation at 4°C. Following this blocking step, nuclei were pelleted and resuspended in FACS Block/Stain Buffer containing 1:1000 dilution of mouse anti-NeuN-Alexa488 (Millipore, MAB377X). An isotype control Mouse IgG-Alexa488 (Life Technologies, MA518167) was included as a negative control along with an unstained sample. Samples were incubated in antibody mix for 1 hour with gentle rotation at 4°C. Nuclei were washed 1X with FACS Block/Stain Buffer, and DRAQ5 nuclear dye (Invitrogen) was added (1:500) prior to sorting. NeuN-highexpressing nuclei were separated from NeuN-low-expressing nuclei using a SONY SH800.

Instrument SONY SH800
Software Sony SH800Z Cell Sorter software was used during acquisition of data. Data were subsequently analyzed using FlowJo (10.0.8r1)

Cell population abundance
Singlet DRAQ5-positive nuclei represented roughly 25% of the initial population. NeuN+ nuclei gated from the singlet DRAQ5positive population represented roughly 60% of the population. Cre-mCherry+ and ΔCre-GFP+ nuclei abundance depended on the viral injection and tissue microdissection and ranged from 20% to 75% of the singlet DRAQ5-positive population. See Extended Data Figs. 11d,e and 13a and Supplementary Fig. 2 for gating strategy.
nature portfolio | reporting summary

March 2021
Gating strategy For NeuN-FACS: Unstained sample without DRAQ5 nuclear dye was used to establish the APC-Cy7-positive gate for DRAQ5positive nuclei. Nuclei stained with DRAQ5 were initially selected based on APC-Cy7 signal, followed by selection of nuclei with linearly proportional FSC area and height signal to isolate singlet nuclei. NeuN+ gate was determined using both a DRAQ5-stained sample that was not stained with Mouse anti-NeuN-Alexa488 (no primary control) and a DRAQ5-stained sample that was stained with a Mouse IgG-Alexa488 isotype control. See Extended Data 13a and Supplementary Fig. 2 for gating strategy. For mCherry and GFP FACS: Unstained sample without DRAQ5 nuclear dye was used to establish the APC-Cy7-positive gate for DRAQ5-positive nuclei. Nuclei stained with DRAQ5 were initially selected based on APC-Cy7 signal, followed by selection of nuclei with proportional APC-Cy7 area and SSC signal to isolate singlet nuclei. mCherry+ and GFP+ gates were determined using a DRAQ5-positive sample from an uninfected mouse. See Extended Data 11d,e and Supplementary Fig. 2 for gating strategy.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.