Androgen Receptor and Histone Variant H2A.Z Interact to Regulate Fear Memory and Memory-Related Gene Transcription

Firyal Ramzan

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy (Ph.D.) Psychology University of Toronto

Androgen Receptor and Histone Variant H2A.Z Interact to Regulate Fear Memory and Memory-Related Gene Transcription

Firyal Ramzan

Doctor of Philosophy (Ph.D.)

Psychology University of Toronto

2019 Abstract

Sex hormones are potent regulators of memory, but the role of androgen receptors in males is poorly characterized compared to the role of estrogens in females. Thus, the goal of my thesis is to characterize the role of AR in fear memory and the mechanistic basis of this effect in male mice. In Chapter 2, I utilized transgenic male mice that overexpress the androgen receptor

(AR) to elucidate its effects on fear memory. I showed that AR overexpression impairs fear memory and that this effect is regulated by circulating androgens. Moreover, I showed that AR regulates the expression of several memory-related genes, including the epigenetic memory suppressor, histone H2A.Z. Our lab previously identified the histone variant H2A.Z as a negative modulator of fear memory through its effects on transcription. Given that AR is a transcription factor that interacts with H2A.Z in prostate cancer, I next characterized the functional and mechanistic interaction between the two.

In Chapters 4 and 5, I used inducible-conditional H2A.Z knockout mice to delete the two genes (H2afz and H2afv) that encode H2A.Z in excitatory neurons in adult mice. I showed that simultaneous deletion of both H2A.Z-encoding genes enhanced fear memory, which extends our prior findings that virally-mediated depletion of H2afz alone enhances memory. Next, I showed that H2A.Z deletion blocked the effects of androgen receptor manipulation on fear memory, ii

suggesting that AR and H2A.Z interact at a functional level. To test the mechanistic basis of this interaction, I utilized cultured hippocampal neurons to show that manipulating AR activation alters H2A.Z binding to DNA, suggesting that AR regulates behaviour through H2A.Z.

Given its interaction with AR, I next investigated whether H2A.Z deletion produces unique effects in males and females. In contrast to males, H2A.Z deletion did not enhance fear memory in females, but it did serve as a protective factor against stress-enhanced fear memory (a

PTSD-modeling paradigm). Overall, these data suggest that AR and H2A.Z interact at the chromatin level to regulate fear memory and that interactions with sex hormones may produce sex-specific effects of H2A.Z on fear memory.

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Acknowledgments

Professional:

Dr. Iva B. Zovkic: Thank you for believing in this collaborative study and being excited to take it on. Without your support and guidance, I would not have had the opportunity to do such an exciting study. Thank you also for your continuous push for me to be a better scientist, without which I would not have learned the meaning of hard work. Your excitement for research continues to inspire me and I can only hope to one day be as excited about science as you.

Dr. D. Ashley Monks: Thank you for believing in me as a scientist and helping me get started as one. Your support and guidance from my undergraduate studies until now have been invaluable and have helped me build a foundation for future scientific endeavours.

Dr. Alison Fleming and Dr. Veronica Afonso: the two people who really got me started on the path of scientific research by studying rats and changes in their sexual behaviour in response to stress during the Psychobiology Lab Course. Thank you for showing me that scientific research is a lot more than the dense articles we were otherwise made to read in undergraduate lectures.

I would also like to thank Dr. Loren J. Martin, for his helpful comments and guidance through my Ph.D. committee meetings.

I am truly grateful towards Dr. Ashlyn Swift-Gallant, a friend and former graduate student buddy, who taught me so much, from technical research skills to being a good lab mate.

To Ilapreet Toor, fellow graduate student and sister from another mother but same dungeon, thank you for your constant support and positivity.

I would also like to thank Dr. Melissa M. Holmes, for being the role model of how I aspire to be, and for making the basement lab space a welcoming and inclusive place throughout my time in the dungeons, from undergraduate thesis studies until now.

Personal:

I am especially grateful to my parents for being supportive and understanding of my choice to do a Ph.D. Without their sacrifices in immigrating to Canada, and their tireless support for their children to obtain more than them, I would not be where I am now.

To Irshad, my husband, without your support throughout this journey I might have remained holed in the basement at UTM and remained unaware of the world around me. Thank you for sticking by me through the ups and downs of this rollercoaster ride. I am truly grateful for your constant love and support, it has helped me in more ways than you know.

Contributions

Firyal Ramzan (author) solely prepared this thesis. All aspects of this body of work, including the planning, execution, analysis, and writing of all original research and publications was performed in whole or in part by the author. The following contributions by other individuals are formally and inclusively acknowledged:

Dr. Iva B. Zovkic (Primary Supervisor and Thesis Committee Member) – mentorship; laboratory resources; guidance and assistance in planning, execution, and analysis of experiments as well as manuscript/thesis preparation

Dr. D. Ashley Monks (Co-Supervisor and Thesis Committee Member) – mentorship; laboratory resources; guidance and assistance in planning, execution, and analysis of experiments as well as manuscript/thesis preparation

Dr. Loren J. Martin (Thesis Committee Member) – mentorship; guidance in the interpretation of results as well as thesis preparation

Dr. Ashlyn Swift-Gallant – teaching me the surgical skills required for experiments in Chapters 3 and 4

Amber B. Azam, M.Sc. – teaching me the behavioural testing skills and assistance in the execution of experiments for Chapter 3

Cindy Tao, M.A. – assistance in the execution of experiments for Chapter 4

Klotilda Narkaj – assistance in the execution of experiments for Chapters 4 and 5

Meaghan Hall – assistance in the execution of experiments for Chapter 4

Dr. Gilda Stefanelli – guidance in the execution of experiments for Chapter 4

Amanda Facciol – guidance in data analysis of experiments for Chapter 5

Table of Contents

Acknowledgments...... iv

Contributions...... vi

Table of Contents ...... vii

List of Tables ...... xi

List of Figures ...... xii

Chapter 1 Introduction and General Aims ...... 1

1.1 Preamble ...... 1

1.2 Thesis Organisation and General Aims ...... 2

1.2.1 Thesis Organisation ...... 2

1.2.2 General Aims ...... 3

Chapter 2 General Introduction ...... 4

2.1 Memory Formation and Consolidation ...... 4

2.1.1 Behavioural Paradigms to Study Memory ...... 5

2.2 Hormonal Effects on Learning and Memory ...... 7

2.2.1 Overview of Steroidogenesis ...... 7

2.2.2 Behavioural effects of sex steroid hormones on learning and memory ...... 9

2.2.3 Cellular effects of androgens on hippocampal structure and function ...... 12

2.2.4 Androgen receptor (AR) and memory ...... 13

2.3 Epigenetic Effects on Learning and Memory ...... 15

2.3.1 Overview of Neuroepigenetics ...... 15

2.3.2 Histone Post-translational Modifications ...... 16

2.3.3 Histone Variants...... 19

2.3.4 Histone variant H2A.Z and transcription ...... 20

2.3.5 Histone variant H2A.Z and memory ...... 23

2.3.6 H2A.Z Knockout Mouse Model ...... 27 vii

2.4 AR and H2A.Z Interaction ...... 28

2.4.1 Evidence of AR and H2A.Z interaction in AR-regulated gene transcription ...... 29

2.5 Thesis Aims and Hypotheses ...... 32

Chapter 3 Androgen Receptor is a Negative Regulator of Contextual Fear Memory in Male Mice...... 34

3.1 Introduction ...... 34

3.2 Methods...... 37

3.2.1 Animals ...... 37

3.2.2 Genotyping ...... 37

3.2.3 Fear Conditioning ...... 38

3.2.4 Flutamide administration ...... 38

3.2.5 Gonadectomy and testosterone (T) replacement ...... 38

3.2.6 Tissue collection ...... 39

3.2.7 Western Blotting ...... 39

3.2.8 mRNA expression and RT-PCR ...... 39

3.2.9 Statistics ...... 40

3.3 Results ...... 41

3.3.1 Validation of CMV-AR in the hippocampus ...... 41

3.3.2 CMV-AR reduces fear memory ...... 41

3.3.3 Gonadectomy removes genotype effects and testosterone restores them ...... 42

3.3.4 Effects of AR on gene expression in area CA1 of the hippocampus ...... 44

3.4 Discussion ...... 47

3.4.1 Effects of AR on gene expression ...... 48

3.4.2 Limitations ...... 50

3.5 Conclusions ...... 52

Chapter 4 H2A.Z Interacts with Androgenic Mechanisms to Regulate Fear Memory ...... 53

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4.1 Introduction ...... 53

4.1.1 Effect of both H2A.Z variants on fear memory ...... 53

4.1.2 Mechanism of interaction between H2A.Z and AR...... 55

4.2 Methods...... 57

4.2.1 Animals ...... 57

4.2.2 Genotyping ...... 57

4.2.3 Tamoxifen ...... 58

4.2.4 Contextual Fear Conditioning ...... 58

4.2.5 Gonadectomy and dihydrotestosterone (DHT) replacement ...... 59

4.2.6 Flutamide administration ...... 59

4.2.7 Tissue collection ...... 59

4.2.8 Western Blotting ...... 60

4.2.9 Primary hippocampal neuronal culture ...... 60

4.2.10 mRNA expression and Quantitative-PCR (qPCR) ...... 61

4.2.11 Chromatin Immunoprecipitation (ChIP) and qPCR ...... 61

4.2.12 Statistics ...... 62

4.3 Results ...... 63

4.3.1 Validation of H2A.Z KO in the hippocampus using gene expression and protein analyses ...... 63

4.3.2 Conditional-inducible H2A.Z deletion improves fear memory without affecting fear extinction in male mice ...... 63

4.3.3 H2A.Z KO improves fear memory and prevents androgenic effects on fear memory ...... 64

4.3.4 H2A.Z KO increases AR protein in the hippocampus ...... 68

4.3.5 DHT impairs depolarization-induced H2A.Z removal ...... 68

4.4 Discussion ...... 71

4.5 Conclusions ...... 75

Chapter 5 H2A.Z has Sex-Specific Effects on Memory ...... 77

5.1 Introduction ...... 77

5.2 Methods...... 79

5.2.1 Animals ...... 79

5.2.2 Genotyping ...... 79

5.2.3 Tamoxifen ...... 80

5.2.4 Stress-Enhanced Fear Learning (SEFL) ...... 80

5.2.5 Elevated Plus Maze (EPM) ...... 81

5.2.6 Open Field (OF) ...... 81

5.2.7 Tissue Collection ...... 82

5.2.8 mRNA Expression and RT-PCR ...... 82

5.2.9 Statistics ...... 83

5.3 Results ...... 83

5.3.1 Fear memory is unaffected in females with H2A.Z deletion ...... 83

5.3.2 H2A.Z deletion has a protective effect on stress-enhanced fear in female mice ...85

5.3.3 Measures of anxiety are unaffected by H2A.Z deletion ...... 87

5.4 Discussion ...... 89

5.5 Conclusions ...... 92

Chapter 6 Unifying Conclusions and Discussion ...... 93

6.1 Concluding Summary ...... 93

6.2 Unifying Discussion...... 95

6.3 Future Directions ...... 99

References ...... 101

List of Tables

3-1. DNA primers for genotyping. 3-2. cDNA primers used in gene expression studies. 4-1. DNA primers used for genotyping H2A.Z KO mice. 4-2. cDNA primers used in gene expression studies. 4-3. Genomic DNA primers used for ChIP. 5-1. DNA primers use for genotyping H2A.Z KO mice. 5-2. cDNA Primers used to confirm H2A.Z knockout.

List of Figures

2-1. Cholesterol metabolites associated with sex steroid hormones.

3-1. AR activation impairs fear memory in CMV-AR mice. 3-2. Gonadectomy removes difference between genotypes on fear memory and T replacement restores them. 3-3. Effects of genotypes, flutamide, and gonadectomy on gene expression in area CA1 of the hippocampus.

4-1. H2A.Z gene and protein expression is reduced in the hippocampus of H2A.Z KO mice.

4-2. H2A.Z KO improves fear memory but not fear extinction in males.

4-3. Effects of androgenic manipulation on fear memory in H2A.Z KO mice.

4-4. AR protein expression in the hippocampus of H2A.Z KO mice.

4-5. Effects of DHT and depolarization in primary hippocampal neurons.

5-1. H2A.Z KO does not affect fear memory in females.

5-2. Stress-enhanced fear learning is affected sex-specifically by H2A.Z deletion.

5-3. Measures of anxiety are unaffected by H2A.Z deletion.

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Chapter 1 Introduction and General Aims 1.1 Preamble

The acquisition, consolidation, and maintenance of memory are vital processes in the way that organisms learn to interact with their environments by forming associations that build expectations and regulate future behaviour. One of the classic observations of associative learning was that of Pavlov’s dogs. During his studies of animal digestive processes, Pavlov noticed that his dogs began salivating in response to the technician who brought them food before seeing the food. To follow up this curious observation, he proceeded with a series of experiments in which he presented the dogs with an auditory stimulus before presenting them with food. These were some of the pioneering experiments exploring classical conditioning, a process in which a conditioned stimulus is associated with an unconditioned stimulus to elicit a conditioned response (Pavlov, 2010).

For associative memories to become long lasting, they must undergo a process of consolidation. During the initial stages of memory consolidation, the hippocampus plays a significant role in memory acquisition. Here, within a time window of up to 7 days after a learning experience, the hippocampus processes and prepares memory for long-term storage in the cortex. Several molecular changes occur within both the hippocampus and the cortex that allow for associative memory to transition to the cortex and enter the maintenance phase of systems consolidation. Thus, the hippocampus is required for the acquisition of memory, but not required for long-term storage of it (reviewed in Frankland & Bontempi, 2005; Walters & Zovkic, 2015).

Classical fear conditioning is a particularly useful model of associative memory for several reasons. First, this type of learning is highly rapid, which allows for an explicit exploration of molecular changes time-locked to the learning event. Second, fear memory, in particular, is dysregulated in several forms of pathological conditions, most notably post-traumatic stress disorder (PTSD). A central aspect of PTSD is the re-experiencing of a traumatic memory, whereby a range of stimuli may serve as “reminders” of the initial trauma that invoke intrusive fear memories (Lissek et al., 2005). Women are more likely to develop PTSD compared to men, and their experience of PTSD is more severe, debilitating, and persistent (Breslau, Davis,

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Andreski, Peterson, & Schultz, 1997; Seedat, Stein, & Carey, 2005), suggesting that androgens may play a protective role against fear based disorders. In a population of Dutch military personnel deployed for four months to a combat zone, lower pre-deployment plasma testosterone levels were predictive of increased severity of self-reported PTSD-related symptoms one year after deployment and vice versa (Reijnen, Geuze, & Vermetten, 2015). These data suggest a potential role of androgenic mechanisms on regulating fear memory. One way through which androgens enact effects is through the androgen receptor (AR), which is a steroid hormone receptor that acts as a transcription factor when bound by androgens.

Learning-induced changes in gene expression are necessary for memories to become long lasting (Walters & Zovkic, 2015) and transcriptional regulation is heavily regulated by epigenetic mechanisms, which include potentially-stable modifications on DNA and associated histones. Neuroepigenetics is the study of epigenetic changes in the nervous system and progress in this field has demonstrated an essential role for various epigenetic modifications in memory regulation (Zovkic & Sweatt, 2013). Of particular relevance to this thesis is the histone variant H2A.Z, whose depletion improves fear memory in male mice (Zovkic, Paulukaitis, Day, Etikala, & Sweatt, 2014). Although the relationship between AR and H2A.Z has not been studied in the brain, a growing body of evidence in prostate cancer indicates that the two proteins interact to regulate transcription. Thus, the goal of this thesis is to characterize how these two factors interact to regulate fear memory.

1.2 Thesis Organisation and General Aims

1.2.1 Thesis Organisation

This thesis is organized to best express the order in which experiments were conducted, based on the experimental questions that were posed as a result of each prior study. Chapter 2 consists of a general literature review meant to provide overarching background and context for all studies in the subsequent chapters. Chapter 3 is reformatted from a primary publication of the first study in this thesis. Chapters 4 and 5 are written in thesis format and describe follow up experiments. Finally, Chapter 6 briefly summarizes the key findings and general conclusions of the thesis and outlines ongoing and future directions.

1.2.2 General Aims

The overarching goal of this thesis was to characterize the androgenic contribution to fear memory and elucidate the underlying mechanism of this contribution by focusing on interactions between AR and the histone variant H2A.Z. Based on evidence for their interaction, I also characterized sex differences in H2A.Z effects on fear memory.

1. Characterize the role of androgens in fear memory and transcription by using a transgenic mouse line that overexpresses AR.

2. Characterize interactions between H2A.Z and AR in fear memory

a. First, characterize fear memory in mice with conditional-inducible knockout of both genes encoding H2A.Z

b. Next, characterized the functional interactions between AR and H2A.Z in fear memory

3. Characterize the mechanism by which AR and H2A.Z interact under basal and activity-induced conditions in hippocampal neurons

4. Given that AR acts as a regulator of H2A.Z, determine if effects of H2A.Z on behaviour are sex-specific

Chapter 2

General Introduction

2.1 Memory Formation and Consolidation

A complex set of neural networks and mechanisms are involved in memory consolidation, the process by which memories are rendered stable and long lasting. Müller and Pilzecker first proposed the theory of memory consolidation a century ago (Lechner, Squire, & Byrne, 1999; McGaugh, 2000; Müller & Pilzecker, 1900). Through numerous studies, they showed that memory of newly learned information is disrupted by learning of other information shortly after the initial learning event, whereas performing similar interference tasks at later time points had no impact on memory. This finding suggested that new memories are initially labile and must undergo a transient biological process that renders them stable and long lasting. The first step in achieving long-lasting contextual fear memories is cellular consolidation, which occurs immediately after learning and lasts for several hours. Once stabilized, recently acquired memories are dependent on the hippocampus for approximately 7 days after learning, after which they become gradually dependent on the prefrontal cortex. This “transfer” of memories to the cortex is termed systems consolidation and is followed by memory maintenance (Frankland & Bontempi, 2005).

Cellular consolidation is triggered by the learning event and involves fast changes in synaptic activation, recruitment of transcription factors and, ultimately, protein synthesis required for structural changes (Frankland & Bontempi, 2005). The hippocampus has been implicated as the crucial brain region for this initial phase of consolidation through studies on brain injury patients (Rempel-Clower, Zola, Squire, & Amaral, 1996; Scoville & Milner, 2000; Squire, Slater, & Chace, 1975) and lesion studies in rodents (Anagnostaras, Maren, & Fanselow, 1999; Cho, Beracochea, & Jaffard, 1993; Clark, Broadbent, Zola, & Squire, 2002). The cellular consolidation phase is brief, as manipulations blocking memory formation during the first few hours after learning (e.g. protein synthesis and transcription inhibitors) are ineffective at blocking

5 memory formation when administered upto 6 hours after learning (reviewed in Walters & Zovkic, 2015).

Systems consolidation, on the other hand, is a longer-lasting, more protracted process and refers to the gradual reorganization of brain regions that support memory (Frankland & Bontempi, 2005). It occurs over multiple stages during which memories transition from the largely hippocampal-dependent recently acquired stage (up to 7 days in rodents and ~2 years in humans) to cortical-dependent remote (≥7 days in rodents and >~2 years in humans) stages of long-term memory maintenance (reviewed in Walters & Zovkic, 2015).

2.1.1 Behavioural Paradigms to Study Memory

Numerous behavioural paradigms have been developed to elucidate the contribution of different brain regions and molecular mechanisms in memory processes. Fear conditioning has been particularly useful for studying the temporal changes in the molecular basis of memory consolidation due to the ability of rodents to acquire this task within a single training session. In the contextual fear conditioning variant of the task, a novel chamber acts as a neutral conditioned stimulus (CS) and is paired with a mild foot shock, which acts as an aversive unconditioned stimulus (UCS). After a delay, animals are re-exposed to the CS (i.e., the training context) without shock and freezing (defined as a lack of movement except breathing) is recorded as a measure of memory. In cued fear conditioning, the UCS is preceded or proceeded by a tone, which acts as the conditioned stimulus. If learning occurs, subsequent exposure to the tone elicits both the emotional and physiological responses produced by the aversive UCS. Due to the emotionally aversive component of this test, the basolateral amygdala is required for encoding the formed association (particularly tone-UCS pairing in cued fear conditioning) in long-term memory (Rudy, Huff, & Matus-Amat, 2004).

I specifically utilized contextual fear conditioning because of its reliance on the hippocampus, a brain region that is crucial for memory consolidation in humans (Eichenbaum, 2013). The hippocampus forms connections between aspects of the context and, upon shock, strengthens connections with the basolateral amygdala, which plays a role in modulating memory consolidation for the context in the hippocampus. In contextual fear conditioning, the amygdala is required for memory formation and storage, unless the rodent is pre-exposed to the context a day before the shock, in which case context representation is established within the hippocampus

6 prior to shock exposure (reviewed in Rudy et al., 2004). The hippocampus is also a significant contributor to the robustness of fear memory due to associating the aversive stimulus with place/context cues (Rudy et al., 2004). Specifically, area CA1 in the hippocampus is strongly activated during both acquisition and recall of contextual fear conditioning, suggesting that it has a critical role in processing contextual fear memories. The cortex, on the other hand, is important specifically with regards to the expression of fear memory at remote time points, and its inactivation results in impaired remote fear memory (Frankland, Bontempi, Talton, Kaczmarek, & Silva, 2004; Walters & Zovkic, 2015).

Other memory tests exploit rodents’ innate preference to explore novelty to measure memory. One such test is the object recognition (OR) test, which measures object recognition memory. This test is dependent on both hippocampal as well as in the insular cortex and ventromedial prefrontal cortex (Akirav & Maroun, 2005; Balderas et al., 2008; Bermudez-Rattoni, Okuda, Roozendaal, & McGaugh, 2005). In this paradigm, the rodent is exposed to two objects in the testing chamber. After some time (90min-4hr for short-term memory or 24+ hr for long-term memory) the rodent is re-exposed to the chamber with a familiar and a novel object and the time spent with each object is measured. If the rodent explores the novel object more, it is said to show learning in the form of recognition of object novelty (Denninger, Smith, & Kirby, 2018; Leger et al., 2013; Vogel-Ciernia & Wood, 2014). Another similar test is object location (OL) memory, which increases reliance on spatial information. This is a measure of hippocampal- dependent spatial memory. The experimental setup for this test is identical to that of OR up until the testing phase where, instead of replacing one object, it is simply moved to another location within the testing chamber. Similar to NOR, if the animal spends more time exploring the novel location, it is said to show learning in the recognition of location novelty (Denninger et al., 2018; Vogel-Ciernia & Wood, 2014).

A similar memory task is the Object-in-Place (OiP) test. OiP associative recognition memory requires the subject to make an association between an object and place in which it was previously encountered. In this test, the rodent is exposed to numerous objects within the testing chamber during the training phase. During the testing phase, however, the objects on one side of the chamber are switched in location and the amount of time spent exploring the side with the switched objects (novel object orientation) versus the side with the familiar object orientation is measured. Increased time spent exploring objects on the novel side indicates successful task

7 completion and learning. In rats, OiP memory formation has also been shown to depend critically on the hippocampus, which has been shown to functionally interact with both the perirhinal cortex (PRH) and medial prefrontal cortex (mPFC) during OiP memory performance (Barker & Warburton, 2015).

One advantage of these tasks is that they are single-trial tasks. In other words, animals can generally learn the task within a single trial, enabling experimenters to investigate memory formation at specific time points. For example, by altering the length of time between the training and testing phases, one can examine effects on cellular consolidation, as well as systems consolidation.

2.2 Hormonal Effects on Learning and Memory

2.2.1 Overview of Steroidogenesis

Cholesterol is the precursor of all steroid hormones. The gonads and adrenal cortex are the primary sources of testosterone in both sexes of most vertebrate species, though smaller amounts of steroids, including testosterone, are also synthesized de novo from cholesterol or steroid precursors in the brain and are referred to as neurosteroids (Baulieu, Robel, & Schumacher, 2001; Melcangi, Garcia-Segura, & Mensah-Nyagan, 2008).

Typically, in mitochondria and smooth endoplasmic reticulum of cells, cholesterol is converted to numerous molecular precursors before being converted to androgens or estrogens (other final steroids include cortisol and aldosterone). Cholesterol is first converted to pregnanolone which can be further converted to progesterone or 17-hydroxy-pregnanolone. 17-hydroxy-pregnanolone is then converted to dehydroepiandrosterone (DHEA), which is further converted to androstenedione, which can be converted to either estrone or testosterone. Estrone can be converted to estradiol (E), ending its conversion pathway. Testosterone (T), however, can undergo two processes. First, it can be further reduced to dihydrotestosterone (DHT) by 5α- reductase, a significantly more potent androgen than T, a process observed in both neurons and glia (Celec et al., 2015; Melcangi et al., 2008). Or it can be aromatized to estradiol by aromatase, observed in numerous brain regions including the hippocampus (Atwi, McMahon, Scharfman, & MacLusky, 2016; Celec, Ostatní-ková, & Hodosy, 2015).. Going back to progesterone in the pathway, progesterone can be further converted to 17α-hydroxypreganolone then

8 androstenedione, and follow the remainder of the pathway as described above (for review, see McHenry, Carrier, Hull, & Kabbaj, 2014)(Fig. 2-1). Estrogens can act upon numerous estrogen receptors (ERs) including ERα, ERβ, and GPER (G-protein coupled ER), while androgens act upon androgen receptors (ARs) of which there are two main kinds, the membrane AR (approximately 56kDA) and AR (2 isoforms of 110kDa or 80kDa).

Figure 2-1. Cholesterol metabolites associated with sex steroid hormones.

Numerous studies have elucidated the actions of each of these steroid hormones. While androgens primarily act upon the androgen receptor (AR) to affect the androgenic pathway, this is not always the case. As mentioned earlier, testosterone can be aromatized to estradiol within the cell. Further, a small percentage of DHT can be reversibly converted to androstane-3α, 17β- diol (3α-diol) in astrocytes, which is a weak ligand for nuclear AR and ER, but a potent modulator of GABA’s effects on GABAA-Rs (Cunningham & Huckins, 1979; Frye, Van Keuren, & Erskine, 1996; Reddy, 2004). DHT can also be metabolized to androstane-3β, 17β-diol (3β- diol) which is a ligand for ERβ (Handa, Sharma, & Uht, 2011). Thus, exposing the brain to testosterone potentially exposes the hippocampus to an array of sex hormones, all of which may

9 contribute to observed responses (reviewed in (MacLusky, Hajszan, Prange-Kiel, & Leranth, 2006)).

Additionally, to study the effects of blocking AR function, a few different AR antagonists can be used. We use flutamide, which is a potent AR antagonist. One potential confound of using flutamide, however, is that it can act upon GABAA-benzodiazepine-chloride channel to affect phenotype (Ahmadiani, Mandgary, & Sayyah, 2003).

2.2.2 Behavioural effects of sex steroid hormones on learning and memory

Sherwin performed one of the first studies investigating the effects of sex hormones on learning and memory in 1988 (Sherwin, 1988). In this study, premenopausal women who underwent oophorectomy and hysterectomy, i.e. the surgical removal of both ovaries and uterus, were given either estrogen only, androgen only, a combined estrogen-androgen replacement, or placebo treatments. She then tested cognitive abilities including STM, LTM, and logical reasoning. Interestingly, placebo-treated women exhibited lower scores on all cognitive tests compared to hormone-treated women. Further, women with hysterectomy only, i.e. surgical removal of the uterus with intact ovaries, showed comparable cognitive abilities to hormone-treated oophorectomized women. These results indicated the importance of ovarian hormones in memory and other cognitive abilities.

Since these initial findings, the effects of steroid hormones on memory have become heavily studied, particularly regarding estrogenic effects on memory in females. Estrogen receptors (ERs) are heavily expressed in brain regions implicated in memory. While ERα, ERβ, and the GPER are all expressed in the hippocampus, ERβ, in particular, is much more prevalent (reviewed in (Weiser, Foradori, & Handa, 2008)). Generally, estradiol acts to enhance or maintain memory in women if the replacement occurs soon after ovariectomy (Sherwin, 2005). Several studies have described enhancing effects of estradiol on memory, specifically when using two object-based one-trial learning tasks such as OR and OL (Frick, Tuscher, Koss, Kim, & Taxier, 2017). For example, several studies have found that pre-training administration of estradiol or agonists of the various ERs given either systemically or directly into the hippocampus can rapidly enhance various forms of hippocampal-mediated memories, including spatial, object, and social memories (Daniel, 2006; Gabor, Lymer, Phan, & Choleris, 2015; Luine, 2014; Lymer et al., 2018; Phan et al., 2015). Studies using post-training treatments were

10 conducted to pinpoint the effects of estradiol on consolidation. Several labs have consistently found that estradiol significantly enhances hippocampal-dependent spatial and object recognition memory consolidation in young male and female mice and rats (reviewed in (Daniel, 2006; Frick, 2015; Luine, 2014)), whereby application of estradiol immediately, but not 1-3 hours after training enhances memory, as measured by the Morris Water Maze (MWM), OR, and OL tasks. While most of these studies used ovariectomized females, similar results were found in gonadally intact males (Packard, Kohlmaier, & Alexander, 1996). Estradiol’s enhancing effects on memory have been observed across multiple labs and between both males and females (Daniel, 2006; Frick, 2015; Luine, 2014).

Effects of estradiol on fear memory are likely mediated by all three isoforms of ER, but ERβ appears to be particularly important, consistent with the abundance of this receptor in the hippocampus in comparison to ERα (Weiser et al., 2008). In particular, both male and female ERβ knockout mice exhibit impaired contextual fear memory, whereas cued fear memory was impaired only in male KO mice (M. Day, Sung, Logue, Bowlby, & Arias, 2005). These findings directly implicate ERβ in contextual fear conditioning in females and both cued and contextual fear conditioning in males.

In contrast, the effects of androgens on memory and related mechanisms are not so heavily studied. While there is evidence of substantial androgenic effects on memory, it is difficult to draw firm conclusions as results within the literature are quite varied. However, evidence does suggest that androgens regulate various forms of memory. For example, various forms of spatial working memory, as indexed by different versions of the radial arm maze and T-maze, are impaired by castration (Gibbs & Johnson, 2008; Spritzer, Gill, Weinberg, & Galea, 2008), but testosterone replacement in castrated males has conflicting results. Whereas testosterone replacement in castrated male rats did not restore spatial memory on the radial arm maze (Gibbs & Johnson, 2008), it did restore performance on a T-maze alteration task and a water radial arm maze (Bimonte-Nelson et al., 2003; Kritzer, McLaughlin, Smirlis, & Robinson, 2001). Thus, the role of androgens on short-term working memory is unclear and may be dependent on the type of task used.

Similarly, testosterone’s effects on spatial memory are also unclear. For instance, studies using the radial arm maze indicate testosterone impairs spatial memory, while studies investigating

11 androgenic effects on performance in the MWM exhibit highly mixed results. In this case, differences may be due to differences in gonadal status and hormone treatment. Specifically, gonadectomy followed by a single hormone injection just prior to training resulted in improved MWM performance (Khalil, King, & Soliman, 2005), while intact males given empty or testosterone silastic implants (Goudsmit, Van De Poll, & Swaab, 1990) or intrahippocampal testosterone injections exhibited impaired MWM performance (Naghdi, Nafisy, & Majlessi, 2001). On the other hand, castration with or without testosterone treatment 35 minutes prior to the first trial (Naghdi, Mohaddess, Khamnei, & Arjomand, 2005) and castration versus sham- surgery (Spritzer et al., 2008) exhibited no difference in performance. Thus, the effects of androgens on spatial memory are heavily dependent on timing and method of androgenic manipulation.

One of the ways through which testosterone exerts its effects on behaviour is through the androgen receptor (AR). AR is expressed in brain regions that regulate memory, including in areas CA1 and CA3 of the hippocampus (Kerr, Allore, Beck, & Handa, 1995; Raskin et al., 2009). Studies have reported mixed effects of AR and its ligands, testosterone and dihydrotestosterone (DHT), on fear memory. For example, some studies report no effects of gonadectomy on fear memory in male rodents (Anagnostaras et al., 1998), whereas others report distinct effects of androgenic manipulations on contextual and cued fear memory (Chen et al., 2014; Edinger, Lee, & Frye, 2004; Frye, Edinger, & Sumida, 2008; MacLusky, Hajszan, & Leranth, 2005; Rubin, 2011; Q. Zhang et al., 2014). Moreover, some evidence suggests that castration reduces fear memory in male rodents (Edinger et al., 2004; McDermott, Liu, & Schrader, 2012) and that this deficit is rescued by treatment with either testosterone or 17β estradiol, but not with DHT (Edinger et al., 2004). Given that DHT is a direct AR agonist, whereas testosterone can be further aromatized into 17β estradiol to activate estrogen receptors (Hojo et al., 2004; Mukai et al., 2010; Ooishi et al., 2012), these data suggest that testosterone may influence fear memory through AR-independent actions. In contrast, both DHT and testosterone improved performance on the inhibitory avoidance test, in which mice learn to avoid an environment associated with shock (Edinger et al., 2004), supporting a direct role of AR in regulating fear-based tasks. There are a few potential reasons behind this variability in the literature. These include differences in the method of testosterone administration (e.g. silastic implant, systemic injections, intracerebral infusions, among others), time of treatment (before or

12 after training), location of treatment (systemic or local), and dose. This is further complicated by the fact that many studies have not measured circulating T levels post-treatment, as sub- and supra-physiological doses of testosterone or DHT may have differing effects on behaviour.

2.2.3 Cellular effects of androgens on hippocampal structure and function

Sex hormones have complex effects on morphology and functional plasticity in the hippocampus. Sexual dimorphisms within the hippocampal structure initially hinted at differences in hormonal effects. For example, male rats have a larger and more asymmetric dentate gyrus than females (Roof, 1993; Roof & Havens, 1992). Furthermore, there are sex differences in the apical dendritic structure and dendritic branching patterns of CA3 pyramidal neurons, which is consistent with the observation that males display a significantly higher density of mossy fiber synapses (Parducz & Garcia-Segura, 1993). Additionally, there is evidence that hippocampal synaptogenesis, as well as hippocampal excitability, fluctuates across the estrus cycle in female rodents, such that they may be comparable to males at certain stages (e.g. diesterus and metestrus) (Prange-Kiel, Fester, Zhou, Jarry, & Rune, 2009; Scharfman, Mercurio, Goodman, Wilson, & MacLusky, 2003; Warren, Humphreys, Juraska, & Greenough, 1995). In male rodents, androgenic manipulations specifically have dissociable effects on excitability, spine density, and neurotrophin production, with evidence for both androgenic and estrogenic mechanisms of gonadal testosterone.

Male mice demonstrate a significant increase in dendritic spine density on apical dendrites of CA1 and CA3 neurons around the time of puberty, perhaps through organizational effects during the pubertal increase in circulating testosterone levels, as pre-pubertal gonadectomy reverses these effects (Meyer, Ferres-Torres, & Mas, 1978). In females, estrogens cause a very significant increase in spine and spine synapse density in the CA1 (Gould, Woolley, Frankfurt, & McEwen, 1990; Leranth, Shanabrough, & Horvath, 2000), suggesting that testosterone may have similar effects in males, perhaps through aromatization. While there is evidence to suggest this may partially be the case in males (Leranth, Petnehazy, & MacLusky, 2003; MacLusky, Hajszan, & Leranth, 2004), it is evident that different mechanisms may be involved in males and females. For example, in adult males, estradiol treatment does not seem to have a significant effect on CA1 pyramidal cell spine synapse density (Leranth et al., 2003), while both testosterone and estradiol increased spine synapse density in ovariectomized females (Leranth, Hajszan, &

MacLusky, 2004). Further, treatment with letrozole, an aromatase inhibitor (blocks estradiol synthesis), did not affect males (Fester et al., 2012). Finally, testosterone and dihydrotestosterone (DHT) treatment rescued gonadectomy-induced reductions in synapse density (Leranth et al., 2003) and rapidly increased spine density (Hatanaka et al., 2015) in area CA1 of male rodents. This is consistent with evidence that male testicular feminization mutant (Tfm) rats, which have a dysfunctional AR, exhibit reduced spine density in the medial prefrontal cortex compared to wild-type male rats (Hajszan, MacLusky, Johansen, Jordan, & Leranth, 2007). These data together provide evidence that estrogen biosynthesis is not required for androgenic effects on hippocampal spines and, further, that androgenic and estrogenic effects on hippocampal structure differ significantly between males and females.

Androgenic effects on hippocampal function are also unclear. Studies looking at electrophysiology within the hippocampus have found mixed effects of androgens. Specifically, in one instance, testosterone application to rat adult male hippocampal slices resulted in improved Shaffer collateral transmission in area CA1 (M. . Smith, Jones, & Wilson, 2002), suggesting that androgens potentiate CA1 plasticity. On the other hand, prepubertal gonadectomy (i.e. testosterone removal) also facilitated long-term potentiation (LTP) in area CA1 of adult male rats, suggesting the opposite, such that androgens reduce CA1 plasticity (Harley, Malsbury, Squires, & Brown, 2000). Given that AR is a transcription factor, with a subset having rapid non-genomic effects through membrane-bound AR, these data suggest that some downstream targets of AR may include synaptic proteins, as evidenced by the effects of AR on synaptic function.

2.2.4 Androgen receptor (AR) and memory

Testosterone primarily exerts its androgenic effects through actions on the androgen receptor (AR), but relatively little is known about the influence of AR on memory formation. AR is expressed in the cortex and the hippocampus, primarily in the CA1 and CA3 regions (Kerr et al., 1995; Raskin et al., 2009). As with testosterone, studies that investigated the role of AR in memory have produced mixed results. One study of women with Complete Androgen Insensitivity Syndrome (CAIS), in which the AR is non-functional in individuals with XY karyotype, compared their performance on a spatial learning task (a virtual Morris Water Maze) to control men and women. They found that women with CAIS performed comparably to control

14 women but slower than men, suggesting that the AR plays some role in regulating spatial memory in humans (Mueller, Verwilst, Van Branteghem, T’Sjoen, & Cools, 2016). In rodents, male Tfm mice, which have a non-functional AR protein, have impaired performance on the Morris Water Maze compared to female Tfm mice, which carry one normal AR allele, suggesting that AR has an important role in spatial learning and memory in male mice (Rizk, Robertson, & Raber, 2005). However, since the Tfm female mice were gonadally intact, the contribution of ovarian hormones such as estradiol cannot be excluded.

Similarly, neural AR deletion impaired performance of male mice on temporal order memory for visual objects (a test for an animal’s familiarity with the order of objects presentation) and also impaired NMDA-Receptor (NMDA-R) function in area CA1 (Picot et al., 2016). However, intra- CA1 injection of either testosterone (AR activation) or flutamide (AR antagonist) resulted in impaired learning in the MWM in rats (Naghdi, Majlessi, & Bozorgmehr, 2005), suggesting that AR may have an inverted U effect on memory. In a recent study, inhibiting estrogen synthesis (via aromatase blockade) in the dorsal hippocampus resulted in impaired performance on both NOR and OL memory tasks in gonadectomized male mice, but not intact males (Koss & Frick, 2019). Memory impairment on these tasks was recapitulated when intact males were treated with flutamide alone, as well as a combination of flutamide and letrozole (Koss & Frick, 2019). These data suggest a protective role of androgens on memory consolidation, as well as a potential interaction between AR and estradiol in mediating memory consolidation in intact male mice (Koss & Frick, 2019). Thus, research results with regards to androgens and AR’s effects on memory and learning are mixed. Although a role for AR in fear conditioning has not been directly measured, some data suggest that chronic treatment with anti-androgenic bisphenol-A results in reduced fear memory in male mice (Q. Zhang et al., 2014).

Transcription factors such as the androgen receptor largely affect phenotype through their actions on gene transcription. Specifically, the AR has been shown to regulate the transcription of numerous genes (Bolton et al., 2007). AR’s ability to transcribe specific genes has further been shown to be regulated by interactions with epigenetic factors on the genome (Dryhurst, McMullen, Fazli, Rennie, & Ausió, 2012). As such, the proceeding section provides an overview of epigenetic effects on memory, followed by an overview of the interaction between AR and epigenetic mechanisms.

2.3 Epigenetic Effects on Learning and Memory

2.3.1 Overview of Neuroepigenetics

Epigenetics is classically defined as the mitotically heritable changes in gene transcription caused by modifications upon the genome that do not alter the underlying genetic structure. Epigenetic marks, particularly DNA methylation, play a role in maintaining cell fate during development, during which phenotypically different body cells are produced from genetically identical cells (e.g. stem cells). Since this cell fate persists over many rounds of cell division throughout an organism’s lifespan, the self-perpetuating nature of epigenetic marks has been referred to as “epigenetic memory” as a way to describe how the cell “remembers” its identity (Bird, 2002). Neuroepigenetics is a term given to the study of epigenetic mechanisms within post-mitotic neuronal cells. Initial interest in epigenetic mechanisms in the brain originated from the self-perpetuating characteristic of certain epigenetic marks that made them an attractive candidate mechanism for maintaining long-lasting memories (Crick, 1984; Miller & Sweatt, 2007). However, many studies have shown that epigenetic marks in the brain can be either transient or stable. In particular, DNA methylation and histone post-translational modifications (PTMs; described in detail below) are dynamically regulated in the brain in response to external stimuli (Levenson & Sweatt, 2005, 2006; Stefanelli, Walters, et al., 2018; Sweatt, Nestler, Meaney, & Akbarian, 2013) and play a crucial role in learning and memory across species (Levenson & Sweatt, 2005, 2006).

DNA methylation is one of the best-known epigenetic mechanisms as it occurs directly on the genome and can be stable. It is the addition of a methyl group to a cytosine pyrimidine residue, generally within dinucleotide CpG sequences (wherein a cytosine nucleotide is followed by a guanine nucleotide). In mammalian genomes, about 1% of cytosines and 70-80% of CpG dinucleotides are methylated (Bird, 2002; Woldemichael, Bohacek, Gapp, & Mansuy, 2014). CpG dinucleotides occur in high densities in CpG islands, which are generally unmethylated, perhaps due to evolutionary reasons as methylated cytosines are more likely to undergo mutations (Woldemichael et al., 2014). Further, CpG islands are commonly found within promoter regions of genes and are generally minimally methylated. Increased DNA methylation typically results in transcriptional repression and is associated with gene silencing (Bird, 2002). DNA methylation can either prevent binding of DNA-binding factors by blocking their DNA

16 recognition sites, or it can recruit binding proteins such as methyl-CpG-binding protein 2 (MeCP2) and transcription repressors to CpG islands, altering chromatin structure and making it less accessible to transcriptional machinery (Jones & Takai, 2001; Klose & Bird, 2006). Although reversible, CpG-methylation can be a stable mark that persists across mitosis and, in some cases, meiosis (Gräff & Mansuy, 2008). It was recently discovered that learning-induced DNA methylation in the brain is highly dynamic and reversible (Miller & Sweatt, 2007). This finding challenged the dogma that epigenetic change is primarily immutable in order to preserve cellular identity. It is now accepted that many epigenetic changes that occur within mature cells are dynamic and continuously adapt to environmental stimuli, such as immune cells producing antibodies to different pathogens (Fernández-Morera, Calvanese, Rodríguez-Rodero, Menéndez- Torre, & Fraga, 2010) or skeletal muscle adapting to training (Lindholm et al., 2014).

DNA is organized into chromatin. DNA is compacted into chromosomes and stored in the cell nucleus, awaiting transcriptional activation by transcriptional machinery. Stretches of approximately 147 bp of DNA are packaged into nucleosomes, which consist of a histone protein octamer containing two dimers of canonical histones H2A-H2B and a tetramer of H3-H4. Linker histone H1 connects repeating nucleosomes to form the building blocks of chromatin (Wolffe & Hayes, 1999). The organization of chromatin into nucleosomes results in genome compaction, and the obstruction of genes from transcriptional machinery (Campos & Reinberg, 2009; Li, Carey, & Workman, 2007). Modifications of histone proteins can alter gene expression by regulating gene accessibility. More specifically, heterochromatin (highly compacted nucleosome structure) is associated with transcriptional repression, while euchromatin (open nucleosome structure) is associated with transcriptional activation.

2.3.2 Histone Post-translational Modifications

Histones are highly alkaline proteins with high affinity for DNA and work together to package DNA into nucleosomes. More specifically, since DNA is negatively charged due to phosphate groups, it easily wraps around histones as they are positively charged proteins due to the prevalence of arginine and lysine amino acids. Histones are classified into two major classes: core histones (H2A, H2B, H3, and H4) and linker histones (H1/H5). Core histones can exist in a canonical or a variant form. The canonical form is replication dependent, meaning that they are only expressed/incorporated during the synthesis phase of the cell cycle when DNA is

17 undergoing replication (Akey & Luger, 2003; J. Jin et al., 2005; Talbert & Henikoff, 2010). Genes encoding for the canonical histones of animals are localized in repeat arrays on the same chromosome and are transcribed during DNA replication in the S phase of mitosis. On the other hand, genes encoding for histone variants are found singly in the genome, with different promoters, may be on separate chromosomes, and are constitutively expressed. Variants differ from their canonical forms in primary amino acid sequence. Canonical histones function primarily in genome packaging and gene regulation. On the other hand, histone variants have more diverse roles, including transcription initiation and termination, DNA repair, meiotic recombination, chromosome segregation, sex chromosome condensation, and sperm chromatin packaging (Talbert & Henikoff, 2010).

Histones primarily participate in epigenetic regulation through post-translational modifications (PTMs). The N- and C-terminal histone tails extend outside the nucleosome core and are the target of numerous PTMs, including acetylation, methylation, and phosphorylation. Since PTMs are largely covalent proteins, one of their primary functions is to alter gene accessibility by modifying histone-DNA interaction by neutralizing the electrostatic attraction between DNA and histones. , Another way histone PTMs regulate gene accessibility is by recruiting non-histone proteins that regulate transcription (Kouzarides, 2007). Both functions affect numerous cellular processes, primarily chromatin compaction and gene expression. These processes are affected differently by distinct PTMs.

While there is a large and growing list of known histone PTMs, the specific PTMs implicated in memory formation and synaptic plasticity consist of histone acetylation, phosphorylation, methylation, and poly-ADP ribosylation (Woldemichael et al., 2014). For the purposes of transcription, these can be divided into those that correlate with activation and those that correlate with repression. In general, PTMs linked with activation include but are not limited to acetylation, methylation, phosphorylation, and ubiquitylation, while those implicated in repression include methylation, ubiquitylation, sumoylation, deamination, and proline isomerization (Kouzarides, 2007). Though, individual PTMs can affect both activation or repression depending on the location and context of the PTM.

Some histone PTMs have been linked specifically to transcriptional activation (e.g. trimethylation of histone three at lysine 4; H3K4me3) (Bernstein et al., 2002) or repression (e.g.

H3K27me3) (Ringrose, Ehret, & Paro, 2004); however, histone PTMs generally exert combinatorial effects on gene expression. Indeed, certain combinations of PTMs tend to co- occupy the same nucleosome (E. T. Wang et al., 2008). For example, individually, H3K4me3 and H3K27me3 are respectively associated with transcriptional activation and repression. However, their co-occurrence on the same nucleosome serves as a dual mark and is associated with poised genes, whereby this dual mark resolves once a decision is made about gene transcription of the associated gene (Bernstein et al., 2006). In addition to this, the location of a histone PTM within a gene may also affect the outcome of transcription. For instance, di-/trimethylation of H3K9 at gene promoters is associated with transcriptional repression, while recent evidence suggests that tri-methylation at the coding region may be associated with activation (Kouzarides, 2007; Vakoc, Mandat, Olenchock, & Blobel, 2005). Thus, the same histone PTM does not always generate the same transcriptional outcome, and the combinatorial effects of various modifications add complexity to gene regulation.

PTMs are deposited on and removed from histone tails by numerous enzymes (Bourtchouladze et al., 2003). While histones can undergo numerous PTMs, the most commonly studied are acetylation, phosphorylation, and methylation. Histone acetylation/deacetylation involves the addition/removal of an acetyl group on lysine (K) residues on histone tails (Hebbes, Thorne, & Crane-Robinson, 1988). This process is catalyzed by histone acetyl-transferase (HAT) and histone deacetylase (HDAC) enzymes which add or remove acetyl groups, respectively. Typically, acetylation is associated with transcriptional facilitation by reducing histone-DNA interactions and by recruiting chromatin-remodeling proteins and RNA polymerase II to initiate transcription (Mujtaba, Zeng, & Zhou, 2007). Histone phosphorylation, on the other hand, primarily occurs on serine and threonine residues and is mediated by numerous nuclear kinases and phosphatases (Chwang, 2006; Koshibu, Gräff, & Mansuy, 2011). Histone phosphorylation, too, is primarily associated with transcriptional activation (Macdonald et al., 2005).

On the other hand, histone methylation, the addition of a methyl group to lysine or arginine residues, can result in either transcriptional activation or repression. For instance, while methylation of lysine 4 on histone H3 (H3K4me3) is associated with transcriptional activation, methylation of lysine 9 (H3K9me) is associated with repression (Bernstein et al., 2002; Ringrose et al., 2004). Moreover, different amounts of methylation, such as mono, di-, or tri-, on the same residue, can have a different transcriptional outcome (Shilatifard, 2006). Deposition and removal

19 of methyl groups on histone tails are regulated by histone methyl transferase (HMT) and demethylase (HDM) enzymes. Numerous HMTs and HDMs have been characterized, and some are linked with memory formation, including Mll (mixed leukemia lineage 1 methyltransferase), an H3K4-specific HMT particularly important for hippocampal function (Kerimoglu et al., 2013).

Finally, histone PTMs can also affect gene accessibility by recruiting proteins that can have either a positive or negative effect on transcription. For example, methylation of lysine 4 on histone 3 (H3K4me) can prevent the binding of HDAC enzymes, potentially resulting in increased acetylation. On the other hand, acetylation at H3K18 can favour the binding of HAT proteins and other transcription factors (such as CREB-binding protein; CBP), thus promoting acetylation and transcription (Jenuwein & Allis, 2001; Kouzarides, 2007; Strahl & Allis, 2000). However, it is a challenge to define the individual impact of histone PTMs due to the existence of a multitude of PTMs that, additionally, occur and exert effects in a combinatorial fashion ((Young, DiMaggio, & Garcia, 2010) for review). To further complicate matters, PTMs have different effects dependent on their location on the histone tail. Specifically, the displacement of histones has been shown to result in repression of basally transcribed genes (Strahl & Allis, 2000).

2.3.3 Histone Variants

One of the lesser studied branches of epigenetics is histone variant exchange, in which canonical histones can be replaced by non-allelic histone variants. Histones and their variants are highly conserved across species. Several variants have been identified for histones H2A, H2B, and H3, while a lack of evidence exists for the presence of H4 variants in mammals (Banaszynski, Allis, & Lewis, 2010). In contrast to canonical histones, histone variants are replication-independent and, as such, they can be synthesised and incorporated into nucleosomes irrespective of cell division (Weber & Henikoff, 2014). The ubiquitous expression of histone variants throughout the cell cycle suggests a unique role in postmitotic cells, such as neurons.

The incorporation of histone variants into the nucleosome is highly dynamic and can be regulated through histone turnover or histone variant exchange. Histone turnover involves the replacement of removed histone proteins with newer identical proteins (J. Jin et al., 2005; Maze et al., 2015), whereas histone variant exchange is the replacement of one histone type with

20 another (J. Jin et al., 2005; Maze et al., 2015). Initially, histone turnover was found to be relatively slow, with a half-life of 117 days for histones in the liver (Commerford, Carsten, & Cronkite, 1982). More recently, however, studies have shown that the half-life of histone variants H3.3 and H2A.Z in neurons is approximately 3.5 days under basal conditions and 0.7 days under induced conditions (Maze et al., 2015). Together, this evidence suggests that histones can be rapidly replaced in the brain. Both histone variant exchange and histone turnover have been implicated in neuronal function and plasticity (Maze et al., 2015; Zovkic et al., 2014) and, recently, histone variant H2A.Z has been implicated in memory formation and learning-induced gene transcription (Narkaj et al., 2018; Stefanelli, Azam, et al., 2018; Zovkic et al., 2014).

The mechanism of histone variant exchange and turnover is facilitated by the existence of a soluble histone pool within the nucleus. There are two distinct but interconnected histone fractions within the nucleus, one that is insoluble and is bound to chromatin, and a second, that is soluble. The soluble histone pool comprises of free, unbound histones that largely consist of newly synthesised as well as recently evicted histones which are guarded by chaperone proteins to prevent binding to DNA (Alvarez et al., 2011; Cook, Gurard-Levin, Vassias, & Almouzni, 2011; Mendiratta, Gatto, & Almouzni, 2019). At any given time, approximately 85% of histones are bound to chromatin, while the remaining 15% is in the soluble pool, awaiting incorporation (Galvani & Thiriet, 2015). Upon stimulation, such as a learning event, stimulus-induced changes in histone levels are observed in both pools, contributing to dynamic histone variant exchange and turnover (Maze et al., 2015). This soluble pool is essential for successful histone exchange/turnover, as evidenced by impaired stimulus-induced histone exchange within the chromatin and transcription in response to low levels of histones in the soluble pool (Maze et al., 2015). This is particularly important for H2A.Z because it is one of the most dynamic histones and undergoes stimulus-induced eviction from chromatin as a crucial step in transcription initiation during learning (Stefanelli, Azam, et al., 2018; Zovkic et al., 2014).

2.3.4 Histone variant H2A.Z and transcription

H2A.Z, a variant of canonical histone H2A, is a significant regulator of transcription initiation. H2A.Z shares only 60% sequence identity with H2A (J. Jin et al., 2005). Intriguingly, studies of H2A.Z are complicated by the fact that, in vertebrates, it is encoded by two separate genes on separate chromosomes that produce proteins differing by three amino acids (Matsuda et al.,

2010). As such, it is impossible to study differences between the two proteins using antibody methods as they cannot distinguish between the two gene products, but differences in their expression levels can be captured with gene expression studies.

In invertebrates, H2A.Z is encoded by one gene (Dryhurst et al., 2009) and the emergence of two genes in vertebrates likely occurred due to duplication of the whole genome at the beginning of vertebrate evolution. Each H2A.Z encoding gene has a unique promoter, 5’ UTR (untranslated regions), and intron/exon organization, yet they produce proteins that differ by only three amino acids (Coon et al., 2005; Dryhurst et al., 2009). Except for yeast, H2A.Z has been shown to be crucial for survival in several organisms, including the ciliate protozoan Tetrahymena (Liu, Li, & Gorovsky, 1996), Drosophila (Clarkson, Wells, Gibson, Saint, & Tremethick, 1999), Xenopus (Ridgway, Brown, Rangasamy, Svensson, & Tremethick, 2004), and mice (Faast et al., 2001). In mice, deletion of only one gene (H2afz, H2A.Z-1) results in lethality, with dramatic resorption of mutant blastocysts during the transition from the blastocyst to the embryonic stage starting at 4.5 days post coitum (Faast et al., 2001). This finding points to the idea that the protein products (H2A.Z-1, H2A.Z-2) of the two genes (H2afz, H2afv) may have differing functions in vertebrates.

H2A.Z is deposited into nucleosomes by ATP-dependent chromatin remodelling complexes such as SNF2-related CBP activator protein (SRCAP; Swr1 in yeast) and p400, which exchange H2A.Z-H2B dimers for H2A-H2B dimers (Choi, Heo, & An, 2009). H2A.Z-containing nucleosomes can either be homotypic (H2A.Z-H2A.Z) or hybrid (containing one H2A and one H2A.Z), with hybrid nucleosomes conferring reduced stability in comparison to homotypic H2A.Z nucleosomes and more stability than H2A-only nucleosomes (Ishibashi et al., 2009). H2A.Z also interacts with other histones/variants within the nucleosome to affect nucleosome stability, as revealed by increased instability in nucleosomes with both H2A.Z and H3.3 in human cells (C. Jin et al., 2009), as well as increased resistance of H2A.Z nucleosomes to affiliate with H1 linker histones (Thakar et al., 2009).

H2A.Z flanking the transcription start site (TSS) of genes has been associated with promoting efficient RNA polymerase II (RNAPII) recruitment in both yeast and human cells (Adam, Robert, Larochelle, & Gaudreau, 2001; Hardy et al., 2009). In D. Melanogaster, H2A.Z at the +1 nucleosome (directly downstream of the TSS) has been correlated with RNAPII pausing

22 downstream of the TSS, a process in which RNAPII halts prior to transcriptional activation (Mavrich et al., 2008). The functional relevance of this process is seen in activity-induced neuronal immediate early genes (IEGs). RNAPII pauses proximal to the promoter of several IEGs until an activity signal is received (Saha et al., 2011). This activity-induced signal releases the paused RNAPII, which then transcribes the genes in question, resulting in rapid transcription of IEGs. H2A.Z located at the nucleosome directly downstream of the TSS (+1) has also been associated with TSS selection, where RNAPII selects a position within the promoter region to begin transcription (Jiang & Pugh, 2009). Considering the increased stability of H2A.Z- containing nucleosomes, it makes sense that +1 nucleosomes with H2A.Z are correlated with RNAPII pausing, as a stable nucleosome serves as a barrier to transcription by preventing unraveling of heterochromatin.

Additionally, transcription is associated with reduced H2A.Z occupancy at promoter regions (Sutcliffe et al., 2009; Zlatanova & Thakar, 2008) as well as gene bodies, where the eviction of randomly incorporated H2A.Z is preferentially replaced with H2A in both plants and animals (Hardy et al., 2009). On the other hand, H2A.Z may also prevent promoters from being methylated (Hardy et al., 2009), as H2A.Z and methylation are mutually antagonistic chromatin marks (Zilberman, Coleman-Derr, Ballinger, & Henikoff, 2008). In animals, methylation at gene bodies may prevent H2A.Z incorporation as well as random transcription at cryptic promoter sites present within gene bodies. Specifically, H2A.Z serves to poise inactive genes for activation, while methylation largely serves to repress genes from being activated. Interestingly, in human cells, H2A.Z nucleosomes in upstream promoter regions contribute to both positive and negative gene regulation (Gévry et al., 2009; Gévry, Ho, Laflamme, Livingston, & Gaudreau, 2007).

As is the case with canonical histones, H2A.Z function is modified by PTMs, with unique modifications producing distinct effects on transcription. Genome-wide analyses in various organisms (yeast (Millar, Xu, Zhang, & Grunstein, 2006), chicken (Bruce et al., 2005), & various prostate cancer cell lines (Valdes-Mora et al., 2012)) have shown that acetylated H2A.Z is enriched at actively transcribed genes, whereas non-acetylated H2A.Z is present at promoters of genes not being actively transcribed. Moreover, other epigenetic markers of transcriptional activation, such as tri-methylated H3 lysine 4 (H3K4me3) (Sarcinella, Zuzarte, Lau, Draker, & Cheung, 2007), are often enriched at H2A.Z-containing nucleosomes, indicating that non-

23 acetylated H2A.Z is a marker of poised genes. In contrast, H2A.Z ubiquitylation is associated with facultative heterochromatin and plays a role in the inactivation of the X-chromosome in human female cells (Sarcinella et al., 2007), indicating that ubH2A.Z is a repressive mark.

Finally, H2A.Z can also affect transcription differently through distinct functions of its two isoforms, H2A.Z-1 and H2A.Z-2, which may be related to differences in their protein structure, as indexed by different mobility on SDS polyacrylamide gel electrophoresis. In addition, the two may exert distinct functional (Dryhurst et al., 2009, 2012) properties upon the nucleosome, that may result in differing effects on gene transcription. For example, in a study utilizing rat cortical neurons, gene transcription of the IEG Arc was impaired by depletion of H2A.Z-2 but not H2A.Z-1(Dunn et al., 2017). In addition, castration resistant lymph node carcinoma of the prostate (LNCaP) xenograft tumours is associated with a specific increase in H2A.Z-1 and not H2A.Z-2 levels (Dryhurst et al., 2012), pointing to a distinct function of H2A.Z-1 in the transition of prostate cancer from an androgen-dependent to independent stage compared to H2A.Z-2. Nevertheless, direct experimental characterization of the two H2A.Z variants is currently limited.

2.3.5 Histone variant H2A.Z and memory

Since the establishment of neuroepigenetics as a field, the majority of research about memory formation has focused on the role of DNA methylation and histone PTMs. Histone variants, on the other hand, have been largely understudied and certain variants have been recently found to play a substantial role in memory formation (Maze et al., 2015; Zovkic et al., 2014). One such variant is H2A.Z, a variant of H2A.

Genome-wide studies of H2A.Z have recently implicated H2A.Z as a negative modulator of fear memory and have also characterized H2A.Z binding in the mouse hippocampus. In previous studies from our lab, we specifically examined H2A.Z binding at the promoter regions of relevant genes. More specifically, we looked at nucleosomes directly upstream (-1) and downstream (+1) of the TSS. This is functionally relevant because the +1 nucleosome is classically the greatest barrier to transcriptional machinery and changes at that nucleosome may be arguably more applicable to changes in transcription (Subramanian, Fields, & Boyer, 2015). Furthermore, we have observed learning-induced changes of H2A.Z binding to be larger at the +1 nucleosome than at the -1 nucleosome, increasing its relevance to study memory (Zovkic et

24 al., 2014). Additionally, transcription factors largely bind at promoter and enhancer regions upstream of the TSS, making the -1 nucleosome relevant for transcription factor studies.

H2A.Z Binding We recently identified H2A.Z as a novel regulator of memory formation and maintenance in mice (Zovkic et al., 2014). We found that, similar to findings in non-neuronal literature, H2A.Z was enriched at gene bodies and promoters, with promoter enrichment far exceeding gene body enrichment. Consistent with data across species and multiple cell types (Coleman-Derr & Zilberman, 2012; Ku et al., 2012), as well as previous findings in our lab (Zovkic et al., 2014), H2A.Z is strongly positioned at sites flanking the TSS. More specifically, we found that H2A.Z occupancy was higher at the +1 nucleosome than it was at the -1 nucleosome of the TSS under basal conditions. However, both nucleosomes showed equivalent and substantial reduction of H2A.Z binding after fear conditioning and increased in binding with age (Stefanelli, Azam, et al., 2018). Thus, even though H2A.Z binding is preferentially increased at the +1 nucleosome at the TSS both the +1 and -1 nucleosome are equally sensitive to learning- and age-related regulation.

Learning-induced changes in H2A.Z binding. Specifically, we showed that fear conditioning resulted in the removal of H2A.Z from the first nucleosome downstream (+1 nucleosome) of the transcription start site (TSS) with an associated increase in expression of several memory-related immediate early genes in the hippocampus (Zovkic et al., 2014). Similar to other epigenetic mechanisms, H2A.Z removal in the hippocampus was transient and returned to basal levels by 2 hours after training (Zovkic et al., 2014). In the mPFC (a region associated with the maintenance of remote memory), H2A.Z eviction during the cellular consolidation window (2 hours after training) was also transient, while its incorporation in the first nucleosome upstream (-1 nucleosome) of the TSS was detected seven days after training and returned to baseline by 30 days. These data provided an initial indication that H2A.Z binding is subject to dynamic stimulus-induced regulation, but that it can also support ongoing processing required for systems consolidation.

H2A.Z’s effects on memory. We further showed that viral depletion of H2A.Z in the hippocampus enhanced recent memory, while depletion in hippocampus or mPFC enhanced remote memory, implicating H2A.Z as a memory suppressor (Zovkic et al., 2014). However, the mechanism by which H2A.Z impacts memory is unclear. H2A.Z depletion only impacted the

25 expression of approximately 400 genes at baseline and enhanced the learning-induced expression of only a small fraction of the memory-related genes studied, suggesting that effects of H2A.Z on transcription may be particularly prominent in response to stimulation.

Changes in H2A.Z binding with age. Considering the contribution of H2A.Z on memory and related transcription in the hippocampus and mPFC, and the general decline of memory with age, we investigated the effects of age on H2A.Z binding and memory (Stefanelli, Azam, et al., 2018). We found that H2A.Z accumulates with age within chromatin in the mouse hippocampus, such that a substantial percentage of genes that were common to both young (4 months) and old (15.5 months) mice showed increased H2A.Z binding in old mice compared to only 1.4% of common genes showing reduced binding in old mice. While this age-related accumulation of H2A.Z was unrelated to memory (both young and old mice showed similar levels of post-shock learning and 24-hour recall), old mice exhibited widespread H2A.Z eviction in response to learning, similar to our previous findings. Similarly, H2A.Z binding was positively correlated with steady-state transcription but negatively correlated with learning-induced transcription. However, 30 minutes after learning, both young and old mice showed up regulation of transcription in genes with reduced H2A.Z binding, consistent with the idea of H2A.Z eviction being important for stimulus-induced gene transcription. Moreover, since viral depletion of H2A.Z did not recapitulate these effects, this indicates that dynamic H2A.Z removal is required for the induction of certain memory-related genes, as opposed to constitutive removal. This is consistent with similar evidence that dynamic H3.3 (a variant of H3) turnover is essential for gene induction (Maze et al., 2015).

Overall, in the hippocampus, H2A.Z is highly enriched at nucleosomes flanking the TSS and its eviction from the -1 or +1 nucleosome is associated with increased stimulus-induced gene expression. Additionally, learning results in H2A.Z eviction from distinct genes in young and aged mice despite similar levels of memory formation. And, finally, H2A.Z accumulates within these regions with age, indicating that replication-independent histone variants become overrepresented in neural chromatin during aging.

H2A.Z deposition and acetylation. Recently, we used a pharmacological approach to investigate the effects of acetylation and histone deposition on H2A.Z’s role in memory, specifically within the longer timeframe of systems consolidation and cortex (Narkaj et al.,

2018). Mice were treated with either a HIV-1TAT interactive protein 60 (Tip60) inhibitor or a histone deacetylase inhibitor (HDACi). Tip60 is a histone acetyl transferase (HAT) that has not only been shown to acetylate histones including H2A.Z, but is also part of a protein complex involved in H2A.Z deposition (Kusch, 2004; Sapountzi, Logan, & Robson, 2006). Using these drugs allowed us to achieve temporally-specific inhibition of incorporation and/or acetylation of H2A.Z, or deacetylation of acH2A.Z. At baseline, hippocampal levels of acH2A.Z binding were increased at both the +1 and -1 nucleosomes of memory-related genes, while H2A.Z binding was unaffected. However, 30 days after learning, cortical levels of both H2A.Z and acH2A.Z were elevated around the TSS. Thus, both H2A.Z and acH2A.Z are implicated in memory maintenance and can be stably altered in the cortex, implicating H2A.Z at all phases of memory formation and storage.

Interestingly, Tip60 inhibition not only reduced H2A.Z binding in hippocampal neurons, but also increased acH2A.Z binding around the TSS of memory-related genes. This suggests that Tip60 influences H2A.Z-related gene transcription more so through H2A.Z incorporation rather than acetylation. Further reinforcing this idea, Tip60 (but not HDAC) inhibition 23 hours after fear learning in mice improved remote memory (7 days post training) and did not affect recent memory (24-hour recall). Tip60 inhibition 30 days post-training resulted in impaired recall 1 hour after the injection but protected the memory from further decline 24 hours and 7 days after the injection, indicating effects of Tip60 are transient. Together, these data indicate that Tip60’s H2A.Z-mediated effects on memory and gene transcription are dependent on its function in depositing H2A.Z into the nucleosome as opposed to its HAT activity. Keeping in mind that Tip60 has many effects unrelated to H2A.Z, our conclusion of H2A.Z’s effects is reinforced by the fact that Tip60 inhibition reduced H2A.Z binding and enhanced remote memory consistent with our previous observations with viral H2A.Z depletion. Interestingly, the fact that HDAC inhibitor treatment did not improve memory despite increased acetylation further supports the hypothesis that the HAT activity of Tip60 is not a key contributor to its effects on memory and its effects may be related to other functions such as H2A.Z deposition. These findings further implicate H2A.Z in memory throughout the prolonged process of systems consolidation and memory maintenance, not just recent memory as we had initially discovered.

2.3.6 H2A.Z Knockout Mouse Model

The previously discussed studies have depleted H2A.Z either using adeno-associated viral (AAV) depletion of H2A.Z1 in the CA1 or have done so with pharmacological inhibition of its deposition. However, one of the consequences of using the AAV approach is that we focused on only one of the genes encoding for H2A.Z (H2afz), while the pharmacological approach may have had other downstream effects resulting from Tip60 inhibition. One of the ways to circumvent this is to use a transgenic mouse model of H2A.Z knockout, in which both H2A.Z- encoding genes are floxed and are knocked out either ubiquitously or upon treatment with the inducing agent.

As of now, there is one study that has used a conditional model of H2A.Z KO to study its effects in vivo, and to characterize effects on embryonic neural development and behaviour in adulthood, though, again, they only focused on H2A.Z1 (H2afz) KO. Shen and colleagues (Shen et al., 2018) crossed H2A.Z1 floxed animals with Nestin-cre animals to produce animals that either did not have the knockout or had the H2A.Z1 gene knocked out in neural tissue. This worked because the Nestin-cre induced knockout is not only neural tissue-specific instead of global, but it also begins taking effect around embryonic day 11 (E11) (Jax website https://www.jax.org/strain/003771), while H2A.Z1 deletion at the blastocyst stage (approximately E5) of embryonic development is fatal (Faast et al., 2001). They discovered that these mice had deficits in exploratory activity, social behaviour, memory, and anxiety-related behaviour. Specifically, the mice showed increased anxiety in both the open field and elevated plus maze tests and increased helplessness on the forced swim test (defined by increased time spent immobile). They also showed impaired social interaction on the three-chamber social arena assay, where they did not show a preference for the familiar or novel mouse. Moreover, they showed impaired working memory on the Y-maze test (defined as reduced time spent in the new arms). Interestingly, these mice also showed impaired fear memory on both contextual fear conditioning and cued fear conditioning. These data suggest that H2A.Z function changes over development, such that knocking out H2A.Z in the brain early in development has significant consequences on the development of various aspects of cognition.

They further found that, at E13, several developmental genes were dysregulated in the brain, several of which are essential for neural differentiation and neurogenesis. Specifically, Nkx2-4, a

28 gene essential for regionalization of the forebrain and embryonic neurogenesis (Sugahara et al., 2016), was downregulated in H2A.Z1 KO E13 brains. The transgenic mice also showed abnormally branched processes at P0. Indeed, knocking down H2A.Z-1 or H2A.Z-2 in E16 brains reduced the number of newly born neurons, indicating an impairment in neurogenesis. Thus, H2A.Z plays a vital role in neural development in vivo, with far-reaching behavioural consequences in adulthood.

Given that our prior studies reported suppressive effects of region-selective depletion of H2afz on memory, it was not clear whether dual deletion of both genes would have the same effect. To address this issue, we used the same conditional H2A.Z knockout mouse as Shen et al. (2018). However, both H2A.Z-encoding genes (H2afz, H2afv) were floxed and, further, instead of crossing them with Nestin-Cre mice to create a conditional knockout in the brain, we crossed them with a CamkIIα-creERT2 line to produce an inducible model that produces excitatory neuron-specific H2A.Z deletion upon treatment with tamoxifen. This technique allows for the control of H2A.Z knockout at any age, circumventing confounding effects of H2A.Z deletion on development. We specifically chose CamKIIα promoter as a driver of CRE because CamKIIα- containing neurons are generally important for learning and memory processes (Shonesy, Jalan- Sakrikar, Cavener, & Colbran, 2014).

2.4 AR and H2A.Z Interaction

Sex interacts with the epigenome to affect sex differences within the brain. Histone modifications are involved in X-chromosome inactivation in females (Shilatifard, 2006). Additionally, sex differences and hormonally induced changes in H3 acetylation of lysine 9/14 (associated with gene transcription) and lysine 9 trimethylation (associated gene inactivation) have been reported in the developing mouse hippocampus and cortex (Tsai, Grant, & Rissman, 2009). There were no influences of sex or hormones on these histone PTMs, however, in the sexually dimorphic preoptic area (POA) (Tsai et al., 2009). Interestingly, increased maternal licking of male pups’ anogenital area in comparison to female pups’, results in increased masculinization of the POA in males (Champagne et al., 2006), as well as increased ERα promoter methylation and reduced ERα gene expression in the POA (Kurian, Olesen, & Auger, 2010). This is interesting as, similar to the AR, ERα is also a transcription factor. Transcription factors further interact with the epigenome to regulate gene transcription, another method in

29 which the various sex steroid receptors may interact with the epigenome in a sex-specific way. Considering the role of sex on the epigenome, and the importance of transcription factors and hormones on sex differences and the epigenome, we decided to study the potential interaction between the androgen receptor and the histone variant H2A.Z, as they may affect fear memory in a sex-specific way.

Indeed, it seems as if the AR and H2A.Z have robust molecular interactions within prostate cancer literature, in which the prostate specific antigen (PSA) gene is a heavily studied and well- characterized model AR-regulated gene (Cleutjens et al., 1997; Cleutjens, van Eekelen, van der Korput, Brinkmann, & Trapman, 1996).

2.4.1 Evidence of AR and H2A.Z interaction in AR-regulated gene transcription

AR is a ligand-activated transcription factor that, upon androgen binding, is transported to the nucleus where it binds at androgen responsive elements (AREs) to affect transcription (Celec et al., 2015; Michels & Hoppe, 2008). The transcriptional activity of AR is influenced by several co-activators and epigenetic factors, including the histone variant H2A.Z, which was recently identified as a negative regulator of fear memory in rodents (Stefanelli, Azam, et al., 2018; Zovkic et al., 2014). Although the interaction between H2A.Z and AR has not been studied in the brain, studies in prostate cancer show that AR interacts with H2A.Z to regulate gene expression (Draker, Sarcinella, & Cheung, 2011). Indeed, treating prostate cancer cells with the synthetic androgen metribolone R1881 results in increased expression of H2A.Z-1, one of the genes that encode H2A.Z (Dryhurst et al., 2012), suggesting that one mechanism through which AR may regulate memory is by influencing the transcription of H2A.Z.

The androgen receptor and histone variant H2A.Z are inextricably linked. Studies show a direct interaction between H2A.Z and AR to regulate gene expression. In general, H2A.Z eviction is associated with activation of gene transcription (Zovkic & Walters, 2015) and numerous coactivators and epigenetic factors regulate AR's capacity as a transcription factor, including chromatin-remodeling complexes, histone acetyl transferases, CREB-binding factors, among others.

Moreover, a group of coactivators that facilitate AR’s transcriptional activity also regulate H2A.Z’s functions. One such coactivator is Tip60, which directly acetylates AR (Sapountzi et al., 2006), promoting its transcriptional activity (Gaughan, Logan, Cook, Neal, & Robson, 2002; Sapountzi et al., 2006). Additionally, there is evidence of Tip60 regulating AR transcriptional activity in conjunction with histone deacetylase 1 (HDAC1) such that they form a trimeric protein complex upon the ARE. Here, they regulate AR’s transcriptional activity through its acetylation status; specifically Tip60 promotes AR transcriptional activity through acetylation, while HDAC1 inhibits it through deacetylation (Gaughan et al., 2002). Tip60 also acts as a histone acetyl transferase (HAT) of several histones, including H2A.Z (Ikura et al., 2000; Kusch, 2004; J. Wang & Chen, 2010; Yamamoto & Horikoshi, 1997) and has been implicated in H2A.Z deposition (Dryhurst & Ausió, 2014), suggesting that AR and H2A.Z share common upstream regulators. Further evidence in prostate cancer cell lines indicates that H2A.Z and acetylated H2A.Z are heavily present at the enhancer and promoter regions of the prostate-specific antigen (PSA) gene, however levels decrease during repeated gene transcription cycles (Dryhurst et al., 2012).

Another AR coactivator is ubiquitin-specific protease 10 (USP-10), which also de-ubiquitylates H2A.Z (Draker et al., 2011). Similar to acetylated H2A.Z, ubiquitylated H2A.Z is also enriched at PSA (also known as kallikrein-3/KLK3) and kallikrein-2 (KLK2) gene regulatory regions, both of which are part of the KLK cluster (a cluster of genes encoding for various serine proteases implicated in numerous tissues, including the prostate, semen, epidermis, and central nervous system). Ubiquitylated H2A.Z plays a role in repressing gene expression (Draker & Cheung, 2009; Sarcinella et al., 2007). USP10 de-ubiquitylates H2A.Z, facilitating its eviction from the nucleosome. Functionally, USP10 depletion blocks H2A.Z eviction from PSA and KLK2 regulatory regions. Similarly, depletion of ubiquitylated H2A.Z correlated with transcriptional activation of PSA, implicating a negative modulatory role of ubiquitylated H2A.Z in AR-regulated gene transcription (Draker et al., 2011). Indeed, it was found that USP10 and H2A.Z are both required for transcriptional activation of AR-regulated PSA and KLK3 genes. Together, these results suggest that specific PTMs on H2A.Z (e.g. ubiquitylation and acetylation) poise the PSA gene for AR-mediated transcription, suggesting that H2A.Z may be a critical regulator of AR effects. These factors function together such that de-ubiquitylation of H2A.Z by

USP10 and acetylation of histones neighbouring the AREs by Tip60 are critical for the events leading up to gene induction (Dryhurst & Ausió, 2014).

Other AR coactivators are also involved with the incorporation of histones and histone variants into chromatin. One such coactivator is SRCAP (SNF-2-related CREB-binding protein activator protein), an ATP-dependent chromatin remodeling complex. SRCAP has been shown to interact with AR on AREs on the PSA promoter and, further, its depletion results in reduced AR-induced transcriptional activity (Slupianek, Yerrum, Safadi, & Monroy, 2010). Furthermore, in conjunction with Tip60, SRCAP catalyzes the incorporation of H2A.Z-H2B dimers into chromatin at both promoters and enhancers (Krogan et al., 2004; Mizuguchi et al., 2004; Wong, Cox, & Chrivia, 2007). Indeed, these two functions of SRCAP interact with each other to affect transcription of AR-regulated genes (Slupianek et al., 2010).

The AR-mediated poising of the PSA gene depends on H2A.Z incorporation at the enhancer and proximal promoter regions (Dryhurst et al., 2012), and is likely accompanied by DNA demethylation. DNA hypermethylation is a direct antagonist of H2A.Z (Zilberman et al., 2008) and has also recently been associated with a tissue-specific enhancer landscape (Xie et al., 2013). H2A.Z presence in enhancer and promoter regions may further assist in establishing histone H1- depleted regions (an indicator of nucleosome depletion), increasing chromatin accessibility as defined by DNase I hypersensitive (DHS) nucleosome-free regions (Malin, Aniba, & Hannenhalli, 2013; Tewari et al., 2012). DHS sites are DNA regions that are sensitive to cleavage by the DNase I enzyme, where uncondensed chromatin results in exposed and accessible DNA. H2A.Z’s presence also aids with the positioning of nucleosomes at AR-binding sites (ARBs) (Berman, Frenkel, Coetzee, & Jia, 2010; He et al., 2010). All of this is likely to be critical for the nucleosome dynamics required in assisting full assembly of the transcriptional machinery containing RNA pol II.

Induction of DNA hypomethylation at CpG islands of tumour suppressor genes promotes nucleosome eviction (Portela et al., 2013) and enhanced chromatin accessibility (Pandiyan et al., 2013) at previously hypermethylated tumor suppressor genes. Similar processes are regulated by SRCAP-mediated H2A.Z insertion (Yang et al., 2012). Indeed, H2A.Z is regularly found at promoters and enhancers within DHS nucleosome-free regions (Barski et al., 2007; Gross & Garrard, 1987; Malin et al., 2013).

Further, androgen receptor responsive elements (AREs) are present primarily at enhancers and promoters of AR-regulated genes. AREs are sites on the DNA consisting of two palindromic half-sites separated by a three-nucleotide spacer; the presence of multiple AREs in a promoter region increases AR specificity and action (for further information see Gelmann, 2002). For example, the AR-regulated prostate cancer marker, PSA, gene has two AREs in the proximal promoter, and one in the enhancer region (Cleutjens et al., 1997, 1996) In prostate cancer cells, androgen treatment results in the eviction of the central nucleosome (located at the centre of AR- binding motifs within enhancers and promoters), while nucleosomes flanking the central nucleosome do not exhibit this eviction (He et al., 2010), which likely induces the nucleosome depletion associated with these regions (He et al., 2012). Notably, in prostate cancer cells, regions with such nucleosome depletions seem to already exist at some AR-responsive enhancers in the absence of androgens, potentially in a receptive state for histone modifiers. Binding of androgen then shifts the equilibrium towards a nucleosome-depleted state (Andreu-Vieyra et al., 2011).

During AR-induced transcription, ARs bind at multiple AREs present within multiple enhancers associated with a gene, leading to the recruitment of transcriptional complexes at these regions. These are then brought together in the process of chromatin looping to interact with AREs present at the promoter region (Wu, Zhang, Shen, Nephew, & Wang, 2011). This process is heavily regulated by an ATP-dependent chromatin remodeling complex that includes SRCAP (responsible for H2A.Z deposition), p300/CBP (HAT), and FoxA1 (forkhead protein A1). SRCAP results in incorporation of H2A.Z, this H2A.Z incorporation further facilitates histone H1 removal from linker DNA (Thakar et al., 2009), demethylated DNA, and nucleosome depleted regions (Yang et al., 2012). This then leads to FoxA1-mediated initiation of transcription (Sahu et al., 2013; Wu et al., 2011) and eviction of H2A.Z-containing nucleosomes. Overall, these data suggest that H2A.Z is central to AR’s transcriptional activity at the PSA gene, but the extent of this relationship at other genes and in the brain remains to be characterized.

2.5 Thesis Aims and Hypotheses

The primary aim of this thesis is to examine the effects of androgenic mechanisms and histone variant H2A.Z on fear memory. The growing body of literature supporting their individual effects leads to our interest in understanding their combined contributions to fear memory.

Further, considering the evidence of their molecular interaction within prostate cancer literature, we were interested in studying these interactions within neural structures important for fear memory, specifically the hippocampus. Given the role of AR in regulating H2A.Z function, a secondary aim was to investigate the role of sex on fear memory, explicitly pertaining to H2A.Z.

The hypotheses and predictions tested are the following:

Hypotheses & Predictions

1. Androgenic mechanisms, acting through the androgen receptor, affect fear memory (Chapter 3).

a. Androgen receptor (AR) overexpression will influence memory dependent on hormonal activation and AR function.

2. Depletion of both H2A.Z encoding genes promotes fear memory (Chapter 4).

a. H2A.Z deletion improves fear memory.

3. AR and H2A.Z interact to regulate fear memory (Chapters 3 and 4).

a. AR overexpression affects H2A.Z expression (Chapter 3).

b. Effects of androgenic manipulations on memory depend on H2A.Z (Chapter 4).

4. Androgenic mechanisms and epigenetic factors interact to affect chromatin binding and gene expression in neurons (Chapter 4).

a. AR and H2A.Z interact to regulate gene expression of both memory-related and AR-regulated genes in hippocampal neurons.

5. H2A.Z has sex-specific effects on fear memory (Chapter 5).

Chapter 3

Androgen Receptor is a Negative Regulator of Contextual Fear Memory in Male Mice

This chapter is reformatted from an article published in Hormones and Behavior that evaluated the effects of androgen receptor overexpression on fear memory and effects on related gene expression in the CA1 area.

Ramzan, F., Azam, A. B., Monks, D. A., and Zovkic, I. B. (2018) Androgen receptor is a negative regulator of contextual fear memory in male mice. Hormones and Behavior 106, 10-18.

3.1 Introduction

Hormones powerfully regulate emotional and cognitive systems that are involved in establishing both normal and pathological forms of associative fear memory, which manifest in conditions such as post-traumatic stress disorder (PTSD) at different rates in men and women (Aikey, Nyby, Anmuth, & James, 2002; Dalla & Shors, 2009; Edinger & Frye, 2004, 2005; Jovanovic et al., 2015; Kranz et al., 2015; McHenry et al., 2014; Morgan & Pfaff, 2001; Suzuki, Eda-Fujiwara, Satoh, Saito, & Miyamoto, 2013). For example, PTSD is characterized by excessive fear and intrusive memories that disproportionately affect women compared to men who are exposed to trauma (Breslau et al., 1998; Holbrook, Hoyt, Stein, & Sieber, 2002), suggesting that testosterone is protective against PTSD, as it is against other types of affective disorders (Kaminetsky, 2005; Veras & Nardi, 2010). Despite evidence that androgens impact cognition and emotional memory, the nature of their influence is unclear (Burkitt, Widman, & Saucier, 2007; Galea et al., 2008; Gouchie & Kimura, 1991; Janowsky, 2006; Moffat & Hampson, 1996). Indeed, the majority of research on the role of sex hormones has focused primarily on estrogenic mechanisms (Frick et al., 2017; Galea, Frick, Hampson, Sohrabji, & Choleris, 2017; Galea et al., 2008; Sherwin, 1988, 2005), whereas the role of the androgen receptor (AR) in hippocampus-dependent fear memory remains unclear.

AR is expressed in brain regions that regulate contextual fear memory, most notably in areas CA1 and CA3 of the hippocampus (Kerr et al., 1995; Raskin et al., 2009). Studies have reported mixed effects of AR and its ligands, testosterone and dihydrotestosterone (DHT), on fear memory. For example, some studies report no effects of gonadectomy on fear memory in male rodents (Anagnostaras et al., 1998), whereas others report distinct effects of androgenic manipulations on contextual and cued fear memory (Chen et al., 2014; Edinger et al., 2004; Frye et al., 2008; MacLusky et al., 2005; Rubin, 2011; Q. Zhang et al., 2014). Moreover, some evidence shows that gonadectomy reduces fear memory (Edinger et al., 2004; McDermott et al., 2012) and that this deficit is rescued by treatment with either testosterone or 17β estradiol, but not with DHT (Edinger et al., 2004). Given that DHT is a direct AR agonist, whereas testosterone can be further aromatized into 17β estradiol to activate estrogen receptors (Hojo et al., 2004; Mukai et al., 2010; Ooishi et al., 2012), these data suggest that testosterone may influence fear memory through AR-independent actions. In contrast, both DHT and testosterone improved performance on the inhibitory avoidance test, in which rats learn to avoid an environment associated with shock (Edinger & Frye, 2004), supporting a direct role of AR in regulating fear-based tasks.

Androgenic regulation of morphological and functional plasticity in the hippocampus is also complex, with dissociable effects of androgen manipulations on excitability, spine density and neurotrophin production, and evidence for both androgenic and estrogenic mechanisms of gonadal testosterone (see Atwi, McMahon, Scharfman, & MacLusky, 2016 for review). For example, neural-specific AR deletion in mice reduces LTP induced by high-frequency stimulation, without impacting LTP induced by theta-burst stimulation (Picot et al., 2016), indicating that AR has selective effects on different types of hippocampal plasticity. Spine density is reduced in the medial prefrontal cortex (mPFC) of Tfm rats (Hajszan et al., 2007), and either testosterone or DHT maintain CA1 hippocampal spine synapse density in castrated male rats (Leranth et al., 2003). Androgens also increase hippocampal neurogenesis through modulation of survival of new neurons (see Galea et al., 2013 for review). Long-term testosterone/DHT replacement consistently ameliorates the impaired survival of new neurons in castrated males (Hamson et al., 2013; Spritzer, Ibler, Inglis, & Curtis, 2011; Wainwright, Lieblich, & Galea, 2011), while short-term androgen replacement shows mixed results (Carrier & Kabbaj, 2012; Spritzer, Daviau, et al., 2011; Spritzer, Ibler, et al., 2011; Wainwright et al.,

2016). Further, survival is also impaired upon AR blocking with flutamide (Hamson et al., 2013).

Given the complexity of androgenic compound actions, it can be difficult to distinguish between androgenic compounds that act through the AR compared to alternative modes of action. That is, testosterone can activate AR directly, or through its metabolite, DHT. In addition, DHT can be further metabolized to 3α-androstanediol to act on the GABA-A receptors, and testosterone can also be aromatized to estradiol or metabolized to 3β-diol, both of which activate ERs, which typically potentiate fear memory (Maeng & Milad, 2015). Thus, to specifically address the role of AR in fear memory, we utilized an AR-overexpressing mouse line (Swift-Gallant, Coome, Ramzan, & Monks, 2016), which we previously used to reveal novel insights into AR function in several behavioural processes, including the resident-intruder paradigm, sexual and aggressive behaviours, and partner preference (Swift-Gallant, Coome, Ramzan, et al., 2016; Swift-Gallant, Coome, Srinivasan, & Monks, 2016). Whereas loss-of-function mutations are invaluable in determining the necessity of AR, AR overexpressing mice take advantage of increased androgen response as a result of increased receptor expression to elucidate phenotypes that emerge at the high range of androgenic signalling. This approach has revealed functions of high AR levels in disease and sexual differentiation that do not always correlate with predictions made from loss- of-function mutant studies (Coome et al., 2017; Ramzan et al., 2015; Swift-Gallant, Coome, Ramzan, et al., 2016), demonstrating a unique utility of this mouse line for studying AR function in fear memory.

In the present study, our goal was to investigate the role of AR in fear memory by addressing four questions: 1) Does increased AR density affect fear memory; 2) Is this effect mediated by testosterone; 3) Can effects of AR density on fear memory be reversed by blocking AR receptors, either indirectly (through gonadectomy) or directly (using the AR antagonist, flutamide); and 4) Given that AR is a ligand-activated transcription factor, does increased AR density alter the expression of memory-related genes in area CA1 of the hippocampus?

3.2 Methods

3.2.1 Animals

Male C57Bl/6 mice bred in our colony were pair housed after weaning and were assigned to testing groups at 60–90 days of age. Mice were housed in 7.5×11.5×5 in cages and maintained on a 12 h light cycle, with ad libitum access to standard mouse chow (Harlan Teklad, Madison, WI) and water. Transgenic mice containing a CMV-stop-AR transgene were crossed with a CMV-Cre line to obtain tissue-wide AR overexpression, as previously described (Swift-Gallant, Coome, Ramzan, et al., 2016). Briefly, CMV-stop-AR mice contain a cytomegalovirus (CMV) promoter coupled to the human androgen receptor (hAR) gene, separated by a floxed stop sequence (Fig. 1a), which is excised when mice are crossed with the CMV-Cre line to allow for hAR transcription, beginning ~embryonic day 8.5, when CMV expression occurs (Baskar et al., 1996). Details on mouse generation can be found in (Swift-Gallant, Coome, Ramzan, et al., 2016). All of the procedures were approved by the University of Toronto Mississauga animal care committee and complied with institutional guidelines and the Canadian Council on Animal Care.

3.2.2 Genotyping

Genotyping was carried out at weaning for each mouse used in the experiments, as previously described (Swift-Gallant, Coome, Ramzan, et al., 2016). Briefly, the presence of the CMV- STOP-AR transgene was identified by amplifying within the Neo stop sequence. However, because this Neo stop sequence is deleted in mice expressing both CMV-Cre and CMV- STOP- AR, we also amplified a unique portion of the CMV-STOP-AR transgene within the hAR coding region (see Table 2-1 for a list of genotyping primers). To ensure that AR overexpression extends to area CA1 of the hippocampus, we used qPCR to confirm the presence of hAR mRNA in CMV-AR compared to WT mice in this region for each mouse included in the experiments.

Table 3-1. DNA primers for genotyping Gene Forward Reverse neostop 5'-AGGATCTCCTGTCATCTCACCTTGCTCCTG 5'-AAGAACTCGTCAAGAAGGCGATAGAAGGCG AR 5'-ACCGAGGAGCTTTCCAGAAT 5'-CTCATCCAGGACCAGGTAGC Cre 5'-AGGTGTAGAGAAGGCACTTA 5'-CTAATCGCCATCTTCCAGCA

3.2.3 Fear Conditioning

Associative fear memory was assessed with contextual fear conditioning, a hippocampus- dependent task in which mice learn to associate a novel context with exposure to shock. To facilitate habituation to the experimenter and the non-associative aspects of the training procedure, mice were transported to the testing room and handled for 30–60 s daily for 3 days before testing. On the training and test days, mice were transported to the testing room and placed into test chambers (9.8 in boxes; designed for mice) equipped with an electrified grid floor (Coulbourn Instruments, Holliston, MA, USA). During training, mice were given 2 min to explore the apparatus before receiving a foot shock (0.5 mA, 2 s), followed by an additional minute of exploration. Memory was assessed 24 h later by replacing the mouse into the training apparatus without shock and measuring freezing behaviour for a total of 3 min. Freezing was recorded with a camera placed directly in the chamber ceiling and facing down towards the mouse and scored by automated software (FreezeFrame, Coulbourn Instruments).

3.2.4 Flutamide administration

5 days before handling (at 60–90 days of age), a subset of intact male mice were injected subcutaneously with the AR antagonist flutamide (8 mg/day) and injections continued daily for a total of 13 days to ensure continued AR inhibition until tissue collection. Flutamide (Sigma # F9397, Oakville, Canada) was prepared daily immediately prior to use, by first dissolving it in 100% ethanol, then in sesame oil (Sigma # S3547, Oakville, Canada). The ethanol was removed using an Eppendorf Vacufuge Plus (spun for 20 min at 30 °C on the V-AL setting), producing the final concentration of 53 mg/mL. Mice were administered 0.15 mL through subcutaneous injection in the nape of the neck. Vehicle-treated control mice received sesame oil on the same schedule as the flutamide-treated mice.

3.2.5 Gonadectomy and testosterone (T) replacement

At 60–90 days of age, a subset of mice underwent castration under inhalant anesthesia (1–2% isoflurane). A midline incision was made in the scrotum to allow for the removal of both testes, after which the incision was closed with wound clips. Immediately after castration, animals were subcutaneously implanted at the nape of the neck with a Silastic capsule containing T (10mm of

39 crystalline T; Silastic tube 1.02 mm id/2.16 mm od) and sealed with Silastic adhesive, as in (Coome et al., 2017). Using the same implant specifications in C57Bl6 mice, Bowen et al. (2012) found that this replacement protocol results in 15.4 ± 1.2 ng/mL plasma T levels. Control animals received an empty Silastic implant. The animals were administered Anafen (5 mg/ kg) on the day of the surgery and for 2 days after surgery. Wound clips were removed if still present after 7 days. Animals underwent 3 days of handling (30 s/mouse) beginning 11 days after surgery and were trained 14 days after surgery.

3.2.6 Tissue collection

For RNA and western blot tissue analysis, mice were cervically dislocated and decapitated. Animal brains were immediately collected and snap frozen in ice-cold isopentane and stored at −80 °C until processing.

3.2.7 Western Blotting

Tissues were homogenized using a dounce homogenizer in RIPA buffer (50mM Tris HCl pH 7.4, 150mM NaCl, 10% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with Protease Inhibitor Cocktail (Cell Signaling, Danvers, MA, USA). Homogenates were incubated for 20 min on ice and flicked every 5 min, centrifuged at maximum speed at 4 °C for 15 min, and the supernatant was collected. Proteins were separated on 10% SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked in TBS-T (TBS with 0.1% Tween- 20) containing 5% skim milk and incubated with rabbit monoclonal anti-AR (1:500, Abcam, ab133273 RRID, Cambridge, UK) and rabbit anti-β-Actin (1:10.000, Cell Signaling, 4967S, Danvers, MA, USA) overnight at 4 °C. After three 5 min TBS-T washes, membranes were incubated with anti-rabbit- HRP secondary antibody (1:10.000 Life Technologies, ThermoFisher, Waltham, MA, USA) for 1 h at RT. Detection was performed by enhanced chemiluminescence and quantification was done using ImageJ (NIH) to quantify Area Under the Curve, with AR normalized to actin.

3.2.8 mRNA expression and RT-PCR

Mice were killed by cervical dislocation at least 2 weeks after the final training session and area CA1 was dissected. RNA was extracted using BioBasic RNA Extraction Kit (BioBasic #BS82322-250, Amherst, NY). Complementary DNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied BioSystems #4368814, Folster, CA, USA). Primers

40 were designed in the lab and used to detect levels of the indicated transcripts, and data were normalized to GAPDH. Comparison of group differences was conducted using relative enrichment analysis, whereby data for experimental animals were normalized to controls using the following formula: 2^−(ΔΔCT). The list of primer sequences is provided in Table 3-2.

Table 3-2. cDNA primers used in gene expression studies. Gene Forward Reverse Melting Temperature (ᣞC) Actb 5'-AGATCAAGATCATTGCTCCTCCT 5'-ACGCAGCTCAGTAACAGTCC 58 Bdnf4 5'-CCAGAGCAGCTGCCTTGCTGTTTA 5'-TGCCTTCTCCGTGGACGTTTACTT 60 Chrna7 5'-GCAACATCTGATTCCGTGCC 5'-TGATCCTGGTCCACTTAGGC 58 Chrm3 5'-GACAGTCGCTGTCTCCGAAC 5-GGTCATATCTGGCAGCCGTG 58 hAR 5'-CTTCGCCCCTGATCTGGTTT 5'-GAGAGAGGTGCCTCATTCGG 56 Gapdh 5'-GTGGAGTCATACTGGAACATGTAG 5-AATGGTGAAGGTCGGTGTG 60 Gria1 5'-ATGTGGAAGCAAGGACTCCG 5'-ACAGAAACCCTTCATCCGCT 58 Gria4 5'-GGACAAGACGAGTGCCTTGA 5-GCTTCGGAAAAAGTCAGCTTCA 56 Grin1 5-AAACCTCGACCAACTGTCCT 5'-GTCGTCCTCGCTTGCAGAAA 55 H2afz 5-CACCGCAGAGGTACTTGAGTT 5-TCCTTTCTTCCCGATCAGCG 58

3.2.9 Statistics

Analyses were conducted with SPSS Version 24 and consisted of independent-samples t-test for comparison of Genotype (WT, CMV-AR). In cases in which there were 2 independent variables, we used a two-way ANOVA with Genotype (WT, CMV-AR) and Treatment (Control, Testosterone; or Vehicle, Flutamide) as independent variables. To compare freezing before and after-shock during the training session, we used a mixed-measures ANOVA, with Genotype and Treatment as the between group factors and Shock (before shock; after-shock) as the within- group factor. Follow-up analyses were conducted using independent-samples t-test or paired- samples t-test only when the omnibus test was significant, thus precluding the need to correct for multiple comparisons. Effect sizes were calculated using the publicly available effect size calculator, found at http://www.campbellcollaboration.org/escalc/html/EffectSizeCalculator- SMD1.php. Significance was set at p≤0.05.

3.3 Results

3.3.1 Validation of CMV-AR in the hippocampus

Generation and validation of AR overexpression in CMV-AR mice have been previously reported (Swift-Gallant, Coome, Ramzan, et al., 2016). Here, we confirmed that AR overexpression extends to the hippocampus using qPCR for each mouse (see Methods section), as well as immunoblotting (t2=12.4, p=0.006, d=12.74) to confirm differences in protein levels (Fig. 3-1a).

3.3.2 CMV-AR reduces fear memory

To determine if AR influences fear memory, we compared freezing behaviour in WT and CMV- AR mice. We reasoned that any differences between the genotypes that are directly attributable to AR overexpression would be eliminated by blocking the activity of AR, prompting us to compare freezing behaviour in control WT and CMV- AR mice to the behaviour of mice treated with the AR antagonist, flutamide. We found no differences between mice that did not receive any injection and mice that received a vehicle injection during training (F1,22=1.88, p=0.18) or on the test day (F1,22=1.69, p=0.21), so these were combined into a single control group.

We first assessed within-session learning by comparing freezing behaviour before and after exposure to shock during the training day. We found a significant Shock (before shock, after 2 shock)×Treatment (Control, Flutamide) interaction (F1,32=20.86, p < 0.001, ƞp =0.40), whereby freezing increased in the 1 min period after shock exposure compared to the 2-min period before shock exposure in control (t25=7.48, p < 0.0001, d=1.47) and flutamide-treated (t9=10.46, p < 0.0001, d=3.31) mice. In addition, flutamide- treated mice froze more than controls after shock exposure (t34=3.94, p < 0.0001, d=1.46), irrespective of genotype (Fig. 3-1b).

Fear memory was assessed by comparing freezing behaviour in control and flutamide-treated

CMV-AR and WT mice 24 h after training. There was a significant interaction (F1,32=4.54, 2 p=0.04, ƞp =0.12) between Genotype (WT, CMV-AR) and Treatment (Control, Flutamide), whereby Control CMV-AR mice exhibited reduced freezing compared to WT controls (t24=3.91, p=0.001, d=1.53). In contrast, no difference in freezing behaviour between genotypes was found for flutamide-treated mice, such that flutamide increased freezing behaviour compared to control treatment in CMV-AR (t16=3.71, p=0.002, d=1.95), but not in WT mice (Fig. 3-1b). These data

42 demonstrate that AR overexpression reduces fear memory and that this effect is reversed by blocking the AR with flutamide.

Figure 3-1. AR activation impairs fear memory in CMV-AR mice. (a) Representative immunoblot demonstrating that CMV-AR have higher AR protein levels in the hippocampus compared to WT controls. Quantification of the western blot (N=2/ group) is shown on the right. (b) Freezing behaviour during the training session (left) and during the memory test (right), conducted 24 h after training. N=13/group for WT and CMV-AR in the control condition and 5/group for WT and CMV-AR in the flutamide condition. *p≤0.05.

3.3.3 Gonadectomy removes genotype effects and testosterone restores them

To determine if the effects of CMV-AR on fear memory are due to differences in sensitivity to circulating androgens, mice were gonadectomized and implanted with a Silastic tube containing either T or an empty control. Although T can be aromatized into estradiol, we chose to replace T

43 instead of a non-aromatizable androgen as an initial test of whether an androgen would affect the system, and also because our interest was in testing how AR overexpression influences behaviour in response to the dominant source of androgen. During training, all mice exhibited significantly increased freezing after-shock than before shock (Main effect of Shock: 2 F1,48=70.28, p < 0.001; ƞp =0.59), but there were no interactions or main effects of Treatment and Genotype, indicating that these factors did not influence within-session learning (Fig. 3-2).

Gonadectomy (GDX) reversed the effects of AR-overexpression on fear memory 24 h after training, as evidenced by a significant Genotype×Treatment (Control, Testosterone) interaction 2 (F1,48 =5.41, p=0.02, ƞp =0.10), whereby differences between WT and CMV-AR mice were not evident in GDX mice without hormone replacement, suggesting that the removal of circulating AR ligands (i.e., testosterone) eliminates the effect of CMV-AR on fear memory. Consistent with this observation, T replacement reduced fear memory only in CMV-AR mice (t17=5.05, p < 0.0001, d=2.31), thus reinstating the difference between genotypes, whereby T-replaced CMV-

AR mice froze less than T-replaced WT mice (t21=2.07, p=0.05, d=0.94). Overall, these data suggest that testosterone-mediated AR activation reduces fear memory (Fig. 3-2).

Figure 3-2. Gonadectomy removes differences between genotypes on fear memory and T replacement restores them. Freezing behaviour during the training session (left) and during the memory test (right), conducted 24 h after training. N=9– 19/group (Vehicle treated: N=19 WT and 10 CMV- AR; Testosterone treated: N=14 WT and 9 CMV-AR). *p≤0.05.

3.3.4 Effects of AR on gene expression in area CA1 of the hippocampus

Given that AR is a ligand-activated transcription factor, we explored potential transcriptional outcomes from all the groups that were utilized in behavioural studies, including intact (i.e., control or flutamide-treated mice that were not gonadectomized) and gonadectomized (with or without T replacement) mice. We focused on genes that were previously implicated in memory formation, including H2afz, a gene encoding the histone variant H2A.Z, which we recently identified as a memory suppressor (Stefanelli, Azam, et al., 2018; Zovkic et al., 2014), and which is positively regulated by AR in prostate cancer (Dryhurst & Ausió, 2014). For intact mice, H2afz levels depended on both Genotype and Treatment (Genotype×Treatment interaction: 2 F3,24=4.87, p=0.04, ƞp =0.17), whereby H2afz expression was elevated in control-treated CMV-

AR compared to WT mice (t16=2.44, p=0.03, d=1.16). In contrast, the effect of genotype was not evident in mice treated with flutamide, as H2afz expression was reduced to WT levels in CMV-

AR mice treated with flutamide compared to CMV-AR controls (t13=3.03, p=0.01, d=1.66). Similarly, the effect of genotype in gonadectomized mice depended on hormonal status 2 (Genotype×Treatment interaction: F1,36=8.10, p=0.007, ƞp =0.18), whereby differences between the genotypes were not evident in gonadectomized mice without hormone replacement, and were restored with T replacement. Specifically, T-replaced CMV-AR mice had higher H2afz expression compared to T-treated WT mice (t17=2.83, p=0.01, d=1.30), indicating that high levels of AR promote H2afz expression in the hippocampus (Fig. 3-3).

Some evidence suggests that testosterone mediates cholinergic receptors (Bleisch, Harrelson, & Luine, 1982), leading us to investigate effects on a muscarinic (Chrm3) and nicotinic (Chrna7) acetylcholine receptor expression. In intact mice, the interaction of Genotype and Treatment 2 approached significance (F1,23=3.77, p=0.06, ƞp =0.14) for Chrm3, leading us to conduct follow- up analyses to test our hypothesis that differences in expression between genotypes would be eliminated by AR blockade. In control mice, Chrm3 levels were higher in CMV-AR compared to

WT mice (t15=2.76, p=0.02, d=1.34), whereas this difference was not found in flutamide-treated mice. Similarly, Chrm3 expression in gonadectomized mice depended on hormonal status 2 (Genotype×Treatment interaction: F1,37=5.06, p=0.03, ƞp =0.12), whereby gonadectomized mice without T replacement had similar Chrm3 expression. T treatment only increased Chrm3 expression in CMV-AR mice (t17=3.25, p=0.005, d=1.49), such that T-treated CMV-AR mice had higher Chrm3 expression than T-treated WT (t17=2.46, p=0.025, d=1.13). For Chrna7, no

45 differences were found in intact mice, but there was a significant increase in Chrna7 expression in response to T treatment in gonadectomized mice (Main effect of Treatment: F1,37=7.86, p=0.008).

There is also evidence for a potential influence of T on glutamatergic transmission in the hippocampus (Picot et al., 2016), leading us to investigate a potential role of CMV-AR on the expression of Gria1 (encodes glutamate receptor AMPA1 subunit), Gria4 (encodes glutamate receptor AMPA4 subunit), and Grin1 (encodes the NMDA1 receptor subunit). There were no differences in Gria1 or Gria4 expression in intact mice, but for both genes, T replacement in gonadectomized mice increased gene expression, irrespective of genotype (Gria1: F1,37=10.56, 2 2 p=0.002, ƞp =0.22; Gria4:F1,37=6.30, p=0.02, ƞp =0.15). For Grin1 expression in intact mice, 2 there was a Genotype×Treatment interaction (F1,24=5.25, p=0.03, ƞp =0.18), whereby Grin1 expression was higher in control-treated CMV-AR compared to control-treated WT mice

(t16=4.12, p=0.001, d=1.98). There were no differences between genotypes when AR was blocked with flutamide. Differences between genotypes were also not evident when mice were gonadectomized without hormone treatment, but were restored by T replacement 2 (Genotype×Treatment interaction: F1,37=5.44, p=0.03, ƞp =0.13), such that T-replaced CMV-AR mice had higher Grin1 expression than T-replaced WT mice (t17=2.72, p=0.015, d=1.25) (Fig. 3). Finally, androgens have also been implicated in regulating BDNF expression (Skucas et al., 2013), leading us to examine the expression of BDNF exon 4 (Bdnf4). In intact mice, a main 2 effect of Treatment (F1,24=8.13, p=0.009, ƞp =0.25) demonstrated reduced levels of Bdnf4 in response to AR blockade with flutamide. In gonadectomized mice, Bdnf4 expression depended on genotype and hormone treatment (Genotype×Treatment interaction: F1,37=7.60, p=0.009, 2 ƞp =0.17), such that T only increased Bdnf4 expression in CMV-AR mice (t17=5.80, p < 0.0001, d=2.67), although the difference between WT and CMV-AR mice treated with T missed significance (t17=2.03, p=0.059, d=0.93) (Fig. 3-3).

Figure 3. Effects of genotypes, flutamide, and gonadectomy on gene expression in area CA1 of the hippocampus. Gene expression is shown as relative enrichment for intact (left) and gonadectomized (right) mice for H2afz, Chrm3, Chrna7, Gria1, Gria4, Grin1, and Bdnf4. The Y-axis indicates the gene name for which relative enrichment was calculated. *p≤0.05.

3.4 Discussion

Different lines of evidence presented here converge on the conclusion that AR is a negative regulator of fear memory. Specifically, as differences between genotypes in gonadectomized mice were restored with equivalent replacement dose of testosterone, our data suggest that reduced freezing in CMV-AR mice is due to differential responses to circulating androgens rather than to different levels of circulating androgens in the two groups. This conclusion is reinforced by our prior reports of distinct behavioural outcomes in WT and CMV-AR mice despite similar levels of circulating testosterone (Swift-Gallant, Coome, Ramzan, et al., 2016; Swift-Gallant, Coome, Srinivasan, et al., 2016).

Indeed, the lack of sensitivity to gonadectomy in WT mice is consistent with others who found that gonadectomy did not affect fear memory (Anagnostaras et al., 1998). Nonetheless, androgen receptor overexpression reduced fear memory only in the presence of ligand, suggesting that sufficiently high androgenic signalling can inhibit fear memory. A key implication of these data is that the ability of AR to modulate fear memory may at least in part depend on individual differences in AR density. In support of this hypothesis, a study of predator scent-induced fear memory showed that rats with an extreme response to this type of stress had lower levels of AR in area CA1 compared to rats that had a minimal response to predator stress (Fenchel et al., 2015), suggesting that individual differences in AR density may alter sensitivity to fear-inducing stimuli. Moreover, the same study showed that testosterone treatment reduced contextual fear memory, but only when administered 7 days after predator scent exposure. If testosterone was administered shortly after stress exposure (within 1 h), it increased fear memory, suggesting that the timing of testosterone treatment is important. In our study, mice received chronic testosterone replacement, indicating that AR can also inhibit memory under conditions of stable activation. Altered sensitivity to predator scent is particularly interesting in light of our previous data, which showed that CMV-AR males have reduced aggression compared to WT mice (Swift-Gallant, Coome, Ramzan, et al., 2016), as evidenced by reduced bouts of chasing and tumbling behaviours in response to a male intruder. In light of the current finding of reduced fear memory, these findings suggest a potential relationship between reduced fear, sensitivity to predator odour, and aggression.

Our results indicate that increased androgen signalling via AR overexpression impairs memory potentially through effects on various memory-related genes, including synaptic genes and the histone variant H2A.Z. This is in stark contrast with data from studies of estradiol, which typically produces enhanced memory in females through actions on the estrogen receptor β (ERβ) (Frick et al., 2017; Kim & Frick, 2017). Although estradiol similarly regulates memory in males, little is known about the mechanisms by which this occurs and whether these effects are distinct from females (Frick et al., 2017). Interestingly, the histone variant H2A.Z is a vital modulator of estrogen and androgen receptor signalling (Gévry et al., 2009), suggesting that both steroid receptors may interact with similar epigenetic factors, though the nature of their interaction is likely to differ.

Although fear memory provides an index of hippocampal function, it also serves as an index of PTSD (Zovkic & Sweatt, 2013), such that AR modulation of fear memory may be distinct from AR modulation of other forms of memory. Indeed, testosterone has been reported to have anxiolytic effects (Edinger & Frye, 2004, 2005; Fenchel et al., 2015; Frye et al., 2008) and as such, it may have an inhibitory effect on fear memory while promoting other forms of memory. Other studies have reported that testosterone improves non-fear based hippocampus-dependent memory, such as the Morris water maze and memory for temporal order in which objects are presented (Naghdi, Majlessi, et al., 2005; Naghdi et al., 2001; Picot et al., 2016), suggesting that AR may be protective against fear memory.

3.4.1 Effects of AR on gene expression

Contextual fear memory is heavily reliant on the hippocampus. Given that AR is a transcriptional regulator, we examined the effects of CMV-AR on mRNA levels of several memory-related genes in area CA1. CMV-AR had a robust effect on H2afz, a gene encoding the histone variant H2A.Z. We previously showed that H2A.Z is a memory suppressor, whereby virally-mediated depletion of H2afz enhanced fear memory (Stefanelli, Azam, et al., 2018; Zovkic et al., 2014). Here, we show that H2afz expression is elevated in CMV-AR mice and that this difference is not evident when AR activity is blocked by gonadectomy or by flutamide, and is restored by testosterone replacement, thus providing converging evidence for AR-mediated upregulation of H2A.Z levels. These findings are consistent with reports in prostate cancer, where high levels of AR activity (via the AR agonist R1881) enhance H2A.Z expression (Dryhurst et al., 2012).

Given that H2A.Z depletion (Stefanelli, Azam, et al., 2018; Zovkic et al., 2014) promotes fear memory, these data generate the hypothesis that at least some of the effects of AR overexpression on memory may be mediated by heightened H2A.Z expression.

Cholinergic receptors are also important regulators of hippocampal fear memory (e.g. Sahdeo et al., 2014) and testosterone mediates muscarinic receptor expression in the rat epididymis, hypothalamus, and amygdala, as well as in vocalization-related regions of the quail brain (Al- Daham & Thomas, 1987; Ball & Balthazart, 1990; Maróstica, Avellar, & Porto, 2005). Here, we show that Chrm3, a gene encoding the M3 muscarinic receptor, is upregulated in CMV-AR mice and that this difference is eliminated by manipulations that block AR activity and is restored with testosterone replacement, indicating that high AR density promotes Chrm3 expression. In contrast, Chrna7, a gene that encodes the nicotinic receptor 7 alpha, was not affected by CMV- AR, but was elevated by testosterone independently of genotype. These findings are consistent with studies in muscle that show reduced density of nicotinic receptors in response to testosterone deprivation (Souccar, Yamamoto, Gonçalo, & Lapa, 1991) and suggest that muscarinic and nicotinic receptors may be differentially sensitive to regulation by AR, whereby the nicotinic receptor may be affected by testosterone in an AR-independent manner. 17β estradiol is a potent regulator of nicotinic receptors in tumour tissues (Lee et al., 2011), suggesting that aromatized testosterone may be acting through estrogen receptors to promote Chrna7 expression.

Interestingly, Gria1 and Gria4, genes that encode AMPA receptor subunits, were similar to the nicotinic receptor, in that we did not find any differences associated with genotype, but the expression of both increased with testosterone replacement. In contrast, Grin1 (glutamate ionotropic receptor NMDA subunit 1) expression was higher in CMV-AR mice, this difference was eliminated by blocking AR activity, and restored by testosterone replacement, directly implicating AR in promoting NMDA receptor expression. Previous studies suggest that AR deletion impacts hippocampal plasticity through effects on NMDA receptor function, although no differences were found in receptor density in that study (Picot et al., 2016). However, AR stimulation with DHT increased NMDA receptor number in area CA1 (Romeo et al., 2005), which is consistent with increased Grin1 expression in CMV-AR mice. However, the lack of effect in WT mice suggests that these transcriptional effects occur selectively in cases of high

AR sensitivity, or by alternate mechanisms associated with potential organizational effects of germline AR overexpression.

Finally, the neurotrophic factor BDNF exon 4 expression is reduced by flutamide treatment irrespective of genotype but is increased by testosterone only in CMV-AR mice, suggesting that AR activation promotes Bdnf4 expression. This contrasts with evidence that gonadectomy decreases BDNF protein levels in the mossy fibre pathway from the dentate gyrus to the CA3 (Skucas et al., 2013), although studies in birds have documented a positive effect of testosterone on BDNF expression in several brain regions (rev in Brenowitz, 2013).

Overall, the use of CMV-AR mice allowed us to identify 2 major categories of genes that exhibit differential sensitivity to AR regulation: 1) genes that are upregulated selectively by CMV-AR, suggesting that their expression is increased by the presence of high levels of AR; and 2) genes that are not affected by AR density, but are nevertheless sensitive to testosterone treatment, implicating alternate pathways by which testosterone may influence their expression, such as the activation of estrogen receptors. These AR-sensitive genes, particularly genes encoding H2A.Z and NMDA receptor subunits, provide strong candidates for investigating the mechanism by which AR influences fear memory. In particular, we are actively pursuing the link between AR and H2A.Z, given the strong evidence for the regulation of this memory suppressor by AR.

3.4.2 Limitations

We focused our investigation of gene expression on the hippocampus, but we cannot exclude the possible contribution of other brain regions or non-neural tissues to mediating behavioural difference in fear memory, given that AR in this model is overexpressed across brain regions and tissue types. Indeed, interpretations of our data would be greatly improved by direct manipulations of AR in specific brain regions of interest, particularly the hippocampus, which is a target of future studies in our lab. Moreover, we cannot exclude potential developmental factors as mediators of differences between genotypes. Indeed, some studies have found opposite LTP effects in brain-specific AR knockout mice compared to pharmacological inhibition of AR (Harley et al., 2000; Picot et al., 2016), indicating that AR overexpression may have unique effects on the adult brain as a result of altered development.

Considering that AR is overexpressed globally and starting at embryonic development, the behavioural effects seen are likely a result of a combination of organizational and activational effects. Indeed, we have previously observed a masculinized cell number and size in the spinal nucleus of the bulbocavernosus (SNB) of female CMV-AR mice in comparison to controls (Coome et al., 2017). Since SNB cell number is heavily influenced by organizational effects and size by activational effects, it is highly conceivable that effects seen on behaviour are affected by a combination of both. However, by performing gonadectomy with or without hormone replacement, we show that the impairment in fear memory in CMV-AR male mice is reversible and at least partially mediated through activational effects. On the other hand, an overall susceptibility to fear memory impairment may be established during organizational periods.

Furthermore, our studies focused specifically on fear conditioning in male mice, so it is not clear if our findings reflect a generalized learning deficit, or a specific reduction in memory for aversive stimuli. Indeed, castration impairs working memory in the Morris Water Maze, without affecting spatial reference memory, or memory in tests of novel object recognition and passive avoidance. In addition, DHT replacement selectively recovered spatial memory in castrated male mice without impacting other forms of memory (Benice & Raber, 2009). Additionally, androgens promote neurogenesis and spine growth in area CA1 of the hippocampus (Leranth et al., 2003; MacLusky et al., 2006; Swift-Gallant et al., 2018), suggesting that androgens have a complex role on memory-related systems, which can translate to unique behavioural outcomes for different forms of memory.

Ultimately, our goal is to draw conclusions about the function of AR during the normal formation of fear memory, particularly in relation to individual and sex differences. The extent to which our data reflect such endogenous variability in AR-related outcomes is not clear, but our findings provide an important first step to demonstrate the capacity of AR to inhibit the formation of fear memory. In upcoming studies, we hope to strengthen our findings of AR- mediated regulation of fear memory by focusing on individual differences in fear memory in relation to variable AR expression in strains with varying magnitudes of fear memory formation (Brinks, de Kloet, & Oitzl, 2007; Siegmund & Wotjak, 2007).

3.5 Conclusions

The differences in fear memory observed in groups with differing levels of AR expression have implications for certain types of resilience, particularly as a protective factor against PTSD. Although fear conditioning is a widely-used index of hippocampus-dependent associative memory, it is also utilized as a model of PTSD because of the strong aversive memory that is produced by exposure to a single traumatic event (Zovkic & Sweatt, 2013). In Dutch military veterans, low testosterone levels pre-deployment predicted increased likelihood of developing PTSD 1–2 years post-deployment (Reijnen et al., 2015), consistent with the observation that PTSD is more common among women than men exposed to trauma (Maddox et al., 2018). In addition, testosterone has been associated with reduced anxiety in humans (McHenry et al., 2014) and in rodent models (Edinger & Frye, 2004; Frye et al., 2008; McDermott et al., 2012), as well as reduced fear responses to a predator scent (J. A. King, De Oliveira, & Patel, 2005). Moreover, individual differences in sensitivity to predator scent exposure are associated with differences in AR in CA1 (Fenchel et al., 2015), suggesting that high levels of AR expression may protect against the development of fear-related disorders.

Chapter 4

H2A.Z Interacts with Androgenic Mechanisms to Regulate Fear Memory

4.1 Introduction

4.1.1 Effect of both H2A.Z variants on fear memory

Epigenetic mechanisms regulate various aspects of memory formation, and this effect is especially well characterized for DNA methylation and histone acetylation (Jarome, Thomas, & Lubin, 2014; Sweatt, 2013). In contrast, the role of histone variant exchange, the process in which a canonical histone is replaced by a histone variant, has only recently garnered attention. Work from our lab has implicated the histone variant H2A.Z as a negative modulator of fear memory, whereby H2A.Z depletion in area CA1 or mPFC enhanced contextual fear memory (Stefanelli, Azam, et al., 2018; Zovkic et al., 2014). This effect is related to the dynamic removal of H2A.Z from gene bodies and gene promoters, which correlates with increased transcription (Stefanelli, Azam, et al., 2018; Zovkic et al., 2014). Thus, endogenous H2A.Z likely suppresses fear memory formation by providing a physical barrier to gene expression.

H2A.Z is a variant of histone H2A and is encoded by two genes, H2afz and H2afv, which are located on separate chromosomes with an independent set of promoters. They encode for variations of H2A.Z protein that differ by only three amino acids (H2A.Z-1 and H2A.Z2, respectively) (Dryhurst et al., 2009; Matsuda et al., 2010). Thus far, studies regarding H2A.Z’s effects on memory have investigated the effects of H2afz only (Stefanelli, Azam, et al., 2018; Zovkic et al., 2014). However, considering the relative abundance of both gene products within the brain, as well as their structural similarity (Dryhurst et al., 2009), it is possible that H2afv may contribute to fear memory as well. Indeed, a recent study showed distinct effects of H2afz and H2afv depletion in cortical neurons on activity-induced gene expression (Dunn et al., 2017). Specifically, IEG Arc transcription in response to neuronal activity was impaired by H2afv depletion, but not H2afz depletion, indicating a greater role of H2afv regulating learning-induced

54 gene transcription. However, under a different context (treatment with tetrodotoxin), Arc transcription is impaired by individual depletion of both variants, H2afz and H2afv. Similarly, context-specific effects of the two variants were also found in numerous activity-dependent IEGs. These data suggest a context-specific role of both variants, H2afz and H2afv, in mediating learning-induced neuronal gene transcription. Further, H2afz and H2afv were shown to regulate basal transcription of mainly non-overlapping gene sets, which included genes encoding for numerous synaptic proteins. These data suggest similar yet distinct roles for both H2A.Z variants in regulating learning-induced neuronal gene transcription.

Despite growing evidence that H2A.Z removal plays an important role in memory, very little is known about how H2A.Z is regulated. In a previous study, we showed that H2A.Z expression in area CA1 of the hippocampus is influenced by manipulations of the androgen receptor (AR), suggesting that AR may also influence H2A.Z expression (Ramzan, Azam, Monks, & Zovkic, 2018). Specifically, AR overexpression resulted in increased levels of H2afz, and this increase was blocked by the AR antagonist flutamide and by the removal of circulating androgens through gonadectomy. Moreover, higher H2afz levels in AR overexpressing mice were restored by testosterone replacement, suggesting that AR potentiates H2A.Z expression. Consistent with a suppressive role of H2afz on fear memory, AR overexpression reduced memory recall, indicating that AR and H2A.Z may interact to regulate memory. Indeed, studies of prostate cancer literature suggest that AR and H2A.Z are required for AR-dependent gene expression (Draker et al., 2011; Dryhurst & Ausió, 2014), suggesting that AR-mediated regulation of fear memory may also require H2A.Z. Elucidating this relationship is especially important for understanding how sex hormones interact with epigenetic factors to regulate fear memory-related conditions, such as post-traumatic stress disorder that disproportionately affects women compared to men (Seedat et al., 2005; Shansky, 2015).

Having previously characterized a link between H2A.Z and AR in an AR-overexpressing mouse model, our primary goal here was to test the hypothesis that AR-mediated effects on fear memory require H2A.Z. To this end, we utilized a conditional-inducible knockout mouse line that results in H2A.Z deletion in CamKIIα containing neurons in adult male mice. However, we previously characterized the behavioural effects of only H2afz, such that observations of improved memory may not extend to the deletion of both genes. Indeed, the H2A.Z protein encoded by H2afv is structurally distinct from H2afz and produces distinct effects on neuronal

55 gene expression (Dryhurst et al., 2009; Dunn et al., 2017; Matsuda et al., 2010). Thus, as a first step, I characterized how the dual depletion of both H2A.Z-encoding genes in CamKIIα neurons influences memory formation, in conjunction with androgenic manipulation.

4.1.2 Mechanism of interaction between H2A.Z and AR

H2A.Z likely affects fear memory through regulating transcription of memory-associated genes. It does so by regulating chromatin accessibility to transcriptional machinery. Indeed, chromatin structure acts as a gatekeeper of transcriptional activity by providing a physical barrier to the underlying DNA. Three main mechanisms act to overcome this barrier, including histone post- translational modifications (PTMs), chromatin remodeling, and histone variant exchange, which work together to modulate transcription (Sweatt et al., 2013). The interaction between H2A.Z and AR is an excellent example of all three processes in action. Specifically, the incorporation of the histone variant H2A.Z into chromatin, via chromatin remodeling complexes and histone acetyl transferases, primarily acts to enhance basal transcription as well as poises the gene for future stimulus-induced activation (Stefanelli, Azam, et al., 2018; Subramanian et al., 2015; Zovkic et al., 2014). Some of these chromatin remodeling complexes (e.g. SRCAP) and HATs (e.g. Tip60) act as co-activators for the transcriptional activity of the AR, likely by facilitating the incorporation and acetylation of H2A.Z, as well as directly modifying the AR itself (Dryhurst & Ausió, 2014).

Studies in prostate cancer suggests that AR and H2A.Z interact through common upstream regulators, including SRCAP, Tip60, and USP10. AR is a transcription factor that translocates to the nucleus upon androgen binding, where it recruits coactivator proteins and binds androgen responsive elements (AREs) upstream of the transcription start site (TSS) (Bennett, Gardiner, Hooper, Johnson, & Gobe, 2010). AR activity is regulated through several cofactors that also regulate the activity of H2A.Z. For example, SRCAP (which interacts with AR at ARE on the PSA to enhance its transcriptional activity), an ATP-dependent chromatin remodeling complex that induces structural changes (e.g., chromatin looping) by incorporating H2A.Z-H2B dimers into chromatin (Dryhurst & Ausió, 2014; Slupianek et al., 2010; Wong et al., 2007).

H2A.Z is associated with poising genes for activation (Subramanian et al., 2015) and its enrichment at TSSs of genes is associated with higher transcription under baseline conditions (Stefanelli, Azam, et al., 2018; Zovkic et al., 2014). This role of H2A.Z may be mediated by its

56 recruitment of RNA polymerase II and other transcriptional coactivators to DNA (Adam et al., 2001; Draker et al., 2012). For example, H2A.Z enrichment at the promoter and enhancer regions of the prostate specific antigen (PSA) gene poises the gene for AR-induced activation. H2A.Z enrichment is also associated with hypomethylation and its eviction from the PSA promoter and enhancer is associated with PSA gene upregulation in response to androgenic stimulation (Dryhurst & Ausió, 2014; Dryhurst et al., 2012), suggesting that H2A.Z mediates the effects of AR.

As with canonical histones, the effect of H2A.Z on transcription is influenced by its post- translational modifications (PTMs) (Ku et al., 2012). Most notably, H2A.Z acetylation is associated with a destabilized nucleosome and promotes transcription (Bruce et al., 2005; Ishibashi et al., 2009; Millar et al., 2006; Valdes-Mora et al., 2012) and some of the acetyltransferases that acetylate H2A.Z also act as transcriptional coactivators of AR. Specifically, Tip60 acetylates both H2A.Z and AR (Gaughan, Brady, Cook, Neal, & Robson, 2001; Gaughan et al., 2002; Halkidou et al., 2003; Sapountzi et al., 2006; J. Wang & Chen, 2010), and AR’s acetylation status directly affects its activity, such that acetylated AR exhibits increased activity while de-acetylation of AR inhibits AR activity, as measured through luciferase assay (Gaughan et al., 2002). We recently implicated Tip60’s interaction with H2A.Z in fear memory, such that reducing H2A.Z incorporation via Tip60 inhibition enhanced fear memory (Narkaj et al., 2018), suggesting that regulation of this factor can indeed impact fear memory. In addition, AR and H2A.Z share another upstream regulator, ubiquitin-specific protease 10 (USP-10). USP-10 directly de-ubiquitylates H2A.Z and is required for AR-mediated gene activation (Draker et al., 2011). Ubiquitylated H2A.Z is associated with transcriptional repression, such that deubiquitylation by USP10 facilitates H2A.Z-associated transcriptional activation as well as AR-mediated gene transcription (Draker et al., 2011; Sevilla & Binda, 2014).

Given our previous observation that AR-is a negative modulator of memory and its overexpression upregulates H2afz (Chapter 3), the second goal of this experiment was to investigate the potential mechanism behind this effect by studying the effects of AR manipulations on H2A.Z binding in cultured hippocampal neurons under basal conditions and in response to neuronal activity, as well as effects on gene transcription.

4.2 Methods

4.2.1 Animals

Behaviour Experiments. Transgenic C57Bl/6 mice containing both Z1(H2afz)-flox and Z2(H2afv)-flox transgenes were purchased from Riken (Riken RBRC # 05765). They were crossed with a tamoxifen-inducible CamkIIα-cre/ERT2 mouse line (The Jackson Laboratory Stock # 012362) to produce mice with both floxed genes with either Cre (H2A.Z KO) or NoCRE littermate controls. Male mice bred in our colony were group-housed after weaning and were assigned to testing groups at 60–90 days of age. Mice were housed in 7.5×11.5×5 in cages and maintained on a regular 12 h light cycle, with ad libitum access to standard mouse chow (Harlan Teklad, Madison, WI) and water.

Cell Culture Experiments. Male and female C57Bl/6 mice were ordered from Jax at 60–90 days of age and group-housed. They were allowed to acclimate to the mouse room for two weeks before breeding. Mice were bred in a harem of 2 females and one male and were separated after three days. The separation day was considered embryonic day 0 (E0) and pregnant mice were cervically dislocated and the embryos removed on E17 for neuronal plating. Mice were housed in 7.5×11.5×5 in cages and maintained on a 12 h light cycle, with ad libitum access to standard mouse chow (Harlan Teklad, Madison, WI) and water. All animal procedures were performed in accordance with the Local Animal Care Committee (LACC) at the University of Toronto Mississauga (UTM).

4.2.2 Genotyping

Genotyping was carried out at weaning for all mice used in behaviour experiments. Animals were ear notched and DNA was extracted using the “HotSHOT” method (Truett et al., 2000). The Cre transgene was identified by amplifying a unique portion within the Cre transgene. The individually floxed genes (H2afz (H2A.Z1) and H2afv (H2A.Z2)) were identified by amplifying the Z1 and Z2 genes, such that the floxed genes resulted in a larger PCR product (see Table 4-1 for a list of genotyping primers and expected band PCR product size, provided by Riken). In addition, we used qPCR to confirm the reduction in mRNA of both Z1 and Z2 in the mPFC region of each mouse included in the experiments.

Table 4-1. DNA primers used for genotyping H2A.Z KO mice. PCR Product Gene Forward Reverse Size Jax Internal Control 5'-CTAGGCCACAGAATTGAAAGATCT 5'-GTAGGTGGAAATTCTAGCATCATCC 324 bp Cre 5'-AGCTCGTCAATCAAGCTGGT 5'-CAGGTTCTTGCGAACCTCAT 184 bp 326 bp (WT) H2A.Z1 5'-CGCCTTGGTAATTCTATCTTCTCC 5'-CGCCAGTTAACACACATGTGATC 448 bp (flox) 570 bp (WT) H2A.Z2 5'-GCCTCAGATCATCCAGTC 5'-GGCTCTGAATTCCCAATGTAG 700 bp (flox)

4.2.3 Tamoxifen

Tamoxifen (Sigma T5648-5G, final concentration 2mg/mL) was dissolved in ethanol (40mg/mL) and mixed with corn oil (375uL tamoxifen in EtOH/750uL corn oil) in 1.5mL microcentrifuge tubes. After thorough vortexing, ethanol was evaporated by spinning in Eppendorf Vacuufuge Plus on V-AL (vacuum-alcohol) setting at 30ᣞC for 20 min (or until all EtOH had evaporated). Mice received 5 daily i.p. injections of tamoxifen (2mg/g) at 60-90 days of age and were given 10 days to recover prior to testing, injections, or surgical procedures. All animals were treated with tamoxifen, such that floxed mice without Cre were utilized as control. During tamoxifen treatment, mice were housed in ventilated cages (5 x 7 x 14 in.) in a closed-circuit atmosphere rack (Techniplast Easy Flow #BOX110EFUL).

4.2.4 Contextual Fear Conditioning

To facilitate habituation to the experimenter and non-associative aspects of the training procedure, mice were transported to the testing room and handled for 30–60 s daily for 3 days before testing. On the training and test days, mice were transported to the testing room and placed into test chambers (9.8 in boxes; designed for mice) equipped with an electrified grid floor (Coulbourn Instruments, Holliston, MA, USA). During training, mice were given 2 min to explore the apparatus before receiving a foot shock (0.5 mA, 2 s), followed by an additional minute of exploration. Memory was assessed 24 h later by replacing the mouse into the training apparatus without shock and measuring freezing behaviour for 3 min. For extinction trials, animals were re-exposed to the training apparatus without shock for 10 min each over 5 consecutive days immediately following the 24 h test. Freezing was recorded with a camera placed directly in the chamber ceiling and scored by automated software (FreezeFrame, Coulbourn Instruments).

4.2.5 Gonadectomy and dihydrotestosterone (DHT) replacement

At 60–90 days of age and 10 days after the last tamoxifen injection, a subset of mice underwent castration under inhalant anesthesia (1–2% isoflurane). A midline incision was made in the scrotum to allow for the removal of both testes, after which the incision was closed with wound clips. Immediately after castration, animals were subcutaneously implanted at the nape of the neck with a Silastic capsule containing DHT (10mm of crystalline T; Silastic tube 1.02 mm id/2.16 mm od) and sealed with Silastic adhesive, as in Coome et al. (2017). Control animals received an empty Silastic implant (Blank). Sham controls were anaesthetized and given two incisions, one in the scrotum and the other in the nape, which were then sealed similarly to gonadectomized animals. The animals were administered Anafen (5 mg/ kg) on the day of the surgery and for 2 proceeding days. Wound clips were removed if still present after 7 days. Animals underwent 3 days of handling (30 s/mouse) beginning 11 days after surgery and were trained 14 days after surgery.

4.2.6 Flutamide administration

5 days before handling (at 60–90 days of age), a subset of intact male mice was injected subcutaneously with the AR antagonist flutamide (8 mg/day) or vehicle (sesame oil) and injections continued daily for a total of 16 days to ensure continued AR inhibition until tissue collection. Flutamide (Sigma # F9397, Oakville, Canada) was prepared daily immediately prior to use, by first dissolving it in 100% ethanol, then in sesame oil (Sigma # S3547, Oakville, Canada). The ethanol was removed using an Eppendorf Vacufuge Plus (spun for 20 min at 30 °C on the V-AL setting), producing the final concentration of 53 mg/mL. Mice were administered 0.15 mL through subcutaneous injection in the nape of the neck. Vehicle-treated control mice received sesame oil on the same schedule as the flutamide-treated mice.

4.2.7 Tissue collection

4.2.8 Western Blotting

Tissues were homogenized using a dounce homogenizer in RIPA buffer (50mM Tris HCl pH 7.4, 150mM NaCl, 10% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with Protease Inhibitor Cocktail (Cell Signaling, Danvers, MA, USA). Homogenates were incubated for 20 min on ice and flicked every 5 min, centrifuged at maximum speed at 4°C for 20 min, and the supernatant was collected. Proteins were separated on 8% SDS-PAGE for AR or 15% SDS- PAGE for H2A.Z, then transferred to a PVDF membrane. Membranes were blocked in TBS-T (TBS with 0.1% Tween- 20) containing 5% skim milk and incubated with rabbit monoclonal anti-AR (1:500, Abcam, ab133273 RRID, Cambridge, UK), rabbit polyclonal anti-H2A.Z (C- term) (1:2000, Millipore ABE1348), and rabbit anti-β-Actin (1:5000, Cell Signaling, 4967S, Danvers, MA, USA) overnight at 4 °C. After three 5 min TBS-T washes, membranes were incubated with anti-rabbit-HRP secondary antibody (1:10,000 Life Technologies, ThermoFisher, Waltham, MA, USA) for 1 h at RT. Detection was performed by enhanced chemiluminescence and quantification was done using ImageJ (NIH) to quantify Area Under the Curve, with AR or H2A.Z normalized to actin from the associated membrane.

4.2.9 Primary hippocampal neuronal culture

6-well plates were coated with poly-lysine (0.1mg/mL; Sigma P2636) for a minimum of one hour before use. Cultures of hippocampal neurons were prepared from E17 C57Bl6/J mice of either sex. The hippocampi were first washed using Hank’s Balanced Salt Solution (1x HBSS Gibco #14175-095), then trypsinated for 30 seconds (0.25% Trypsin (Life Technologies 15050065) and washed with HBSS. Protease activity of trypsin was stopped using dissecting medium (10% normal horse serum (Life Technologies 26050070) in DMEM (Life Technologies 11995065)). The neurons were dissociated by triturating through a 1000uL pipette tip 10-15 times. The dissociated neurons were diluted in Neurobasal Medium (L-Glutamine) (Gibco 21103-049) supplemented with 20mM L-glutamine (Gibco 25030-081), 1x Penicillin- Streptomycin (Sigma P4333-100ML), and 1x B27 supplement (Gibco 17504-044) and plated in 6-well plates (3 embryos/plate). Neurons were used at 10 days in vitro (DIV). At DIV10, neurons were treated with either 1nM DHT (dissolved in dimethyl sulfoxide; DMSO) or with an equivalent volume of DMSO. 24 hr later (DIV11), they were treated with 55mM KCl for 20 min to induce depolarization. Neurons were then processed for either RNA extraction or ChIP.

4.2.10 mRNA expression and Quantitative-PCR (qPCR)

Mice used for behaviour experiments were euthanized by cervical dislocation at least 2 weeks after the final training session and area CA1 was dissected. Tissue was stored at -80ᣞC until ready for RNA extraction. For cell culture experiments, hippocampal neurons were washed with ice cold HBSS two times, after which RNA was extracted.

RNA was extracted using BioBasic RNA Extraction Kit (BioBasic #BS82322-250, Amherst, NY). Complementary DNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied BioSystems #4368814, Folster, CA, USA). Primers were designed in the lab and used to detect levels of the indicated transcripts, and data were normalized to the geographic mean Gapdh and β-actin. Comparison of group differences was conducted using relative enrichment analysis (2^−ΔΔCT) and data for experimental animals were normalized to controls (NoCRE GDX+Blank) and data for cell culture experiments were normalized to DMSO-treated controls. The list of primer sequences is provided in Table 4-2.

Table 4-2. qPCR primers used in gene expression studies. Melting Gene Forward Reverse Temperature (ᣞC) Actin 5'-AGATCAAGATCATTGCTCCTCCT 5'-ACGCAGCTCAGTAACAGTCC 58 Gapdh 5'-GTGGAGTCATACTGGAACATGTAG 5-AATGGTGAAGGTCGGTGTG 60 H2afz 5-CACCGCAGAGGTACTTGAGTT 5-TCCTTTCTTCCCGATCAGCG 58 H2afv 5'-CAAGGCTAAGGCGGTGTCTC 5'-CTGCTAACTCCAACACCTCAGC 58 Fkbp5 5’-TGGTGTTCGTTGTTGGGGAA 5’-CCAAAACCATAGCGTGGTCC 58 Th 5’-GAGGTATACGCCACGCTGAA 5’-GGAAGCCAGTCCGTTCCTTC 60 B2m 5’-TGCTATCCAGAAAACCCCTCA 5’-TTTCAATGTGAGGCGGGTGG 58 Fos 5’-GGCACTAGAGACGGACAGAT 5’-ACAGCCTTTCCTACTACCATTC 60

4.2.11 Chromatin Immunoprecipitation (ChIP) and qPCR

Primary hippocampal neurons were incubated in 1% formaldehyde for 10 min at 37°C, when the reaction was quenched with 1.25 M glycine. Samples were washed with PBS and lysis buffer

(50mM NaCl, 10mM PIPES pH 6.8, 5mM MgCl2, 1mM CaCl2) was added to all samples before sonication (25% power, 3x for 10s on 30s off; Thermo Fisher Scientific). Samples were pre- warmed at 37ᣞC for 5min, after which 1µL of MNase (Cell Signaling) was added to each sample. Samples were incubated at 37ᣞC for 20 min. EDTA (0.83mM) and SDS (1%) were added to the samples, which were then aliquoted and stored at -80ᣞC until use.

Samples were thawed on ice and diluted with ChIP dilution buffer (16 mM Tris pH 8, 0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl). 20 µL of Millipore Protein G magnetic beads were added to each sample, with either 1.4 µg of H2A.Z (polyclonal anti-rabbit; Millipore ABE1348), or 1 µg AR (polyclonal anti-mouse; Santa-Cruz sc-7306 X) antibody overnight at 4°C. The next day, samples were washed sequentially with low-salt, high-salt, LiCl (Millipore), and Tris-EDTA (TE; 10mM Tris, 1mM EDTA) buffers and rotated at 4ᣞC for 5 min per wash. Immune complexes from both ChIP and input samples were de-crosslinked and extracted in de- crosslinking buffer (1% SDS, 0.5% Proteinase K, in TE buffer) and heated at 65°C for 2 hr, followed by 95°C for 10 min before purification with a PCR Purification Kit (Bio Basic). Primers were designed to detect specific sequences (Table 5-1), and ChIP data were calculated as the percentage of input, then normalized against the control group (DMSO-treated samples).

Table 4-3. Genomic DNA primers used for ChIP-qPCR. Melting Gene Forward Reverse Temperature (*C) -1 Fos 5'-AGGAGACCCCCTAAGATCCC 5'-CTGTCGTCAACTCTACGCCC 64 -1 Th 5'-CCCTGTCTTCATGTCGTGTCT 5'-GAGGCCTCCGTCCCATTAG 58 -1 B2m 5'-AAGGGTTGAGTTCTGCCAGTT 5'-GGGTGTGCTCTTGAGTTTGG 58 -1 Fkbp5 5'-GTGTCAGTCAGCTTCCTCCA 5'-TTCGTTTGCATCTCCGCCT 58

4.2.12 Statistics

Analyses were conducted with SPSS Version 24 and consisted of independent-samples t-test for comparison of Genotype (NoCRE, H2A.Z KO). In cases in which there were 2 independent variables, we used a two-way ANOVA with Genotype (NoCRE, H2A.Z KO) and Treatment (Control, DHT; or Vehicle, Flutamide) as independent variables. To compare freezing before and after-shock during the training session, we used a mixed-measures ANOVA, with Genotype and Treatment as the between group factors and Shock (before shock; after-shock) as the within- group factor. Follow-up analyses were conducted using independent-samples t-test or paired- samples t-test only when the omnibus test was significant, thus precluding the need to correct for multiple comparisons.

For cell culture experiments, analyses consisted of a two-way ANOVA with Hormone treatment (DMSO, DHT) and Stimulation (vehicle, KCl) as independent variables. Follow-up analyses

63 were conducted using independent-samples t-test only when the omnibus test was significant, thus precluding the need to correct for multiple comparisons.

4.3 Results

4.3.1 Validation of H2A.Z KO in the hippocampus using gene expression and protein analyses

We confirmed that both H2A.Z encoding genes are reduced in H2A.Z KO male mouse CA1 region (H2afz: t(20) = 5.317, p=0.000, H2afv: t(20) = 2.644, p=0.016; Fig 4-1a). We further confirmed that H2A.Z protein is also reduced in H2A.Z KO male mouse CA3 region (we used CA3 for protein as the CA1 was used for qPCR analyses; t(10) = 2.593, p=0.027; Fig 4-1b).

Figure 4-1. H2A.Z gene and protein expression are reduced in the hippocampus of H2A.Z KO mice. a) H2afz and H2afv gene expression is reduced in area CA1 in H2A.Z KO mice, b) H2A.Z protein expression is reduced in area CA3 in H2A.Z KO mice.

4.3.2 Conditional-inducible H2A.Z deletion improves fear memory without affecting fear extinction in male mice

We previously showed that depleting H2Afz enhances fear memory for up to 30 days (Zovkic et al., 2014), but the extent to which this memory is resistant to extinction is not clear. To test whether knocking out both H2A.Z-encoding genes would affect fear memory, mice first underwent fear conditioning and memory was measured after 24 hr. Consistent with H2afz depletion alone, conditional-inducible knockout of both H2A.Z-enocoding genes resulted in enhanced fear memory compared to NoCRE controls (t(27) = 2.487, p=0.019) (Fig. 4-2b).

Extinction training occurred over 5 days and involved the re-exposure of mice to the training context for 10 min each day without additional exposure to shock. Freezing was compared

64 during the first minute of daily exposure to provide an index of the initial fear reaction to the box, and for the total 10 min period to get an index of overall extinction rates. For the first minute, a mixed measures ANOVA of Extinction Day (Days 1-5) x Genotype (H2A.Z KO, NoCRE) revealed a main effect of Extinction Day (F(4,60) = 6.737, p=0.000), with follow up analyses revealing a significant drop in percent freezing between days 2 and 3 (t(16) = 3.376, p=0.004). A mixed measures ANOVA on percent freezing through the 10 minute session similarly revealed a main effect of Extinction Day only (F(4,60) = 3.122, p=0.021), suggesting that despite of enhanced fear memory, H2A.Z deletion does not impair or enhance fear extinction (Fig. 4-2c).

Figure 4-2. H2A.Z KO improves fear memory but not fear extinction in males. a) Timeline of fear conditioning and extinction trials, b) percent freezing at 24 hr recall after train day, c) fear recall during the first minute and total test period of extinction trials. *p≤0.05

4.3.3 H2A.Z KO improves fear memory and prevents androgenic effects on fear memory

To characterize the effect of the dual deletion of both H2A.Z encoding genes on fear memory, we compared freezing behaviour in inducible-conditional H2A.Z KO mice to NoCRE controls.

In parallel studies, we investigate the role of androgenic mechanisms in H2A.Z-KO and control mice by exposing each group to gonadectomy with or without DHT replacement. We did not find any differences between intact mice and sham controls on any measure of fear learning and memory (Train before shock: F(1,24) = 0.412, p=0.527, after shock: F(1,24) = 0.523, p=0.477, 24 h recall: F(1,24) = 0.043, p=0.838), such that these groups were collapsed for statistical analyses.

Training. We first assessed within-session learning by comparing freezing behaviour before and after exposure to shock during training. A mixed measures ANOVA of Shock (Before, After) X Genotype (NoCRE, H2A.Z KO) X Hormone (control, GDX + DHT, GDX + Blank) revealed a significant effect of Shock, such that all groups showed increased freezing after than before shock (F(1,58) = 45.242, p=0.000; Fig 4-3b).

24h memory recall. A Genotype X Hormone ANOVA revealed significant effects of Genotype (F(1,57) = 5.669, p=0.021) and Hormone (F(2,57) = 3.543, p=0.035) on 24h recall of fear memory, although the interaction did not reach significance (F(2,57) = 2.366, p=0.103). Follow- up analyses with independent t-tests were performed to confirm whether deletion of both H2A.Z- encoding genes does improve fear memory, and to clarify the observed main effects. In control mice (I.e., no surgery + sham control), H2A.Z knockout mice had enhanced fear memory compared to NoCRE controls (t(23) = 2.388, p=0.026), suggesting that dual deletion of both H2A.Z encoding genes enhances fear memory. These differences between genotypes were no longer evident when mice were gonadectomized, suggesting a role for gonadal hormones in mediating the effect of H2A.Z on fear memory in male mice.

Treatment with DHT restored genotype effects in gonadectomized mice, such that H2A.Z KO mice again had enhanced fear memory compared to NoCRE controls (t(18) = 2.773, p=0.013). Further analyses showed that H2A.Z KO mice were not influenced by any of the treatment conditions (i.e. gonadectomy or DHT replacement), whereas NoCRE controls exhibited reduced freezing in response to DHT treatment compared to gonadectomized mice without DHT replacement (t(18) = 3.274, p=0.004; Fig 4-3c). These data are consistent with our previous evidence that AR inhibits fear memory (Ramzan et al., 2018) and suggest that H2A.Z deletion reduces sensitivity to androgenic manipulations.

Blocking AR improves fear memory in NoCRE mice

Training. To validate the genotype-specific effect of AR manipulations on fear memory, we next tested the effect of AR antagonism via flutamide. During training, a Shock (Before, After) X Genotype (NoCRE, H2A.Z KO) X Hormone (Vehicle, Flutamide) ANOVA revealed a significant effect of Shock (F(1,34) = 57.879, p=0.000), as well as a significant interaction between shock and hormone (F(1,34) = 5.232, p=0.029). Follow up analyses using a 2-way ANOVA revealed that flutamide treated animals, regardless of genotype, froze more before (F(1,34) = 20.578, p=0.000) and after (F(1,34) = 17.160, p=0.000; Fig 4-3e) shock.

24h memory recall. At the 24 hr recall memory test, a 2-way ANOVA revealed an interaction between Genotype and Hormone (F(1, 34) = 7.124, p=0.012). Follow up analyses using independent samples T-test revealed improved fear memory in vehicle-treated H2A.Z KO mice compared to NoCRE controls (t(19) = 2.339, p=0.030). When mice were treated with flutamide, these differences were no longer evident. As with DHT, flutamide treatment only influenced fear memory in NoCRE-controls, whereby flutamide treatment increased freezing compared to vehicle (t(16) = 2.770, p=0.014; Fig 4-3f). These data reinforce the inhibitory effect of AR on fear memory (Ramzan et al., 2018) and suggest that androgenic effects are dependent upon H2A.Z status.

Figure 4-3. Effects of androgenic manipulations on fear memory in H2A.Z KO mice. a) Experimental timeline for gonadectomy experiments, b) within session learning on training day showed increased percent freezing in response to shock, c) at 24 hr recall, control H2A.Z KO mice showed improved fear memory, which was maintained with hormone replacement in gonadectomized animals, and reversed in animals without replacement driven by an increase in freezing by NoCRE animals. d) Experimental timeline for AR antagonist (flutamide)-treatment experiments, e) on training day, within session learning occurred such that all groups showed increased percent freezing after shock, with flutamide-treated animals showing increased % freezing overall, f) percent freezing at 24 hr recall is increased with AR antagonism in NoCRE animals but not H2A.Z KO mice.

4.3.4 H2A.Z KO increases AR protein in the hippocampus

We analyzed AR protein levels in area CA3 of the hippocampus based on previous evidence that AR overexpression results in increased H2afz transcription in area CA1 (Ramzan et al., 2018). H2A.Z KO resulted in increased AR protein (t(10) = 2.926, p=0.015; Fig 4-3b).

Figure 4-4. AR protein expression in the hippocampus of H2A.Z KO mice. AR protein is increased in area CA3 of H2A.Z KO mice.

4.3.5 DHT impairs depolarization-induced H2A.Z removal

To validate our DHT treatment, we first quantified AR protein levels in response to DHT and showed a significant increase in AR protein (t(10) = 3.77, p = 0.004). To assess effects of DHT on basal and activity-induced H2A.Z binding, we used ChIP to investigate H2A.Z levels at synaptic and AR-regulated genes. Beta-2-microglobulin (B2M) is a previously identified AR- regulated gene that has been shown to have AR binding at an ARE upstream of the TSS as well as increased transcription in response to androgen treatment in androgen responsive prostate cancer cells (Romanuik et al., 2009; Yoon & Wong, 2006). B2M is a serum protein associated with MHC class 1 molecules. A significant Stimulation (Veh, KCl) X Hormone (DMSO, DHT) interaction (F1,8 = 6.36, p = 0.036) showed that depolarization resulted in significantly reduced H2A.Z binding only in control neurons, whereas DHT treatment blocked H2A.Z eviction from B2M promoter. DHT treatment resulted in a trend for reduced H2A.Z under basal conditions (binding in DHT compared to control neurons under unstimulated conditions (t(4) = 2.67, p = 0.056). Depolarization resulted in H2A.Z removal from the B2M promoter in control cells (t4=7.72, p = 0.002), but DHT-treated depolarized neurons did not differ from DHT-treated unstimulated neurons, suggesting that DHT treatment reduces stimulus-induced H2A.Z

69 dynamics. Similarly, DHT treatment produced a Hormone X Stimulation interaction for mRNA expression of B2M (F1,12 = 5.43, p = 0.038), whereby depolarization only produced a significant decrease in B2M expression in control depolarized compared to non-depolarized neurons (t6=4.13, p = 0.006). However, both DHT-treated groups (depolarized and not) had significantly lower B2M expression than non-stimulated controls (both p < 0.05).

FK506 Binding Protein 51 (Fkbp5) is another AR-regulated gene implicated in glucocorticoid receptor signaling (Blouin, Sillivan, Joseph, & Miller, 2016; Bolton et al., 2007; Magee, Chang, Stormo, & Milbrandt, 2006). There was no depolarization-induced removal of H2A.Z in any group, but DHT resulted in higher levels of H2A.Z binding compared to control-treated neurons (Main effect of Hormone: F1,8 = 5.63, p = 0.045). This difference was not associated with altered Fkbp5 transcription.

TH encodes the catecholamine precursor tyrosine hydroxylase and has been implicated as an AR target in human neuroblastoma cells as well as a murine dopaminergic cell line (Jeong, Kim, Kwon, Kim, & Seol, 2006). DHT treatment did not affect H2A.Z binding, although depolarization induced H2A.Z removal from the TH promoter (main effect of Stimulation: F1,8 = 30.55, p = 0.001). Similarly, depolarization increased TH expression independently of DHT (main effect of Stimulation: F1,12 = 5.56, p = 0.04), suggesting that activity-induced regulation of TH is not dependent on AR in cultured hippocampal neurons.

Finally, Fos is an immediate-early gene that plays a crucial role in neural plasticity (Jaworski, Kalita, & Knapska, 2018). DHT treatment resulted in higher overall levels of H2A.Z binding at the Fos promoter (main effect of Hormone: F1,8 = 5.39, p = 0.049). Additionally, stimulation resulted in significant H2A.Z removal from the Fos promoter irrespective of hormone treatment (main effect of Stimulation: F1,8 = 8.59, p = 0.02), suggesting that DHT treatment alters H2A.Z binding, but does not disrupt dynamics. Fos expression increased with Stimulation (F1,12 = 22.16, p = 0.001) irrespective of hormone treatment.

Figure 4-5. Effects of DHT and depolarization in primary hippocampal neurons. a) DHT treatment increased AR protein 24 hours after treatment in primary hippocampal neuronal culture, b) Top row depicts H2A.Z binding levels as normalized to vehicle- treated controls (depicted by the dashed line). Bottom row depicts gene expression as relative enrichment in comparison to vehicle-treated controls (dashed line). Fkbp5 = FK506 binding protein 51, B2m = beta-2-microglobulin, Th = tyrosine hydroxylase, Fos = c-fos proto oncogene immediate early gene. Dashed line = Vehicle controls with no hormone or KCl treatment (everything is normalized to this), * (star) = significantly different than dashed control, & = main effect of depolarization/stimulation, # = main effect of hormone. p≤0.05

4.4 Discussion

Activity-induced H2A.Z removal is emerging as an important step in memory formation, suggesting that H2A.Z is a negative regulator of fear memory (Stefanelli, Azam, et al., 2018; Zovkic et al., 2014). In the present study, we found deleting both H2A.Z-encoding genes in CamKIIα neurons enhances fear memory recall, suggesting that mature neurons tolerate the loss of both H2A.Z-encoding genes and that such loss has a positive effect on fear memory. Interestingly, H2A.Z deletion does not seem to affect extinction in males. Specifically, we see that, with every exposure to the context, males freeze less over time, expressing extinction. More specifically, when looking at the first minute, we see a drastic drop in freezing from days 2 to 3, indicating that this is likely the timepoint at which the greatest learning of extinction occurs, consistent with results from other labs (Lacagnina et al., 2019).

Second, we show that H2A.Z KO mice have reduced sensitivity to AR manipulations compared to controls, suggesting that AR and H2A.Z interact to regulate fear memory. Additionally, reducing AR activity through gonadectomy or treatment with an AR antagonist results in improved fear memory only in mice with intact H2A.Z levels, suggesting that AR regulates memory through actions on H2A.Z. Finally, we show that these effects on memory may in part be influenced by changes in DHT-induced H2A.Z binding, and a concomitant modification in expression of AR-regulated genes, hinting at changes in H2A.Z eviction. Collectively, these results provide evidence that AR and H2A.Z interact to regulate fear memory, potentially through regulation of gene transcription.

H2A.Z deletion enhances fear memory

Results from the present study extend our previous finding that H2A.Z depletion or the pharmacological inhibition of H2A.Z binding enhances fear memory (Narkaj et al., 2018; Stefanelli, Azam, et al., 2018; Zovkic et al., 2014) by demonstrating a similar effect when both H2A.Z-encoding genes are deleted in CamKIIα neurons throughout the brain. This finding further supports the role of H2A.Z as a negative modulator of memory as has been previously established in our lab. Importantly, by deleting both H2A.Z-encoding genes, we show that deletion of both genes in select neurons throughout the brain and during adulthood does not result in an obvious negative outcome for mice, indicating that mature rodents are able to tolerate H2A.Z deletion. Indeed, this finding is particularly intriguing because H2A.Z-1 and H2A.Z-2

72 have distinct effects during development (Faast et al., 2001) and regulate distinct transcriptional profiles in response to neuronal activity in cultured cortical cells (Dunn et al., 2017), suggesting that they may have distinct effects on behaviour. However, our data suggest that the loss of total H2A.Z has similar effects as region-specific depletion of only H2afz on memory, such that the loss of H2afv does not disrupt the memory-potentiating effect of H2afz loss. Given that both genes were deleted in this study, it is possible that loss of H2afz is masking potentially unique effects of H2afv on memory. Indeed, in the adult human brain, the relative abundance of total H2A.Z is 10-15% of all H2A (Weber & Henikoff, 2014) and H2AFZ (H2A.Z-1 transcript) is approximately 4 times more abundant than H2AFV (H2A.Z-2 transcript) (Dryhurst et al., 2009). As such, the memory-enhancing effects of co-deletion of both variants may reflect the dominant function of H2afz, but it nevertheless supports the hypothesis that the loss of total H2A.Z in CamKIIα neurons is well tolerated.

Effects of H2A.Z in the adult brain appear to be distinct from its effects in the developing brain. Using a similar floxed mouse line with the selective deletion of H2Afz driven by the Nestin promoter (expressed in neural and glial precursors throughout the nervous system (https://www.jax.org/strain/003771), rather than a subpopulation of neurons within the brain, as is the case with CamKIIα), Shen and colleagues (2018) showed that neural-specific H2A.Z-1 deletion around E16 impairs brain development and cognitive function in adult mice, establishing the importance of H2A.Z as a neurodevelopmental factor. Specifically, they show that embryonic H2A.Z-1 deletion results in impaired performance on measures of anxiety, motor behaviour, social interaction, depressive behaviours, and memory (Shen et al., 2018). Combined with the results of the current study, these data suggest that the role of H2A.Z changes during maturation, such that its loss is selectively detrimental during early stages of development. While the timing of such a switch is not known, our data suggest that H2A.Z levels accumulate with age (Stefanelli, Azam, et al., 2018), such that its function in behaviour may be plastic over time.

Effects of AR on behavior depend on H2A.Z

Blocking AR activity selectively improved fear memory in mice with normal levels of H2A.Z which is consistent with our previous evidence that AR is a negative regulator of fear memory (Ramzan et al., 2018). These effects were not evident in H2A.Z KO mice despite increased AR protein expression, suggesting that AR requires H2A.Z to exert effects on behaviour, a

73 hypothesis that was tested in Chapter 3. An alternative explanation is that too much or too little AR protein may have adverse effects. Specifically, in earlier studies using this mouse model of AR overexpression, we have seen reduced masculinization of aggressive behaviours and olfactory preference in male mice (Swift-Gallant, Coome, Ramzan, et al., 2016; Swift-Gallant, Coome, Srinivasan, et al., 2016). These results suggest a substantial overexpression of the AR protein may result in impaired AR function, in the shape of an inverted U-shape effect, and improved memory when there is an unextreme increase in AR protein (as in the H2A.Z KO mice). However, considering we did not see any AR-mediated changes in H2A.Z KO mice, it is more likely that AR affects fear memory through interactions with H2A.Z and, when H2A.Z is removed, it is unable to mediate AR’s effects on fear memory.

In comparison to the previous study in which we used a ubiquitous model of AR overexpression from early development, using a conditional inducible H2A.Z knockout mouse model allowed us to induce the knockout in adult mice. This allowed us to complement the previous study by examining effects after the major organizational periods have passed. Thus, effects of hormones seen are more likely to reflect activational effects. This is further supported by gonadectomy resulting in improved fear memory in NoCRE controls but not in H2A.Z KO mice, indicating that activational effects of androgens on fear memory in males are prevented by H2A.Z deletion.

H2A.Z and the AR may influence fear memory through common upstream regulators, such as the histone acetyltransferase Tip60 (Sapountzi et al., 2006). Tip60 directly acetylates AR to promote its effects on transcription (Gaughan et al., 2001, 2002) and also affects H2A.Z function through its acetylation and by contributing to its deposition into the nucleosome (Choi et al., 2009; Gaughan et al., 2002; Halkidou et al., 2003). In prostate cancer, androgen withdrawal upregulates Tip60 and results in its accumulation within the nucleus, whereas androgen withdrawal downregulates Tip60 in both AR-expressing and AR-non-expressing prostate cancer cell lines (Halkidou et al., 2003), suggesting that androgens may affecting Tip60 levels through both AR-dependent and independent mechanisms. As Tip60 is a coactivator for class 1 hormone receptors, which includes not only AR but also estrogen- (ERs) and progesterone- (PRs) receptors (Gaughan et al., 2001), these results indicate a potential disruption in nuclear receptor function.

Specifically, we found that promoters of AR-regulated genes (specifically Th and B2m) as well as the IEG Fos showed increased H2A.Z eviction in response to stimulus-induced depolarization. This is consistent with previous findings of increased H2A.Z eviction at gene promoters in response to a learning event (Stefanelli, Azam, et al., 2018). Interestingly, H2A.Z is also evicted at the B2m gene promoter in response to DHT treatment or AR activation. This is consistent with findings from Slupianek and colleagues (2010) in which DHT treatment resulted in reduced H2A.Z binding at the PSA promoter (Slupianek et al., 2010). However, the other two AR- regulated genes studied, Fkbp5 and Th, do not show an androgen-induced eviction of H2A.Z from their promoter region. In fact, Fkbp5 exhibits increased H2A.Z binding in response to DHT treatment, while Th exhibits equivalent amounts of H2A.Z binding in DMSO-only and DHT- only treated groups. This is in contrast with findings regarding H2A.Z-mediated changes in AR- regulated gene transcription. However, considering this is the first study to investigate the effect of H2A.Z binding on these genes in response to both AR activation and neuronal stimulation in neuronal cells, it is likely that these genes are regulated differently in neurons than in prostate cancer cells. Moreover, H2A.Z binding at the Th, B2m, and Fkbp5 genes have not previously been investigated, particularly in response to AR activation. As a result, it is highly possible that these genes are not as clearly regulated through AR and H2A.Z interaction as the PSA gene is.

Intriguingly, the majority of the genes studied exhibit gene expression corresponding to their H2A.Z binding profile. The one exception is B2m, which not only overall exhibits reduced H2A.Z binding in comparison to the DMSO-only group, but also reduced gene expression. The other genes (Th, Fkbp5, and Fos), exhibit increased gene expression when H2A.Z binding is reduced, and decreased expression when H2A.Z binding is increased, as would be expected based on previous studies (Stefanelli, Azam, et al., 2018; Zovkic et al., 2014). B2m is an immune factor that has been shown to accumulate with age in the bloodstream and hippocampus and, further, is associated with impaired contextual fear conditioning in male mice (L. K. Smith et al., 2015). This suggests that B2m expression may be lower initially and may increase with age. Further, B2m expression may not be directly affected by H2A.Z eviction.

There are, however, potential confounds that may have minimal effects on the data — specifically, we cultured primary hippocampal neurons from a mixture of male and female embryos. The heterogeneity of primary hippocampal neuron cultures may contribute to potential confounds, particularly as a study on immortalized mouse hippocampal female-derived (E14)

75 and male-derived (E18) showed that the female-derived neurons showed AR expression while the male-derived cells did not (Gingerich et al., 2010). However, due to the heterogeneity of the cell culture, this effect may be diluted and, further, DHT treatment results in such a large magnitude of AR expression in comparison to DMSO-treated neurons that this effect may be washed out. On the other hand, we used neurobasal medium that included the estrogen mimic phenol red, suggesting that there may be an underlying but minimal activation of the estrogenic pathways. Given that H2A.Z also regulates ER function (Gévry et al., 2009), there is a possibility of interactions between the androgenic and estrogenic mechanisms. However, considering that we used the non-aromatizable androgen, DHT, in treating neurons, it is unlikely that the treatment itself may have interacted with estrogenic mechanisms, thus minimizing the potential effects of phenol red. Finally, we did not use charcoal-stripped horse serum. Thus, there may be other endogenous hormones that may have potentially affected the data. However, these effects are at the very least consistent between experiments. Further, as the experimental effects seen are highly robust and specific to stimulation or DHT treatment, it is unlikely that these variables caused a large enough effect to significantly affect the findings of this study directly.

4.5 Conclusions

Overall, the results suggest that H2A.Z variants may have distinct developmental roles such that deletion during early development is fatal, while deletion during adulthood is tolerated with an improvement in fear memory. Further, we show through androgenic manipulations that AR affects fear memory in male mice through interaction with histone variant H2A.Z. Indeed, these effects may occur through interaction of AR with H2A.Z, resulting in modulation of AR- regulated gene transcription, supported by the evidence in prostate cancer literature indicating their interaction to regulate AR-regulated gene transcription activation and repression (Draker et al., 2011; Gaughan et al., 2002).

Our findings lend some support to the model that AR activation through DHT results in coactivator mediated eviction of H2A.Z from the promoter regions of affected genes, alongside increased transcription of these genes. While not identical to findings in prostate cancer literature regarding the PSA gene, this is a novel finding within hippocampal neurons and sets the basis for

76 future studies investigating effects on gene expression and further mechanisms of AR’s function and effects on behaviour.

Considering this interaction between AR and H2A.Z in regulating fear memory and knowing that H2A.Z also similarly interact with the estrogen receptor, in the next chapter we sought to further elucidate the contributions of sex on H2A.Z’s effects on fear memory.

Chapter 5

H2A.Z has Sex-Specific Effects on Memory

5.1 Introduction

In the previous chapters, we showed that H2A.Z is a negative regulator of fear memory and that the androgen receptor (AR) produces effects on behaviour in an H2A.Z-dependent manner. Given that AR is a sex steroid receptor that is more highly expressed in the male than the female brain (Lu, McKenna, Cologer-Clifford, Nau, & Simon, 1998; Tsai, Taniguchi, Samoza, & Ridder, 2015), this interaction generated the hypothesis that effects of H2A.Z on memory may be sex-specific. Indeed, H2A.Z interacts with ERα, and ERα-regulated genes (Gévry et al., 2009) and estrogens and ERs have a well-established role in memory (Frick et al., 2017), leading us to compare fear memory between male and female rodents.

There are known sex differences in fear conditioning behaviour. Although both sexes exhibit learning, male rodents tend to freeze more than females during recall and extinction trials (Chang et al., 2009; R. R. Gupta, Sen, Diepenhorst, Rudick, & Maren, 2001; Maren, De Oca, & Fanselow, 1994; Pryce, Lehmann, & Feldon, 1999). This observation contrasts the increased susceptibility of women to fear-related disorders, including anxiety and post-traumatic stress disorder (PTSD) (Haskell et al., 2010). There are also reports of sex differences in the experience of PTSD symptoms, whereby men and women report different and even opposing symptoms regarding emotional distress and distractibility. For example, among U.S. military veteran men and women with the same overall post-traumatic stress severity, women tended to report more frequent concentration difficulties and distress from reminders, while men tended to report more frequent nightmares, emotional numbing, and hypervigilance (M. W. King, Street, Gradus, Vogt, & Resick, 2013). Some of the variations in the clinical population, however, may also be due to different types of trauma suffered by men versus women, and, further, a difference in susceptibility to stress symptoms in response to specific types of trauma. For example, more female than male victims of violent assault experienced a pervasive disturbance, and victims of

78 violent assault are also more likely to report more severe distress than victims of non-assaultive violence (Chung & Breslau, 2008).

Stress-enhanced fear learning (SEFL) is a rodent model of acute stress that mimics several symptoms of PTSD. It utilizes a modification of the contextual fear conditioning paradigm to study the effect of an intense traumatic experience (multiple footshocks) on fear memory in a distinct context with a milder exposure to a similar experience (one footshock) in a different context (Perusini et al., 2016; Rajbhandari, Gonzalez, & Fanselow, 2018; Rau, DeCola, & Fanselow, 2005). This task models the exaggerated reaction to a new mild stressor or reminders of trauma in PTSD patients, whereby the exaggerated fear response to the novel context is more appropriate to the original traumatic context than the novel one. The effect of the multiple-shock stressor is nonassociative as it occurs in a novel context and only after the rodent received a milder form of the stressor (1 shock). Sensitization is a nonassociative process whereby there is increased reactivity to a potent stimulus after repeated exposure to the stimulus, producing a lowered activation threshold for subsequent stimulation. This effect may be similar to that in PTSD patients in whom exposure to a traumatic event causes a sensitized reaction to less intense but qualitatively similar stressors (Dykman, Ackerman, & Newton, 1997).

Another key feature of PTSD is that the potentiated fear reaction persists for a much longer time frame than contextual fear conditioning alone. According to the DSM-V, symptoms must be present for a minimum of 30 days for a PTSD diagnosis. SEFL is a similarly long-lasting phenomenon. In fact, in SEFL, rats have been observed to maintain the enhanced fear reaction to the novel context up to 90 days after the initial exposure to multiple shocks (Rau & Fanselow, 2009). Additionally, eliminating fear of the traumatic context after exposure to multiple shocks (i.e. eliminating freezing in the traumatic context through extinction) does not diminish the enhanced fear learning in response to a mild stressor in the novel context (Rau et al., 2005). This suggests that extinguishing fear of trauma-related events/contexts may not reduce the nonassociative effects of the trauma, and may correspond to the reduced effectiveness of extinction in treating PTSD (Peri, Ben-Shakhar, Orr, & Shalev, 2000).

This is a robust model of PTSD, as the Fanselow group has observed that 90% of rats develop the PTSD phenotype (reviewed in Perusini et al., 2016). Thus, we chose to use this model as it extends findings from the fear conditioning paradigm, which we have used thus far, to include

79 the instance of a much stronger stressor. Here, we explore the hypothesis that H2A.Z deletion has sex-specific effects on fear memory and, further, on stress-enhanced fear learning. Given that H2A.Z is a negative modulator of fear memory in male mice, we hypothesized that male H2A.Z KO mice would show an enhanced PTSD-like phenotype on the SEFL test.

5.2 Methods

5.2.1 Animals

Male and female C57Bl/6 mice bred in our colony were group-housed after weaning and assigned to testing groups at 60–90 days of age. Mice were housed in ventilated cages (5 x 7 x 14 in.) in a closed-circuit atmosphere rack (Techniplast Easy Flow #BOX110EFUL) and maintained on a 12 h light cycle, with ad libitum access to standard mouse chow (Harlan Teklad, Madison, WI) and water. Transgenic C57Bl/6 mice containing both Z1(H2afz)-flox and Z2(H2afv)-flox transgenes were purchased from Riken (Riken RBRC # 05765). They were crossed with a tamoxifen-inducible CamkIIα-cre/ERT2 mouse line (The Jackson Laboratory Stock # 012362) to produce mice with both floxed H2A.Z genes and either Cre (H2A.Z KO) or NoCRE littermate controls.

5.2.2 Genotyping

Genotyping was carried out at weaning for all mice used in the experiments. Animals were ear notched and DNA was extracted using the “HotSHOT” method (Truett et al., 2000). The cre transgene was identified by amplifying a unique portion within the cre transgene. The individually floxed genes (Z1 and Z2) were identified by amplifying the Z1 and Z2 genes, such that the floxed genes produced a larger PCR product (see Table 5-1 for a list of genotyping primers and expected band PCR product size). In addition, we used qPCR to confirm the reduction in mRNA of both Z1 and Z2 in the mPFC region of each mouse included in the experiments.

Table 5-1. DNA primers use for genotyping H2A.Z KO mice. PCR Product Gene Forward Reverse Size Jax Internal Control 5'-CTAGGCCACAGAATTGAAAGATCT 5'-GTAGGTGGAAATTCTAGCATCATCC 324 bp Cre 5'-AGCTCGTCAATCAAGCTGGT 5'-CAGGTTCTTGCGAACCTCAT 184 bp 326 bp (WT) H2A.Z1 5'-CGCCTTGGTAATTCTATCTTCTCC 5'-CGCCAGTTAACACACATGTGATC 448 bp (flox) 570 bp (WT) H2A.Z2 5'-GCCTCAGATCATCCAGTC 5'-GGCTCTGAATTCCCAATGTAG 700 bp (flox)

5.2.3 Tamoxifen

5.2.4 Stress-Enhanced Fear Learning (SEFL)

The mouse version of the SEFL paradigm was adapted from Rajbhandari et al. (2018). The chambers and electric shock boxes were purchased from Coulbourn Instruments (Holliston, MA, USA) and the chambers were modified to create 2 distinct training and testing contexts. Context A was differentiated by the following cues: the chamber was wiped down and scented with 1% acetic acid and was illuminated by white light. In addition, green plexiglass inserts were placed within the chamber such that the colour of the chamber had a green hue and a triangular shape (17.5 cm x 17.5 cm x 23 cm diagonal), and the furniture within the testing room was placed in a triangular format. Context B was illuminated by a red light and cleaned and scented with 5% ammonium hydroxide (Sigma# 221228-500ML-A), the furniture was rearranged along the walls, and no inserts were used such that the chamber was in an original manufacturer shape (9.8 inch boxes; designed for mice).

Mice were handled in the testing room for 3 days before testing. On each day, they were transported to the room in their home cages and allowed to habituate to the hallway for 30 min. They were then transported to the room in their home cages where they were handled for 30-60

81 sec each. On day 1, all testing was conducted in Context A. Mice were placed in different cages (7.5×11.5×5 in) and transported on a metal cart to a room (also illuminated with white light) across from the testing room and allowed to habituate for 30 min. They were then placed into individual black wooden boxes (8.5 x 8.5 x 8.5 in.) and transported to the testing room, similar to context A in Rau et al. (2005). These procedures were done to reduce chances of generalization between the two contexts. They were then placed in the chamber and exposed to 10 randomly- spaced 1 mA 1 sec shocks over a 1 hr period to mimic a traumatic experience. All mice were then returned to their animal holding room and home cages.

On day 2, all mice were trained with a mild fear conditioning protocol in Context B (Train Day). Mice remained in their home-cages and were transported on a plastic cart to the hallway outside of the testing room and allowed to habituate for 30 min. Mice were then transported to the testing room two at a time and placed in Context B for 2 min of exploration, followed by a single foot shock (0.5mA, 2 sec), then another minute of exploration. Memory for context B was tested on days 3 and 9 (24 Hr and 7 Day Recall, respectively), wherein mice underwent the same procedures as during the Context B Training day but were exposed to Context B for 4 minutes without shock. In all cases, percent freezing was scored by automated software (FreezeFrame, Coubourn Instruments).

5.2.5 Elevated Plus Maze (EPM)

Mice were transported to the room in their home cages and were allowed to habituate to the room for 30 min. They were then placed in the EPM apparatus (arm width 5cm, arm length from the middle to end: 35cm, closed arm wall height: 15.25cm, elevation from floor: 63cm) always facing one of the closed arms and allowed to explore for 10 min. Video was recorded by a Sony HD HandyCam (HDR-CX405) and scored manually by an experimenter blind to the experimental conditions and sex of the animals. Number of entries into the closed and open arms, as well as time spent in closed vs open arms were scored. Light levels in the open arms were recorded to be 162 lux and in the closed arm 141 lux (Light Meter (LuxMaster 11010067)).

5.2.6 Open Field (OF)

To acclimatize mice to transportation and handling, animals were transported to the behavior testing room in their home cages daily for 3 days before testing, where they were allowed to

82 habituate to the room for 30 min, then handled by the experimenter for 30-60s each. On the day of open field testing, mice were transported to the room in their home cages and allowed to habituate to the room for 30 min before testing. They were then placed in the testing chamber (45cm x 45cm x 45cm) and allowed to explore for 10 min. They were then placed back into their home cages. Video was recorded using a camera (Microsoft LifeCam Studio) and scored using EthoVision XT 8.5. Time spent in the outer region along the wall (10cm from the wall) and in the centre (25cm x 25cm) was scored using EthoVision XT 8.5 (Noldus Information Technology, Wageningen, The Netherlands). Light levels were recorded to be 10.4 lux at the bottom of the chamber.

5.2.7 Tissue Collection

For RNA tissue analysis, mice were cervically dislocated and decapitated. Animal brains were immediately collected and snap frozen in ice-cold isopentane and stored at −80 °C until processing.

5.2.8 mRNA Expression and RT-PCR

Mice were euthanized by cervical dislocation at least 2 weeks after the final training session and mPFC was dissected. RNA was extracted using BioBasic RNA Extraction Kit (BioBasic #BS82322-250, Amherst, NY). Complementary DNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied BioSystems #4368814, Folster, CA, USA). Primers were designed in the lab and used to detect levels of the indicated transcripts, and data were normalized to Actin and Gapdh. Comparison of group differences was conducted using relative enrichment analysis, whereby data for experimental animals were normalized to controls using the following formula: 2^−(ΔΔCT). The list of primer sequences is provided in Table 5-2.

Table 5-2. cDNA Primers used to confirm H2A.Z knockout. Melting Gene Forward Reverse Temperature (ᣞC) Actin 5'-AGATCAAGATCATTGCTCCTCCT 5'-ACGCAGCTCAGTAACAGTCC 58 Gapdh 5'-GTGGAGTCATACTGGAACATGTAG 5-AATGGTGAAGGTCGGTGTG 60 H2afz 5-CACCGCAGAGGTACTTGAGTT 5-TCCTTTCTTCCCGATCAGCG 58 H2afv 5'-CAAGGCTAAGGCGGTGTCTC 5'-CTGCTAACTCCAACACCTCAGC 58

5.2.9 Statistics

Analyses were conducted with SPSS Version 24 and consisted of independent-samples t-test for comparison of Genotype (NoCRE, H2A.Z KO). For studies with 2 independent variables, we used a two-way ANOVA with Genotype (NoCRE, H2A.Z KO) and Sex (Male, Female) as independent variables. To compare freezing before and after shock during the training session, we used a mixed-measures ANOVA, with Genotype and Treatment as the between group factors and Shock (before shock; after shock) as the within-group factor. Follow-up analyses were conducted using independent-samples t-test or paired-samples t-test only when the omnibus test was significant, thus precluding the need to correct for multiple comparisons.

5.3 Results

5.3.1 Fear memory is unaffected in females with H2A.Z deletion

We previously showed that conditional-inducible H2A.Z deletion results in enhanced fear memory in male mice. To test within session learning, we compared freezing before and after shock exposure during the training session (Fig. 5-1b). A Shock (before, after) x Genotype (NoCRE, H2A.Z KO) ANOVA revealed a main effect of Shock (F(1,13) = 11.196, p=0.005) as well as a main effect of genotype (F(1,13) = 8.892, p=0.011), such that all females showed increased freezing after shock, but H2A.Z KO females froze less overall (t(13) = 3.199, p=0.007). During the 24 hr recall test, there was no difference in fear memory between H2A.Z KO and NoCRE females (t(13) = 0.066, p=0.946) (Fig. 5-1c), suggesting that H2A.Z may not have equivalent roles in fear memory in male and female rodents.

Since Tamoxifen is a selective estrogen receptor modulator (SERM) with potentially enhancing effects in the brain and since estradiol has memory potentiating effects in both females and males, it is possible that tamoxifen treatment in both groups masked effects of H2A.Z deletion. To test this possibility, H2A.Z-Cre female mice were injected with either tamoxifen or vehicle (corn oil) as a control (Fig. 5-1d). During training session, a Shock (before, after) x Treatment (oil, tamoxifen) ANOVA revealed a main effect of Shock (F(1,10) = 15.962, p=0.003) and an approaching significant effect of treatment (F(1,10) = 4.607, p=0.057) (Fig. 5-1e). Memory recall was tested after 24 h or 7 days. The effect of Treatment (oil, tamoxifen) was not significant

84 on either day (Fig. 5-1f), suggesting that tamoxifen treatment does not seem to affect fear memory in female mice.

Figure 5-1. H2A.Z KO does not affect fear memory in female mice. a) Timeline of fear conditioning trials in tamoxifen-treated H2A.Z KO and NoCRE females, b) percent freezing during the training session, c) percent freezing during the 24 hr recall. d) Timeline of fear conditioning test in H2A.Z KO females treated with vehicle or tamoxifen, e) percent freezing during the training session, f) percent freezing during the 24 hr and 7 day recall sessions. *p≤0.05

5.3.2 H2A.Z deletion has a protective effect on stress-enhanced fear in female mice

Since there are sex difference and potential endocrine effects on fear memory (as in posttraumatic stress disorder), both males and females of both genotypes were tested for stress- enhanced fear learning. The two sexes were separately analyzed statistically since they were tested in separate cohorts. A mixed measures ANOVA of shock (before, after) x genotype (NoCRE, H2A.Z KO) on training day in context B revealed a main effect of Shock in males (F(1,22) = 47.911, p=0.000; Fig 5-2b) and females (F(1,20) = 46.199, p=0.000; Fig. 6-3d), suggesting that both sexes showed within-session learning for training in context B.

A Recall Day (24 hr, 7 Day) x Genotype (H2A.Z KO, NoCRE) ANOVA revealed a only a main effect of Recall Day in males (F(1,16) = 2.933, p=0.000; Fig. 5-2e), whereas the same analysis in females revealed both a main effect of Recall Day (F(1,20) = 9.319, p=0.006) and Genotype (F(1,20) = 4.635, p=0.044) (Fig. 5-2c). Specifically, both males and females showed reduced freezing at 7-day recall compared to 24 hr recall, suggesting extinction over time. Follow-up analyses with a one-way ANOVA in females revealed that, at 24 hr recall, H2A.Z KO females froze less than NoCRE females (F(1,20) = 5.461, p=0.030), while this difference disappeared by 7 days (1.418, p=0.248). This suggests that conditional-inducible H2A.Z KO does not mediate stress-enhanced fear learning, whereas H2A.Z deletion may serve as a protective factor in females at 24 hr recall.

Figure 5-2. Stress-enhanced fear learning is affected sex-specifically by H2A.Z deletion. a) Timeline of stress-enhanced fear learning, b) females percent freezing during the context B training session and c) percent freezing at 24 hr and 7 day recall in context B. Males d) percent freezing during context B training session and c) percent freezing at 24 hr and 7 day recall. *p≤0.05

5.3.3 Measures of anxiety are unaffected by H2A.Z deletion

To ensure that effects on memory, particularly fear memory, are not affected by changes in anxiety or motor behaviour, we measured behaviour on the elevated plus maze (EPM) and open field (OF), both of which are commonly used tests for anxiety that also include indices of motor behaviour.

In the OF, a Sex (male, female) x Genotype (H2A.Z KO, NoCRE) ANOVA on time spent in the outside region (10 cm from the wall) and on distance traveled revealed no significant main effects or interaction (Fig. 5-3b). indicating that neither factor influences basal anxiety on this measure (Fig. 5-3c).

On the EPM, there were no effects of Sex or Genotype on amount of time spent in the open or closed arms, suggesting a lack of genotype or sex effects on this particular measure of anxiety (Fig. 5-3e). Number of entries into both open or closed arms is a measure of motor activity. Similar to distance traveled in the OF, a two-way ANOVA revealed no significant main effects of sex or genotype, or interaction between the two factors. This provides further evidence for the idea that H2A.Z deletion does not affect fear memory through changes in anxiety or motor behaviour (Fig. 5-3f).

Figure 5-3. Measures of anxiety are unaffected by H2A.Z deletion. a) Open Field apparatus schematic, b) time spent in the outside region by male and female mice, c) total distance traveled by male and female mice. d) Elevated Plus Maze apparatus schematic, e) time spent in open and closed arms by male and female mice, and f) number of entries into open and closed arms by male and female mice.

5.4 Discussion

In this study, we find numerous instances of sex differences in H2A.Z’s effects on memory, most notably that in contrast to males, H2A.Z deletion does not enhance fear memory in females. Sex differences in fear memory are not caused by confounding effects of tamoxifen treatment on ER activity in females (Baez-Jurado et al., 2019), as tamoxifen-treated females do not behave differently from oil-treated females. Tamoxifen is a non-steroidal analog of estradiol and an SERM that has about 100- and 1000-times higher affinity for ER than estrogens and can cross the blood brain barrier. It has ERα antagonistic effects in breast tissue (hence its use in breast cancer treatment), while having agonistic effects in the reproductive system and even enhances the risk of developing endometrial cancer (Hu, Hilakivi-Clarke, & Clarke, 2015). It binds the ERα, inducing a conformational change that prevents the receptor from binding with its coactivators, thus preventing genomic effects in breast cancer (Gévry et al., 2009), and thus potentially acting through non-genomic pathways and the GPER to affect endometrial cancer (Hu et al., 2015). Tamoxifen’s effects in the nervous system are less well-defined; however, evidence indicates that it has significant effects on neuronal structure and function. Specifically, it has neuroprotective effects in hippocampal neurons (Mosquera et al., 2014; Sharma & Mehra, 2008), promotes axonal outgrowth (Tian et al., 2009) and neuronal survival (Y. Zhang et al., 2005), and further improves the cholinergic system enhancing memory (Newhouse et al., 2013). However, given that effects of H2A.Z deletion were not found in oil-treated or tamoxifen treated mice, we conclude that H2A.Z deletion has sex-specific effects on fear memory.

The basis for sex-specific effects of H2A.Z deletion on memory is not clear. Based on our previous findings (Chapter 3 and 4), the androgen receptor regulates H2A.Z dynamics in males, suggesting that genotype effects in males are in part driven by effects of AR. Although we have not investigated the effects of androgenic manipulations on fear memory in H2A.Z KO females, there is some evidence to suggest that AR may affect hippocampal-dependent object location memory in females. Specifically, transgenic females overexpressing AR showed impairment in novel location recognition when irradiated, while wild-type controls with and without irradiation and transgenic mice without radiation were successful in this task. This suggests that, while AR overexpression did not impair baseline novel location memory in female mice, upon treatment with a biological stressor such as irradiation, it inhibits memory (Acevedo, Tittle, & Raber, 2008). On the other hand, spatial memory as tested by the MWM was improved in AR transgenic

90 females in comparison to wild-type females, suggesting that AR may improve spatial memory in females. Thus, AR likely plays a complex role in female hippocampal-dependent memory regulation (Acevedo et al., 2008). This connection, however, is largely understudied and firm conclusions are impossible to draw.

Additionally, H2A.Z’s effects on memory in females may occur through a different mechanism than in males. Specifically, previous data suggest that H2A.Z also regulates estrogen receptor signaling in non-neuronal cells (Gevry et al 2009). Similar to AR, H2A.Z is actively recruited to the proximal promoter of various genes upon cellular treatment with estradiol and, further, H2A.Z associates to ERα-responsive enhancers and is required for association of certain transcriptional factors (Gévry et al., 2009), suggesting that H2A.Z influences ERα function in non-neuronal tissue. As a result, it is likely that one of the ways that H2A.Z deletion has differing effects on fear memory in males and females is through differential effects on the transcriptional activity of the AR and ERs, though at this point the literature investigating the interactions between H2A.Z and the hormone receptors is limited, and non-existent when considered with memory. There is, however evidence of estradiol’s interactions with histone acetylation and deacetylation in influencing novel object recognition (Zhao, Fan, Fortress, Boulware, & Frick, 2012; Zhao, Fan, & Frick, 2010), as well as with DNA methylation in affecting memory (Fan et al., 2010; Fernandez et al., 2008). Briefly, estradiol induces an increase in H3 acetylation in the dorsal hippocampus in females, and this is necessary for estradiol- dependent enhancement of NOR memory consolidation. Additionally, the increase of DNA methylation may be essential for estradiol’s enhancing effect on memory consolidation on the NOR task (reviewed in Frick, 2013). However, comparable levels of the three estrogen receptors in the hippocampus of male and female rats (Hutson et al., 2019) suggest that ERs may not be the source of sex differences in the acute fear conditioning paradigm. Indeed, only a high dose of EB replacement resulted in potentiated fear memory in female mice (Y. K. Matsumoto, Kasai, & Tomihara, 2018), suggesting a minimal impact of normal doses of estrogen on acute training.

In contrast to acute fear memory, we found that H2A.Z deletion resulted in reduced susceptibility of female mice to fear conditioning in a new context after previous shock exposure in the stress- enhanced fear learning protocol, suggesting that loss of H2A.Z may be protective against PTSD- like symptoms in females, but not males. Both males and females showed reduced freezing after re-exposure to the context 7 days after the initial test, indicating that extinction of the enhanced

91 fear memory occurs regardless of sex and H2A.Z status. Consistent with our findings in NoCRE mice, other labs also did not report sex differences in SEFL (Poulos, Zhuravka, Long, Gannam, & Fanselow, 2015). Other animal models of PTSD study fear learning in previously stressed rodents, by using the following as previous stressors: physical stressors, social stressors, and psychological stressors (Whitaker, Gilpin, & Edwards, 2014), all of which disrupt fear extinction in male rodents, consistent with the idea of impaired extinction in PTSD patients being due at least in part to trauma exposure (reviewed in Shansky, 2015). More specifically, in eyeblink conditioning studies, prior exposure to tail shock stress produces different effects in males and females. Specifically, males show an increased conditioned response after stress exposure, while females show estradiol-dependent reduction in responding (Wood & Shors, 1998). This is similar to our finding in which H2A.Z deletion reduced female responding to a mild stressor (in comparison to NoCRE controls).

However, given that H2A.Z had distinct effects on initial vs. stress-enhanced fear learning in males and females, our data suggest that distinct mechanisms influence initial fear learning (i.e., without SEFL) and the consolidation of fear learning after SEFL; and that H2A.Z selectively inhibits SEFL in females. In the human population, the majority of people experience at least one traumatic event in their lifetime, yet the prevalence of PTSD is only ~7% (Kessler, Chiu, Demler, & Walters, 2005). This suggests that protective factors, whether environmental or molecular, may offer resilience to the majority of people, while other factors may confer vulnerability in developing PTSD. Considering our findings, H2A.Z deletion may confer protective effects specifically in females against developing PTSD symptoms. Furthermore, there is evidence the intergenerational epigenetic effects, such as maternal stress and early-life adversity, may lead to altered stress responses later in life (Daskalakis, Bagot, Parker, Vinkers, & de Kloet, 2013; Vaiserman, 2015; Yehuda et al., 2015). Although researchers are only beginning to identify neuroepigenetic factors involved in SEFL, a few studies have done so. Specifically, SPS (stress-potentiated startle) before fear conditioning has a similar effect to the Fanselow SEFL paradigm. A study utilizing this technique found that the delayed extinction profile can be rescued using HDAC inhibitor, implicating a role for histone acetylation in maintaining stress- potentiated learning (Y. Matsumoto et al., 2013).

DNA methylation can also contribute to the PTSD symptoms in SEFL. Specifically, Toda and colleagues (2014) found that a SEFL model involving maternal separation was associated with

92 increased DNA methylation at the Fkbp5 gene, which is a critical regulator of the glucocorticoid receptor (GR) and, thus, affects the sensitivity of the hypothalamic pituitary axis to mediate stress responses. This directly translates to the clinical population as a reduction in FKBP5 levels have been observed in PTSD patients (Yehuda et al., 2009) and changes in its methylation status have also been reported in Holocaust survivors and their offspring (Yehuda et al., 2016).

While currently understudied, epigenetic mechanisms have a great potential in understanding and explaining perseverant effects of stress. Since they are modifications on the genome without altering the underlying DNA, are the primary modulator of environmental effects on gene transcription and, moreover, can be long-lasting and passed along from mother to offspring, they are a prime candidate for understanding the perseverant nature of stress-enhanced memories in PTSD patients as well as intergenerational effects of traumatic experiences.

Given that ERβ facilitates fear memory in females (Zeidan et al., 2011), it is possible that the loss of H2A.Z may interact similarly with ERβ to potentially affect female susceptibility to stress-enhancement of fear learning.

Finally, to account for any potential changes in anxiety or motor behaviour induced by H2A.Z deletion, we performed the EPM and OF tests. Here, we found that H2A.Z deletion did not affect both anxiety and motor activity measures on both tests in both males and females. Thus, contributions of these factors can be excluded from the results we see.

5.5 Conclusions

Overall, the results of this study indicate that H2A.Z has differential effects on fear memory in males and females. Further, it inhibits enhanced fear learning in response to an acute traumatic stressor in females but may not be affecting stress-enhanced fear learning in males through a similar mechanism. These are important findings that indicate the interaction of sex with epigenetic mechanisms in affecting memory. Specifically, basic mechanisms of epigenetics and gene transcription may differ according to sex due to difference in hormone receptors, among other factors. Further, fundamental mechanisms may differ between males and females and result in different phenotypes in the two sexes.

Chapter 6

Unifying Conclusions and Discussion

6.1 Concluding Summary

Memory is a key part of adapting to our environment, especially in relation to avoiding environments that have produced negative outcomes. However, in anxiety- and fear-associated disorders, such as PTSD, fear memory has gone awry due to factors such as biological sex, hormones, and epigenetics. Years of research have revealed sex differences in memory formation and recall (Cherrier et al., 2001; Dalla & Shors, 2009; Janowsky, 2006; Maddox et al., 2018; Mueller et al., 2016; Newhouse et al., 2013; Sherwin, 2005) and the underlying effects of sex steroid hormones have been elucidated, with studies largely focusing on the contributions of estradiol in females (Frick, 2015; Frick et al., 2017; Sherwin, 2005). Finally, study of the effects of epigenetics on memory formation, consolidation, and recall have recently come to the forefront, with a major focus on DNA methylation and more recent interest in histone dynamics (J. J. Day & Sweatt, 2011; S. Gupta et al., 2010; Jarome et al., 2014; Miller, Campbell, & Sweatt, 2008; Miller & Sweatt, 2007). However, the combinatorial effects of these three factors on memory have not been extensively studied. Relatively recent work from the Frick Lab have, however, begun to scratch the surface of these interactions in relation to novel object recognition memory in females, and more specifically in relation to estradiol and histone H3 acetylation (Fortress & Frick, 2014; Frick, 2013). In my thesis, I aimed to add to the literature by investigating the interactions of androgenic mechanisms with neuroepigenetics in the context of fear memory in male mice. A further goal was to investigate the interaction of neuroepigenetics on sex-specific effects on fear memory.

In my work, I reveal the contributions of androgenic mechanisms, acting particularly through the androgen receptor, on fear memory in male mice. Specifically, I show that AR overexpression results in a ligand- and AR-activity dependent impairment in fear memory as well as a corresponding increase in H2A.Z gene expression in area CA1, in addition to other gene expression changes. This implicates AR as a negative regulator of memory, perhaps through interactions with H2A.Z. Next, I show that the epigenetic regulator histone variant H2A.Z (a

94 negative modulator of memory) interacts with androgenic mechanisms to also affect fear memory. More precisely, brain-specific deletion of H2A.Z results in improved fear memory, which is unaffected by systemic androgenic manipulations, suggesting that knocking out H2A.Z prevents androgen depletion or AR blockade from further improving fear memory (and from DHT-treatment to impair fear memory in gonadectomized animals). To elucidate the mechanism of this interaction, I used cell culture and chromatin immunoprecipitation experiments to show that the two interact to regulate H2A.Z-chromatin binding at promoter regions of memory- related and AR-regulated genes within hippocampal neurons. I further explore the interaction of neuroepigenetics and sex on fear memory. Specifically, I show that the histone variant H2A.Z affects fear memory differentially dependent on sex, such that its deletion improves fear memory in males (without affecting extinction) but has no effect in females. Further, H2A.Z mediates stress-enhancing effects on fear learning differentially in males and females, such that its deletion does not affect males, but protects females from exhibiting an exaggerated fear response to a novel context. These data indicate that H2A.Z plays a role in maintaining baseline fear memory formation in males by suppressing enhanced fear memory formation. In females, on the other hand, rather than affecting baseline fear memory formation, H2A.Z plays more of a role in increasing rather than suppressing, fear learning in response to acute traumatic stress. These data indicate that H2A.Z plays sex-specific roles in affecting fear memory.

Overall, the findings of this thesis have very significant implications. Neuroepigenetics is an expanding field of research and only recently have interactions with memory been examined. As such, it is even more recent that interactions of neuroepigenetics with sex and hormones, specifically in regulating memory, have been investigated. Indeed, current findings, in conjunction with previous findings from our lab, implicate a histone variant in fear memory formation through regulation of gene transcription. Evidence throughout my dissertation points to the androgen receptor acting on and interacting with H2A.Z to affect gene transcription and, further, to affect fear memory in male mice. It is very likely that other transcription factors (such as progesterone receptor and estrogen receptors) interact with the epigenome in a similar way to affect memory and other behaviours. These findings implicate H2A.Z as a potential therapeutic target for aberrant fear learning, particularly in relation to AR in males, and PTSD in females.

6.2 Unifying Discussion

There are a few unifying themes throughout this thesis, particularly the study of androgenic hormones, neuroepigenetics, and sex in the context of fear memory. To outline the significance of these findings, this section will include a discussion of all three factors as well as a comparison with similar findings regarding estrogenic hormones, neuroepigenetics, and sex.

The finding of the interaction between AR and H2A.Z in affecting fear memory in male mice leads us to consider the potential effect of sex on H2A.Z’s effects on fear memory. Cancer studies have shown interactions between H2A.Z and both sex steroid hormone receptors, AR and ERα, indicating potentially different mechanisms of H2A.Z’s effects in males and females (Dryhurst & Ausió, 2014; Gévry et al., 2009; Peters et al., 2009). Seeing the sex-specific effects of H2A.Z on memory in Chapter 5 suggests H2A.Z may be a mediator between hormonal effects on memory and, more broadly, that steroid hormone receptors act upon the epigenome to cause their effects.

While the study of epigenetic contributions to sex differences is relatively recent, there are indications that hormones interact with the epigenome to affect sexual differentiation and sexual dimorphisms. In particular, hormones exert effects on the epigenome to affect sexual differentiation of neural structures as well as their susceptibility to response to hormones throughout the lifespan. The sexually dimorphic nucleus of the preoptic area (SDN-POA) is a classic sexually dimorphic structure located within the hypothalamus and both much larger in males than females and also has larger soma size in male than female rodents (Davis, Popper, & Gorski, 1996; Sickel & McCarthy, 2000). Recent studies show overall increased DNA methylation on the ERα promoter in the SDN-POA of 1-day old male pups in comparison to female controls (Kurian et al., 2010), while there is a more specific increase in DNA methylation on specific CpG sites of the ERα promoter in the SDN-POA of 1-2 day old female pups in comparison to male pups (McCarthy et al., 2009). This effect was reversed with estradiol treatment (which also results in masculinization of the female SDN-POA (Gilmore, Varnum, & Forger, 2012; Orikasa & Sakuma, 2010)), indicating a complex and potentially organizational role of hormones interacting with epigenetic mechanisms to affect sexual differentiation. Similarly, there are sex differences in epigenetic modifications in the principle nucleus of the bed nucleus of the stria terminalis (BNSTp). Specifically, the BNSTp is both larger in volume and

96 contains more cells in male than female mice. This sexual dimorphism is established by testosterone-induced rescue of cells in males during the early postnatal period. Evidence indicates that histone acetylation is critical in maintaining this sex difference by preventing cell death (i.e. preventing testosterone’s masculinization effect) in male mice (Murray, Hien, De Vries, & Forger, 2009). There is further evidence of sex differences in histone modifications in the hippocampus and cortex. Specifically, in comparison to neonatal females, neonatal male mice exhibit increased histone 3 lysine 9 trimethylation (H3K9me3) and acetylation on lysine residues 9 and 14 of histone 3 (H3K9/14Ac) in both the cortex and hippocampus (Tsai et al., 2009). While these studies did not asses the effects of sexually dimorphic epigenetic mechanisms on behaviour or memory, they provide an initial indication of the interaction between sex steroid receptors with epigenetic factors in affecting sexually dimorphic phenotypes.

Recent research has provided further support for the hypothesis of epigenetic factors acting as mediators for hormonal effects on memory. Studies were performed based on the basic finding of estradiol’s enhancing effect on novel object recognition in females (e.g. (Fernandez et al., 2008)). Building off this finding, they investigated the effects of estradiol or HDAC inhibitor (HDACi) on acetylation of histones H3 and H4 in the dorsal hippocampus and, following this, whether histone acetylation was necessary for estradiol’s memory-enhancing effects. They found, first, that acetylation of both H3 and H4 was increased with HDACi treatment, as expected. However, estradiol only increased acetylation of H3 but not H4 (Zhao et al., 2010) This was followed up with a study in which they inhibited histone acetylation in the dorsal hippocampus using a HAT inhibitor at a dose low enough to not alter memory by itself (Zhao et al., 2012). They found that estradiol was unable to facilitate novel object recognition memory when mice were infused with this low dose of HAT inhibitor immediately after training (Zhao et al., 2012). Consistent with their previous finding, this low dose also prevented estradiol-induced acetylation of H3 (Zhao et al., 2012), indicating that histone H3 acetylation in the dorsal hippocampus is essential for the memory-enhancing effects of estradiol in females and, further, estradiol likely specifically regulates H3-associated genes. These data provide evidence that the rapid non-genomic effects of estradiol interact with the epigenome to affect memory in females.

Moreover, estradiol treatment also reduced levels of HDAC2 protein in the dorsal hippocampus, consistent with previous findings of HDAC2 being a negative modulator of memory (Guan et al., 2009; Haettig et al., 2011; Zhao et al., 2012, 2010). These findings together suggest that estradiol

97 directly interacts with epigenetic mechanisms, specifically by increasing H3 acetylation and reducing HDAC2 levels, to mediate memory enhancement in female mice. These studies further found that inhibiting DNA methylation immediately after training also prevented estradiol’s memory enhancing effects (Zhao et al., 2010). Thus, estradiol likely acts on both DNA methylation and histone acetylation in the dorsal hippocampus to affect gene transcription in order to affect novel object recognition memory in females. Notably, these studies of estradiol’s interactions with epigenetic mechanisms in females are particularly challenging to do because most of the work elucidating the roles of epigenetics in learning and memory has been done in male mice. As a result, this requires a set of experiments first confirming the epigenetic effect in females, which is then followed by the study of estrogenic interactions.

Analogous to these findings, in my study, I find that AR overexpression increases H2A.Z levels in the CA1 and impairs fear memory and that H2A.Z deletion prevents AR-mediated changes in fear memory. Specifically, when H2A.Z is deleted from excitatory neurons within the brain, androgenic manipulations are unable to exert the effects on fear memory that are observed in NoCRE animals. Moreover, H2A.Z seems to have sex-specific effects in modulating fear memory and stress-induced enhancement of fear learning. In context with findings regarding estradiol and histone acetylation, these findings lend proof to the idea that hormones affect memory through intermediary interaction with epigenetic mechanisms and, importantly, these interactions may differ between males and females, whether it be the type of interaction or the degree to which that mechanism manifests within the individual sexes.

Studies of hormonal effects on memory, as mediated through epigenetic mechanisms provide much needed understanding of mechanisms underlying sex differences in normal cognitive processes and disease susceptibility. There are numerous fear and memory-related disorders that manifest sex-specifically within the clinical population. Disorders such as Alzheimer’s Dementia and PTSD exhibit a significantly higher incidence in women than men (Dalla & Shors, 2009) and the incidence of Alzheimer’s and other age-related diseases is correlated with declining hormone levels (Luine, 2014). Although age-related decline of hormones has been associated with Alzheimer’s, the causal link is unclear and as such, treatment with hormone replacement is not always successful or sufficient in preventing cognitive decline (Sherwin, 2005). However, by understanding the interaction between hormones and epigenetic mechanisms, we may be able to discover novel therapeutic targets that may be more precise targets to specifically maintain or

98 improve cognition rather than an overall hormone replacement therapy, which comes with its own set of side effects, specifically off-target effects as is the case with tamoxifen treatment for breast cancer simultaneously increasing the risk for endometrial cancer (Hu et al., 2015).

Similarly, PTSD is a disorder that is much more likely to manifest in women who have experienced a traumatic event as compared to men (Dalla & Shors, 2009). While this may not be wholly related to hormones as other factors come into play (e.g. type of trauma, the incidence of traumatic events), hormones play a contributary role. A Dutch study of male soldiers found that men with higher levels of testosterone before being deployed were less likely to report PTSD- like symptoms compared to men with lower pre-deployment levels of testosterone (Reijnen et al., 2015), indicating a potential protective role of testosterone against stress-potentiated fear memory formation. This is also supported by our findings of impaired fear memory in males with increased AR expression.

Overall, the findings of this thesis provide evidence of hormonal regulation of memory occurring through interactions with neuroepigenetic mechanisms. Previously, the interaction of AR and H2A.Z was only studied in prostate cancer in the context of finding therapeutic targets for prostate cancer treatment. We show that a similar mechanism of interaction may exist within hippocampal neurons and, further, that this interaction likely directly affects fear memory in male mice, perhaps through changes in memory-related and AR-regulated gene expression.

6.3 Future Directions

This study has not only resulted in a deeper understanding of androgenic contributions to fear memory through interacting with epigenetic factors but has also opened a new avenue of research and a plethora of questions that need to be further studied. While this study aimed to gain a greater understanding of the interactions between the androgen receptor and histone variant H2A.Z in fear memory, we did not study this interaction in females. For future studies, it would be pertinent to investigate the degree to which this interaction between AR and H2A.Z affect memory in female mice. This, in combination with a study of H2A.Z’s interactions with ERα (Gévry et al., 2009) on fear memory in both male mice and female mice, would provide a greater understanding of the extent to which the two sex steroid systems interact with histone variants to regulate memory in the two sexes. This would further the understanding of whether steroid hormone receptor and histone variant interactions are specific or not (i.e. answering questions such as whether AR only interacts with H2A.Z, and ER only interacts through histone acetylation).

Additionally, because our androgenic manipulations were always systemic (i.e. global AR overexpression, gonadectomy with systemic hormone replacement), conclusions regarding the hormonal manipulations cannot be targeted to the brain, or, more specifically, to memory-related structures such as the hippocampus. This makes it difficult to pinpoint a direct causal link between AR in the brain and behaviour, as it is possible for ARs outside of the nervous system to affect behaviour as well (Ramzan et al., 2015; Swift-Gallant, Coome, Ramzan, et al., 2016; Swift-Gallant, Coome, Srinivasan, et al., 2016). While this can be partially ramified through the brain-specific deletion of H2A.Z, future studies would benefit from targeting manipulations to the hippocampus, perhaps through stereotaxic surgery and intracerebral infusions of hormones or AR overexpression/deletion through viral vectors. Targeting the effects to specific brain regions will lead to more causal and direct links between the site of action and memory formation.

Pursuing the study of the interaction at a molecular level has allowed us to gain a deeper understanding of the interaction of AR and H2A.Z at the chromatin level in hippocampal neurons. Further, we were able to investigate the contributions of the genomic effects of AR in studying chromatin binding. Studying the contributions of non-genomic AR effects on fear memory and interactions would provide a more holistic understanding of AR’s effects within the

100 brain. This is supported by the contribution of ERs non-genomic cell signaling effects in influencing H3 acetylation (Zhao et al., 2010). AR has non-genomic effects through cell signal cascades by effects on protein kinases including MAPK (ERK), protein kinase C (PKC), protein kinase A (PKA), and Akt (summarised in Bennett et al., 2010). These effects are rapid in comparison to genomic effects, as the processes of transcription and translation are unneeded. Given the evidence that one of the ways in which estradiol affects H3 acetylation is through non- genomic interactions with ERK (Zhao et al., 2010), it is plausible that AR may have similar interactions.

Given that fear conditioning is used both as an index of normal memory and of PTSD, we would glean further understanding of H2A.Z’s effects on memory by utilizing memory tests without an aversive component. One such test is the Object in Place (OiP) task. In this task, rodents are exposed to multiple objects within the testing arena during the training phase. Objects on one side of the arena are switched in location during the test phase, and amount of time spent exploring the objects is scored. Increased time spent exploring the side with the switched objects indicates successful task completion. This is an excellent hippocampal-dependent task that would test for memory without the confounding variable of negative emotional valence. In fact, our lab is currently undertaking this task and will soon be able to elucidate the effects of H2A.Z deletion on hippocampal dependent location memory.

As we have recently identified H2A.Z as a negative modulator of fear memory in area CA1 in male mice (Stefanelli, Azam, et al., 2018; Zovkic et al., 2014), it is a potential therapeutic target for treating memory-related disorders. This is particularly relevant knowing that H2A.Z accumulates in the hippocampus with age, and is correlated with age-related memory decline (Stefanelli, Azam, et al., 2018). Elucidating its role in affecting memory in both males and females and, further, the mechanisms through which it affects memory in both, is vital to further understanding the role of epigenetic factors in affecting fear memory, and, further, whether they can be utilized in a clinical setting to develop more precise treatments for patients.

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