Profiling DNA break sites and transcriptional changes in response to contextual fear learning

Neuronal activity generates DNA double-strand breaks (DSBs) at specific loci in vitro and this facilitates the rapid transcriptional induction of early response genes (ERGs). Physiological neuronal activity, including exposure of mice to learning behaviors, also cause the formation of DSBs, yet the distribution of these breaks and their relation to brain function remains unclear. Here, following contextual fear conditioning (CFC) in mice, we profiled the locations of DSBs genome-wide in the medial prefrontal cortex and hippocampus using γH2AX ChIP-Seq. Remarkably, we found that DSB formation is widespread in the brain compared to cultured primary neurons and they are predominately involved in synaptic processes. We observed increased DNA breaks at genes induced by CFC in neuronal and non-neuronal nuclei. Activity-regulated and proteostasis-related transcription factors appear to govern some of these gene expression changes across cell types. Finally, we find that glia but not neurons have a robust transcriptional response to glucocorticoids, and many of these genes are sites of DSBs. Our results indicate that learning behaviors cause widespread DSB formation in the brain that are associated with experience-driven transcriptional changes across both neuronal and glial cells.

30 Subsequently, stimulation of the rodent brain was found to generate DSBs following seizures [6] 31 or behavioral manipulation [2,4]. While wakefulness in zebrafish [5], or wakefulness with 32 exploration in fruit flies and mice [9], increased DSBs in neurons that were reduced during sleep.

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One source of genomic stress in the brain is its high transcriptional output; neurons 34 respond in real-time to environmental changes and this activity necessitates continual modulation 35 of transcription [1]. We made the unexpected discovery that stimulating the activity of primary 36 cortical neurons generates DSBs specifically at the rapidly induced early response genes (ERGs), 37 and this promotes their expression [4]. Increases in γH2AX at some of these ERGs was later 38 observed in the brain during fear learning [7] or following memory retrieval [10]. In other 39 contexts of gene induction, including through transcriptional induction mediated by nuclear 40 receptors [11][12][13][14] or heat shock and serum-stimulation [15], DSBs appear to facilitate gene 41 induction. Within the complex milieu of the brain, it is therefore likely that different upstream 42 pathways contribute to the generation of DSBs, yet their locations and their relation to brain 43 function is an open question. As DSBs pose a threat to genomic integrity [4], understanding the 44 genome-wide DSB landscape of the brain would facilitate our understanding of how the brain 45 balances timely transcriptional responses with the generation of DSBs, while revealing sites of 46 genomic stress that could seed DNA lesions detrimental to neuronal function and contribute to 47 brain aging and neurodegenerative diseases. 48 We set out to understand the in vivo landscape of DSBs in the brain during learning and 49 how they correspond with gene expression changes occurring in neurons and glia. We find fear 50 learning paradigm-induced genes are overrepresented amongst those genes with the highest 51 levels of DSBs in the medial prefrontal cortex and hippocampus. These genes are downstream of 52 pathways that are shared in part by neurons and non-neurons, and in other cases unique to each 53 group of cells. Surprisingly, we find potential glia-enriched DSB hotspots at genes that have a 54 robust transcriptional response to glucocorticoid receptor signaling in glia.
134 Because the peak of ERG induction occurred as early as 10 minutes or as late as 30 minutes after 135 CFC, we included both time points in our sequencing analyses (Fig S2B 147 genes at 30 minutes) (Fig S3F). Non-neuronal nuclei also exhibited considerable transcriptional 148 changes in response to CFC, but with more comparable numbers of upregulated genes between 149 brain areas and with a large overlap occurring at 30 minutes (34 genes at 10 minutes and 242 150 genes at 30 minutes) ( Fig S3G). Further, we found biological processes related to synaptic 151 structure and function were amongst the most enriched GO categories in the upregulated genes 152 of neurons -mirroring our γH2AX ChIP-Seq ( Fig 1C). In contrast, neuronal downregulated 153 genes had minimal enrichment for biological processes (a single significantly enriched term: 154 "cell-cell adhesion via plasma-membrane adhesion molecules"; adjusted p-value = 2.4x10 -3 ).

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To assess the relationships between activity-induced DSBs and gene expression in the 156 brain, we compared the ChIP-seq and RNA-seq data. First, examining a specific genomic locus 157 of the ERG Arc revealed increases in γH2AX signal with concomitant upregulation in both 158 neurons and non-neurons ( Fig 1D). Globally, we find four categories of γH2AX-associated genes 159 whose expression was altered after CFC: those upregulated exclusively in neurons (56 HIP and 160 114 mPFC), genes upregulated in both neurons and non-neurons (12 HIP and 28 mPFC), genes 161 upregulated specifically in non-neurons (19 HIP and 12 mPFC), and a small subset of 162 downregulated genes (16 HIP and 15 mPFC) (categories denoted by "Differential Grouping" 163 row) (Fig 1E and 1F). Overall, we find transcriptional changes are more strongly associated with 164 γH2AX in the brain than anticipated. Previously, we observed twenty gene-associated γH2AX 165 loci following stimulation of cultured neurons [4], while in the HIP and mPFC we see more than 166 100-150 gene-associated γH2AX loci that are transcriptionally induced (Fig 1E and 1F).
167 Activity-dependent genes are a source of DNA breaks in the brain 168 We next sought to understand the overlap between CFC-upregulated genes and γH2AX 169 peaks. Overall, we found that γH2AX peaks were over-represented with genes upregulated by In HIP and mPFC we found multiple genes with γH2AX peaks that were induced after  Table) [55,56]. Interestingly, we found that many of the 289 γH2AX-containing genes that were responsive to CFC only in non-neuronal nuclei are 290 coincident with genes annotated to a GR-binding site ( Fig 4A).  We tested whether a subset of these genes can be induced by the GR-specific agonist 310 dexamethasone in cultured primary glia. In contrast to Actb which is not a known target of GR, 311 we found dexamethasone induced the expression of Ddit4, Sgk1, and Glul, genes that were 312 specifically upregulated in non-neuronal nuclei during CFC and annotated to a GR-binding site 313 ( Fig 4B). Thus, our findings implicated the GR in mediating gene induction in glia after fear 314 learning. Next, to assess whether GR activity is sufficient to increase DSBs at these genes, we 315 treated cultured primary glia with dexamethasone and measured γH2AX enrichment by ChIP-316 qPCR. The genes Ddit4, Glul, and Sgk1, alongside the canonical GR-inducible gene Mt1 [58], 317 showed significant increases in γH2AX enrichment ( Fig 4C). Arc, with similarly high γH2AX 318 levels following CFC, alongside the housekeeping gene B2m, did not exhibit γH2AX enrichment 319 in response to dexamethasone (Fig S9B).

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Our RNA-seq data from sorted nuclei showed upregulation of the γH2AX-associated 321 gene Ddit4 only in non-neuronal nuclei following CFC, a similar pattern for many of our other 322 putative and confirmed GR-regulated genes (Fig 4D).  Table) 329 [55,56]. Strikingly, both the anterior cingulate cortex (ACC) of the medial prefrontal cortex, and 330 the hippocampal area Cornu Ammonis 1 (CA1) showed higher baseline acetylation around GR 331 peaks in non-neurons vs. neurons ('Naive') ( Fig 4E and Fig S9C). We then examined H3K27Ac 332 signal in CA1 under additional experimental conditions including 'context' (exposure to the 333 context without a foot shock) and 'shock' (context paired with a foot shock). We found that 334 H3K27Ac signal at GC peaks in the neuronal fraction increased similarly after exposure to either 335 context or shock, suggesting a generalized enhancer activation in response to exploratory 336 behavior that may be independent of stress. In contrast, the non-neuronal fraction showed 337 increases in H3K27Ac after shock, demonstrating that these enhancers are responsive to the 338 stressful condition in non-neurons but not in neurons (Fig 4E; Fig S9D, intergenic peaks).

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Our findings identified a group of CFC-responsive non-neuronal genes that are likely 340 regulated by GR signaling (Fig 4A-4E). We checked gene expression of the GR gene, Nr3c1, 341 finding that neurons express Nr3c1 at approximately half the level of non-neurons (Fig S9E).
342 The differing GR expression levels could be one of the reasons why these same genes did not 343 exhibit induction or increased enhancer activity in neurons (Fig 4A). Therefore, we verified 344 whether GR nuclear translocation occurs in response to receptor agonism in both cell types. We 345 measured GR nuclear intensity in mouse brain after treatment with corticosterone, the

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We next sought to understand how well GR-mediated gene induction could explain the 416 glia-specific DSBs seen in vivo, and whether genes regulated through this pathway in neurons 417 incur DSBs. Examining all γH2AX-containing genes that were also upregulated in one of the 418 cell types after corticosterone, we found that the vast majority (32/43; 74%) are regulated only in 419 glia (Fig 5D) 525 Treatment and control groups were euthanized in a staggered manner to minimize circadian 526 differences between groups. Naive mice remained in their home cages prior to euthanasia. For 527 tissue collection, the animals were sacrificed by cervical dislocation and the brains were rapidly 528 extracted and submerged in ice-cold PBS. To isolate the medial prefrontal cortex and 529 hippocampus, the brain was placed ventral side up in an Alto coronal 0.5mm mouse matrix 530 resting on ice. Three coronal cuts were administered with razor blades, one separating the PFC 531 from the olfactory bulb, one placed approximately around the optic chiasm to separate the PFC 532 from the hippocampus, and one placed within the cerebellum for stability. The pieces containing 533 the mPFC and hippocampi were placed in an ice-cold PBS-filled dish for isolation with a 534 dissection microscope. To isolate the mPFC, a horizontal cut was administered just above the 535 anterior olfactory nucleus with a razor blade. 571 Whole-cell mRNA processing 572 Extraction of mRNA from whole tissue and cultured mixed glia was performed with the RNeasy 573 mini kit (Qiagen). For brain tissue, homogenization was performed by aspirating the tissue in 574 RLT Plus buffer through a 20-gauge needle and syringe approximately 10 times until 575 homogenized. For cell culture, the media was aspirated before RLT Plus was added and 576 distributed with rocking. Purification proceeded as described by the manufacturer. Isolated RNA 577 was quantified on a NanoDrop spectrophotometer (Thermo Fisher Scientific) and 1ug RNA was 578 used to make cDNA with the OligodT RNA to cDNA EcoDry Premix (Takara) according to the 579 manufacturer's instructions, before proceeding to qPCR analysis. 580 581 qPCR 582 For qPCR analysis, diluted cDNA or genomic DNA was subjected to quantitative real-time PCR 583 in triplicate with the indicated primers using Ssofast EvaGreen Supermix (Bio-Rad) in a CFX 584 Connect Real-Time System (Bio-Rad). For gene expression analysis, normalization was against 585 Hprt using the ΔΔCT method. For ChIP, normalization was against Input. Primer sequences can 586 be found in S1 Table. 587 588 Flow Cytometry 589 Fixed brain nuclei (see 'Tissue Homogenization') were resuspended in 1mL 0.5% BSA in PBS 590 (IgG-Free, Protease-Free; Jackson ImmunoResearch). Nuclei were stained in Eppendorf tubes 591 with the relevant antibodies rocking for 30-60 minutes at 4°C. For neuron and glia isolation, 592 nuclei were stained with NeuN AF488 (1:1000; clone MAB377X; Millipore). For neuron and