This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Cellular/Molecular The DNA repair associated protein Gadd45׆ 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, Paul R. Marshall, Laura J. Leighton, Esmi L. Zajaczkowski, Ziqi Wang, Sachithrani U. Madugalle, Jiayu Yin, Timothy W. Bredy and Wei Wei Cognitive Neuroepigenetics Laboratory, Queensland Brain Institute, The University of Queensland, St Lucia, QLD 4072, Australia https://doi.org/10.1523/JNEUROSCI.2024-18.2018 Received: 7 August 2018 Revised: 11 November 2018 Accepted: 4 December 2018 Published: 13 December 2018 Author contributions: X.L., P.M., T.B., and W.W. designed research; X.L., P.M., L.L., E.Z., Z.W., S.M., J.Y., and W.W. performed research; X.L., P.M., T.B., and W.W. analyzed data; X.L. and P.M. wrote the first draft of the paper; L.L., T.B., and W.W. edited the paper; W.W. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. The authors gratefully acknowledge grant support from the NIH (5R01MH105398-TWB) and the NHMRC (APP1033127-TWB). XL was supported by postgraduate scholarships from the University of Queensland, the ANZ Trustees Queensland for Medical Research and University of Queensland development fellowship. PRM is supported by postgraduate scholarships from NSERC and the University of Queensland. LJL is supported by postgraduate scholarships from the Westpac Bicentennial Foundation and the University of Queensland. We would also like to thank Ms. Rowan Tweedale for helpful editing of the manuscript. Correspondence should be addressed to Please send correspondence to: Dr. Wei Wei, [email protected] or Dr. Timothy W. Bredy, [email protected] Cite as: J. Neurosci 2018; 10.1523/JNEUROSCI.2024-18.2018 Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors ͳ ʹ ͵ The DNA repair associated protein Gadd45 regulates the temporal coding of immediate Ͷ early gene expression within the prelimbic prefrontal cortex and is required for the ͷ consolidation of associative fear memory ͸ ͹ ͺ ͻ Xiang Li*, Paul R. Marshall*, Laura J. Leighton, Esmi L. Zajaczkowski, Ziqi Wang, ͳͲ Sachithrani U. Madugalle, Jiayu Yin, Timothy W. Bredy# and Wei Wei# ͳͳ ͳʹ Cognitive Neuroepigenetics Laboratory, Queensland Brain Institute, ͳ͵ The University of Queensland, St Lucia, QLD 4072, Australia ͳͶ ͳͷ ͳ͸ *These authors contributed equally to this work. ͳ͹ ͳͺ #Please send correspondence to: ͳͻ ʹͲ Dr. Wei Wei ʹͳ [email protected] ʹʹ or ʹ͵ Dr. Timothy W. Bredy ʹͶ [email protected] ʹͷ ʹ͸ Acknowledgements: The authors gratefully acknowledge grant support from the NIH ʹ͹ (5R01MH105398-TWB) and the NHMRC (APP1033127-TWB). XL was supported by ʹͺ postgraduate scholarships from the University of Queensland, the ANZ Trustees ʹͻ Queensland for Medical Research and University of Queensland development fellowship. ͵Ͳ PRM is supported by postgraduate scholarships from NSERC and the University of ͵ͳ Queensland. LJL is supported by postgraduate scholarships from the Westpac Bicentennial ͵ʹ Foundation and the University of Queensland. We would also like to thank Ms. Rowan ͵͵ Tweedale for helpful editing of the manuscript. ͵Ͷ ͵ͷ ͵͸ ͵͹ ͵ͺ ͵ͻ ͶͲ Ͷͳ ͳ Ͷʹ Ͷ͵ Abstract ͶͶ Ͷͷ We have identified a member of the growth arrest and DNA damage (Gadd45) protein Ͷ͸ family, Gadd45, which is known to be critically involved in DNA repair, as a key player in the Ͷ͹ regulation of immediate early gene (IEG) expression underlying the consolidation of Ͷͺ associative fear memory in adult male C57/Bl6 mice. Gadd45 temporally influences Ͷͻ learning-induced IEG expression in the prelimbic prefrontal cortex (PLPFC) through its ͷͲ interaction with DNA double-strand break (DSB)-mediated changes in DNA methylation. Our ͷͳ findings suggest a two-hit model of experience-dependent IEG activity and learning that ͷʹ comprises 1) a first wave of IEG expression governed by DSBs and followed by a rapid ͷ͵ increase in DNA methylation, and 2) a second wave of IEG expression associated with the ͷͶ recruitment of Gadd45 and active DNA demethylation at the same site, which is necessary ͷͷ for memory consolidation. ͷ͸ ͷ͹ Significance statement: How does the pattern of immediate early gene (IEG) transcription ͷͺ in the brain relate to the storage and accession of information, and what controls these ͷͻ patterns? This paper explores how Gadd45, a gene that is known to be involved with DNA ͸Ͳ modification and repair, regulates the temporal coding of IEGs underlying associative ͸ͳ learning and memory. We reveal that, during fear learning, Gadd45 serves to act as a ͸ʹ coordinator of IEG expression and subsequent memory consolidation by directing temporally ͸͵ specific changes in active DNA demethylation at the promoter of plasticity-related IEGs. ͸Ͷ ʹ ͸ͷ Introduction ͸͸ ͸͹ Memory consolidation requires learning-induced gene expression, protein synthesis, and ͸ͺ changes in synaptic plasticity (Izquierdo & McGaugh, 2000; Kandel, 2001). For decades, ͸ͻ models of cued and contextual fear conditioning have been used to advance the ͹Ͳ understanding of the cellular, molecular and circuit-level mechanisms of learning and ͹ͳ memory. Although the amygdala and hippocampus have historically been the primary focus ͹ʹ of studies on fear-related learning, it is becoming evident that the prefrontal cortex (PFC) is ͹͵ also involved in the initial encoding of fear memories, with the dorsomedial or prelimbic ͹Ͷ region of the PFC playing a particularly important role (Giustino & Maren, 2015; Rizzo et al., ͹ͷ 2017; Santos, Kramer-Soares, Favaro, & Oliveira, 2017; Klavir, Prigge, Sarel, Paz, & Yizhar, ͹͸ 2017; Widagdo et al., 2016; Dejean et al., 2016). With respect to genomic responses to ͹͹ learning, several members of a particularly unique class of inducible genes known as the ͹ͺ immediate early genes (IEGs) are rapidly and transiently expressed in response to neural ͹ͻ activity, and are thought to be important for information processing in the brain due to the ͺͲ tight temporal relationship between their expression and learning. Specifically, activity- ͺͳ regulated cytoskeleton-associated protein (Arc), fos proto-oncogene (c-Fos), and neuronal ͺʹ PAS domain protein 4 (Npas4) have all been shown to be critically involved in memory ͺ͵ formation (Gallo, Katche, Morici, Medina, & Weisstaub, 2018; Minatohara, Akiyoshi, & ͺͶ Okuno, 2016; Sun & Lin, 2016). However, a detailed understanding of the molecular ͺͷ mechanisms by which these IEGs are regulated during learning and the formation of ͺ͸ memory remains to be developed. ͺ͹ ͺͺ It has become increasingly evident that there is an important epigenetic layer of regulatory ͺͻ control over gene expression and memory, which includes DNA modification (Baker- ͻͲ Andresen, Ratnu, & Bredy, 2013). DNA methylation and active DNA demethylation are ͻͳ directly involved in gene expression underlying memory formation (Li et al., 2014), and ͻʹ several recent studies have extended these findings to include IEGs. For example, RNA- ͻ͵ directed changes in DNA methylation regulate c-Fos expression by directing DNMT activity ͻͶ to the c-Fos promoter (Baker-Andresen, Ratnu, & Bredy, 2013). DNA hydroxymethylation ͻͷ regulates both c-Fos and Arc expression in the hippocampus (Li et al., 2014), and chronic ͻ͸ drug exposure increases DNA methylation at the c-Fos and Arc promoters, which has a ͻ͹ profound effect on learning and memory (Baker-Andresen et al., 2013; Li et al., 2014; Miller ͻͺ et al., 2010). Several active DNA demethylation pathways have been proposed, and each ͻͻ has been shown to be involved in regulating gene expression related to plasticity and ͳͲͲ memory (Li, Wei, Ratnu, & Bredy, 2013, Kaas et al., 2013; Li et al., 2014; Rudenko et al., ͳͲͳ 2013). Perhaps most direct pathway involves independent members of the Gadd45 family of ͵ ͳͲʹ DNA repair proteins (D, E and J), where each has been shown to form a complex with DNA ͳͲ͵ repair enzymes to guide the removal of 5-mC by either base or nucleotide excision repair ͳͲͶ (Barreto et al., 2007; Ma, Guo, Ming, & Song, 2009; Rai et al., 2008). ͳͲͷ ͳͲ͸ Early reports indicated that in sensory and motor neurons, Gadd45D is induced by nerve ͳͲ͹ injury (Befort, Karchewski, Lanoue, & Woolf, 2003), whereas Gadd45E was found to be ͳͲͺ activity-dependent and involved in neurogenesis (Ma et al., 2009). With respect to ͳͲͻ experience-dependent plasticity in the adult brain, Gadd45E has also been shown to ͳͳͲ influence memory formation, although reports differ with regards to whether its knockdown ͳͳͳ leads to enhancement (Sultan, Wang, Tront, Liebermann, & Sweatt, 2012) or impairment of ͳͳʹ contextual fear memory (Leach et al., 2012). Although fear-related learning led to a ͳͳ͵ significant increase in Gadd45J mRNA expression in the hippocampus and amygdala ͳͳͶ (Sultan et al., 2012), and the expression of Gadd45β, Gadd45γ and Arc and a relationship ͳͳͷ with DNA methylation in a mouse model of depression has been reported (Grassi et al., ͳͳ͸ 2017), whether any members of the Gadd45 family of proteins contribute to memory ͳͳ͹ formation through direct effects on IEG expression has yet to be determined. ͳͳͺ ͳͳͻ Materials and Methods ͳʹͲ Mice: 9-11 week-old C57BL/6J (Animal Resource Centre, WA, Australia) male mice were ͳʹͳ housed four per cage, maintained on a 12hr light/dark schedule, and allowed free access to ͳʹʹ food and water. All testing was conducted during the light phase in red-light-illuminated ͳʹ͵ testing rooms following protocols approved by the Animal Ethics Committee of the University ͳʹͶ of Queensland.