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. 2019 Feb 6;39(6):970-983.
doi: 10.1523/JNEUROSCI.2024-18.2018. Epub 2018 Dec 13.

The DNA Repair-Associated Protein Gadd45γ Regulates the Temporal Coding of Immediate Early Gene Expression within the Prelimbic Prefrontal Cortex and Is Required for the Consolidation of Associative Fear Memory

Xiang Li  1 , Paul R Marshall  1 , Laura J Leighton  1 , Esmi L Zajaczkowski  1 , Ziqi Wang  1 , Sachithrani U Madugalle  1 , Jiayu Yin  1 , Timothy W Bredy  2 , Wei Wei  2
Affiliations

The DNA Repair-Associated Protein Gadd45γ Regulates the Temporal Coding of Immediate Early Gene Expression within the Prelimbic Prefrontal Cortex and Is Required for the Consolidation of Associative Fear Memory

Xiang Li et al. J Neurosci. .

Erratum in

Abstract

We have identified a member of the growth arrest and DNA damage (Gadd45) protein family, Gadd45γ, which is known to be critically involved in DNA repair, as a key player in the regulation of immediate early gene (IEG) expression underlying the consolidation of associative fear memory in adult male C57BL/6 mice. Gadd45γ temporally influences learning-induced IEG expression in the prelimbic prefrontal cortex (PLPFC) through its interaction with DNA double-strand break (DSB)-mediated changes in DNA methylation. Our findings suggest a two-hit model of experience-dependent IEG activity and learning that comprises (1) a first wave of IEG expression governed by DSBs and followed by a rapid increase in DNA methylation, and (2) a second wave of IEG expression associated with the recruitment of Gadd45γ and active DNA demethylation at the same site, which is necessary for memory consolidation.SIGNIFICANCE STATEMENT How does the pattern of immediate early gene transcription in the brain relate to the storage and accession of information, and what controls these patterns? This paper explores how Gadd45γ, a gene that is known to be involved with DNA modification and repair, regulates the temporal coding of IEGs underlying associative learning and memory. We reveal that, during fear learning, Gadd45γ serves to act as a coordinator of IEG expression and subsequent memory consolidation by directing temporally specific changes in active DNA demethylation at the promoter of plasticity-related IEGs.

Keywords: DNA double-strand break; DNA methylation; DNA repair; immediate early gene; memory consolidation; neuroepigenetic.

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Figures

Figure 1.
Figure 1.
Gadd45γ expression is activity-dependent and necessary for cued fear learning in the PLPFC. A–C, Fold-change for fear conditioned animals which is calculated relative to each group's own context control at that time point. A, Following cued fear conditioning there was no significant change in Gadd45α or (B) Gadd45β mRNA levels. C, There was a significant increase in Gadd45γ 3 and 5 h post-conditioning and decrease at 12 h time point. D, Time course of behavioral training and shRNA infusion. E–G, Representative images show the location of infused lentivirus expressing shRNA knockdown effect at ∼55, 50, and 40% for (H) Gadd45α, (I) Gadd45β, and (J) Gadd45γ, separately, at the protein level. There were no significant differences in percentage freezing during the fear test for animals infused with either Gadd45α shRNA (K) or Gadd45β shRNA lenti-virus (L) compared with the control, which has no specificity to any known mouse transcript. M, There was a significant decrease in the percentage freezing for animals infused with Gadd45γ shRNA compared with control at test (N = 8–14 per group). FC, Fear conditioned; CTX, context exposure; CS, conditioned stimulus (tone); US, unconditioned stimulus (shock); PreCS, contextual freezing before first CS; AvgCS, average freezing at test. Error bars represent SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001.
Figure 2.
Figure 2.
Control experiments showing no nonspecific effect of shRNA lentivirus injection on behavior and gene expression. A, Time course of behavioral training. Gadd45γ knockdown had no effect on fear acquisition (B) and short memory when tested 2 h post-training (C; N = 8 per group). Open-field test results show that there is no effect of on generalized anxiety as measured by time spent in the center of the open field (D) or the number of entries to the center zone (E); likewise, there is no effect on locomotion (F; N = 7–8 per group). G, Time course of behavioral training. Compared with PBS injection, lenti-viral transduction itself had on effect on mRNA expression of (H) Gadd45α, (I) Gadd45β, and (J) Gadd45γ (N = 4 per group). K, Additionally, Gadd45α shRNA had no effect on other two family members. L, Gadd45β shRNA had no effect on other two family members and (M) Gadd45γ shRNA had no effect on other two family members (N = 3 per group for K–M). PreCS, Contextual freezing before first CS; AvgCS, average freezing at test. Error bars represent SEM. * p < 0.05, ** p < 0.01.
Figure 3.
Figure 3.
Fear learning leads to a distinct pattern of IEG expression in the PLPFC regulated by DSBs. All panels show fold-change for fear conditioned animals which is calculated relative to each group's own context control at that time point. A, There was a significant increase in mRNA expression 0 and 5 h post-fear conditioning for Arc, (B) c-Fos, (C) Npas4, and (D) Cyr61. E, The IEG mRNA induction was associated with a significant increase in RNA Pol II occupancy 0 and 5 h as well post-fear conditioning for Arc, (F) c-Fos, (G) Npas4, and (H) Cyr61. I, There was also a significant increase in γH2A.X occupancy immediately following cued fear conditioning for Arc, (J) c-Fos, (K) Npas4, and (L) Cyr61. M, We also observed a significant increase in Topo IIβ binding immediately following cued fear conditioning for Arc, (N) c-Fos, (O) Npas4, and (P) Cyr61. N = 4–6 per group for all panels. FC, Fear conditioned; CTX, context exposure. Error bars represent SEM. * p < 0.05, ** p < 0.01.
Figure 4.
Figure 4.
Fear conditioning associated DSBs are followed by time-dependent increases in DNA methylation, and later by Gadd45γ recruitment. There was a significant increase in Gadd45γ occupancy at 5 h for (A) Arc, (B) c-Fos, (C) Npas4, and (D) Cyr61. E, A significant increase in DNMT3A occupancy was observed 1 h after fear conditioning for Arc, (F) c-Fos, (G) Npas4, and (H) Cyr61. I, A significant increase in 5-mC levels as measured by methylated DNA immunoprecipitation was found post-fear conditioning for Arc, (J) c-Fos, (K) Npas4, and (L) Cyr61. N = 6 per group for all panels. FC, Fear conditioned; CTX, context exposure. Error bars represent SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 5.
Figure 5.
Gadd45γ regulates learning-induced IEG expression in a temporally specific manner through interaction with DNA methylation. Presence of Gadd45γ shRNA led to significantly less Gadd45γ occupancy at (A) Arc, (B) c-Fos, (C) Npas4, and (D) Cyr61. Gadd45γ knockdown also led to a significant decrease in mRNA expression at 5 h for (E) Arc, (F) c-Fos, (G) Npas4, and (H) Cyr61, without any influence on mRNA expression of Arc, c-Fos, Npas4, and Cyr61 at 0 h time point (E–H). Gadd45γ shRNA infusion result in remaining high level of promoter DNA methylation at 5 h for (I) Arc, (J) c-Fos, (K) Npas4, and (L) Cyr61. N = 4–6 per group for all panels. FC, Fear conditioned; CTX, context exposure. Error bars represent SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 6.
Figure 6.
Gadd45γ is necessary for cued fear learning in a temporally specific manner. A, Time course of behavioral training and anti-Gadd45γ antibody infusion. B, There was a significant decrease in the percentage freezing for animals infused with Gadd45γ-blocking antibody compared with control at test (N = 7–8 per group). Following antibody injection, there was a significant reduction in Gadd45γ occupancy at the promoter of (C) Arc, (D) c-Fos, (E) Npas4, and (F) Cyr61. The induction of IEG mRNA expression was blocked at 5 h post-training compared with control (G) Arc, (H) c-Fos, (I) Npas4, and (J) Cyr61. N = 4 per group for C–J. FC, Fear conditioned; CTX, context exposure; CS, conditioned stimulus (tone); US, unconditioned stimulus (shock); PreCS, contextual freezing before first CS; AvgCS, average freezing at test. Error bars represent SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 7.
Figure 7.
Cyr61 mRNA expression within PLPFC is necessary for cued fear learning. A, Time course of behavioral training and Cyr61 DsiRNA infusion. B, PLPFC infusion of Cyr61 DsiRNA (100 nm) administered 3 h after fear training blocks fear memory consolidation (N = 7–8 per group). C, Cyr61 DsiRNA (100 nm) also significantly reduced Cyr61 mRNA expression at 5 h post-fear training (n = 5 per group). D, Time course of behavioral training and shRNA infusion. E, There were no significant differences between the Cyr61 shRNA and control groups during fear acquisition. F, There was a significant decrease in the percentage freezing for animals infused with Cyr61 shRNA compared with control at test (N = 7–8 per group). FC, Fear conditioned; CTX, context exposure. Error bars represent SEM. * p < 0.05.
Figure 8.
Figure 8.
Etoposide infusion into the PLPFC inhibits fear memory formation by blocking repair of DSBs. A, Time course of behavioral training and etoposide infusion. B, There were no significant differences between etoposide (0.1 μg/side) and control groups in percentage freezing during fear acquisition. C, There was a significant decrease in the percentage freezing for animals infused with etoposide compared with control at test (N = 9–12 per group). D, Etoposide infusion blocked the temporal accumulation of 5-mC within the promoter region of Arc, (E) c-Fos, (F) Npas4, and (G) Cyr61. H, Etoposide infusion also inhibited Gadd45γ occupancy at promoter of Arc at the 5 h time point, (I) c-Fos, (J) Npas4, and (K) Cyr61. Etoposide led to a significant decrease in mRNA at 5 h post-training (L) Arc, (M) c-Fos, (N) Npas4, and (O) Cyr61. Etoposide infusion had no effect on Arc, c-Fos, Npas4, and Cyr61 mRNA expression at the 0 h time point (L–O). N = 4 per group for D–O. FC, Fear conditioned; CTX, context exposure. Error bars represent SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 9.
Figure 9.
Fear learning leads to a rapid induction in IEG expression in the PLPFC that is mediated by Topo2β associated DSBs within their promoter region, and which is followed by an increase in DNMT3a-mediated DNA methylation at the same locus. The increased deposition of 5-mC results in the recruitment of the DNA repair associated protein Gadd45γ, which is then necessary for the induction of a second peak of IEG expression and the consolidation of fear memory.
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